the prokaryotes || the family streptomycetaceae, part i: taxonomy

CHAPTER 1.1.7The Family Streptomycetaceae, Part I: Taxonomy

The Family Streptomycetaceae, Part I: Taxonomy

PETER KÄMPFER

Phylogeny and Taxonomy

The family Streptomycetaceae was created byWaksman and Henrici (1943). Originally thisfamily harbored only the type genus Streptomy-ces. Zhang et al. (1997) proposed that the genusKitasatospora be included, and recently, a thirdgenus, Streptacidiphilus, was added (Kim et al.,2003).

Description of the family StreptomycetaceaeWaksman and Henrici 1943 emend, Kim et al.(2003) (Strep.to.my.ce.ta’ce.ae. ending to denotea family; M.L. masc. n. Streptomyces, type genusof the family) is based on data taken from Will-iams et al. (1989), Zhang et al. (1997) and Kimet al. (2003). These aerobic, Gram-positive, non-acid-alcohol fast actinomycetes form an exten-sively branched substrate mycelium that rarelyfragments. The aerial mycelium forms chains ofthree to many spores. Members of a few speciesbear short chains of spores on the substratemycelium. The organisms produce a wide rangeof pigments responsible for the color of thesubstrate and aerial mycelium. The organismsgrow within different pH ranges, namely 5.5–9(Kitasatospora), 5–11.5 (Streptomyces), and 3.5–6.0 (Streptacidiphilus). They are chemoorgan-otrophic with an oxidative type of metabolism.The substrate mycelium contains either LL-(Streptacidiphilus and Streptomyces) or meso-(Kitasatospora) diaminopimelic acid as thepredominant diamino acid; aerial or submergedspores contain LL-diaminopimelic acid. Inwhole-organism sugar profiles, either majoramounts of galactose or galactose and rhamnose(Kitasatospora and Streptacidiphilus) can bedetected. Lipid profiles typically contain hexa-and octa-hydrogenated menaquinones with nineisoprene units as the predominant isopreno-logues. The polar lipid profiles are composedof diphosphatidylglycerol, phosphatidylethano-lamine, phosphatidylinositol, and phosphatidyli-nositol mannosides. Fatty acids are complexmixtures of saturated, iso- and anteiso-fattyacids. Mycolic acids are not present. The mol%G

+ C of the DNA ranges generally between 66and 74%. Members of all three taxa are widelydistributed in terrestrial habitats, especially soil.

Very few species are pathogens for animals(including man) and plants.

A phylogenetic tree showing selected repre-sentatives of all three genera (all species of Strep-tacidiphilus and Kitasatospora and selectedStreptomyces “species”) representing the clustersof the numerical taxonomic study of Williams etal. (1983a) is shown in Fig. 1. The genera aredifficult to differentiate on the basis of phenotypicfeatures (including chemotaxonomic markers).Some characteristic features are shown in Table 1.

History

Early investigations of actinomycetes, includingstreptomycetes, were dominated by a strongemphasis of morphology and the high degree ofmorphological diversity was subsequently con-sidered to be sufficient for their assignment togenera and families (Waksman, 1961; Cross andGoodfellow, 1973). A short summary of earlyclassification systems of actinomycetes is given inIntroduction to the Classification of the Actino-myces in this Volume. Streptomycetes are theproducers of more than 5000 known bioactivecompounds (Anderson and Wellington, 2001),and estimates of the total number of antimicro-bial compounds produced by representatives ofStreptomyces screened for new antibiotics are ofthe order of 100,000 (Watve et al., 2001). In addi-tion, not only has the overall versatility of thesecompounds been studied in great detail, but alsoa high proportion of them have known biologicaleffects, which is unparalleled in the living world(Kieser et al., 2000).

The family Streptomycetaceae was originallyproposed by Waksman and Henrici (1943) andcontained at that time only two genera: the genusStreptomyces and the genus Micromonospora.Streptomyces was described as “Streptomyceta-ceae,” forming spores in chains on aerial hyphae.Spores are apparantly endogeneous in origin,formed by a segregation of protoplasm withinthe hyphae into a series of round oval or cylin-drical bodies. Chains of spores are often spirallycoiled. Sporophores may be simple or branched(Waksman and Henrici, 1943). Figure 2 showsthe morphology of the aerial mycelium of

Prokaryotes (2006) 3:538–604DOI: 10.1007/0-387-30743-5_22

CHAPTER 1.1.7 The Family Streptomycetaceae, Part I: Taxonomy 539

three streptomycetes. Although they are impor-tant (see Table 2 of The Family Nocardiopsaceaein this Volume), morphological differencesbetween members of the actinomycete genera donot represent the extensive diversity of generaand species of the sporoactinomycetes.

Beginning in the 1950s, new developments inthe application of numerical phenetics, numeri-cal taxonomy, chemosystematics, and finallymolecular systematics revolutionized the classifi-cation of actinomycetes. These developments

have been excellently reviewed for actinomycetesystematics by Goodfellow et al. (1999).

The determination of major cell wall sugarsand peptidoglycan composition (Lechevalierand Lechevalier, 1970; Schleifer and Kandler,1972) led to a classification system of well-characterized chemotypes and peptidoglycantypes. The pioneering work of Lechevalier andcoworkers (Becker et al., 1964; Lechevalier andLechevalier, 1970) clearly showed that Strepto-myces and the other genera of the family Strep-

Fig. 1. Phylogenetic analysis based on 16S rRNA gene sequences available from the European Molecular Biology Laboratorydata library (accession numbers are given in brackets) constructed after multiple alignment of data. Calculations of distances(distance options according to the Kimura-2 model) and clustering with maximum parsimony method were performed usingthe software package Mega (Molecular Evolutionary Genetics Analysis) version 2.1. Bootstrap values based on 1000 repli-cations are listed as percentages at the branching points.

A B C D E F G H I J K

1448bp v DSM 40277 ATCC 25493 ISP 5277 A 18 I 11 009 1-19 FU-1 L2

1448bp v DSM 40206 ATCC 15870 ISP 5206 A 18 I 11 009 1-19 L2


1483bp DSM 40547 ATCC 27466 ISP 5547 A 38 009 1-19

1449bp v DSM 40228 ATCC 23871 ISP 5228 A 18 I 11 009 1-19 FU-1 L2

1450bp v DSM 40185T ATCC 14925 ISP 5185 A 18 I 11 061 1-22 La12 L2


1448bp DSM 40227 ATCC 11416 ISP 5227 A 18 009 1-19 FU-1


1448bp v DSM 40129T ATCC 19743 ISP 5129 A 18 I 11 009 1-19 FU-1 L2

1448bp v DSM 40310T ATCC 25489 ISP 5310 A 18 I 11 009 1-19 L2


1519bp v DSM 40003T ATCC 23875 ISP 5003 A 12 I 07 006 1-18 KA-F


1450bp DSM 40531T ATCC 27457 ISP 5531 A 18 046 1-19 La14 L2

1448bp v DSM 40154 ATCC 19904 ISP 5154 A 18 I 11 1-7 1-19


1476bp v DSM 40394 ATCC 25495 ISP 5394 A 1A I 01 1-1 1-1 KA-D

1476bp v DSM 40233T ATCC 23899 ISP 5233 A 1A I 01 1-1 1-1 FU-1 KA-D

1476bp v DSM 40455T ATCC 25422 ISP 5455 A 1A I 01 1-1 1-1 un FU-1 OC-non KA-D

1476bp v DSM 40131T ATCC 19778 ISP 5131 A 1A I 01 1-1 1-1 FU-1 KA-D

1476bp v DSM 40001T ATCC 19736 ISP 5001 A 1A I 01 1-1 1-1 KA-D

1448bp v DSM 40499T ATCC 14511 ISP 5499 A 18 I 11 1-5 011 L2

1476bp v DSM 40347 ATCC 6246 ISP 5347 A 1A I 01 1-1 1-1 KA-D

1448bp v DSM 40133T ATCC 19804 ISP 5133 A 18 I 11 009 1-19 FU-12a L2


1485bp v DSM 40546T ATCC 27451 ISP 5546 A 34 III 06 1-5 009 La14 OC-III

1531bp v DSM 40014 ATCC 14975 ISP 5014 A 23 I 20 1-5 009



1474bp v DSM 40372T ATCC 3329 ISP 5372 A 1A I 01 1-1 1-1 La3 KA-D

1520bp v DSM 40564 ATCC 27420 ISP 5564 A 39 II 10 052 017 L2

1486bp v DSM 40579T ATCC 23385 ISP 5579 A 36 II 09 021 002

1358bp v DSM 40313T ATCC 3004 ISP 5313 A 16 I 09 032 027 FU-6 OC-non

1518bp v DSM 40221T ATCC 23934 ISP 5221 F 55 Sv. 03 22-1 040 L4

1489bp v DSM 40268 ATCC 25184 ISP 5268 B 42 I 19 035 1-33

1461bp v DSM 40187 ATCC 23904 ISP 5187 A 32 I 16 041 012 FU-6

1483bp v DSM 40318 ATCC 25473 ISP 5318 A 32 I 16 051 018 L1

1488bp v DSM 40492 ATCC 12757 ISP 5492 A 29 I 15 025 109


1449bp v DSM 40090T ATCC 19775 ISP 5090 A 18 I 11 016 1-19 La21 L2

1484bp v DSM 40494 ATCC 19006 ISP 5494 C 45 II 13 1-7 1-15



1448bp DSM 40432 ATCC 19842 ISP 5432 A 18 009 1-19 L2

1448bp DSM 40140 ATCC 23614 ISP 5140 A 18 009 1-19 L2

1473bp v DSM 40127T ATCC 10762 ISP 5127 A 14 II 04 22-4 043 OC-I




1450bp v DSM 40073T ATCC 3338 ISP 5073 A 40 I 18 009 0-19 La1 OC-II

1531bp v DSM 40262T ATCC 25435 ISP 5262 A 19 I 12 009 1-19

1448bp v DSM 40013 ATCC 19748 ISP 5013 A 18 I 11 1-6 1-10 FU-1 L2

1329bp v DSM 40069T ATCC 8664 ISP 5069 F 61 I 22 22-3 042 FU-12b OC-I L3/L5

1516bp v DSM 40445 ATCC 27467 ISP 5445 F 61 22-3 042 L5

1444bp v DSM 40108T ATCC 14923 ISP 5108 A 18 009 1-19 L2

1484bp v DSM 40226 ATCC 11009 ISP 5226 A 15 I 08 1-5 011 KA-B

1485bp v IFO 13550

1532bp v DSM 40395 ATCC 25497 ISP 5395 A 1B I 02 1-3 1-2 KA-B

1518bp v DSM 40323T ATCC 25452 ISP 5323 A 1C I 03 1-2 015 FU-19b KA-B

1426bp v DSM 40163T ATCC 19913 ISP 5163 H Sm III 23 22-3 1-08 un OC-II

1517bp v DSM 40581T ATCC 11062 ISP 5581 F 64 III 21 22-4 1-07 un OC-II

1483bp v DSM 40230T ATCC 10712 ISP 5230 A 06 I 05 002 1-7 un KA-C

A length of the 16S rRNA partial sequence B v = name if validly published C DSMZ: Deutsche Sammlung von Mikroorganismen und Zellkulturen, Braunschweig, Germany; ATCC: American Type Culture Collection Rockville, MD, U.S.A.; IFO: Institute of Fermentation, Osaka, Japan; ISP = Inernational Streptomyces Project Number; T = Type strain. D Cluster and subcluster numbers according to Williams et al., (1983a) E Assignment of strains to species according to Bergey´s Manual of Systematic Bacteriology, Vol. 4. For the genus Streptomyces, species categories I to IV are shown, with the species number within the category according to Williams et al. (1989), for species category IV the ´series´assignment is also given, according to spore color. For species previously assigned to the genus Streptoverticillium (now reclassified to Streptomyces, species numbers are according to Locci and Schofield (1989). F Cluster and subcluster numbers according to Kämpfer et al. (1991). The first number is that of the UPGMA/SSM analysis, the second number that to the UPGMA/SJ analysis. G Clusters according ot Lanoot et al. (2002) on the basis of protein profiles. H Cluster number according to Fulton et al. (1995)on the basis of fingerprints of the 16S rRNA operons. I Groups based on primary structures of N termini of AT-L30 proteins according to Ochi (1995). J Clusters based on the phylogenetic tree constructed from the 120 bp alpha-region according to Kataoka et al. (1997). Meanwhile more than 450 Streptomyces 120 bp alpha regions have been sequences. K Strains included into DNA-DNA hybridization studies: L1: Labeda and Lyons (1991a), L2: Labeda and Lyons (1991b); L3: Labeda (1998); L4: Labeda (1996); L5: Labeda (1993).

Streptomyces roseoviolaceus (AJ399484)

Streptomyces janthinus (AJ399478)

Streptomyces luteogriseus (AJ399490)

Streptomyces steffisburgensis (AB045889)

Streptomyces afghaniensis (AJ399483)

Streptomyces bellus (AJ399476)

Streptomyces coerulescens (AJ399462)

Streptomyces thermotolerans (AJ399482)

Streptomyces iakyrus (AJ399489)

Streptomyces collinus (AJ306623)

Streptomyces purpurascens (AB045888)

Streptomyces caelestis (AJ399467)

Streptomyces albogriseolus (AJ494865)

Streptomyces coeruleorubidus (AJ306622)

Streptomyces pallidus (AJ399492)

Streptomyces fumanus (AJ399463)

Streptomyces arenae (AJ399485)

Streptomyces sampsonii (D63871)

Streptomyces coelicolor (Z76678)

Streptomyces albidoflavus (Z76676)

Streptomyces limosus (Z76679)

Streptomyces canescens (Z76684)

Streptomyces griseochromogenes (AJ399491)

Streptomyces odorifer (Z76682)

Streptomyces resistomycificus (AJ310926)

Streptomyces coeruleofuscus (AJ399473)

Streptomyces nogalater (AB045886)

Streptomyces eurythermus (D63870)

Streptomyces gougerotii (Z76687)

Streptomyces rutegersensis ssp. rutgersensis (Z76688)

Streptomyces intermedius (Z76686)

Streptomyces bluensis (X79324)

Streptomyces thermonitrificans (Z68098)

Streptomyces albus (X53163)

Streptomyces mashuense (X79323)

Streptomyces albofaciens (AB045880)

Streptomyces hygroscopicus (AJ391821)

Streptomyces melanosporofaciens (AJ391837)

Streptomyces albulus (AB024440)

Streptomyces cinnabarinus (AJ399487)

Streptomyces lanatus (AJ399469)

Streptomyces capoamus (AB045877)

Streptomyces curacoi (AJ399471)

Streptomyces longisporus (AJ399475)

Streptomyces indigocolor (AJ399464)

Streptomyces bicolor (AJ276569)

Streptomyces aureofaciens (bobili) (AB045876)

Streptomyces chartreusis (AJ399468)

Streptomyces pseudovenezuelae (AJ399481)

Streptomyces griseorubiginosus (AJ399488)

Streptomyces phaeochromogenes (AF500071)

Streptomyces bottropensis (AB026217)

Streptomyces echinatus (AJ399465)

Streptomyces lavendulae ssp. lavendulae (D85116)

Streptomyces subrutilus (X80825)

Streptomyces cyaneus (AJ399460)

Streptomyces argenteolus (AB045872)

Streptomyces griseus ssp. griseus (AF056711)

Streptomyces setonii (D63872)

Streptomyces flavogriseus (AJ494864)

Streptomyces lateritius (AF454764)

Streptomyces bikiniensis (X79851)

Streptomyces venezuelae (AB045890)

Streptacidiphilus albus (AF074415)

Streptacidiphilus carbonis (AF074412)

Streptacidiphilus neutrinimicus (AF074410)

Kitasatosporia azatica (U93312)

Kitasatosporia mediocidica (U93324)

Kitasatospora cystarginea (AB022872)

Kitasatosporia cochleata (AB022871)

Kitasatosporia paracochleata (U93328)

Kitasatosporia griseola (AB022870)

Kitasatosporia phosalacinea (AB022869)

Kitasatosporia cheerisanensis (AF050493)

Kitasatosporia setae (AB022868)

Kitasatospora cineracea (AB022875)

Kitasatospora niigatensis (AB022876)

63

89

25

99

63

28

48

78

48

55

89

43

73

45

58

64

42

52

30

70

21

47

25

18

53

35

43

78

61

70

68

58

99

78

28

31

55

19

3

18

42

5

67

9

1

10

25

1

1

6

16

83

37

28

21

10

540 P. Kämpfer CHAPTER 1.1.7

Table 1. Chemotaxonomic, morphological and physiological characteristics of Kitasatospora, Streptacidiphilus and Strepto-myces strains.

Symbols and abbreviations: +, present; 2c, fatty acid group sensu Kroppenstedt (1985); DPG, diphosphatidylglycerol; PE,phosphatidylethanol; PI, phosphatidylinositol; PIMs, phosphatidylinositol mannosides; MK-9(H6, H8), hexa-and octa-hydro-genated menaquinones with nine isoprene units; and ND, not determined.aRhamnose was detected in whole-organism hydolysates of Kitasatospora mediocidica (Labeda, 1988).bAerial and submerged spores contain ll-A2pm and vegetative mycelia meso-A2pm.cAlkalophilic strains, which grow between pH 8.0 and 11.5, have an optimum at pH 9–9.5 (Mikami et al., 1982).From Kim et al. (2003) and the previous studies of Shirling and Gottlieb (1976), mura et al. (1989), Lonsdale (1985),Williams et al. (1989) and Nakagaito et al. (1992).

Characteristics Streptomyces Kitasatospora Streptacidiphilus

Long chains of spores formed on aerial hyphae

+ + +

Major menaquiones MK-9(H6, H8) MK-9(H6, H8) MK-9(H6, H8)Predominant phospholipids DPG, PE, PI, and PIMs DPG, PE, PI, and PIMs DPG, PE, PI, and PIMsDiagnostic sugars in whole-organism

hydrolysatesNone Galactosea Galactose and rhamnose

Fatty acid patternd 2c 2c 2cG

+C content of DNA (mol %) 66–73 70–74 70–72Isomer(s) of diaminopimelic acids in whole-

organism hydrolysatesLL-A2pm LL-/mesoA2pm

b LL-A2pm

Optimal pH range 6.5–8.0c ND 4.5–5.5pH range for growth 5.0–11.5 5.5–9.0 3.5–6.0

O

A

C

B

Fig. 2. Morphology of the aerial mycelium of three strepto-mycetes. (A) A Streptomyces species: sympodially branchedaerial hyphae; spore chains form spirals with up to 10 turns.(B) A Streptoverticillium species: spore chains arranged intypical verticils along straight, long aerial hyphae; the end ofthe spore chain is sometimes hook-like or forms one to twoturns. (C) “Streptomyces pallidus”: despite the verticil-likearrangement of spore chains, this organism was described asStreptomyces by Shirling and Gottlieb (1972). All photos:

×250.


Tabl

e 2.

Key

bio

chem

ical

mar

kers

of

Stre

ptom

ycet

acea

e ge

nera

and

som

e ot

her

sele

cted

act

inom

ycet

e ge

nera

(be

long

ing

to d

iffe

rent

fam

ilies

) pr

oduc

ing

an a

eria

l myc

eliu

m.a

Sym

bols

and

abb

revi

atio

ns:

+,pr

esen

t;

-,no

t ap

plic

able

for

ll-

A2p

m;I

PB

; int

erpe

ptid

e br

idge

; A, a

rabi

nose

and

gal

acto

se; B

, mad

uros

e; C

, no

diag

nost

ic s

ugar

s; D

, ara

bino

se a

nd x

ylos

e; E

,rh

amno

se a

nd g

alac

tose

; PI,

phos

phat

idyl

glyc

erol

(va

riab

le);

PII

, onl

y ph

osph

atid

yeth

anol

amin

e, P

III,

phos

phat

idyl

chol

ine

(wit

hph

osph

atid

ylet

hano

lam

ine,

pho

spha

tidy

lmet

hyle

than

ola-

min

e an

d ph

osph

atid

ylgl

ycer

ol v

aria

ble

and

no p

hos p

holip

ids

cont

aini

ng g

luco

sam

ine)

; PIV

, pho

spho

lipid

s co

ntai

ning

glu

cosa

min

e (w

ith

phos

phat

idyl

etha

nola

min

e an

d ph

osph

atid

ylm

eth-

ylet

hano

lam

ine

vari

able

); M

enaq

uino

nes:

num

ber

indi

cate

s nu

mbe

r of

isop

rene

uni

ts, H

xin

dica

tes

pres

ence

of

x hy

drog

enat

ed m

enaq

uino

nes;

and

ND

, not

det

erm

ined

.a T

he g

enus

Kin

eosp

oria

does

not

pro

duce

aer

ial m

ycel

ium

.b D

ata

from

Goo

dfel

low

(19

89).

c Dat

a fr

om L

eche

valie

r et

al.

(197

7, 1

981)

.d D

ata

from

Kro

ppen

sted

t (1

985)

.e D

ata

from

Kro

ppen

sted

t (1

987)

and

R. K

ropp

enst

edt

(per

sona

l com

mun

icat

ion)

.f D

ata

from

Bow

en e

t al

. (19

89).

g For

deta

ils, s

ee I

ntro

duct

ion

to t

he C

lass

ifica

tion

of

the

Act

inom

yce s

.h D

ata

from

Ito

h et

al.

(198

9).

i Dat

a fr

om R

. Kro

ppen

sted

t (p

erso

nal c

omm

unic

atio

n).

j Dat

a fo

r m

enaq

uino

nes

from

How

arth

et

al. (

1986

).M

odifi

ed a

ccor

ding

to

Kor

n-W

endi

sch

and

Kut

zner

(19

92).

Fam

ily a

nd g

enus

Dia

min

opim

elic

acid

(A

2pm

)bG

lyci

nein

IP

Bb

Pep

tido

glyc

anty

peb

Suga

rty

peb

Pho

spho

lipid

type

cM

ycol

icac

idsb

Fatt

y ac

idpa

tter

ndM

enaq

uino

nese

G

+C c

onte

nt(m

ol%

)

Stre

ptom

ycet

acea

eK

itasa

tosp

ora

LL/m

eso

+A

3

gC

/EP

II

-2c

9(H

6)/(

H8)

66–7

3St

rept

omyc

esL

L

+A

3

g

-P

II

-2c

9(H

6)/(

H8)

69–7

8St

rept

acid

ophi

lus

LL

+A

3

gE

PII

-2c

9(H

6)/(

H8)

70–7

2P

seud

onoc

ardi

acea

eA

myc

olat

opsi

sm

eso

-A

1

gA

PII

-3f

9(H

4)(H

2)66

–69

Kib

delo

spor

angi

umf

mes

o

-A

1

gA

PII

-3f

ND

66P

seud

onoc

ardi

am

eso

-A

1

gA

PII

I

-2f

8(H

4)79

Sacc

haro

poly

spor

am

eso

-A

1

gA

PII

I

-2c

/3e

9(H

4)/1

0(H

4)/9

(H2)

70–7

2Sa

ccha

rom

onos

pora

mes

o

-A

1gA

PII

-2a

9(H

4)/8

(H4)

69–7

4A

ctin

opol

yspo

ram

eso

-A

1gA

PII

I-

2c9(

H6)

/9(H

4)64

Gen

era

belo

ngin

g to

diff

eren

t fa

mili

es o

fth

eA

ctin

obac

teri

ag

Spor

icht

hya

LL

+A

3g-

ND

-3a

9(H

6)/9

(H8)

ND

Kin

eosp

oria

hL

L/m

eso

(+)

ND

CP

III

-1

9(H

4)69

Noc

ardi

oide

sL

L+

A3g

-P

I-

3c8(

H4)

66–7

3A

ctin

omad

urai

mes

o-

A1g

BP

I-

3a9(

H6)

/(H

4)/(

H8)

66–7

2M

icro

tetr

aspo

rai

mes

o-

A1g

BP

IV-

3c9(

H4)

/(H

2)/(

H0)

66–6

9G

lyco

myc

esm

eso

+N

DD

PI

-2c

9(H

4)/1

0(H

4)71

–73

Sacc

haro

thri

xm

eso

-N

DC

/EP

II-

3f9(

H4)

/10(

H4)

70–7

6N

ocar

dij

mes

o-

Alg

AP

II+

1bcy

clo

8(H

4)/9

(H2)

64–7

2N

ocar

diop

sis

mes

o-

ND

CP

III

-3d

10(H

2)/(

H4)

/(H

6)64

–69

Stre

ptoa

llote

ichu

sm

eso

-N

DC

PII

-N

D9(

H6)

/10(

H6)

ND


tomycetaceae, proposed by then, contained LL-diaminopimelic acid (LL-A2pm) in its pepti-doglycan (cell wall type I), whereas meso-A2pmwas found in most of the other actinomycetesdescribed at that time. The genera containingLL-A2pm in their peptidoglycan contained aninterpeptide bridge composed of a glycine resi-due (type A3γ of Schleifer and Kandler, 1972).In addition to these chemical traits, which hadthe advantage of higher genetic stability incomparison with morphological features, thepattern of sugars in whole-cell hydrolysates(Lechevalier and Lechevalier, 1970), phospho-lipids (Lechevalier et al., 1977a), fatty acids(Kroppenstedt, 1985), menaquinones (Aldersonet al., 1985; Kroppenstedt, 1985), and acetylatedmuramic acid residues (Uchida and Aida, 1977)were shown to be of essential importance for theclassification of actinomycetes. Major chemotax-onomic markers of the genus Streptomyces andother actinomycete genera are given in Table 2.In combination with other phenotypic proper-ties, like physiological and biochemical charac-teristics, these traits were also helpful in defininggenera within the family Streptomycetaceae.This resulted in the reclassification of sixadditional genera (Actinopycnidium, Actinospo-rangium, Chainia, Elytrosporangium, Kitasatoaand Microellobosporia), described mainly on thebasis of morphological features, to the genusStreptomyces (Williams et al., 1983a; Goodfellowet al., 1986b; Goodfellow et al., 1986c; Goodfel-low et al., 1986d; Goodfellow et al., 1986e).

The application of 16S rRNA oligonucleotidecataloguing (Stackebrandt and Woese, 1981) andsubsequently the sequencing of the 16S rRNAgenes provided a basis for studies of the naturalrelationships among actinomycetes and relatedorganisms (for details, see Stackebrandt et al.,[1997] and Introduction to the Classification ofthe Actinomyces in this Volume). On the basis ofthese data, the description of the family Strepto-mycetaceae was emended by Wellington et al.(1992) and Witt and Stackebrandt (1990), whoproposed the unification of the genera Kita-satosporia and Streptoverticillium with the genusStreptomyces, and more recently by Stacke-brandt et al. (1997), who excluded the genusSporichthya. Zhang et al. (1997) demonstrated,however, that the genus Kitasatosporia formeda stable subbranch in Streptomyces, whensequences from the almost complete 16S rRNAgenes were compared. In addition, members ofthe genus Kitasatosporia can be distinguishedfrom Streptomyces by the ratio of meso-DAP toLL-DAP and the presence of galactose in whole-cell hydrolysates (Zhang et al., 1997; Table 1).The genera Kineosporia and Sporichthya, bothsharing chemotaxonomic similarities with mem-bers of the genus Streptomyces and considered

to be members of this genus (Logan, 1994), havebeen shown by 16S rRNA sequencing to be inde-pendent genera: Sporichthya is a member of thefamily Sporichthyaceae of the suborder Franki-neae (Stackebrandt et al., 1997), and the genusKineosporia is grouped together with Kineococ-cus (Kudo et al., 1998) into the tentative family“Kineococcaceae” (see Introduction to the Clas-sification of the Actinomyces in this Volume).Recently the genus Streptacidiphilus has beenproposed by Kim et al. (2003) to accommodateacidophilic actinomycetes forming a distinctclade within the family Streptomycetaceae.

Note that although 16S rRNA sequence anal-yses have provided a framework for prokaryoticclassification, the current classification systembased on this molecule has not yet solved thetaxonomic problems within the genera (espe-cially within the genus Streptomyces). Severalstudies have attempted to use sequence datafrom variable regions of 16S rRNA to establishtaxonomic structure within the genus, but thevariation is too limited to resolve problems ofspecies differentiation (see Witt and Stacke-brandt [1990], Stackebrandt et al., [1991], Stack-ebrandt et al., [1992], Anderson and Wellington[2001], and the references therein ).

The discovery of antibiotics produced bystreptomycetes in the 1940s, which led to exten-sive screening for novel bioactive compounds,and the subsequent need for patenting, whichled to an extreme overclassification of the genus,complicated the situation. Producers of novelnatural products were described as new speciesand patented. Species described within thegenus Streptomyces increased from approxi-mately 40 to over 3000 (Trejo, 1970). The currentstatus of streptomycete taxonomy including phy-logeny has been summarized by Anderson andWellington (2001) and will be treated briefly inthe next sections. Of the 539 species and subspe-cies listed under the List of Bacterial Nameswith Standing in Nomenclature as of December9, 2003, 376 are on the Approved lists (Tables 3and 4).

Genus Kitasatospora

Zhang et al. (1997) revived the genus Kita-satospora to accommodate actinomycete strainsforming a stable, separate subbranch on the basisof phylogenetic analyses within the family Strep-tomycetaceae and containing major amounts ofmeso-DAP in their whole-cell hydrolysates. Phy-logenetic trees were also constructed by using16S-23S rRNA gene spacers, leading to group-ings similar to those based on 16S rRNAsequence data (Zhang et al., 1997).

The substrate mycelium of members of Kita-satospora is as well developed as the Streptomy-


ces substrate mycelium. The aerial myceliumbears long spore chains with more than 20spores. Galactose is present in whole-cellhydrolysates of Kitasatospora. Specific nucle-otide signatures in the sequences of both 16SrRNA and 16S-23S rRNA gene spacers can dif-ferentiate Kitasatospora from Streptomyces (fordetails, see Zhang et al., 1997); however, pheno-typic differences between Streptomyces and Kita-satospora are not pronounced so that theseparate genus status of Kitasatospora may bequestioned.

To date, eleven species of the genus Kita-satospora have been recognized: Kitasatosporasetae (Omura et al. 1982), Kitasatospora phosa-lacinea (Takahashi et al., 1984a), Kitasatosporagriseola (Takahashi et al., 1984a), Kitasatosporamediocidica (Labeda, 1988), Kitasatosporacystarginea (Kusakabe and Isono, 1988), Kita-satospora cochleata (Nakagaito et al., 1992b;Zhang et al., 1997), Kitasalospora paracochleata(Nakagaito et al., 1992b; Zhang et al., 1997),Kitasatospora azatica (Nakagaito et al., 1992b;Zhang et al., 1997), Kitasalospora cheerisanensis(Chung et al., 1999), Kitasatospora cineracea(Tajima et al., 2001) and Kitasatospora niigaten-sis (Tajima et al., 2001).

The designations of some species to this genusare open to discussion. Kitasatospora cys-targinea, Kitasatospora griseola, Kitasatosporamediocidica, Kitasatospora phosalacinea andKitasatospora setae are synonyms of Streptomy-ces cystargineus, Streptomyces griseolosporeus,Streptomyces mediocidicus, Streptomyces phosa-lacineus and Streptomyces setae, respectively.For these species, and according to scientificopinion, an author may use Kitasatospora orStreptomyces. See the List of Bacterial Nameswith Standing in Nomenclature for detailedcomments.

Genus Streptacidophilus

The genus Streptacidophilus was proposed byKim et al. (2003) to accommodate acidophilicactinomycetes isolated from acidic soils and litter.On the basis of 16S rRNA sequence analysis, itcould be shown that the 11 isolates formed astable clade within the family Streptomycetaceae.These organisms showed a distinctive pH profile,showed a unique 16S rDNA signature, and con-tained major amounts of LL-diaminopimelicacid, galactose and rhamnose in whole-cellhydrolysates (Kim et al., 2003). The members ofthe genus form an extensively branched, nonfrag-menting mycelium carrying long chains of sporesin aerial mycelia at maturity (Kim et al., 2003).To date, three species have been recognized:Streptacidophilus albus, Streptacidophilus neu-trinimicus and Streptacidophilus carbonis.

Similar to Kitasatospora, Streptomyces andStreptacidophilus have no pronounced pheno-typic differences, so that a separate genus statusalso of Streptacidophilus may be questioned.

Genus Streptomyces

The genus Streptomyces Waksman and Henrici(1943) is the type genus of the family. Most ofthe general characteristics described below alsoapply to members of the genera Kitasatosporaand Streptacidiphilus, unless stated otherwise.

General Characteristics Streptomycetes areGram-positive aerobic members of the orderActinomycetales within the class Actinobacteria(Stackebrandt et al., 1997) and have a DNA G+Ccontent of 69 ± 78 mol%. The vegetative hyphae(0.5–2.0 µm in diameter) produce an extensivelybranched mycelium that rarely fragments. Theaerial mycelium at maturity forms chains ofthree to many spores. Some species may bear

Table 3. Studies on numerical classification of streptomycetes.

Abbreviation: ISP, International Streptomyces Project.aFourteen and 168 were obtained by the Wroclaw taxonomy method (dendrite method); 21 and 37 were obtained by thecentrifugal correlation method. Modified according to Korn-Wendisch and Kutzner (1992).

Nature of materialNumber of

strains

Number ofcharacters(features)

Numberof clusters

Number ofunclustered

strains References

“Species” 159 105 24 16 Silvestri et al., 1962Isolates 18 46 5 Williams et al., 1969ISP “species” 448 31 14/21 168/37 Kurylowicz et al., 1975a

ISP “species” 618 24 15 218 Gyllenberg, 1976Streptomyces with verticils and

pseudoverticils, formerlyStreptoverticillium

111 185 24 Locci et al., 1981

394 ISP species plus others 475 139 73 28 Williams et al., 1983a394 ISP species plus others 821 329 15 (major) 40 Kämpfer et al., 1991

34 (minor)

Tabl

e 4.

Stre

ptom

yces

spec

ies

liste

d in

alp

habe

tica

l ord

er in

clud

ed in

com

preh

ensi

ve t

axon

omic

stu

dies

sin

ce 1

980.

Spec

ies

nam

e

Stra

in n

umbe

r(s)

Clu

ster

num

bers

of

num

eric

al t

axon

omic

stud

ies

and

Ber

gey

cate

gori

es a

ndsp

ecie

s no

.aG

roup

s ac

cord

ing

to d

iffe

rent

stu

dies

abp

rRN

A d

ata

stra

in n

o.if

dif

fere

nt f

rom

the

first

col

umn

Acc

. no.

(EM

BL

)b

Stra

ins

incl

uded

inD

NA

/DN

Ahy

brid

isat

ion

stud

iesc

12

34

56

78

9

S.ab

ikoe

nsis

vD

SM40

831T

NR

RL

B-2

113T

Ha1

1358

bpX

5316

8L

4S.

abur

avie

nsis

vD

SM40

033T

AT

CC

2386

9IS

P50

33A

02II

0122

-304

3un

OC

-I12

0bp

JCM

4613

D44

265

S.ac

hrom

ogen

esv

DSM

4002

8A

TC

C12

767

ISP

5028

A19

I12

1-1

009

FU

-1K

A-B

120b

pJC

M45

61D

4423

2S.

achr

omog

enes

ssp

.ac

hrom

ogen

esv

S.ac

hrom

ogen

es s

sp.

rubr

adir

isv

DSM

4078

9N

RR

L30

61I

1202

800

9

S.ac

idis

cabi

esv

DSM

4166

8A

TC

C49

003

1530

bpJC

M79

13D

6386

5S.

acid

omyc

etic

usD

SM40

798

AT

CC

1161

122

-304

2D

8510

6S.

acid

ores

ista

nsD

SM40

540

AT

CC

2741

3IS

P55

401-

31-

2S.

acri

myc

ini

vD

SM40

135T

AT

CC

1988

5IS

P51

35IV

04 (

gree

n se

ries

)1-

301

012

0bp

JCM

4339

D44

060

S.ac

tuos

usD

SM40

337

AT

CC

2542

1IS

P53

371-

71-

19L

2S.

acul

eola

tus

vD

SM41

644

S.af

ghan

iens

isv

DSM

4022

8A

TC

C23

871

ISP

5228

A18

I11

009

1-19

FU

-114

49bp

AJ3

9948

3L

2S.

alan

osin

icus

vD

SM40

606T

AT

CC

1571

0IS

P56

06IV

01 (

gray

ser

ies)

009

1-19

120b

pJC

M47

14D

4430

4S.

alba

dunc

usv

DSM

4047

8TA

TC

C14

698

ISP

5478

IV02

(gr

ay s

erie

s)00

61-

1012

0bp

JCM

4715

S.al

biax

ialis

vD

SM41

799

S.al

bido

chro

mog

enes

vD

SM41

800

S.al

bido

flavu

sv

DSM

4045

5TA

TC

C25

422

ISP

5455

A1A

I01

1-1

1-1

unF

U-1

OC

-non

KA

-D14

76bp

Z76

676

S.al

bido

chro

mog

enes

vD

SM40

880

I01

020

032

Z76

683

S.al

bidu

sD

SM40

320

AT

CC

2542

31-

31-

2S.

albi

dus

DSM

4079

3N

RR

LB

-167

2IS

P53

20A

1B1-

31-

2S.

albi

dus

DSM

4086

91-

11-

1S.

albi

flavi

nige

rN

RR

LB

-135

6T14

66bp

AJ3

9181

2S.

albi

retic

uli

vD

SM40

051T

AT

CC

1972

1IS

P50

51F

SMv.

1107

606

9H

a512

0bp

JCM

4116

D44

009

S.al

boci

nere

scen

sD

SM40

794

NR

RL

3419

002

1-7

S.al

bocy

aneu

sD

SM40

197

AT

CC

1584

5IS

P51

97A

Sm00

700

3S.

albo

faci

ens

vD

SM40

268

AT

CC

2518

4IS

P52

68B

42I

1903

51-

3314

89bp

JCM

4342

AB

0458

80S.

albo

flavu

sv

DSM

4004

5TA

TC

C12

626

ISP

5045

E54

III

2003

31-

33un

OC

-IV

120b

pJC

M46

15D

4426

6S.

albo

flavu

sv

DSM

4076

1N

CIB

9453

III

2003

51-

33S.

albo

gris

eolu

sv

DSM

4000

3TA

TC

C23

875

ISP

5003

A12

I07

006

1-18

KA

-F15

19bp

AJ4

9486

5S.

albo

helv

atus

DSM

4041

0A

TC

C19

820

ISP

5410

22-3

1-08

S.al

bolo

ngus

vD

SM40

570

AT

CC

2741

4IS

P55

70F

63II

1522

-404

312

0bp

JCM

4716

D44

306

L4

S.al

boni

ger

vD

SM40

043T

AT

CC

1246

1IS

P50

43A

1BI

021-

61-

31S.

albo

rubi

dus

DSM

4046

5A

TC

C23

612

ISP

5465

A12

066

034

S.al

bosp

inus

vD

SM41

422

IV03

(gr

ay s

erie

s)01

31-

19S.

albo

spor

eus

vD

SM40

795T

AT

CC

1539

4IV

01 (

red

seri

es)

063

049

La1

120b

pJC

M41

35D

4401

3S.

albo

spor

eus

ssp .

albo

spo

v12

0bp

JCM

4135

D44

013

S.al

bosp

oreu

s ss

p .la

bilo

mv

DSM

4167

2

S.al

bove

rtic

illat

usv

DSM

4167

8TH

a6S.

albo

vina

ceus

vD

SM40

136T

AT

CC

1582

3IS

P51

36A

1BI

021-

300

8K

A-B

120b

pJC

M43

43D

4406

3S.

albo

viri

dis

vD

SM40

326

AT

CC

2542

5IS

P53

26A

1BI

021-

31-

2K

A-B

120b

pJC

M44

49D

4414

6

S.al

bulu

sv

DSM

4049

2A

TC

C12

757

ISP

5492

A29

I15

025

109

1488

bpA

B02

4440

S.al

bus

vD

SM40

313T

AT

CC

3004

ISP

5313

A16

I09

032

027

FU

-6O

C-n

on13

58bp

X53

163

S.al

bus

vD

SM40

652

I09

030

1-34

S.al

bus

vD

SM40

763

IMR

U38

88I

0903

01-

34S.

albu

sv

DSM

4078

5I

0906

603

4S.

albu

sv

DSM

4083

2N

RR

L24

90I

0901

402

1S.

albu

sv

DSM

4089

0I

091-

11-

114

76bp

Z76

689

S.al

bus

vD

SM40

946

I09

030

1-34

S.al

bus

vD

SM40

947

I09

030

1-34

S.al

bus

vD

SM40

948

I09

030

1-34

S.al

bus

vD

SM40

949

I09

030

1-34

S.al

bus

vD

SM40

950

I09

22-5

039

S.al

bus

vD

SM40

951

I09

030

1-34

S.al

bus

vD

SM40

963

I09

030

1-34

S.al

bus

vD

SM40

964

I09

030

1-34

S.al

bus

vD

SM40

965

I09

030

1-34

S.al

bus

ssp.

alb

usv

1485

bpJC

M10

204

AB

0458

84S.

albu

s ss

p. p

atho

cidi

cus

vS.

alm

quis

tiiv

DSM

4044

7A

TC

C61

8IS

P54

47A

I09

030

1-34

121b

pJC

M44

51D

4414

8S.

alni

DSM

4055

7A

TC

C27

415

ISP

5557

A16

1-1

1-1

S.al

thio

ticus

vD

SM40

092

AT

CC

1972

4IS

P50

92A

08I

0700

61-

1812

1bp

JCM

4344

AB

0182

05S.

amak

usae

nsis

vD

SM40

219T

AT

CC

2387

6IS

P52

19B

12II

I12

079

063

unO

C-I

120b

pJC

M46

17D

4426

8L

2S.

ambo

faci

ens

vD

SM40

053T

AT

CC

2387

7IS

P50

53A

SmI

2000

61-

18F

U-6

121b

pJC

M46

18D

4426

9S.

amin

ophi

lus

vD

SM40

186T

AT

CC

1496

1IS

P51

86A

16I

0903

11-

34L

a512

1bp

JCM

4275

D44

040

S.an

andi

iv

DSM

4053

5A

TC

C19

388

ISP

5535

B42

I19

021

1-05

S.an

thoc

yani

cus

vD

SM41

422T

121b

pJC

M50

58D

4442

7S.

antib

iotic

usv

DSM

4023

4TA

TC

C86

63IS

P52

34A

31I

211-

71-

15O

C-I

V12

1bp

JCM

4620

D44

270

S.an

timyc

otic

usv

DSM

4028

4TA

TC

C23

880

ISP

5284

IV05

(gr

ay s

erie

s)05

101

812

1bp

JCM

4228

D44

034

S.an

ulat

usv

DSM

4036

1TA

TC

C27

416

ISP

5361

A1B

I02

047

1-35

La2

2O

C-I

KA

-B12

0bp

JCM

4721

D44

309

S.ar

abic

usv

DSM

4025

2A

TC

C23

881

ISP

5225

A12

I07

006

1-18

121b

pJC

M46

22D

4427

1S.

ard

usv

DSM

4052

7A

TC

C27

417

ISP

5527

Sv.0

322

-104

0H

a2L

4S.

ard

usv

DSM

4052

5IS

P55

2512

0bp

JCM

4543

D44

223

S.ar

enae

vD

SM40

293T

AT

CC

2542

8IS

P52

93A

18I

1100

91-

1914

47bp

AJ3

9948

5L

2S.

arge

nteo

lus

vD

SM40

226

AT

CC

1100

9IS

P52

26A

15I

081-

501

1K

A-B

1484

bpJC

M46

23A

B04

5872

S.ar

men

iacu

sv

DSM

4312

5T15

32bp

JCM

3070

AB

0180

92S.

asch

abad

icus

NR

RL

B-5

643

L2

S.as

iatic

usv

DSM

4176

1T14

83bp

A14

P1

AJ3

9183

0S.

aspe

rgill

oide

sSM

4056

5A

TC

C14

808

ISP

5565

F59

Sv.0

822

-104

0H

a13

S.as

tero

spor

usv

S.at

ratu

sv

DSM

4167

3T12

0bp

JCM

3386

D43

986

S.at

roau

rant

iacu

sv

S.at

rofa

cien

sD

SM40

475

AT

CC

2741

8IS

P54

75A

3300

91-

09S.

atro

oliv

aceu

sv

DSM

4013

7TA

TC

C19

725

ISP

5137

A03

II20

006

0-10

La2

3O

C-I

S.at

rovi

rens

v

S.au

rant

iacu

sv

DSM

4041

2TA

TC

C19

822

ISP

5412

C45

II13

012

019

La1

OC

-non

120b

pJC

M44

53D

4415

0S.

aura

ntio

gris

eus

vD

SM40

138T

AT

CC

1988

7IS

P51

38A

SmII

I09

1-5

011

La1

6O

C-I

V12

0bp

JCM

4346

D44

065

S.au

reoc

ircu

latu

sv

DSM

4038

6A

TC

C19

823

ISP

5386

A03

II20

033

1-33

120b

pJC

M44

54D

4415

1S.

aure

ofac

iens

vD

SM40

127T

AT

CC

1076

2IS

P51

27A

14II

0422

-404

3O

C-I

1473

bpJC

M46

24 8

1A

B04

5876

S.au

reof

asci

culu

sD

SM40

414

AT

CC

1982

4IS

P54

14E

5403

31-

33S.

aure

omon

opod

iale

sD

SM40

416

AT

CC

1982

5IS

P54

1603

31-

33S.

aure

omon

opod

iale

sD

SM40

914

011

1-20

Spec

ies

nam

e

Stra

in n

umbe

r(s)

Clu

ster

num

bers

of

num

eric

al t

axon

omic

stud

ies

and

Ber

gey

cate

gori

es a

ndsp

ecie

s no

.aG

roup

s ac

cord

ing

to d

iffe

rent

stu

dies

abp

rRN

A d

ata

stra

in n

o.if

dif

fere

nt f

rom

the

first

col

umn

Acc

. no.

(EM

BL

)b

Stra

ins

incl

uded

inD

NA

/DN

Ahy

brid

isat

ion

stud

iesc

12

34

56

78

9

(Con

tinue

d)

S.au

reor

ectu

sv

S.au

reov

ersi

lisv

DSM

4038

7TA

TC

C15

853

ISP

5387

Sv. 0

522

-104

0H

a712

1bp

JCM

4457

D44

154

L4

S.au

reov

ertic

illat

usv

DSM

4008

0A

TC

C15

854

ISP

5080

A10

I06

033

1-33

121b

pJC

M43

47D

4406

6L

3S.

aure

us14

48bp

B73

19T

AY

0943

68S.

aure

us14

11bp

B-C

R4

AY

0943

69S.

aure

usD

SM40

862

AT

CC

3309

1-3

1-20

S.au

reus

DSM

4086

7A

TC

C15

437

1-7

1-19

S.av

ella

neus

vD

SM40

554

AT

CC

2373

0IS

P55

54II

1700

21-

712

0bp

JCM

4725

D44

312

S.av

erm

ectin

ius

v14

85bp

MA

-468

0A

B07

8897

S.av

erm

itilis

v15

17bp

MA

-468

0TA

F14

5223

S.av

idin

iiv

DSM

4052

6A

TC

C27

419

ISP

5526

F56

023

004

120b

pJC

M47

26D

4431

3S.

azat

icus

(K

. aza

ticus

)v

1481

bpU

9331

2S.

azur

eus

vD

SM40

106

AT

CC

1492

1IS

P51

06A

18I

1100

91-

19F

U-1

AJ3

9947

0L

2S.

baar

nens

isv

DSM

4023

2A

TC

C23

885

ISP

5232

A1B

I02

006

1-2

KA

-B12

0bp

JCM

4349

D44

067

S.ba

cilla

ris

vD

SM40

598

AT

CC

1585

5IS

P55

98A

1BI

021-

31-

2K

A-B

120b

pJC

M47

27D

4431

4S.

badi

usv

DSM

4013

9TA

TC

C19

888

ISP

5139

CSm

III

151-

11-

1un

OC

-I12

0bp

JCM

4350

D44

069

S.ba

ldac

cii

vD

SM40

845T

AT

CC

2365

4Sv

.01

22-1

040

Ha7

FU

-12b

1349

bpX

5316

4L

4S.

bam

berg

iens

isv

DSM

4059

0TA

TC

C13

879

ISP

5590

ASm

III

1007

51-

25L

a20

OC

-non

120b

pJC

M47

28D

4431

5S.

beiji

ange

nsis

v14

99bp

YIM

6A

F38

5681

S.be

llus

vD

SM40

185T

AT

CC

1492

5IS

P51

85A

18I

1106

11-

22L

a12

1450

bpA

J399

476

L2

S.bi

colo

rD

SM40

140

AT

CC

2361

4IS

P51

40A

1800

91-

1914

48bp

AJ2

7656

9L

2S.

biki

nien

sis

vD

SM40

581T

AT

CC

1106

2IS

P55

81F

64II

I21

22-4

1-07

unO

C-I

I15

17bp

X79

851

S.bi

vert

icill

atus

vD

SM40

272

AT

CC

2361

5IS

P52

72Sv

.01

22-1

040

Ha7

120b

pJC

M44

31D

4413

9L

4S.

blas

tmyc

etic

usv

DSM

4002

9TA

TC

C19

731

ISP

5029

F58

Sv.0

222

-104

0H

a312

0bp

JCM

4184

D44

025

L4

S.bl

uens

isv

DSM

4056

4A

TC

C27

420

ISP

5564

A39

II10

052

017

1520

bpX

7932

4L

2S.

bobi

liv

DSM

4005

6TA

TC

C33

10IS

P50

56IV

02 (

whi

te s

erie

s)1-

71-

1512

0bp

JCM

4627

D44

274

S.bo

ttrop

ensi

sv

DSM

4026

2TA

TC

C25

435

ISP

5262

A19

I12

009

1-19

1531

bpA

B02

6217

S.br

asili

ensi

sv

DSM

4315

9T13

56bp

X53

162

S.br

unne

us (

K. b

runn

ea)

vIF

O14

627T

1475

bpU

9331

4S.

bung

oens

isv

S.ca

caoi

ssp.

caca

oiv

DSM

4005

7TA

TC

C30

82IS

P50

57A

16I

0903

11-

34L

a512

1bp

JCM

4352

D44

070

S.ca

cois

sp.a

soen

sis

vS.

cael

estis

vD

SM40

084

AT

CC

1508

4IS

P50

84A

18I

1100

91-

1914

48bp

AJ3

9946

7L

2S.

cael

icus

DSM

4083

5N

RR

L29

5703

91-

28L

a21

S.ca

erul

eus

vD

SM40

292

ISP

4292

La1

912

0bp

JCM

4670

D44

236

S.ca

erul

eus

vD

SM40

103T

AT

CC

2742

1IS

P51

03IV

07 (

gray

ser

ies)

058

050

La1

9S.

caes

ius

DSM

4041

9A

TC

C19

828

ISP

5419

A21

006

1-18

S.ca

espi

tosu

mD

SM40

603

AT

CC

2744

2IS

P56

03Sv

.22

-104

0S.

calif

orni

cus

vD

SM40

058T

AT

CC

3312

ISP

5058

A09

II02

1-3

030

La2

2F

U-6

OC

-I12

0bp

JCM

4567

D44

236

S.ca

lifor

nicu

sv

DSM

4080

1A

TC

C15

436

II02

1-3

1-2

S.ca

lvus

vD

SM40

010

AT

CC

1338

2IS

P50

10A

12I

0700

61-

1812

1bp

JCM

4628

D44

275

S.ca

nadi

ensi

sD

SM40

837

AT

CC

1777

602

01-

20

Spec

ies

nam

e

Stra

in n

umbe

r(s)

Clu

ster

num

bers

of

num

eric

al t

axon

omic

stud

ies

and

Ber

gey

cate

gori

es a

ndsp

ecie

s no

.aG

roup

s ac

cord

ing

to d

iffe

rent

stu

dies

abp

rRN

A d

ata

stra

in n

o.if

dif

fere

nt f

rom

the

first

col

umn

Acc

. no.

(EM

BL

)b

Stra

ins

incl

uded

inD

NA

/DN

Ahy

brid

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ion

stud

iesc

12

34

56

78

9

Tabl

e 4.

Con

tinue

d

S.ca

nari

esv

DSM

4052

8TA

TC

C27

423

ISP

5528

A20

I13

009

1-19

S.ca

ndid

usv

DSM

4014

1TA

TC

C19

891

ISP

5141

A03

002

1-7

120b

pJC

M46

29D

4427

6S.

cane

scen

sv

DSM

4000

1TA

TC

C19

736

ISP

5001

A1A

I01

1-1

1-1

KA

-D14

76bp

Z76

684

S.ca

ngkr

inge

nsis

vD

SM41

769T

1482

bpD

13P

3A

J391

831

S.ca

nife

rus

vS.

canu

sv

DSM

4001

7TA

TC

C12

237

ISP

5017

A25

III

0200

91-

19L

a21

OC

-IV

120b

pJC

M45

69D

4423

8S.

capi

llisp

iral

isv

DSM

4169

5T12

1bp

JCM

5075

D44

439

S.ca

poam

usv

DSM

4049

4A

TC

C19

006

ISP

5494

C45

II13

1-7

1-15

1484

bpJC

M47

34A

B04

5877

S.ca

puen

sis

DSM

4040

2A

TC

C25

436

ISP

5402

B42

035

1-33

S.ca

rnos

usD

SM40

294

AT

CC

2543

7IS

P52

94A

1500

61-

18S.

carp

atic

usv

S.ca

rpin

ensi

sv

DSM

4383

5T12

0bp

JCM

3301

D43

982

S.ca

tenu

lae

vD

SM40

258

AT

CC

1247

6IS

P52

58C

43II

1103

504

112

1bp

JCM

4353

D44

071

S.ca

ttley

a14

84bp

JCM

4925

AB

0458

71S.

cavi

scab

ies

vA

TC

C51

928

1523

bpA

F11

2160

S.ca

vour

ensi

s ss

p.ca

vour

ensi

sv

DSM

4030

0A

TC

C14

889

ISP

5300

A1B

I02

1-3

1-2

FU

-6K

A-A

120b

pJC

M45

55D

4422

8

S.ca

vour

ensi

s ss

p.w

ashi

ngto

nens

isv

S.ce

llost

atic

usv

DSM

4018

9A

TC

C23

894

ISP

5189

A06

I05

007

003

120b

pJC

M46

31D

4427

7S.

cellu

lofla

vus

vD

SM40

839T

AT

CC

2980

6IV

01`(

yello

w s

erie

s)02

003

212

1bp

JCM

4126

D44

011

S.ce

llulo

lytic

usv

S.ce

llulo

sae

vD

SM40

362T

AT

CC

2543

9IS

P53

62A

13II

0300

61-

18L

a15

OC

-non

S.ce

llulo

sae

vD

SM40

802

AT

CC

3313

1-1

1-1

1476

bpZ

7669

0S.

cha

mpa

vatii

vD

SM40

841T

NR

RL

B-5

682

IV02

`(ye

llow

ser

ies)

1-1

1-1

120b

pJC

M50

66D

4443

5S.

cha

rtre

usis

vD

SM40

085T

AT

CC

1492

2IS

P50

85A

18I

1100

91-

1914

48bp

AJ3

9946

8L

2S.

cha

ttano

ogen

sis

vD

SM40

002T

AT

CC

1973

9IS

P50

02un

OC

-non

121b

pJC

M42

99A

L44

047

S. c

hiba

ensi

sv

DSM

4022

0A

TC

C23

895

ISP

5220

A24

II05

009

1-19

120b

pJC

M46

32D

4427

8S.

chr

esto

myc

etic

usv

DSM

4054

5A

TC

C14

947

ISP

5545

B42

I19

035

1-33

121b

pJC

M47

35D

4431

9S.

chr

omof

uscu

sv

DSM

4027

3TA

TC

C23

896

ISP

5273

A15

I08

006

1-18

La6

OC

-III

120b

pJC

M43

54D

4407

2S.

chr

omog

enes

DSM

4076

502

003

2S.

chr

yseu

sv

DSM

4042

0A

TC

C19

829

ISP

5420

A17

I10

22-3

1-08

120b

pJC

M47

37D

4432

1L

3S.

chr

ysom

allu

sV

DSM

4012

8A

TC

C11

523

ISP

5128

A1B

1-3

1-2

FU

-22

KA

-BS.

chr

ysom

allu

sv

DSM

4087

002

003

2S.

chr

ysom

allu

s ss

p.ch

ryso

mal

lus

vD

SM40

685

082

077

120b

pJC

M42

96D

4404

6

S. c

hrys

omal

lus

ssp.

fum

igat

usv

S.ci

nere

orec

tus

vS.

cine

reor

uber

ssp

.ci

nere

orub

erv

DSM

4001

2A

TC

C19

740

ISP

5012

A05

I04

002

038

FU

-612

0bp

JCM

4572

D44

240

S.ci

nere

orub

er s

sp.

fruc

tofe

rmen

tans

vD

SM40

692

NR

RL

2588

I04

006

1-18

S.ci

nere

ospi

nus

vS.

cine

reus

vD

SM43

033T

120b

pJC

M30

40D

4397

4S.

cine

roch

rom

ogen

esv

DSM

4165

1T12

0bp

JCM

3385

D43

985

S.ci

nnab

arin

usv

DSM

4046

7A

TC

C23

617

ISP

5467

A18

I11

009

1-19

1448

bpA

J399

487

L2

S.ci

nnam

omeu

s ss

p .al

bosp

orus

DSM

4089

7A

TC

C25

186

Sv.0

222

-104

0L

4

S.ci

nnam

onen

sis

vD

SM40

803T

AT

CC

1230

8IV

02`(

red

seri

es)

22-3

042

120b

pJC

M40

19D

4398

8S.

cinn

amon

ensi

sv

DSM

4080

4A

TC

C15

413

033

1-33

Spec

ies

nam

e

Stra

in n

umbe

r(s)

Clu

ster

num

bers

of

num

eric

al t

axon

omic

stud

ies

and

Ber

gey

cate

gori

es a

ndsp

ecie

s no

.aG

roup

s ac

cord

ing

to d

iffe

rent

stu

dies

abp

rRN

A d

ata

stra

in n

o.if

dif

fere

nt f

rom

the

first

col

umn

Acc

. no.

(EM

BL

)b

Stra

ins

incl

uded

inD

NA

/DN

Ahy

brid

isat

ion

stud

iesc

12

34

56

78

9

(Con

tinue

d)

S.ci

nnam

oneu

sv

DSM

4000

5TA

TC

C11

874

ISP

5005

F55

Sv. 0

222

-104

0H

a413

45bp

X53

171

L4

S.ci

nnam

oneu

s ss

p.az

ocol

utus

DSM

4064

6A

TC

C12

686

Sv. 0

322

-104

0L

4

S.ci

nnam

oneu

s ss

p.la

nosu

sD

SM40

898

AT

CC

2518

7Sv

. 02

22-1

040

S.ci

nnam

oneu

s ss

p.sp

arsu

sD

SM40

899

AT

CC

2518

5Sv

. 02

22-1

040

S.ci

nner

ocro

catu

sD

SM40

876

076

069

S.ci

rrat

usv

DSM

4047

9A

TC

C14

699

ISP

5479

F62

II14

22-3

042

120b

pJC

M47

38D

4432

2S.

cisc

auca

sicu

sv

DSM

4027

5TIS

P52

7512

0bp

JCM

4384

D44

099

S.ci

treo

fluor

esce

nsv

DSM

4026

5TA

TC

C15

858

ISP

5265

A1B

I02

1-3

1-2

FU

-19b

KA

-B12

0bp

JCM

4356

D44

074

S.ci

treu

sD

SM40

364

AT

CC

2544

1IS

P53

64A

1A1-

11-

1S.

clav

ifer

vD

SM40

843T

120b

pJC

M50

59D

4442

8S.

clav

ulig

erus

vD

SM40

751T

AT

CC

2706

4IV

10 (

gray

ser

ies)

22-5

036

1486

bpJC

M47

10A

B04

5869

S.co

chle

atus

vS.

coel

esce

nsv

DSM

4042

1A

TC

C19

830

ISP

5421

A21

I14

006

1-18

121b

pJC

M47

39D

4432

3S.

coel

iatu

sD

SM40

422

AT

CC

1983

3IS

P54

2200

91-

19S.

coel

icofl

avus

vS.

coel

icol

orv

DSM

4023

3TA

TC

C23

899

ISP

5233

A1A

I01

1-1

1-1

FU

-1K

A-D

1476

bpZ

7667

8S.

coer

ulat

usD

SM40

424

AT

CC

1983

4IS

P54

2400

91-

19S.

coer

uleo

flavu

sv

S.co

erul

eofu

scus

vD

SM40

144

AT

CC

2361

8IS

P51

44A

18I

1100

91-

1914

47bp

AJ3

9947

3L

2S.

coer

uleo

prun

usv

S.co

erul

eoro

seus

NR

RL

B-5

642

L2

S.co

erul

eoru

bidu

sv

NR

RL

1237

2L

2S.

coer

uleo

rubi

dus

vN

RR

L30

45L

2S.

coer

uleo

rubi

dus

vD

SM40

145

AT

CC

1374

0IS

P51

45A

18I

1100

91-

1914

51bp

AJ3

0662

2L

2S.

coer

ules

cens

vD

SM40

146

AT

CC

1989

6IS

P51

46A

18I

1100

91-

1914

50bp

AJ3

9946

2L

2S.

colli

nus

vD

SM40

129T

AT

CC

1974

3IS

P51

29A

18I

1100

91-

19F

U-1

1448

bpA

J306

623

L2

S.co

lom

bien

sis

vD

SM40

558

AT

CC

2742

5IS

P55

58F

61I

2222

-304

2F

U-1

2b12

0bp

JCM

4740

D44

324

L5

S.co

ralu

sD

SM40

256

AT

CC

2390

1IS

P52

56A

1900

91-

19S.

corc

horu

sii

vD

SM40

340

AT

CC

2544

4IS

P53

40A

20I

1300

91-

1912

0bp

JCM

4467

D44

162

S.co

riof

acie

nsD

SM40

485

AT

CC

1415

5IS

P54

85A

1A1-

11-

1S.

cost

aric

anus

vS.

crem

eus

vD

SM40

147

AT

CC

1989

7IS

P51

47A

1BI

0200

21-

7F

U-2

112

0bp

JCM

4362

D44

079

S.cr

etac

eus

DSM

4056

1A

TC

C30

05IS

P55

61A

031-

31-

2K

A-B

S.cr

ysta

llinu

sv

DSM

4094

5IV

03 (

red

seri

es)

009

1-09

120b

pJC

M50

67D

4443

6S.

cura

coi

vD

SM40

107

AT

CC

1338

5IS

P51

07A

18I

1100

90-

1914

48bp

AJ3

9947

1L

2S.

cusp

idos

poru

sv

DSM

4142

5IV

11 (

gray

ser

ies)

22-4

1-06

120b

pJC

M43

16D

4405

2S.

cyan

eofu

scat

usv

DSM

4014

8A

TC

C23

619

ISP

5148

A1B

I02

1-3

1-2

120b

pJC

M43

64D

4408

1S.

cyan

eus

vD

SM40

108T

AT

CC

1492

3IS

P51

08A

1800

91-

1914

44bp

AJ3

9946

0L

2S.

cyan

oalb

usv

DSM

4019

8TA

TC

C15

859

ISP

5198

A37

I17

007

003

La1

712

0bp

JCM

4363

D44

080

S.cy

anoc

olor

DSM

4042

5A

TC

C19

835

ISP

5425

A21

006

1-18

S.cy

anog

enus

DSM

4042

6A

TC

C19

836

ISP

5426

006

1-18

Spec

ies

nam

e

Stra

in n

umbe

r(s)

Clu

ster

num

bers

of

num

eric

al t

axon

omic

stud

ies

and

Ber

gey

cate

gori

es a

ndsp

ecie

s no

.aG

roup

s ac

cord

ing

to d

iffe

rent

stu

dies

abp

rRN

A d

ata

stra

in n

o.if

dif

fere

nt f

rom

the

first

col

umn

Acc

. no.

(EM

BL

)b

Stra

ins

incl

uded

inD

NA

/DN

Ahy

brid

isat

ion

stud

iesc

12

34

56

78

9

Tabl

e 4.

Con

tinue

d

S.cy

anog

lom

erus

ssp

.ce

llulo

seD

SM40

427

AT

CC

1983

7IS

P54

2701

31-

19

S.cy

anog

rise

usD

SM40

534T

HSm

019

023

La2

0S.

cyst

argi

neus

(K. c

ysta

rgin

ea)

v14

82bp

JCM

7356

U93

318

S.da

ghes

tani

cus

vD

SM40

149

AT

CC

2362

0IS

P51

49A

1700

601

012

0bp

JCM

4365

D44

082

L3

S.da

ghes

toni

cus

vN

RR

LB

-271

0L

3

S.di

asta

ticus

vD

SM40

496T

AT

CC

3315

ISP

5496

A19

I12

1-1

1-1

unF

U-1

OC

-non

1354

bpX

5316

1S.

dias

tatic

us s

sp.

arde

siac

usv

S.di

asta

toch

rom

ogen

esv

DSM

4044

9TA

TC

C12

309

ISP

5449

A19

I12

009

1-19

AB

0262

18S.

dias

tato

chro

mog

enes

vD

SM40

700

AT

CC

1230

9I

121-

71-

1515

31bp

D63

867

S.di

stal

licus

vD

SM40

846

NC

IB89

36Sv

. 01

22-1

040

Hal

4L

4S.

djak

arte

nsis

vD

SM40

743T

AT

CC

1344

1IV

12 (

gray

ser

ies)

035

1-33

121b

pJC

M49

57D

4442

0S.

durh

amen

sis

vD

SM40

539

AT

CC

2319

4IS

P55

39A

30II

0600

91-

1912

0bp

JCM

4747

D44

331

S.eb

uros

pore

usD

SM40

944

064

1-30

S.ec

hina

tus

vD

SM40

013

AT

CC

1974

8IS

P50

13A

18I

111-

61-

10F

U-1

1448

bpA

J399

465

L2

S.ec

hina

tus

vD

SM40

730

1-7

1-15

L2

S.ec

hino

rube

rv

DSM

4169

6T12

0bp

JCM

5016

D44

426

S.ed

eren

sis

vD

SM40

741T

AT

CC

1530

4IV

14 (

gray

ser

ies)

013

1-19

120b

pJC

M49

58A

B01

8209

S.eh

imen

sis

vD

SM40

253T

AT

CC

2390

3IS

P52

53Sv

. 09

22-1

040

Hal

120b

pJC

M41

62D

4402

1S.

endu

sv

DSM

4018

7N

RR

L23

39L

a8L

1S.

enis

soca

esili

sv

S.er

umpe

nsv

DSM

4094

1TA

TC

C23

266

IV15

(gr

ay s

erie

s)03

51-

3312

0bp

JCM

5060

D44

429

S.er

ythr

aeus

vS.

eryt

hrog

rise

usv

DSM

4011

6TA

TC

C27

427

ISP

5116

IV04

(re

d se

ries

)07

41-

27L

a15

S.es

pino

sus

NR

RL

5729

1518

bpX

8082

6S.

euro

cidi

cus

vD

SM40

604T

AT

CC

2742

8IS

P56

04F

56Sv

.02

22-1

040

Ha5

120b

pJC

M40

29D

4398

9L

4S.

euro

paei

scab

iei

v14

92bp

CF

BP

4497

AJ0

0742

3S.

eury

ther

mus

vD

SM40

014

AT

CC

1497

5IS

P50

14A

23I

201-

500

915

31bp

D63

870

S.ex

folia

tus

vD

SM40

060T

AT

CC

1262

7IS

P50

60A

05I

0400

21-

7un

OC

-II

KA

-C12

0bp

JCM

4366

D44

083

S.fa

sicu

latu

sD

SM40

054

AT

CC

1975

1IS

P50

54A

2902

500

5S.

felle

usv

DSM

4013

0TA

TC

C19

752

ISP

5130

A1A

I01

1-1

1-1

KA

-D14

76bp

Z76

681

S.fe

lleus

vD

SM40

647

NR

RL

2251

I01

22-3

1-07

S.fe

lleus

vD

SM40

976

I01

002

1-7

S.fe

rven

s ss

p. f

erve

nsv

DSM

4008

6A

TC

C27

429

ISP

5086

Sv.0

122

-104

0L

4S.

ferv

ens

ssp.

mel

rosp

orus

vD

SM40

905T

NR

RL

3117

Sv.0

122

-104

0H

a7S.

filam

ento

sus

vD

SM40

022

AT

CC

1975

3IS

P50

22A

05I

0400

21-

712

0bp

JCM

4576

D44

244

S.fil

ipin

ensi

sv

DSM

4011

2TA

TC

C23

905

ISP

5112

A30

II06

009

1-19

La1

0O

C-I

II12

0bp

JCM

4369

D44

086

S.fim

bria

tus

vD

SM40

942T

AT

CC

1505

1IV

16 (

gray

ser

ies)

006

1-18

1484

bpJC

M49

10A

B04

5868

S.fim

icar

ius

vD

SM40

322

AT

CC

2544

9IS

P53

22A

1BI

021-

31-

2F

U-9

120b

pJC

M44

72D

4416

7S.

finla

yiv

DSM

4021

8TA

TC

C23

340

ISP

5218

ISm

III

2422

-404

3un

OC

-I12

0bp

JCM

4637

D44

279

S.fla

veol

usv

DSM

4006

1TA

TC

C33

19IS

P50

61A

24II

051-

61-

13L

a12

OC

-III

121b

pJC

M45

77D

4424

5S.

flave

scen

sD

SM40

428

AT

CC

1983

8IS

P54

2822

-31-

08S.

flave

usv

DSM

4315

3TL

a21

120b

pJC

M30

35D

4397

1S.

flavi

dofu

scus

vS.

flavi

dovi

rens

vD

SM40

150T

AT

CC

1990

0IS

P51

50IV

03 (

yello

w s

erie

s)02

603

3L

a22

120b

pJC

M44

74D

4416

9S.

flavi

scle

rotic

usv

DSM

4027

0I

0801

700

7K

A-G

121b

pJC

M47

51D

4433

3S.

flavo

chro

mog

enes

DSM

4054

1A

TC

C14

841

ISP

5541

A05

002

1-7

FU

-NC

S.fla

voch

rom

ogen

esD

SM40

651

009

1-19

S.fla

vofu

ngin

iv

DSM

4036

6A

TC

C27

430

ISP

5366

B42

033

1-33

120b

pJC

M47

53D

4433

5

Spec

ies

nam

e

Stra

in n

umbe

r(s)

Clu

ster

num

bers

of

num

eric

al t

axon

omic

stud

ies

and

Ber

gey

cate

gori

es a

ndsp

ecie

s no

.aG

roup

s ac

cord

ing

to d

iffe

rent

stu

dies

abp

rRN

A d

ata

stra

in n

o.if

dif

fere

nt f

rom

the

first

col

umn

Acc

. no.

(EM

BL

)b

Stra

ins

incl

uded

inD

NA

/DN

Ahy

brid

isat

ion

stud

iesc

12

34

56

78

9

(Con

tinue

d)

S.fla

vofu

scus

vS.

flavo

gris

eus

vD

SM40

323T

AT

CC

2545

2IS

P53

23A

1CI

031-

201

5F

U-1

9bK

A-B

1518

bpA

J494

864

S.fla

vogr

iseu

sD

SM40

990

AT

CC

3331

I03

1-4

1-4

S.fla

vope

rsic

usv

DSM

4009

3TA

TC

C19

756

ISP

5093

F56

22-1

040

Ha1

412

0bp

JCM

4307

D44

049

L4

S.fla

votr

icin

iv

DSM

4015

2A

TC

C23

621

ISP

5152

F61

I22

22-3

042

FU

-112

0bp

JCM

4371

D44

087

L3/

L5

S.fla

vova

riab

ilis

vS.

flavo

vire

nsv

DSM

4006

2A

TC

C33

20IS

P50

62A

1C1-

201

526

8bp

U72

171

S.fla

vovi

ridi

sv

DSM

4015

3A

TC

C19

903

ISP

5153

A28

006

1-10

121b

pJC

M43

72D

4408

8S.

flocc

ulus

vD

SM40

327T

AT

CC

2545

3IS

P53

27A

16I

0903

01-

3412

1bp

JCM

4476

D44

171

S.flo

rida

ev

DSM

4093

8N

CIB

9345

IV04

(ye

llow

ser

ies)

1-3

1-2

120b

pJC

M50

68D

4443

7S.

fluor

esce

nsv

DSM

4020

3A

TC

C15

860

ISP

5203

A1B

I02

1-3

1-2

KA

-B12

0bp

JCM

4373

D44

089

S.fr

adia

ev

DSM

4006

3TA

TC

C10

745

ISP

5063

G68

II18

22-5

039

unO

C-I

121b

pJC

M45

79D

4424

6S.

frag

ilis

vD

SM40

044T

AT

CC

2390

8IS

P50

44G

SMII

I22

078

058

OC

-III

120b

pJC

M46

38D

4428

0S.

gris

eopl

anus

DSM

4000

9A

TC

C19

766

ISP

5009

A29

I15

078

060

120b

pJC

M43

00D

4404

8S.

fulv

issi

mus

vD

SM40

593T

AT

CC

2743

1IS

P55

93A

1003

41-

33un

OC

-IV

120b

pJC

M47

54D

4433

6L

3S.

fulv

issi

mus

vD

SM40

767

1-3

1-2

S.fu

lvor

obeu

sv

S.fu

lvov

irid

isD

SM40

210

AT

CC

1586

3IS

P52

10A

031-

31-

20S.

fum

anus

vD

SM40

154

AT

CC

1990

4IS

P51

54A

18I

111-

71-

1914

48bp

AJ3

9946

3S.

fum

igat

iscl

erot

icus

vD

SM43

154T

121b

pJC

M31

01D

4397

9S.

fung

icid

icus

DSM

4002

0A

TC

C27

432

ISP

5020

A16

035

1-33

S.fu

ngic

idic

usD

SM40

811

AT

CC

1385

302

400

5S.

galb

usv

DSM

4008

9A

TC

C23

910

ISP

5089

A15

I08

006

1-10

1517

bpX

7985

2S.

galb

usv

DSM

4048

0A

TC

C14

077

ISP

5480

I08

1-5

011

1517

bpX

7932

5S.

galil

aeus

vD

SM40

481

AT

CC

1496

9IS

P54

81A

19I

121-

71-

1514

84bp

AB

0458

78S.

ganc

idic

usv

DSM

4093

5TN

RR

LB

-187

2IV

17 (

gray

ser

ies)

006

1-18

121b

pJC

M41

71D

4402

2S.

gard

neri

vD

SM40

064

AT

CC

9604

ISP

5064

A04

002

1-07

FU

-23

KA

-C12

0bp

JCM

4375

D44

091

S.ge

latic

usv

DSM

4006

5TA

TC

C33

23IS

P50

65A

SmII

I11

003

1-3

unS.

geld

anom

ycet

icus

NR

RL

3602

T11

29bp

AJ3

9182

4S.

geys

irie

nsis

vD

SM40

742T

AT

CC

1530

3IV

18 (

gray

ser

ies)

006

1-18

121b

pJC

M49

62D

4442

1S.

ghan

aens

isv

DSM

4074

6TA

TC

C14

672

IV05

(gr

een

seri

es)

1-7

1-21

121b

pJC

M49

63D

4442

2S.

gibs

onii

vD

SM40

959

IV05

(w

hite

ser

ies)

030

1-34

121b

pJC

M50

61D

4443

0S.

glau

cesc

ens

vD

SM40

716

1519

bpX

7932

2S.

glau

cesc

ens

vD

SM40

155T

AT

CC

2362

2IS

P51

55A

28II

I05

006

1-10

La1

6O

C-I

IIL

2S.

glau

cogr

iseu

sN

RR

L12

514

L2

S.gl

auco

spor

usv

S.gl

aucu

sv

S.gl

obis

poru

s ss

p.ca

ucas

icus

DSM

4081

4A

TC

C19

907

I02

1-1

1-1

S.gl

obis

poru

s ss

p .fla

vofu

scus

S.gl

obis

poru

s ss

p.gl

obis

poru

sv

DSM

4019

9TA

TC

C15

864

ISP

5199

A1B

I02

11-

31-

2K

A-B

120b

pJC

M43

78D

4409

3

S.gl

obos

usv

DSM

4081

5TA

TC

C14

979

IV19

(gr

ay s

erie

s)22

-304

212

0bp

JCM

4225

D44

032

Spec

ies

nam

e

Stra

in n

umbe

r(s)

Clu

ster

num

bers

of

num

eric

al t

axon

omic

stud

ies

and

Ber

gey

cate

gori

es a

ndsp

ecie

s no

.aG

roup

s ac

cord

ing

to d

iffe

rent

stu

dies

abp

rRN

A d

ata

stra

in n

o.if

dif

fere

nt f

rom

the

first

col

umn

Acc

. no.

(EM

BL

)b

Stra

ins

incl

uded

inD

NA

/DN

Ahy

brid

isat

ion

stud

iesc

12

34

56

78

9

Tabl

e 4.

Con

tinue

d

S.gl

omer

atus

vS.

glom

eroa

uran

tiacu

sv

DSM

4042

9IS

P54

2912

0bp

JCM

4761

D44

340

S.go

bitr

icin

iv

DSM

4170

1T12

0bp

JCM

5062

D44

431

S.go

shik

iens

isv

DSM

4019

0A

TC

C23

914

ISP

5190

F61

I22

22-3

042

120b

pJC

M42

94D

4404

4L

3/L

5S.

goug

erot

iiv

DSM

4032

4TA

TC

C10

975

ISP

5324

A1A

I01

1-1

1-1

KA

-D14

76bp

Z76

687

S.gr

amin

earu

sv

S.gr

amin

ofac

iens

vD

SM40

559T

AT

CC

1270

5IS

P55

59A

26II

I03

004

1-23

OC

-I12

0bp

JCM

4762

D44

341

S.gr

isei

nige

rN

RR

LB

-186

5TT

1495

bpA

J391

818

S.gr

isei

nus

vD

SM40

047

AT

CC

2391

5IS

P50

47A

1BI

021-

31-

2F

U-6

KA

-B12

0bp

JCM

4379

D44

094

S.gr

iseo

aura

ntia

cus

vD

SM40

430

AT

CC

1984

0IS

P54

30A

12I

071-

71-

1512

0bp

JCM

4763

D44

342

S.gr

iseo

brun

neus

vD

SM40

066

AT

CC

1976

2IS

P50

66A

1BI

021-

31-

2F

U-6

KA

-A12

0bp

JCM

4380

D44

095

S.gr

iseo

brun

neus

vD

SM40

915

I02

020

1-08

S.gr

iseo

carn

eus

vD

SM40

004T

AT

CC

1262

8IS

P50

04F

55Sv

. 03

22-1

040

Ha6

unF

U-1

2b15

15bp

X99

943

L4

S.gr

iseo

chro

mog

enes

vD

SM40

499T

AT

CC

1451

1IS

P54

99A

18I

111-

501

114

48bp

AJ3

9949

1L

2S.

gris

eofa

cien

sD

SM40

816

AT

CC

1318

000

21-

7S.

gris

eofla

vus

vD

SM40

456T

AT

CC

2545

6IS

P54

56A

37I

1700

61-

18L

a4O

C-n

on12

1bp

JCM

4479

D44

174

S.gr

iseo

flavu

sv

DSM

4069

8N

RR

L27

17I

171-

71-

14S.

gris

eofu

scus

vD

SM40

191

AT

CC

2391

6IS

P51

91A

12I

071-

61-

16F

U-6

KA

-GS.

gris

eoin

carn

atus

vD

SM40

274T

AT

CC

2362

3IS

P52

74A

13II

0300

61-

18L

a15

121b

pJC

M43

81D

4409

6S.

gris

eola

vend

usD

SM40

385

AT

CC

2545

7IS

P53

8522

-304

2L

5S.

gris

eolo

albu

sv

DSM

4046

8TA

TC

C23

624

ISP

5468

IV05

(ye

llow

ser

ies)

)017

007

121b

pJC

M44

80D

4417

5S.

gris

eolo

spor

eus

vS.

gris

eolu

sv

DSM

4006

7A

TC

C33

25IS

P50

67A

1CI

031-

201

5F

U-2

4K

A-B

120b

pJC

M40

43D

4399

0S.

gris

eolu

sv

DSM

4085

4A

TC

C11

796

I03

023

004

S.gr

iseo

lute

usv

DSM

4039

2TA

TC

C12

768

ISP

5392

C43

II11

1-5

1-16

La2

4O

C-I

II12

0bp

JCM

4765

D44

344

S.gr

iseo

myc

ini

vD

SM40

159

AT

CC

2362

5IS

P51

59A

12I

0700

61-

1012

1bp

JCM

4382

D44

097

S.gr

iseo

rose

usD

SM40

768

AT

CC

1212

500

601

0S.

gris

eoru

bens

vD

SM40

160

AT

CC

1990

9IS

P51

60A

12I

0700

61-

18K

A-F

121b

pJC

M43

83D

4409

8S.

gris

eoru

ber

vD

SM40

281T

AT

CC

2391

9IS

P52

81A

21I

1401

802

3un

OC

-I14

36bp

AY

0945

85S.

gris

eoru

bigi

nosu

sv

DSM

4046

9A

TC

C23

627

ISP

5469

A18

I11

009

1-19

1449

bpA

J399

488

L2

S.gr

iseo

spor

eus

vD

SM40

562

AT

CC

2743

5IS

P55

62A

23I

201-

71-

1912

0bp

JCM

4766

D44

345

S.gr

iseo

stra

min

eus

vD

SM40

161T

AT

CC

2362

8IS

P51

61F

60IV

06 (

gree

n se

ries

)00

61-

1012

1bp

JCM

4385

D44

100

S.gr

iseo

vert

icill

atus

vD

SM40

507T

ISP

5507

F58

22-1

040

Ha4

120b

pJC

M42

02D

4402

8S.

gris

eovi

ridi

sv

DSM

4022

9TA

TC

C23

920

ISP

5229

A17

I10

006

010

La6

OC

-III

120b

pJC

M46

43D

4428

3L

3S.

gris

eus

vD

SM40

855

AT

CC

1013

7I

021-

31-

214

78bp

Y15

501

S.gr

iseu

s ss

p . a

lpha

vD

SM40

937

NR

RL

B-2

249

I02

1-3

1-2

S.gr

iseu

s ss

p . c

reto

sus

vD

SM40

561T

ISP

5561

120b

pJC

M47

42D

4432

6S.

gris

eus

ssp.

far

inos

usv

DSM

4093

2I

021-

31-

2S.

gris

eus

ssp .

gri

seus

vIF

O13

550

1485

bpA

B04

5866

S.gr

iseu

s ss

p . s

olvi

faci

ens

vD

SM40

933

NR

RL

B-1

561

I02

1-1

1-1

S.gr

iseu

s ss

p . g

rise

usv

DSM

4023

6TA

TC

C23

345

ISP

5236

A1B

I02

11-

31-

2L

a22

FU

-19b

KA

-B15

37bp

AF

0567

11S.

hach

ijoen

sis

vD

SM40

114T

AT

CC

1976

9IS

P51

14F

55Sv

.04

22-1

040

Ha4

FU

-NC

120b

pJC

M43

31D

4405

4L

4S.

hals

tedi

iv

DSM

4006

8TA

TC

C10

897

ISP

5068

A1C

I03

1-2

015

FU

-24

OC

-IK

A-B

120b

pJC

M40

52D

4399

1S.

hals

tedi

iv

DSM

4086

3A

TC

C13

449

I03

1-3

1-2

S.ha

wai

iens

isv

DSM

4004

2A

TC

C12

236

ISP

5042

A18

I11

009

1-19

1448

bpA

J399

466

L2

S.he

imi

DSM

4032

8A

TC

C25

460

ISP

5328

1-6

1-14

S.he

liom

ycin

iv

S.he

lvat

icus

vD

SM40

431

AT

CC

1984

1IS

P54

31F

62II

1422

-304

312

0bp

JCM

4768

D44

346

S.he

rbar

icol

orv

DSM

4012

3A

TC

C23

922

ISP

5123

A02

II01

22-4

043

120b

pJC

M41

38D

4401

4S.

hiro

shim

ensi

sv

DSM

4003

7A

TC

C19

772

ISP

5037

F57

Sv.0

122

-104

0H

a7F

U-N

C12

0bp

JCM

4098

D44

005

L4

S.hi

rsut

usv

DSM

4009

5TIS

P50

9512

0bp

JCM

4587

D44

249

S.ho

min

isD

SM40

770

AT

CC

3008

1-1

1-1

S.hu

mid

usv

DSM

4026

3A

TC

C12

760

ISP

5263

A19

I12

009

1-19

269b

pU

7216

9

Spec

ies

nam

e

Stra

in n

umbe

r(s)

Clu

ster

num

bers

of

num

eric

al t

axon

omic

stud

ies

and

Ber

gey

cate

gori

es a

ndsp

ecie

s no

.aG

roup

s ac

cord

ing

to d

iffe

rent

stu

dies

abp

rRN

A d

ata

stra

in n

o.if

dif

fere

nt f

rom

the

first

col

umn

Acc

. no.

(EM

BL

)b

Stra

ins

incl

uded

inD

NA

/DN

Ahy

brid

isat

ion

stud

iesc

12

34

56

78

9

(Con

tinue

d)

S.hu

mif

erD

SM40

602

AT

CC

1374

8IS

P56

02A

1C03

51-

33

S.hu

mif

erus

vD

SM43

030T

121b

pJC

M30

37D

4397

2S.

hydr

ogen

ans

vD

SM40

586

AT

CC

1963

1IS

P55

86A

05I

0400

21-

7S.

hygr

osco

picu

sv

AT

CC

2143

115

17bp

X79

853

S.hy

gros

copi

cus

vN

RR

L14

7714

83bp

AJ3

9181

9S.

hygr

osco

picu

sv

NR

RL

2387

T14

65bp

AJ3

9182

0S.

hygr

osco

picu

s ss

p.as

com

ycet

icus

DSM

4082

2A

TC

C14

891

056

016

La7

S.hy

gros

copi

cus

ssp.

deco

yicu

sv

S.hy

gros

copi

cus

ssp.

geld

anus

NR

RL

3602

L1

S.hy

gros

copi

cus

ssp.

gleb

osus

v

S.hy

gros

copi

cus

ssp.

hygr

osco

picu

sv

IFO

1359

814

85bp

AB

0458

64

S.hy

gros

copi

cus

ssp .

hygr

osco

picu

sv

DSM

4018

7A

TC

C23

904

ISP

5187

A32

I16

041

012

FU

-614

61bp

AJ3

9182

1

S.hy

gros

copi

cus

ssp .

hygr

osco

picu

sv

DSM

4057

8TA

TC

C27

438

ISP

5578

A32

I16

085

012

La8

FU

-6L

1

S.hy

gros

copi

cus

ssp .

ossa

myc

etic

usv

DSM

4082

4A

TC

C15

420

I16

009

1-19

S.ia

kyru

sv

DSM

4048

2A

TC

C15

375

ISP

5482

A18

I11

009

1-19

1448

bpA

J399

489

L2

S.in

diae

nsis

vS.

indi

goco

lor

DSM

4043

2A

TC

C19

842

ISP

5432

A18

009

1-19

1448

bpA

J399

464

L2

S.in

digo

feru

sv

DSM

4012

4TIS

P51

2412

0bp

JCM

4646

D44

285

S.in

doni

ensi

sv

DSM

4175

9T14

81bp

A4

R2

AJ3

9183

5S.

inte

rmed

ius

vD

SM40

372T

AT

CC

3329

ISP

5372

A1A

I01

1-1

1-1

La3

KA

-D14

74bp

Z76

686

S.in

usita

tus

vD

SM41

441T

121b

pJC

M49

88D

4442

4S.

ipom

oeae

vD

SM40

383T

AT

CC

2546

2IS

P53

83IV

02 (

blue

ser

ies)

077

074

L2

S.ip

omoe

aev

DSM

4081

8A

TC

C11

747

IV02

(bl

ue s

erie

s)00

91-

19S.

jant

hinu

sv

DSM

4020

6A

TC

C15

870

ISP

5206

A18

I11

009

1-19

1448

bpA

J399

478

L2

S.ja

vens

isv

DSM

4176

4T14

71bp

B22

P3

AJ3

9183

3S.

kana

myc

etic

usv

DSM

4050

0TIS

P55

00L

a11

120b

pJC

M47

75D

4435

2S.

karn

atak

ensi

sD

SM40

345

AT

CC

2546

3IS

P53

45C

4422

-403

5S.

kash

imir

ensi

sv

DSM

4033

6TIS

P53

36H

a812

0bp

JCM

4776

D44

353

S.ka

suga

ensi

sv

DSM

4081

9TIS

P58

1914

88bp

M33

8-M

1A

B02

4441

S.ka

trae

vD

SM40

550

AT

CC

2744

0IS

P55

50F

61I

2222

-304

212

0bp

JCM

4777

D44

354

L5

S.ke

ntuc

kens

isv

DSM

4005

2A

TC

C12

691

ISP

5052

FSM

Sv.1

122

-104

0H

al4

120b

pJC

M41

53D

4401

9L

4S.

kifu

nens

isv

1481

bpJC

M90

81U

9332

2S.

kish

iwad

ensi

sv

DSM

4039

7TA

TC

C25

464

ISP

5397

Sv.1

522

-104

0H

al1

121b

pJC

M44

86D

4418

0S.

krai

nski

iD

SM40

321

AT

CC

2546

5IS

P53

21A

1A1-

11-

1S.

kres

tom

ycet

icus

DSM

4082

003

51-

33

Spec

ies

nam

e

Stra

in n

umbe

r(s)

Clu

ster

num

bers

of

num

eric

al t

axon

omic

stud

ies

and

Ber

gey

cate

gori

es a

ndsp

ecie

s no

.aG

roup

s ac

cord

ing

to d

iffe

rent

stu

dies

abp

rRN

A d

ata

stra

in n

o.if

dif

fere

nt f

rom

the

first

col

umn

Acc

. no.

(EM

BL

)b

Stra

ins

incl

uded

inD

NA

/DN

Ahy

brid

isat

ion

stud

iesc

12

34

56

78

9

Tabl

e 4.

Con

tinue

d

S.ku

nmin

gens

isv

DSM

4168

1T12

0bp

JCM

7473

D44

441

S.ku

rssa

novi

iv

DSM

4016

2TA

TC

C15

824

ISP

5162

F60

IV20

(gr

ay s

erie

s)02

51-

1512

0bp

JCM

4388

D44

103

S.la

beda

ev

S.la

ceyi

1127

bpC

762

AY

0943

65S.

lace

yi13

89bp

C76

5A

Y09

4366

S.la

ceyi

1446

bpC

7654

TA

Y09

4367

S.la

daka

num

vD

SM40

587T

NR

RL

3191

TH

a12

1357

bpX

5316

7L

4S.

lana

tus

vD

SM40

090T

AT

CC

1977

5IS

P50

90A

18I

1101

61-

19L

a21

1449

bpA

J399

469

L2

S.la

rent

iiD

SM41

684T

120b

pJC

M50

63D

4443

2S.

late

ritiu

sv

DSM

4016

3TA

TC

C19

913

ISP

5163

HSm

III

2322

-31-

08un

OC

-II

1426

bpJC

M43

89A

F45

4764

S.la

vend

ofol

iae

vD

SM40

217T

AT

CC

1587

2IS

P52

17IV

07 (

red

seri

es)

22-3

1-08

120b

pJC

M43

91D

4410

6

S.la

vend

ulae

vIF

O14

028

1514

bpD

8511

4S.

lave

ndul

aev

NR

RL

B-2

343

L3/

L5

S.la

vend

ulae

vN

RR

LB

-240

2L

3/L

5S.

lave

ndul

aev

NR

RL

B-3

080

L3/

L5

S.la

vend

ulae

vD

SM40

748

AT

CC

1415

9I

2222

-304

2F

U-1

2bS.

lave

ndul

aev

DSM

4157

0A

TC

C11

924

I22

22-3

042

D85

109

S.la

vend

ulae

vD

SM41

571

AT

CC

1366

4I

221-

31-

2D

8511

0S.

lave

ndul

aev

DSM

4157

3A

TC

C14

158

I22

22-3

042

D85

111

S.la

vend

ulae

vD

SM41

576

AT

CC

1416

2I

2222

-304

2D

8511

2S.

lave

ndul

ae s

sp.

gras

seri

usv

DSM

4038

5T12

0bp

JCM

4056

D43

992

S.la

vend

ulae

ssp

.av

iren

sN

RR

LB

-165

76L

5

S.la

vend

ulae

ssp

.br

asili

cus

NR

RL

B-2

937T

TL

3/L

5

S.la

vend

ulae

ssp

.in

osito

philu

sN

RR

LB

-390

4TT

L3/

L5

S.la

vend

ulae

ssp

.la

vend

ulae

vD

SM40

069T

AT

CC

8664

ISP

5069

F61

I22

22-3

042

FU

-12b

OC

-I13

29bp

D85

116

L3/

L5

S.la

vend

ulig

rise

usv

DSM

4048

7TA

TC

C13

306

ISP

5487

A34

Sv.0

21-

500

912

0bp

JCM

4545

AJ3

9948

7L

4S.

lave

ndul

ocol

orv

DSM

4021

6A

TC

C15

871

ISP

5216

F61

I22

22-3

1-08

120b

pJC

M43

90D

4410

5L

5S.

levi

sv

S.le

vori

sD

SM40

202

AT

CC

1587

6IS

P52

021-

11-

1S.

liban

iv

DSM

4055

5A

TC

C23

732

ISP

5555

A29

I15

025

005

121b

pJC

M47

81D

4435

7S.

liban

i ssp

. ruf

usv

S.lil

acin

usv

DSM

4025

4TA

TC

C23

930

ISP

5254

Sv.1

622

-104

0H

a812

1bp

JCM

4188

D44

027

S.lim

osus

vD

SM40

131T

AT

CC

1977

8IS

P51

31A

1AI

011-

11-

1F

U-1

KA

-D14

76bp

Z76

679

S.lin

coln

ensi

sv

DSM

4035

5A

TC

C25

466

ISP

5355

A19

I12

009

1-19

1519

bpX

7985

4S.

lineo

myc

ini

vS.

lipm

anii

vD

SM40

070

AT

CC

3331

ISP

5070

A1B

I02

1-3

1-2

FU

-9K

A-B

S.lip

man

iiv

DSM

4075

2A

TC

C27

357

I02

079

061

1484

bpJC

M47

11A

B04

5961

S.lis

teri

DSM

4029

7Sv

.06

703

4S.

litm

ocid

ini

vD

SM40

164

AT

CC

1991

4IS

P51

64A

05I

0400

21-

7K

A-C

120b

pJC

M43

94D

4410

9S.

livid

ans

DSM

4043

4A

TC

C19

844

ISP

5434

A21

006

1-18

FU

-6A

B03

756

S.lo

iden

sis

DSM

4082

5A

TC

C11

415

029

1-32

S.lo

mon

dens

isv

DSM

4142

8IV

03 (

blue

ser

ies)

009

1-19

121b

pJC

M48

66D

4441

5S.

long

ispo

rofla

vus

vD

SM40

165T

AT

CC

1991

5IS

P51

65A

39II

1000

501

0un

OC

-non

120b

pJC

M43

96D

4411

1S.

long

ispo

roru

ber

vD

SM40

599

AT

CC

2744

3IS

P55

99A

10I

0603

31-

33K

A-F

121b

pJC

M47

84D

4435

9S.

long

ispo

roru

ber

vD

SM40

749

AT

CC

1393

1I

0603

31-

33L

3S.

long

ispo

rus

vD

SM40

166T

AT

CC

2393

1IS

P51

66A

18I

1100

91-

1914

48bp

AJ3

9947

5L

2S.

long

issi

mus

DSM

4043

5A

TC

C19

850

ISP

5435

E54

033

1-33

S.lo

ngis

sim

usD

SM40

826

AT

CC

1456

206

41-

30

Spec

ies

nam

e

Stra

in n

umbe

r(s)

Clu

ster

num

bers

of

num

eric

al t

axon

omic

stud

ies

and

Ber

gey

cate

gori

es a

ndsp

ecie

s no

.aG

roup

s ac

cord

ing

to d

iffe

rent

stu

dies

abp

rRN

A d

ata

stra

in n

o.if

dif

fere

nt f

rom

the

first

col

umn

Acc

. no.

(EM

BL

)b

Stra

ins

incl

uded

inD

NA

/DN

Ahy

brid

isat

ion

stud

iesc

12

34

56

78

9

(Con

tinue

d)

S.lo

ngw

oode

nsis

vD

SM41

677T

120b

pJC

M49

76D

4442

3S.

luce

nsis

vD

SM40

317

AT

CC

1780

4IS

P53

17A

31I

211-

51-

1612

0bp

JCM

4490

D44

183

S.lu

ridu

sv

DSM

4008

1TA

TC

C19

782

ISP

5081

F62

II14

22-3

1-08

La1

7O

C-I

I12

0bp

JCM

4591

D44

252

S.lu

sita

nus

vD

SM40

568

AT

CC

1584

2IS

P55

68C

44II

1200

61-

1812

1bp

JCM

4785

D44

360

S.lu

teofl

uore

scen

sD

SM40

398

AT

CC

2546

9IS

P53

9803

802

5S.

lute

ogri

seus

vD

SM40

483

AT

CC

1507

2IS

P54

83A

18I

1100

91-

1914

48bp

AJ3

9949

0L

2S.

lute

olut

esce

nsD

SM40

600

AT

CC

2744

5IS

P56

00A

1B1-

31-

20S.

lute

oret

icul

iD

SM40

509

AT

CC

2744

6IS

P55

09Sv

.1-

81-

17H

a913

54bp

X53

172

L4

S.lu

teos

pore

usv

DSM

4083

3TH

a10

S.lu

teov

ertic

illat

usv

DSM

4003

8TA

TC

C23

933

ISP

5038

F55

Sv. 0

322

-104

0H

a1F

U-1

2b12

0bp

JCM

4099

S.ly

dicu

sv

DSM

4046

1TA

TC

C25

470

ISP

5461

A29

I15

025

005

La9

FU

-21

OC

-non

1481

bpY

1550

7S.

mac

rosp

orus

vD

SM40

096

AT

CC

1978

3IS

P50

96A

3800

601

0S.

mac

rosp

orus

vD

SM41

449T

1484

bpZ

6809

9S.

mac

rosp

orus

vD

SM41

476T

1490

bpZ

6810

0S.

maj

orci

ensi

sN

RR

L15

167

L5

S.m

alac

hitic

usD

SM40

167

AT

CC

1991

8IS

P51

67A

1206

005

1L

a14

S.m

alac

hito

fusc

usv

DSM

4033

2A

TC

C25

471

ISP

5332

006

1-18

120b

pJC

M44

93D

4418

5S.

mal

achi

tore

ctus

DSM

4033

3A

TC

C25

472

ISP

5333

006

1-18

S.m

alac

hito

spin

usv

S.m

alay

sien

sis

vD

SM41

697T

1475

bpA

TB

-11

AF

1173

04S.

mas

huen

sis

vD

SM40

221T

AT

CC

2393

4IS

P52

21F

55Sv

.03

22-1

040

Ha1

115

18bp

X79

323

L4

S.m

ashu

ensi

sv

DSM

4089

6Sv

.03

22-1

040

S.m

assa

spor

eus

vD

SM40

035T

AT

CC

1978

5IS

P50

35D

SMII

I19

015

1-19

La1

2O

C-I

II12

1bp

JCM

4593

D44

253

S.m

aten

sis

vD

SM40

188

AT

CC

2393

5IS

P51

88A

12I

0700

61-

18F

U-1

122b

pJC

M46

51D

4428

6S.

mau

veco

lor

vD

SM41

702T

120b

pJC

M50

02D

4442

5S.

med

ioci

dicu

sv

DSM

4002

1A

TC

C23

936

ISP

5021

FSm

22-1

040

Ha3

120b

pJC

M40

60D

4399

4S.

med

ioci

dicu

sv

DSM

4086

4A

TC

C13

278

1-3

1-2

S.m

edio

cidi

cus

vD

SM40

865

AT

CC

1327

91-

31-

2S.

med

iola

niv

DSM

4105

8T12

0bp

JCM

5076

D44

440

S.m

edite

rran

eiD

SM40

773

064

1-30

S.m

egas

poru

sv

DSM

4147

6T14

90bp

Z68

100

S.m

elan

ogen

esv

DSM

4019

2TA

TC

C23

937

ISP

5192

A33

II07

009

1-09

120b

pJC

M43

98D

4411

3S.

mel

anos

poro

faci

ens

vD

SM40

318

AT

CC

2547

3IS

P53

18A

32I

1605

101

814

83bp

AJ3

9183

7L

1S.

mel

anos

poro

faci

ens

vD

SM40

318T

NR

RL

B-1

2234

1483

bpA

J271

887

S.m

exic

anus

v14

50bp

CH

-M-1

035T

AF

4411

68S.

mic

higa

nens

isv

DSM

4001

5A

TC

C14

970

ISP

5015

A06

I05

005

029

120b

pJC

M45

94D

4425

4S.

mic

rofla

vus

vD

SM40

331T

AT

CC

1323

1IS

P53

31A

23I

201-

31-

2L

a22

OC

-I12

0bp

JCM

4496

D44

188

S.m

inoe

nsis

DSM

4003

1A

TC

C19

787

ISP

5031

A19

009

1-19

S.m

inut

iscl

erot

icus

vD

SM40

301

AT

CC

1775

7IS

P53

01A

15I

0800

61-

18K

A-G

121b

pJC

M47

90D

4436

5S.

mir

abili

sv

DSM

4055

3A

TC

C27

447

ISP

5553

A19

I12

1-7

1-19

1466

bpA

F11

2180

S.m

isak

iens

isv

DSM

4022

2TA

TC

C23

938

ISP

5222

F66

II16

22-4

043

La1

8O

C-n

on12

0bp

JCM

4653

D44

287

S.m

isio

nens

isv

DSM

4030

6A

TC

C14

991

ISP

5306

A31

I21

1-6

1-16

120b

pJC

M44

97D

4418

9S.

mob

arae

nsis

vD

SM40

847T

AT

CC

2903

2Sv

.07

22-1

040

Ha1

2F

U-1

2bL

4S.

mob

arae

nsis

vD

SM40

587

ISP

5587

120b

pJC

M47

78D

4435

5S.

mod

erat

usD

SM40

529

AT

CC

2344

3IS

P55

29A

Sm03

31-

33S.

mon

omyc

ini

v

Spec

ies

nam

e

Stra

in n

umbe

r(s)

Clu

ster

num

bers

of

num

eric

al t

axon

omic

stud

ies

and

Ber

gey

cate

gori

es a

ndsp

ecie

s no

.aG

roup

s ac

cord

ing

to d

iffe

rent

stu

dies

abp

rRN

A d

ata

stra

in n

o.if

dif

fere

nt f

rom

the

first

col

umn

Acc

. no.

(EM

BL

)b

Stra

ins

incl

uded

inD

NA

/DN

Ahy

brid

isat

ion

stud

iesc

12

34

56

78

9

Tabl

e 4.

Con

tinue

d

S.m

oroo

kaen

sis

vD

SM40

503T

AT

CC

1916

6IS

P55

03F

59Sv

. 08

22-1

040

Ha1

3S.

mur

inus

vD

SM40

091

AT

CC

1978

8IS

P50

91A

17I

101-

61-

1612

0bp

JCM

4333

D44

056

L3

S.m

utab

ilis

vD

SM40

169

AT

CC

1991

9IS

P51

69A

12I

0700

61-

18K

A-E

121b

pJC

M44

00D

4411

5S.

mut

omyc

ini

vS.

naga

nish

iiv

DSM

4028

2A

TC

C23

939

ISP

5282

A31

I21

1-6

1-15

S.na

raen

sis

DSM

4050

8A

TC

C13

788

ISP

5508

A1C

003

1-3

S.na

rbon

ensi

sv

DSM

4001

6A

TC

C19

790

ISP

5016

A04

I04

002

1-7

KA

-C12

0bp

JCM

4596

D44

255

S.na

shvi

llens

isv

DSM

4031

4A

TC

C25

476

ISP

5314

A05

I04

002

1-7

120b

pJC

M44

98D

4419

0S.

netr

opsi

sv

DSM

4025

9TA

TC

C23

940

ISP

5259

F56

Sv.0

122

-104

0H

a14

FU

-21

120b

pJC

M40

63D

4399

5L

4S.

neya

gaw

aens

isv

DSM

4058

8A

TC

C27

449

ISP

5588

A18

I11

009

1-19

FU

-24

1449

bpA

B02

6219

L2

S.ni

ger

vD

SM40

302

A40

I18

069

1-26

121b

pJC

M31

58D

4398

0S.

nigr

esce

nsv

DSM

4027

6TA

TC

C23

941

ISP

5276

A29

I15

025

005

La2

121b

pJC

M44

01D

4411

6S.

nigr

ifac

iens

vD

SM40

071

AT

CC

1979

1IS

P50

71A

1CI

031-

201

5K

A-B

120b

pJC

M42

23D

4403

1S.

nitr

ospo

reus

vD

SM40

023T

ISP

5023

120b

pJC

M40

64D

4399

6S.

nive

orub

erv

DSM

4063

8TA

TC

C14

971

IV08

(re

d se

ries

)01

31-

19L

a112

0bp

JCM

4234

D44

035

S.ni

veus

vD

SM40

088T

AT

CC

1979

3IS

P50

88A

1BI

0204

301

3L

a19

120b

pJC

M45

99D

4425

6S.

nobi

lisD

SM40

441

AT

CC

1925

1IS

P54

41A

1003

31-

33S.

nobo

rito

ensi

sv

DSM

4022

3TA

TC

C25

477

ISP

5223

A33

II07

009

1-09

La1

9O

C-I

120b

pJC

M45

57D

4422

9S.

oliv

aceo

viri

dis

vD

SM40

334T

AT

CC

2363

0IS

P53

34A

20I

1300

91-

19L

a21

OC

-III

120b

pJC

M44

99D

4419

1S.

nodo

sus

vD

SM40

109

AT

CC

1489

9IS

P51

09A

35II

0800

61-

1112

0bp

JCM

4656

AF

1140

34S.

noga

late

rv

DSM

4054

6TA

TC

C27

451

ISP

5546

A34

III

061-

500

9L

a14

OC

-III

1485

bpJC

M47

99A

B04

5886

S.no

jirie

nsis

vD

SM41

655T

120b

pJC

M33

82D

4398

4S.

nour

sei

vD

SM40

635T

AT

CC

1145

5IV

23 (

gray

ser

ies)

025

1-09

121b

pJC

M49

22D

4441

9S.

nova

ecae

sare

aev

DSM

4035

8TA

TC

C27

452

ISP

5358

JSm

III

2500

400

6O

C-I

V12

0bp

JCM

4800

D44

371

S.oc

hrac

eisc

lero

ticus

vD

SM40

594T

III

0806

91-

26O

C-n

on12

1bp

JCM

4801

D44

372

S.oc

hrol

eucu

sD

SM40

591

AT

CC

3006

ISP

5591

1-1

1-1

S.od

orif

erv

DSM

4034

7A

TC

C62

46IS

P53

47A

1AI

011-

11-

1K

A-D

1476

bpZ

7668

2S.

olig

ocar

boph

ilus

DSM

4058

9A

TC

C27

453

ISP

5589

A1B

1-3

1-2

S.ol

ivac

eisc

lero

ticus

vD

SM40

595T

AT

CC

1572

2IS

P55

95IV

24 (

gray

ser

ies)

069

1-26

121b

pJC

M48

05D

4437

5S.

oliv

aceu

sv

DSM

4007

2TA

TC

C33

35IS

P50

72A

1CI

0304

201

4L

a23

FU

-1S.

oliv

aceu

sv

DSM

4153

8A

TC

C21

379

I03

1-4

1-4

709b

pA

F31

8046

S.ol

ivoc

hrom

ogen

esv

DSM

4045

1A

TC

C33

36IS

P54

51A

19I

1200

91-

1914

48bp

AY

0943

70S.

oliv

ochr

omog

enes

ssp

.cy

tovi

rinu

sv

DSM

4082

8A

TC

C12

791

I12

009

1-19

FU

-1

S.ol

ivom

ycin

iv

S.ol

ivor

etic

uli

ssp.

oliv

oret

icul

iv

DSM

4010

5TIS

P51

05H

a112

0bp

JCM

4176

D44

023

S.ol

ivov

ertic

illat

usv

DSM

4019

6IS

P51

9612

0bp

JCM

4400

D44

007

S.ol

ivov

ertic

illat

usv

DSM

4025

0TN

RR

LB

-199

4TT

Ha1

8L

4S.

oliv

ovir

idis

vD

SM40

211

AT

CC

1588

2IS

P52

11A

03II

201-

301

012

0bp

JCM

4432

D44

140

S.om

iyae

nsis

vD

SM40

552

AT

CC

2745

4IS

P55

52A

05I

0400

21-

7K

A-C

120b

pJC

M48

06D

4437

6S.

orin

oci

vD

SM40

571T

AT

CC

2320

2IS

P55

71F

58Sv

.17

22-1

040

Ha1

512

0bp

JCM

4546

D44

225

S.or

natu

sD

SM40

307

AT

CC

2326

5IS

P53

07A

1B1-

31-

215

18bp

X79

326

S.os

treo

gris

eus

DSM

4051

1A

TC

C27

455

ISP

5511

A25

025

1-23

S.pa

ctum

vD

SM40

530T

AT

CC

2745

6IS

P55

30C

44II

1222

-403

5L

a11

OC

-II

121b

pJC

M48

09D

4437

7S.

palli

dus

DSM

4053

1TA

TC

C27

457

ISP

5531

A18

046

1-19

La1

414

50bp

AJ3

9949

2L

2S.

para

coch

leat

usv

S.pa

rado

xus

vD

SM43

350T

121b

pJC

M30

52D

4397

5S.

para

guay

ensi

sD

SM40

567

AT

CC

2745

8IS

P55

67C

4603

31-

33S.

parv

ispo

roge

nes

vD

SM40

473T

AT

CC

1256

8IS

P54

73Sv

.02

22-1

040

Ha1

120b

pJC

M46

94D

4430

2S.

parv

ullu

sv

DSM

4072

204

802

6S.

parv

ullu

sv

DSM

4072

8I

0700

61-

18S.

parv

ullu

sv

DSM

4091

201

11-

20

Spec

ies

nam

e

Stra

in n

umbe

r(s)

Clu

ster

num

bers

of

num

eric

al t

axon

omic

stud

ies

and

Ber

gey

cate

gori

es a

ndsp

ecie

s no

.aG

roup

s ac

cord

ing

to d

iffe

rent

stu

dies

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rRN

A d

ata

stra

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o.if

dif

fere

nt f

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first

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umn

Acc

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(EM

BL

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Stra

ins

incl

uded

inD

NA

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Ahy

brid

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ion

stud

iesc

12

34

56

78

9

(Con

tinue

d)

S.pa

rvul

usv

DSM

4004

8TA

TC

C12

434

ISP

5048

A12

006

1-18

La2

4S.

parv

usv

DSM

4034

8A

TC

C12

433

ISP

5348

A1B

I02

1-3

1-2

FU

-6K

A-B

120b

pJC

M40

69D

4399

8

S.pa

rvus

vD

SM40

829

AT

CC

1232

0I

0200

502

9S.

pauc

ispo

roge

num

DSM

4031

5A

TC

C12

596

ISP

5315

F55

Sv. 0

222

-104

0S.

pent

atic

um s

sp. j

enen

seD

SM40

848

Sv. 0

322

-104

0L

4S.

peru

vien

sis

DSM

4059

2A

TC

C27

459

ISP

5592

A18

009

1-19

AJ3

9949

4S.

peuc

etiu

sv

DSM

4075

4N

CIB

1097

2IV

09 (

red

seri

es)

035

1-33

1486

bpJC

M99

20A

B04

5887

S.ph

aeoc

hrom

ogen

esv

DSM

4007

3TA

TC

C33

38IS

P50

73A

40I

1800

90-

19L

a1O

C-I

I14

50bp

AF

5000

71S.

phae

ochr

omog

enes

vD

SM40

788

NR

RL

B-1

266

I18

076

071

S.ph

aeof

acie

nsv

DSM

4036

7TIS

P53

6712

0bp

JCM

4814

D44

381

S.ph

aeol

utei

gris

eus

NR

RL

5182

1479

bpA

J391

815

S.ph

aeop

urpu

reus

vD

SM40

125

AT

CC

2394

6IS

P51

25A

09II

0200

91-

1912

0bp

JCM

4660

D44

290

S.ph

aeov

irid

isv

DSM

4028

5A

TC

C23

947

ISP

5285

A19

I12

009

1-19

120b

pJC

M46

61D

4429

1S.

phos

alac

ineu

sv

1475

bpJC

M33

40U

9333

0S.

pilo

sus

vD

SM40

097T

AT

CC

1979

7IS

P50

97A

37I

1700

61-

1012

1bp

JCM

4403

D44

118

S.pl

aten

sis

vD

SM40

041

AT

CC

1386

5IS

P50

41A

29I

1502

500

5F

U-2

114

88bp

JCM

4662

AB

0458

82S.

plic

atus

vD

SM40

319

AT

CC

2548

3IS

P53

19A

12I

0700

61-

18K

A-E

121b

pJC

M45

04D

4419

4S.

plur

icol

ores

cens

vD

SM40

019

AT

CC

1979

8IS

P50

19A

1BI

021-

31-

2K

A-B

120b

pJC

M46

02D

4425

8S.

poly

chro

mog

enes

vD

SM40

316

AT

CC

1259

5IS

P53

16F

61I

2222

-304

212

0bp

JCM

4505

D44

195

L3/

L5

S.po

onen

sis

vD

SM40

596T

AT

CC

1572

3IS

P55

96A

22II

1907

11-

19L

a4O

C-I

II12

1bp

JCM

4815

D44

382

S.pr

aeco

xv

DSM

4039

3TA

TC

C33

74IS

P53

93IV

08 (

yello

w s

erie

s))1

-31-

212

0bp

JCM

4506

D44

196

S.pr

asin

opilo

sus

vD

SM40

098T

AT

CC

1979

9IS

P50

98A

37I

1700

700

3L

a20

119b

pJC

M44

04D

4411

9S.

pras

inos

poru

sv

DSM

4050

6TA

TC

C17

918

ISP

5506

A38

III

0722

-21-

15L

10O

C-I

II11

9bp

JCM

4816

D44

383

S.pr

asin

usv

DSM

4009

9TA

TC

C19

800

ISP

5099

A37

I17

007

003

120b

pJC

M46

03D

4425

9S.

pris

tinae

spir

alis

DSM

4033

8A

TC

C25

486

ISP

5338

A26

004

1-23

S.pr

unic

olor

vD

SM40

335T

AT

CC

2548

7IS

P53

35A

11II

I01

1-1

1-1

unO

C-I

I12

0bp

JCM

4508

D44

198

S.ps

amm

otic

usv

DSM

4034

1TA

TC

C25

488

ISP

5341

F67

II17

011

1-21

OC

-I12

0bp

JCM

4434

D44

141

S.ps

eudo

echi

nosp

oreu

sv

S.ps

eudo

gris

eolu

sv

NR

RL

3985

1516

bpX

8082

7S.

pseu

dogr

iseo

lus

vD

SM40

026

AT

CC

1277

0IS

P50

26A

12I

0700

61-

18K

A-G

121b

pJC

M40

71D

4399

9S.

pseu

dove

nezu

elae

vD

SM40

212

AT

CC

2395

1IS

P52

12A

18I

1100

91-

1914

50bp

AJ3

9948

1L

2S.

pseu

dove

nezu

elae

vD

SM40

213

I11

22-3

042

L3

S.pu

lche

rD

SM40

566

AT

CC

1384

9IS

P55

66A

1200

61-

18S.

pulv

erac

eus

vD

SM41

657T

120b

pJC

M75

45D

4444

2S.

puni

ceus

vD

SM40

083

AT

CC

1980

1IS

P50

83A

09II

0200

502

912

0bp

JCM

4406

D44

121

S.pu

rpeo

fusc

usv

DSM

4028

3TA

TC

C23

952

ISP

5283

IV26

(gr

ay s

erie

s)22

-304

312

1bp

JCM

4665

D44

294

S.pu

rpur

asce

nsv

DSM

4031

0TA

TC

C25

489

ISP

5310

A18

I11

009

1-19

1448

bpA

B04

5888

L2

S.pu

rpur

eus

vD

SM43

360T

1350

bpX

5317

0S.

purp

ureu

sv

DSM

4336

2TI

2322

-31-

05L

a18

OC

-IS.

purp

urog

enei

scle

rotic

usv

DSM

4027

1A

4006

91-

2612

1bp

JCM

4818

D44

385

S. p

yrid

omyc

etic

usD

SM40

024

AT

CC

2395

3IS

P50

2422

-406

2S.

race

moc

hrom

ogen

esv

DSM

4019

4A

TC

C23

954

ISP

5194

F61

I22

22-3

042

120b

pJC

M44

07D

4412

2L

5

Spec

ies

nam

e

Stra

in n

umbe

r(s)

Clu

ster

num

bers

of

num

eric

al t

axon

omic

stud

ies

and

Ber

gey

cate

gori

es a

ndsp

ecie

s no

.aG

roup

s ac

cord

ing

to d

iffe

rent

stu

dies

abp

rRN

A d

ata

stra

in n

o.if

dif

fere

nt f

rom

the

first

col

umn

Acc

. no.

(EM

BL

)b

Stra

ins

incl

uded

inD

NA

/DN

Ahy

brid

isat

ion

stud

iesc

12

34

56

78

9

Tabl

e 4.

Con

tinue

d

S. r

ameu

sv

DSM

4168

5T12

0bp

JCM

5064

D44

433

S.ra

mul

osus

vD

SM40

100T

AT

CC

1980

2IS

P51

00C

SmII

I16

035

041

unO

C-n

on12

1bp

JCM

4604

D44

260

S.ra

ngoo

nens

isv

DSM

4045

2TA

TC

C68

60IS

P54

52IV

07 (

whi

te s

erie

s)03

01-

3412

0bp

JCM

4510

D44

200

S.re

cife

nsis

vD

SM40

115

AT

CC

1980

3IS

P51

15A

23I

201-

505

912

0bp

JCM

4408

D44

123

S.re

ctiv

ertic

illat

usv

DSM

4043

6TA

TC

C19

845

ISP

5436

F57

Sv.1

822

-104

0H

a7S.

rect

ivio

lace

usv

S.re

galis

DSM

4053

2A

TC

C27

460

ISP

5532

009

1-19

S.re

gens

isv

DSM

4055

1A

TC

C27

461

ISP

5551

A20

I13

009

1-19

120b

pJC

M48

20D

4438

7S.

resi

stom

ycifi

cus

vD

SM40

133T

AT

CC

1980

4IS

P51

33A

18I

1100

91-

19F

U-1

2a14

48bp

AJ3

9947

2L

2S.

retic

uli

DSM

4077

6A

TC

C23

384

1-3

1-2

S.re

ticul

isca

biei

v14

18bp

CF

BP

4531

AJ0

0742

8S.

retic

ulum

DSM

4089

3A

TC

C25

607

Sv.

22-1

040

L4

S.re

ticul

um s

sp.

prot

omyc

icum

DSM

4084

9TSv

. 19

22-1

040

S.rh

izos

phae

ricu

sv

DSM

4176

0T14

80bp

A10

P1

AJ3

9183

4S.

rim

osus

v60

67bp

R6-

554T

X62

884

S.ri

mos

us s

sp.

paro

mom

ycin

usv

S.ri

mos

us s

sp.r

imos

usv

DSM

4026

0TA

TC

C10

970

ISP

5260

B42

I19

035

1-33

La9

OC

-non

1485

bpJC

M46

67A

B04

5883

S.ri

shir

iens

isv

DSM

4048

9A

TC

C14

812

ISP

5489

A19

I12

1-7

1-15

FU

-12a

120b

pJC

M48

21D

4438

8S.

roc

hei

vD

SM40

231T

AT

CC

1073

9IS

P52

31A

12I

0700

61-

18L

a13

OC

-III

KA

-E86

9bp

AJ2

9199

5S.

rosa

DSM

4053

3A

TC

C27

462

ISP

5533

A17

006

010

S.ro

seis

cler

otic

usv

DSM

4030

3A

TC

C17

755

ISP

5303

II19

049

022

121b

pJC

M48

23A

B01

8206

S.ro

seoc

hrom

ogen

esD

SM40

463

AT

CC

1340

0IS

P54

631-

300

4S.

rose

ochr

omog

enes

DSM

4085

6A

TC

C33

471-

31-

2S.

rose

ochr

omog

enes

DSM

4087

9IF

O33

6322

-304

2S.

rose

odia

stat

icus

vD

SM41

703T

120b

pJC

M42

95D

4404

5S.

rose

oflav

usv

1412

bpJC

M41

54A

F36

9704

S.ro

seofl

avus

vA

TC

C19

920

1523

bpA

F29

0616

S.ro

seofl

avus

vD

SM40

536

AT

CC

1316

7IS

P55

36IV

10 (

red

seri

es)

22-5

105

S.ro

seof

ulvu

sv

DSM

4017

2A

TC

C19

921

ISP

5172

A14

II04

002

1-7

120b

pJC

M46

05D

4426

1S.

rose

ogri

seus

DSM

4048

8A

TC

C12

414

ISP

5488

009

1-19

S.ro

seol

ilaci

nus

vD

SM40

173

AT

CC

1992

2IS

P51

73G

68II

1822

-503

912

1bp

JCM

4335

D44

057

S.ro

seol

usv

DSM

4017

4A

TC

C23

210

ISP

5174

A05

I04

002

1-7

120b

pJC

M44

11D

4412

6S.

rose

olut

eus

DSM

4024

0A

TC

C23

975

ISP

5240

A17

1-6

1-16

S.ro

seos

poru

sv

DSM

4012

2A

TC

C23

958

ISP

5122

A05

I04

002

1-7

120b

pJC

M44

12D

4412

7S.

rose

over

ticill

atus

vD

SM40

039T

AT

CC

1980

7IS

P50

39Sv

.01

22-1

040

Ha7

L4

S.ro

seov

ertic

illat

us s

sp.

albo

spor

usv

DSM

4090

0A

TC

C25

189

Sv.0

122

-104

0

S.ro

seov

ertic

illat

usv

IFO

1284

511

97bp

AB

0728

37S.

rose

ovio

lace

usv

DSM

4027

7A

TC

C25

493

ISP

5277

A18

I11

009

1-19

FU

-114

48bp

AJ3

9948

4L

2S.

rose

ovir

idis

vD

SM40

175

AT

CC

2395

9IS

P51

75A

05I

0422

-203

712

0bp

JCM

4414

D44

128

S.ro

seus

DSM

4007

6A

TC

C19

808

ISP

5076

A07

002

1-7

S.ru

ber

vD

SM40

304

IV11

(re

d se

ries

)04

902

212

1bp

JCM

3131

AB

0182

03S.

rube

scen

sD

SM40

777

NR

RL

B-1

519

076

071

S.ru

bigi

noso

helv

olus

vD

SM40

176T

AT

CC

1992

6IS

P51

76IV

12 (

red

seri

es)

006

1-2

120b

pJC

M44

15D

4412

9S.

rubi

gino

sus

vD

SM40

177

AT

CC

1992

7IS

P51

77A

12I

0700

61-

1812

1bp

JCM

4416

D44

130

S.ru

broc

hlor

inus

DSM

4085

0N

RR

LB

-125

58H

a7L

4S.

rubr

over

ticill

atus

DSM

4085

1Sv

.01

22-1

040

Ha1

S.ru

brov

ertic

illat

usD

SM41

489

NR

RL

B-1

6433

L4

S.ru

tger

sens

is s

sp.

cast

elar

ensi

sv

DSM

4083

0A

TC

C15

191

I01

055

018

Spec

ies

nam

e

Stra

in n

umbe

r(s)

Clu

ster

num

bers

of

num

eric

al t

axon

omic

stud

ies

and

Ber

gey

cate

gori

es a

ndsp

ecie

s no

.aG

roup

s ac

cord

ing

to d

iffe

rent

stu

dies

abp

rRN

A d

ata

stra

in n

o.if

dif

fere

nt f

rom

the

first

col

umn

Acc

. no.

(EM

BL

)b

Stra

ins

incl

uded

inD

NA

/DN

Ahy

brid

isat

ion

stud

iesc

12

34

56

78

9

(Con

tinue

d)

S.ru

tger

sens

is s

sp.

rutg

erse

nsis

vD

SM40

077T

AT

CC

3350

ISP

5077

A1A

I01

1-1

1-1

KA

-D14

76bp

Z76

688

S.sa

lmon

isv

1340

bpD

PU

D 0

098T

X53

169

S.sa

lmon

isv

DSM

4089

5TN

RR

LB

-147

2Sv

. 05

22-1

040

Ha7

L4

S.sa

mps

onii

vD

SM40

394

AT

CC

2549

5IS

P53

94A

1AI

011-

11-

1K

A-D

1476

bpD

6387

1

S.sa

nnan

ensi

sv

S.sa

ppor

onen

sis

vD

SM41

675T

Ha4

S.sa

prop

hytic

usD

SM40

537

AT

CC

3351

ISP

5537

A1A

1-1

1-1

S.sa

race

ticus

DSM

4024

1A

TC

C25

496

ISP

5241

A29

050

019

S.sc

abie

i (s

cabi

es)

vA

TC

C49

173

1530

bpD

6386

2S.

scab

iei

vD

SM40

078

AT

CC

2396

2IS

P50

78A

031-

301

0S.

scab

iei

vD

SM40

611

AT

CC

3352

1-4

1-4

S.sc

abie

iv

DSM

4085

9IF

O31

111-

201

5S.

scab

iei

vD

SM40

960

006

1-18

S.sc

abie

iv

DSM

4096

104

51-

24S.

scab

iei

vD

SM40

962

014

021

S.sc

abie

iv

DSM

4099

401

31-

19S.

scab

iei

vD

SM40

995

008

019

S.sc

abie

iv

DSM

4099

600

21-

7S.

scab

iei

vD

SM40

997

009

1-19

S.sc

abie

iv

DSM

4099

800

91-

19S.

scab

iei

vD

SM40

999

008

019

S.sc

abie

iv

DSM

4100

000

91-

19S.

scle

rotia

lus

vD

SM40

269

I18

069

1-26

121b

pJC

M30

39D

4397

3S.

scop

ifor

mis

v14

02bp

A25

TA

F18

4081

S.se

oule

nsis

v14

79bp

IMSN

U 2

126

Z71

365

S.se

ptat

usv

DSM

4057

7TA

TC

C27

464

ISP

5577

F55

Sv.0

222

-104

0H

a6S.

seta

ev

DSM

4386

114

64bp

M55

220

S.se

toni

iv

DSM

4039

5A

TC

C25

497

ISP

5395

A1B

I02

1-3

1-2

KA

-B15

32bp

D63

872

S.sh

owdo

ensi

sv

DSM

4050

4A

TC

C15

105

ISP

5504

A06

I05

22-2

037

120b

pJC

M48

30D

4439

3S.

sind

enen

sis

vD

SM40

255T

AT

CC

2396

3IS

P52

55A

1B1-

31-

2K

A-B

120b

pJC

M46

69D

4429

7S.

sioy

aens

isv

DSM

4003

2A

TC

C13

989

ISP

5032

A29

I15

025

005

121b

pJC

M44

18D

4413

1S.

som

alie

nsis

vD

SM40

760

AT

CC

1481

703

51-

3314

83bp

AJ0

0739

9S.

spad

icis

DSM

4047

6A

TC

C19

017

ISP

5476

A34

1-5

009

S.sp

arso

gene

sv

DSM

4035

6TA

TC

C25

498

ISP

5356

A32

I16

010

1-19

La7

1493

bpA

J391

817

L1

S.sp

ecta

bilis

vD

SM40

512

NR

RL

2792

TIS

P55

1212

1bp

JCM

4832

D44

395

L3

S.sp

eibo

nae

vD

SM41

797T

1490

bpA

F45

2714

S.sp

eleo

myc

ini

vS.

sphe

roid

esv

DSM

4029

2A

TC

C23

965

ISP

5292

A1B

I02

040

048

La1

912

0bp

JCM

4670

D44

298

S.sp

inov

erru

cosu

sv

S.sp

iral

isv

DSM

4383

612

1bp

JCM

3302

D43

983

S.sp

irov

ertic

illat

usv

DSM

4003

6A

TC

C19

811

ISP

5036

A06

I05

002

1-7

KA

-A12

0bp

JCM

4609

D44

263

S.sp

itsbe

rgen

sis

vH

a7

Spec

ies

nam

e

Stra

in n

umbe

r(s)

Clu

ster

num

bers

of

num

eric

al t

axon

omic

stud

ies

and

Ber

gey

cate

gori

es a

ndsp

ecie

s no

.aG

roup

s ac

cord

ing

to d

iffe

rent

stu

dies

abp

rRN

A d

ata

stra

in n

o.if

dif

fere

nt f

rom

the

first

col

umn

Acc

. no.

(EM

BL

)b

Stra

ins

incl

uded

inD

NA

/DN

Ahy

brid

isat

ion

stud

iesc

12

34

56

78

9

Tabl

e 4.

Con

tinue

d

S.sp

orif

erum

DSM

4090

1A

TC

C25

188

Sv. 0

222

-104

0S.

spor

ocin

ereu

sv

S.sp

oroc

livat

usv

S.sp

oroc

ochl

eatu

s(K

. par

acoc

hlea

tus)

vIF

O14

769T

1475

bpU

9332

8

S.sp

oror

aveu

sv

S.sp

orov

erru

cosu

sv

S.st

effis

burg

ensi

sD

SM40

547

AT

CC

2746

6IS

P55

47A

3800

91-

1914

83bp

JCM

4833

AB

0458

89S.

stel

lisca

biei

v14

78bp

CF

BP

452

1A

J007

429

S.st

ram

ineu

sv

DSM

4168

3TH

a16

S.st

rept

omyc

ini

DSM

4020

0A

TC

C15

886

ISP

5200

1-3

1-2

S.su

brut

ilus

vD

SM40

445

AT

CC

2746

7IS

P54

45F

6122

-304

215

16bp

X80

825

L5

S.su

lfon

ofac

iens

vA

TC

C31

892

620b

pA

F31

8042

S.su

lphu

reus

vD

SM40

104T

AT

CC

2746

8IS

P51

04C

SmII

I17

068

002

unO

C-n

on12

0bp

JCM

4835

D44

397

S.sy

ring

ium

vD

SM41

480

Ha1

4

S.ta

itoen

sis

DSM

4149

9N

RR

LB

-164

35L

4S.

taka

taen

sis

DSM

4057

6A

TC

C27

469

ISP

5576

Sv. 0

922

-104

0H

a1L

4S.

tana

shie

nsis

vD

SM40

195T

AT

CC

2396

7IS

P51

95IV

30 (

gray

ser

ies)

002

1-7

120b

pJC

M46

71D

4429

9S.

taur

icus

vD

SM40

560

AT

CC

2747

0IS

P55

60A

1901

201

914

92bp

JCM

4837

AB

0458

79S.

tend

aev

DSM

4010

1TA

TC

C19

812

ISP

5101

A12

I07

006

1-18

La1

4K

A-E

1530

bpD

6387

3S.

tene

brar

ius

DSM

4047

7A

TC

C17

920

ISP

5477

026

027

S.te

rmitu

mv

DSM

4032

9A

TC

C25

499

ISP

5329

A05

I04

22-2

037

120b

pJC

M45

18D

4420

6S.

teta

nuse

mus

DSM

4058

5A

TC

C27

471

ISP

5585

A1A

1-1

1-1

S.th

erm

oalc

alito

lera

nsv

DSM

4174

1T12

86bp

TA

56T

AJ0

0028

4S.

ther

moa

utot

roph

icus

vS.

ther

moc

arbo

xydo

vora

nsv

DSM

4429

415

02bp

U94

487

S.th

erm

ocar

boxy

dovo

rans

vD

SM44

296T

1502

bpU

9448

9S.

ther

moc

arbo

xydu

sv

DSM

4429

315

00bp

U94

490

S.th

erm

ocop

roph

ilus

vD

SM41

700T

1499

bpB

19A

J007

402

S.th

erm

odia

stat

icus

vD

SM40

573T

AT

CC

2747

2IS

P55

73A

1CI

0300

61-

1814

83bp

AB

0180

96S.

ther

mofl

avus

DSM

4057

4A

TC

C27

473

ISP

5574

A36

1-7

1-15

S.th

erm

ogri

seus

v15

40bp

CC

TC

CA

A 9

7A

F05

6712

S.th

erm

ogri

seus

v15

40bp

CC

TC

CA

A 9

7A

F05

6714

S.th

erm

olin

eatu

sv

DSM

4145

1T14

81bp

Z68

097

S.th

erm

onitr

ifica

ns(t

herm

ovul

gari

s)v

DSM

4057

9TA

TC

C23

385

ISP

5579

A36

II09

021

002

1486

bpZ

6809

8

S.th

erm

ophi

lus

DSM

4036

5A

TC

C19

282

ISP

5365

A15

006

1-18

S.th

erm

ospi

nosi

spor

usv

1522

bpA

T 1

0A

F33

3113

S.th

erm

otol

eran

sD

SM40

227

AT

CC

1141

6IS

P52

27A

1800

91-

19F

U-1

1448

bpA

J399

482

S.th

erm

ovio

lace

usv

1350

bpA

B10

6A

Y02

9353

S.th

erm

ovio

lace

us s

sp.

apin

gens

vD

SM41

392T

121b

pJC

M43

12D

4405

0

S.th

erm

ovio

lace

us s

sp.

term

ovio

lace

usv

DSM

4044

3TA

TC

C19

283

ISP

5443

C45

II13

004

006

La1

314

83bp

Z68

096

S.th

erm

ovul

gari

sv

DSM

4044

4TA

TC

C19

284

ISP

5444

A36

II09

021

002

unO

C-n

on14

86bp

Z68

094

S.th

iolu

teus

vD

SM40

027T

AT

CC

1231

0IS

P50

27F

SmSv

..21

22-1

040

Ha1

712

0bp

JCM

4087

D44

001

S.to

rulo

sus

vD

SM40

894T

NR

RL

B-3

889

IV31

(gr

ay s

erie

s)00

91-

1912

1bp

JCM

4872

D44

416

S.to

xytr

icin

iv

DSM

4017

8A

TC

C19

813

ISP

5178

F61

22-3

042

120b

pJC

M44

21D

4413

3L

3/L

5S.

toyo

caen

sis

DSM

4003

0A

TC

C19

814

ISP

5030

A29

013

1-19

S.tr

icol

orv

DSM

4270

412

1bp

JCM

5065

D44

434

S.tr

opic

alen

sis

DSM

4052

0A

TC

C17

963

ISP

5520

F55

Sv..

0322

-104

0H

a6L

4S.

tube

rcid

icus

vD

SM40

261T

AT

CC

2550

2IS

P52

61C

47II

I14

025

005

La2

OC

-non

121b

pJC

M45

58D

4423

0S.

tuir

usv

DSM

4050

5A

21I

1400

61-

1814

55bp

JCM

4846

AF

5034

90

Spec

ies

nam

e

Stra

in n

umbe

r(s)

Clu

ster

num

bers

of

num

eric

al t

axon

omic

stud

ies

and

Ber

gey

cate

gori

es a

ndsp

ecie

s no

.aG

roup

s ac

cord

ing

to d

iffe

rent

stu

dies

abp

rRN

A d

ata

stra

in n

o.if

dif

fere

nt f

rom

the

first

col

umn

Acc

. no.

(EM

BL

)b

Stra

ins

incl

uded

inD

NA

/DN

Ahy

brid

isat

ion

stud

iesc

12

34

56

78

9

(Con

tinue

d)

S.tu

rgid

isca

bies

v15

28bp

S27

AF

3617

82S.

turg

idis

cabi

esv

AT

CC

7023

48T

T18

65bp

AB

0262

21S.

umbr

inus

vD

SM40

278

AT

CC

1992

9IS

P52

78A

05I

041-

61-

1612

0bp

JCM

4521

D44

209

S.um

bros

usD

SM40

242

AT

CC

2550

4IS

P52

42A

331-

61-

12S.

valin

usN

RR

LB

-564

4L

2S.

vari

abili

sv

DSM

4017

9A

TC

C19

930

ISP

5179

A12

I07

006

1-18

KA

-F12

1bp

JCM

4422

D44

134

S.va

rieg

atus

vS.

vars

ovie

nsis

vD

SM40

346T

AT

CC

2550

5IS

P53

46C

46II

I13

037

028

La1

2O

C-I

I12

1bp

JCM

4523

D44

211

S.va

stus

vS.

vello

sus

NR

RL

8037

1520

bpX

9994

2S.

vend

arge

nsis

DSM

4037

9A

TC

C25

507

ISP

5379

035

1-33

S.ve

nezu

elae

vD

SM40

230T

AT

CC

1071

2IS

P52

30A

06I

0500

21-

7un

KA

-C14

83bp

JCM

4526

AB

0458

90S.

vern

eD

SM40

079

AT

CC

3353

ISP

5079

A40

069

1-26

S.ve

rsip

ellis

DSM

4049

1A

TC

C27

475

ISP

5491

ASm

1-7

1-15

S.ve

rtic

illiu

mD

SM40

903

AT

CC

1500

3Sv

. 06

22-1

040

S.vi

nace

usv

DSM

4025

7A

TC

C11

861

ISP

5257

A06

I05

1-3

1-2

S.vi

nace

usv

DSM

4051

5A

TC

C27

476

ISP

5515

A06

I05

22-3

042

KA

-A12

0bp

JCM

4849

D44

405

S.vi

rgin

iae

DSM

4009

4A

TC

C19

817

ISP

5094

F61

I22

22-3

042

FU

-12b

120b

pJC

M44

25D

8512

3L

3/L

5S.

vina

ceus

drap

pus

vD

SM40

470

AT

CC

2551

1IS

P54

70A

12I

0700

61-

18K

A-E

121b

pJC

M45

29D

4421

4S.

viol

aceo

chro

mog

enes

vD

SM40

181T

AT

CC

1993

2IS

P51

81IV

33 (

gray

ser

ies)

009

1-19

121b

pJC

M45

30D

4421

5S.

viol

aceo

latu

sv

DSM

4043

8A

TC

C19

847

ISP

5438

A21

I14

006

1-18

121b

pJC

M45

31D

4421

6S.

viol

aceo

rect

usv

DSM

4027

9A

TC

C25

514

ISP

5279

A05

I04

002

1-7

120b

pJC

M45

32D

4421

7S.

viol

aceo

rube

rv

DSM

4004

9TA

TC

C14

980

ISP

5049

IV34

(gr

ay s

erie

s)06

91-

2612

1bp

JCM

4423

D44

135

S.vi

olac

eoru

ber

(coe

licol

or)

v23

00bp

A(3

)2X

6051

4

S.vi

olac

eus

vD

SM40

082T

AT

CC

1588

8IS

P50

82A

06I

0500

90-

19O

C-I

II12

1bp

JCM

4533

D44

218

S.vi

olac

eusn

iger

vD

SM40

182

I16

009

020

L1

S.vi

olac

eusn

iger

vD

SM40

563T

AT

CC

2747

7IS

P55

63A

32I

1605

101

8L

a7O

C-I

1480

bpA

J391

823

L1

S.vi

olac

eusn

iger

vD

SM40

699

I16

041

012

S.vi

olac

eusn

iger

vD

SM41

598

NR

RL

B-1

356

I16

054

018

L1

S.vi

olac

eusn

iger

vD

SM41

599

NR

RL

B-1

477

I16

053

018

L1

S.vi

olac

eusn

iger

vD

SM41

600

NR

RL

B-1

478

I16

053

018

L1

S.vi

olac

eusn

iger

vD

SM41

602

NR

RL

B-1

6257

I16

054

018

L1

S.vi

olac

eusn

iger

vN

RR

L80

9714

97bp

AJ3

9181

6L

1S.

viol

aceu

snig

erv

DSM

4071

0N

RR

L28

34I

161-

61-

1613

41bp

AJ3

9181

3L

1S.

viol

aceu

snig

erv

DSM

4160

1N

RR

LB

-579

9I

161-

51-

1614

84bp

AJ3

9181

4L

1S.

viol

aceo

rubi

dus

vS.

viol

arus

vD

SM40

205

AT

CC

1589

1IS

P52

05A

18I

1100

91-

1914

47bp

AJ3

9947

7L

2S.

viol

asce

nsv

DSM

4018

3A

TC

C23

968

ISP

5183

A06

I05

002

1-7

120b

pJC

M44

24D

4413

6S.

viol

atus

vD

SM40

209T

AT

CC

1589

2IS

P52

09A

18I

1105

001

9L

a12

1448

bpA

J399

480

S.vi

olen

sv

DSM

4059

7A

TC

C15

898

ISP

5597

A40

I18

069

1-26

121b

pJC

M30

72D

4397

7S.

viol

ochr

omog

enes

DSM

4020

7A

TC

C15

893

ISP

5207

A18

009

1-19

L2

S.vi

rens

v

Spec

ies

nam

e

Stra

in n

umbe

r(s)

Clu

ster

num

bers

of

num

eric

al t

axon

omic

stud

ies

and

Ber

gey

cate

gori

es a

ndsp

ecie

s no

.aG

roup

s ac

cord

ing

to d

iffe

rent

stu

dies

abp

rRN

A d

ata

stra

in n

o.if

dif

fere

nt f

rom

the

first

col

umn

Acc

. no.

(EM

BL

)b

Stra

ins

incl

uded

inD

NA

/DN

Ahy

brid

isat

ion

stud

iesc

12

34

56

78

9

Tabl

e 4.

Con

tinue

d

Abb

revi

atio

ns: E

MB

L, E

urop

ean

Mol

ecul

ar B

iolo

gy L

abor

ator

y; v

, val

id n

ame;

T, t

ype

stra

in; D

SM, D

euts

che

Sam

mlu

ng v

on M

ikro

orga

nism

en u

nd Z

ellk

ultu

ren ;

AT

CC

, Am

eric

an T

ype

Cul

ture

Col

lect

ion;

ISP

, Int

erna

tion

al S

trep

tom

yces

Pro

ject

; NR

RL

,A

RS

Cul

ture

Col

lect

ion,

Nor

ther

n R

egio

nal R

esea

rch

Lab

orat

ory,

U.S

. Dep

artm

ent

of A

gric

ultu

re, P

eori

a, I

llino

is, U

SA; J

CM

, Jap

an C

olle

ctio

n O

f M

icro

orga

nism

s; IM

RU

, Ins

titu

te o

f M

icro

biol

ogy,

Rut

gers

Sta

te U

nive

rsit

y; I

MSN

U, I

nsti

tute

of

Mic

robi

olog

y,Se

oul N

atio

nal U

nive

rsit

y, S

eoul

, Kor

ea; a

nd C

FB

P, C

olle

ctio

n Fr

anca

ise

des

Bac

ter i

es P

hyto

path

ogen

es.

a 1) C

lust

er a

nd s

ubcl

uste

r nu

mbe

rs a

ccor

d ing

to

Will

iam

s et

al.

(198

3a).

2) A

ssig

nmen

t of

str

ains

to

spec

ies

acco

rdin

g to

Ber

gey’

s M

anua

l of

Sys

tem

atic

Bac

teri

olog

y, V

ol. 4

. For

the

gen

us S

trep

tom

yces

,spe

cies

cat

egor

ies

I to

IV

are

sho

wn,

wit

h th

e sp

ecie

snu

mbe

r w

ithi

n th

e ca

tego

ry a

ccor

ding

to

Will

iam

s et

al.

(198

9); f

or s

peci

es c

ateg

ory

IV, t

he ‘s

erie

s’ a

ssig

nmen

t is

als

o gi

ven,

acco

rdin

g to

spo

re c

olor

. For

spe

cies

pre

viou

sly

assi

gned

to

the

genu

s St

rept

over

ticill

ium

(now

rec

lass

ified

to

Stre

ptom

yces

), sp

ecie

snu

mbe

rs a

re a

ccor

ding

to

Loc

ci a

nd S

chofi

eld

(198

9). 3

) C

lust

er a

nd s

ubcl

uste

r nu

mbe

rs a

re a

ccor

d ing

to

Käm

pfer

et

al. (

1991

). T

he fi

rst

num

ber

is t

hat

of t

he U

PG

MA

/SSM

anal

ysis

, the

sec

ond

num

ber

is t

hat

of t

he U

PG

MA

/SJ

anal

ysis

. 4)

Gro

ups

are

acco

rdin

gto

the

stu

dy o

f H

atan

o et

al.

(200

3): H

a1 (

syno

nym

s of

S.a

biko

ensi

s), H

a2 (

syno

nym

s of

S.a

rdus

), H

a3 (

syno

nym

s of

S.b

last

myc

etic

us),

Ha4

(sy

nony

ms

of S

.cin

nam

oneu

s), H

a5 (

syno

nym

s of

S.e

uroc

idic

us),

Ha6

(sy

nony

ms

of S

.gri

seoc

arne

us),

Ha7

(sy

nony

ms

ofS.

hiro

shim

ensi

s), H

a8 (

syno

nym

s of

S.l

ilaci

nus)

, Ha9

(“S

.lut

eore

ticul

i”),

Ha1

0 (S

.lut

eosp

oreu

s), H

a11

(syn

onym

s of

S.m

ashu

ensi

s), H

a12

(syn

onym

s of

S.m

obar

ensi

s), H

a13

(syn

onym

s of

S.m

oroo

kaen

se),

Ha1

4 (s

ynon

yms

of S

.net

rops

is),

Ha1

5 (S

.ori

noki

),H

a16

(S.s

tram

ineu

s), H

a17

(S.t

hiol

uteu

s), a

nd H

a18

(syn

onym

s of

S.v

irid

flavu

s). 5

) C

lust

ers

are

acco

rdin

g to

Lan

oot

et a

l . (2

002)

on

the

basi

s of

pro

tein

pro

files

. 6)

Clu

ster

num

bers

are

acc

ordi

ng t

o F

ulto

n et

al .

(199

5) o

n th

e ba

sis

of fi

nger

prin

ts o

f th

e 16

SrR

NA

ope

rons

. 7)

Gro

ups

base

d on

pri

mar

y st

ruct

ures

of

N t

erm

ini o

f AT-

L30

pro

tein

s ar

e ac

cord

ing

to O

chi (

1995

). 8)

Clu

ster

s ba

sed

on t

he p

hylo

gene

tic

tree

con

stru

cted

fro

m t

he 1

20-b

p al

pha-

reg i

on a

re a

ccor

ding

to

Kat

aoka

et

al. (

1997

). M

ore

than

450

Stre

ptom

yces

120-

bp a

lpha

reg

ions

hav

e be

en s

eque

nced

sin

ce t

hose

rep

orts

.b T

he s

trai

n nu

mbe

rs a

nd t

he a

cces

sion

num

bers

(E

MB

L)

of t

he d

atab

ases

are

giv

en in

the

adj

acen

t co

lum

ns. A

cces

sion

num

bers

(E

MB

L)

of c

ompl

ete

16S

rRN

A s

eque

nces

are

als

o gi

ven

in t

hose

col

umns

in a

ddit

ion

to t

he n

umbe

r of

bas

es in

the

seq

uenc

e .c 9)

Str

ains

incl

uded

in t

he D

NA

-DN

A h

ybri

diza

tion

stu

dies

L1:

Lab

eda

and

Lyo

ns (

1991

a): o

n th

e ba

sis

of 7

0% D

NA

-DN

A s

imila

rity

, S.h

ygro

scop

icus

NR

RL

238

7T a

nd S

.end

usar

e sy

nony

mou

s (t

oget

her

wit

h se

vera

l str

ains

of

S.vi

olac

eusn

iger

;the

rem

aini

ngst

rain

s re

pres

ent

sing

le s

peci

es);

L2:

Lab

eda

and

Lyo

ns (

1991

b):

on t

he b

asis

of

70%

DN

A-D

NA

sim

ilari

ty,

S.be

llus,

S.cu

raco

i,an

dS.

coer

uleo

rubi

dus

are

syno

nym

ous;

S.af

ghan

iens

is,

S.ja

nthi

nus,

S.ro

seov

iola

ceus

,S.

viol

atus

,an

dS.

purp

uras

cens

are

syno

nym

ous;

the

rem

aini

ng s

trai

ns r

epre

sent

sin

gle

spec

ies;

L3:

Lab

eda

(199

8): o

n th

e ba

sis

of 8

0% D

NA

-DN

A s

imila

rity

, S.g

rise

ovir

idis

and

S.d

aghe

ston

icus

are

syno

nym

ous;

S. c

hrys

eus

and

S.lo

ngis

poro

rube

rar

e sy

nony

mou

s; th

e re

mai

ning

str

ains

rep

rese

ntsi

ngle

spe

cies

; L4:

Lab

eda

(199

6): o

n th

e ba

sis

of 7

0% D

NA

-DN

A s

imila

rity

, S.a

biko

ensi

s,S.

wak

sman

ii,an

dS.

taka

taen

sis

are

syno

nym

ous;

S.bi

vert

icill

atus

,S.f

erve

ns,S

.bal

dacc

ii,S.

rose

over

ticill

atus

are

syno

nym

ous;

S.ne

trop

sis,

S.ke

ntuc

kens

is,S

.flav

oper

sicu

sar

e sy

nony

mou

s; S.

cinn

amon

eus

subs

p . a

zaco

luta

,S.h

achi

joen

sis

are

syno

nym

ous;

the

rem

aini

ng s

trai

ns r

epre

sent

sin

gle

spec

ies;

L5:

Lab

eda

(199

3): o

n th

e ba

sis

of 8

0% D

NA

-DN

A s

imila

rity

, S.

lave

ndul

ae,S

.lav

endu

lae

subs

p . a

vire

ns,S

.lav

endu

lae

subs

p .gr

asse

rius

,S.c

olum

bien

sis

are

syno

nym

ous;

and

the

rem

aini

ng s

trai

ns r

epre

sent

sin

gle

spec

ies.

S.vi

rgin

iae

vIF

O37

29T

1514

bpD

8511

9S.

viri

difa

cien

sD

SM40

239

AT

CC

1198

9IS

P52

39F

6622

-404

3S.

viri

difla

vus

vS.

viri

dis

DSM

4038

1A

TC

C15

732

ISP

5381

A27

009

1-19

S.vi

ridi

sD

SM40

637

076

069

S.vi

ridi

viol

aceu

sv

DSM

4028

0TA

TC

C27

478

ISP

5280

IV35

(gr

ay s

erie

s)00

61-

1812

0bp

JCM

4855

D44

408

S.vi

rido

brun

neus

vS.

viri

doch

rom

ogen

esv

1494

bpJC

M50

13A

F04

5858

S.vi

rido

chro

mog

enes

vD

SM40

110T

AT

CC

1492

0IS

P51

10A

27II

I04

009

1-19

OC

-III

121b

pJC

M48

56D

4440

9L

2S.

viri

dodi

asta

ticus

vD

SM40

249T

AT

CC

2551

8IS

P52

49IV

36 (

gray

ser

ies)

006

1-18

121b

pJC

M45

36D

4422

1S.

viri

dofla

vum

DSM

4023

7A

TC

C12

631

ISP

5237

J70

Sv.2

422

-104

0S.

viri

doge

nes

DSM

4045

4A

TC

C33

72IS

P54

54A

0300

61-

4S.

viri

dosp

orus

vD

SM40

243

AT

CC

2747

9IS

P52

43A

15I

0800

61-

1812

1bp

JCM

4859

D44

410

S.vi

tam

inop

hilu

sv

S.vu

lgar

isD

SM40

201

AT

CC

1589

5IS

P52

011-

31-

2S.

wak

sman

iiD

SM40

464

Sv. 0

922

-104

0L

4S.

wed

mor

ensi

sv

S.w

erra

ensi

sv

DSM

4048

6A

TC

C14

424

ISP

5486

A12

I07

006

1-18

KA

-G12

1bp

JCM

4860

D44

411

S.w

illm

orei

vD

SM40

459T

AT

CC

6867

ISP

5459

A1B

I02

1-3

1-2

KA

-B12

0bp

JCM

4861

D44

412

S.xa

ntho

chro

mog

enes

vD

SM40

111T

AT

CC

1981

8IS

P51

11F

63II

1500

502

9L

a23

OC

-I12

0bp

JCM

4612

D44

264

S.xa

ntho

cidi

cus

vD

SM40

575T

AT

CC

2748

0IS

P55

75F

66II

1622

-404

3L

a18

120b

pJC

M48

62A

B01

8208

S.xa

ntho

litic

usv

DSM

4024

4TA

TC

C27

481

ISP

5244

C24

II05

062

024

La2

112

0bp

JCM

4863

D44

413

S.xa

ntho

phae

usv

DSM

4013

4A

TC

C19

819

ISP

5134

F61

I22

084

067

120b

pJC

M44

26D

4413

8L

5S.

yate

nsis

DSM

4177

114

93bp

SFO

Cin

76

AF

3368

00S.

yere

vaen

sis

DSM

4316

7TII

I18

080

066

OC

-IS.

yere

vane

nsis

vD

SM43

167

120b

pJC

M30

65D

4397

6S.

yogy

akar

tens

isv

DSM

4176

6T14

81bp

C4R

3(S3

)A

J391

827

S.yo

kosu

kane

nsis

vD

SM40

224

AT

CC

2552

0IS

P52

24A

30II

0600

91-

1912

0bp

JCM

4559

D44

231

S.yu

nnan

ensi

sv

1521

bpY

IM41

004T

AF

3468

18S.

zaom

ycet

icus

vD

SM40

196

AT

CC

2748

2IS

P51

96A

05I

0400

21-

7K

A-C

120b

pJC

M48

64D

4441

4

Spec

ies

nam

e

Stra

in n

umbe

r(s)

Clu

ster

num

bers

of

num

eric

al t

axon

omic

stud

ies

and

Ber

gey

cate

gori

es a

ndsp

ecie

s no

.aG

roup

s ac

cord

ing

to d

iffe

rent

stu

dies

abp

rRN

A d

ata

stra

in n

o.if

dif

fere

nt f

rom

the

first

col

umn

Acc

. no.

(EM

BL

)b

Stra

ins

incl

uded

inD

NA

/DN

Ahy

brid

isat

ion

stud

iesc

12

34

56

78

9


short chains of spores on the substrate mycelium.Sclerotia, pycnidial-, sporangia-, and synnemata-like structures may be formed by some species.The spores are nonmotile. On complex agarmedia, discrete and lichenoid, leathery orbutyrous colonies are formed. Colonies are ini-tially relatively smooth surfaced, but later theydevelop an aerial mycelium that may appearfloccose, granular, powdery or velvety.

Members of the genus Streptomyces undergoa complex life cyle, which has been studied mostintensively for strain “S. coelicolor” A2(3). Strep-tomyces colonies are multicellular, differentiatedorganisms exhibiting temporal and spatial con-trol of gene expression, morphogenesis, metabo-lism and the flux of metabolites (see chapter 2 ofKieser et al., [2000] for more details).

Strains belonging to the genus Streptomycesmay produce a wide variety of pigments respon-sible for the color of the vegetative and aerialmycelia (Figs. 3 and 4). In addition, colored dif-fusible pigments may also be formed. Note thatthe production of pigments largely depends onthe medium composition and cultivation condi-tions (Figs. 3 and 4). Many strains produce oneor more antibiotics (more details are given inThe Family Streptomycetaceae, Part II: MoleularBiology in this Volume). The metabolism is oxi-dative and chemoorganotrophic. The catalasereaction is positive, and generally, nitrates arereduced to nitrites. Most representatives candegrade polymeric substrates like casein, gelatin,hypoxanthine, starch and also cellulose. In addi-tion, a wide range of organic compounds is usedas sole sources of carbon for energy and growth(Williams et al., 1983a; Kämpfer et al., 1991b;Korn-Wendisch and Kutzner, 1992a). The opti-mum temperature for most species is 25–35°C;however, several thermophilic and psychrophilicspecies are known. The optimum pH range forgrowth is 6.5–8.0.

The Embden-Meyerhof-Parnas (glycolysis)pathway of glucose catabolism has been found inmany streptomycetes (Cochrane, 1961), but alsothe hexose monophosphate shunt (Salas et al.,1984) was detected in S. antobioticus. Severalstreptomycetes are able to switch from glycolysisto the hexose monophosphate shunt duringsecondary metabolism (Kieser et al., 2000). Atpresent, no streptomycete is known to use theEntner-Doudoroff pathway. Sugar transport ismediated in connection with phosphorylation byspecific kinases (Sabater et al., 1972; lkeda et al.,1984). The phosphoenolpyruvate:fructose phos-photransferase system (PTS) for the transportand phosphorylation of fructose has recentlybeen detected in S. coelicolor, S. lividans andS. griseofuscus (Titgemeier et al., 1995). Moredetails about specific metabolic pathways,including nitrogen metabolism and the regula-

tion processes involved, are given in chapter 1 ofKieser et al. (2000) and the references therein.

On the basis of 16S rRNA/DNA sequencecomparisons, members of the genus Streptomycesform a separate line of descent, and Stackebrandtet al. (1997) proposed the emendation of thefamily Streptomycetaceae in the suborder Strep-tomycinae and the order Actinomycetales. Theintrageneric phylogenetic relationships of manyof the 346 recognized species in Bergey’s Manualof Systematic Bacteriology (Williams et al., 1989)inferred from the 350 complete 16S rRNAsequences, however, are clearly restricted by thelimited resolving power of the method to discrim-inate between related species and are often incontrast with a morphologically and physiologi-cally based classification. Though about 350almost complete 16S rRNA sequences are avail-able to date, the high degree of conservationwithin 16S rRNA genes causes problems forresolving phylogenetic relationships at the inter-generic level.

Notably, the different methods used for group-ing of the Streptomyces species often leadto contradictory results. In Table 4, all 376Streptomyces species and subspecies with validnames (as of December 9, 2003; taken from theList of Bacterial Names with Standing inNomenclature); and some additional specieswith names not validly published (but includedin taxonomic studies) are given with their group-ing according to different studies.

The chemotaxonomic features for the identifi-cation of strains at the genus level (for details seebelow) are of high value and can be summarizedas follows: The cell wall peptidoglycan containsmajor amounts of LL-diaminopimelic acid(LL-A2pm). Genus members lack mycolic acids,contain major amounts of saturated, iso- andanteiso-fatty acids, possess either hexa- oroctahydrogenated menaquinones with nine iso-prene units as the predominant isoprenolog, andhave complex polar lipid patterns that typicallycontain diphosphatidylglycerol, phosphatidyle-thanolamine, phosphatidylinositol, and phos-phatidylinositol mannosides (Table 2; Figs. 5 and6). In addition to these traits, the acyl type of themuramyl residues in the cell-wall peptidoglycansis acetyl (Uchida and Seino, 1997). Strains arewidely distributed and abundant in soil, includ-ing composts (see detailed description below). Afew species are pathogenic for animals and man,and others are phytopathogens. The type speciesis Streptomyces albus (Rossi-Doria 1891) Waks-man and Henrici (1943).

Cell Wall Composition

Peptidoglycan The cell walls of streptomycetesshow the typical ultrastructure and chemical


Fig. 3. A–J: Color of the aerial mycelium of Streptomyces strains grown on different agar media after 3 weeks of incubationat 28°C. Left: starch-casein-nitrate agar; middle: GYM agar; right: oatmeal agar (for compositions, see Tables 10 and 12).Species names and strain numbers are given in Table 6.

a

b

c

d

e

f

g

h

i

j


composition of Gram-positive bacteria(Schleifer and Kandler, 1972). They appearunder the electron microscope as homogeneousless electron dense layers of about 16–35 nm.The cell walls have a multilayered structure ofpeptidoglycan strands. The peptidoglycan is aheteropolymer consisting of heteropolysaccha-ride chains cross-linked through short peptideunits. The so-called “sugar back bone” of thepeptidoglycan is constructed of alternating β-1,4-linked units of N-acetylglucosamine and N-acetylmuramic acid. The carboxyl group ofmuramic acid is substituted by an oligopeptideof alternating D- and L-amino acids (Schleiferand Kandler, 1972). Streptomyces is character-ized by the tetrapeptide L-Ala–D-Glu–LL-A2pm–D-Ala. This tetrapeptide is crosslinked bya pentaglycine bridge which extends from the C-terminal D-alanine of the peptide unit to theamino group located on the D carbon of LL-A2pm, resulting in the macromolecule structureforming the cell envelope. This LL-A2pm-Gly5,or A3γ peptidoglycan type (Schleifer andKandler, 1972), is diagnostic for streptomycetesand some other combined-wall chemotype I act-inomycetes (Lechevalier and Lechevalier, 1970).

The aerobic actinomycetes were grouped(using specific amino acids in purified cell walls)into four so-called “wall chemotypes” byLechevalier and coworkers. Cell walls withmeso-DAP and LL-DAP were detected early. Inanother study, Takahashi et al. (1984b) reportedthat strains belonging to this group change cellwall composition during sporulation. They foundin submerged mycelium LL-DAP and glycine(wall chemotype I) whereas in spores, only meso-DAP could be detected (wall chemotype IIIaccording to Lechevalier and Lechevalier, 1970).In 11 streptomycetes, the cell wall compositionsof aerial, substrate and submerged myceliumdiffered in the quantitative distribution ofcell wall amino acids and cell wall sugars. N-Acetylmuramic acid is found in the glycolyl typeof cell wall of Streptomyces, as in all other acti-nomycetes (Uchida and Aida, 1977). Muramicacid phosphate residues are the attachmentpoints to teichoic acids, which are of diagnosticvalue for Gram-positive bacteria. The cell wallteichoic acids (polymeric substances containingrepeating phosphodiester groups) consist ofpolyols (i.e., the sugar alcohols glycerol and rib-itol) or N-acetylamino sugars or both. Theteichoic acids of streptomyces are of the samestructure as those of other Gram-positive bacte-ria, containing either ribitol phosphate orglycerol phosphate polymers; significantly, theteichoic acid of actinomycetes does not containester-bound D-alanine but does have ester-linked acetic acid and sometimes succinic acidresidues (Naumova et al., 1980).

Fig. 4. A–F: Color of the substrate mycelium and solublepigments of Streptomyces strains grown on different agarmedia after 7 days of incubation at 28°C. Left: starch-casein-nitrate agar; middle: GYM agar; right: oatmeal agar (forcompositions, see Tables 10 and 12). Species names and strainnumbers are given in Table 7.

a

b

c

d

e

f


For streptomycetes, the synthesis of either rib-itol phosphate (S. streptomycinii and S. violaceus)or glycerol phosphate polymers (S. thermovul-garis, S. levoris, S. rimosus and S. antibioticus) hasbeen reported (Naumova et al., 1980). In ribitolteichoic acids, positions 1 and 5 of ribitols areconnected to the phosphates; in glycerol teichoicacids, position 1 is commonly connected to 3; andin other types, position 1 connected to 2 (as in S.antibioticus) is not common. The polyol phos-phates can be substituted with various combina-tions of sugars or amino sugars or both, whichare linked to glycerols or ribitols via glycosidicbonds. At present, few strains (species) havebeen investigated in detail and so the role ofteichoic acid in the taxonomy of Streptomyces isnot clear (Naumova et al., 1980).

Cell Wall Polysaccharides These compoundsseem to be of no diagnostic value in those strainswhere LL-DAP is found in whole cell hydroly-sates (Lechevalier et al., 1971). Some of the diag-nostic sugars found in other actinomycetes, likexylose, galactose and arabinose, were reportedoccasionally in streptomycetes. Hundreds ofstreptomyces were analyzed for the presenceof diagnostic sugars (Kroppenstedt, 1977), andmainly ribose, mannose and glucose were usuallyfound in small amounts.

Phospho- and Glycolipids The lipids of strepto-mycetes comprise mainly diphosphatidylglycerol(DPG), phosphatidylethanolamine (PE), phos-phatidylinositol (PI), and phosphatidylinositol-mannosides (PIMs). Lipid composition has beenextensively investigated and summarized byLechevalier et al. (1977b). Glycolipids do notoccur consistently in streptomycetes, and their

qualitative and quantitative lipid compositiondepends largely on culture conditions. Underphosphate limiting conditions, the amount of gly-colipids increase, significantly.

The taxonomic significance of polar lipids inactinomycetes was demonstrated by Lechevalieret al. (1977b). From the phospholipid results of97 actinomycete strains representing 20 genera,Lechevalier et al. (1977b) proposed a classifica-tion of five phospholipid types. These five groupsare based on the presence or absence of certainnitrogenous phospholipids. The marker lipidsof type II (PII) are phosphatidylethanolamine(PE), methyl-PE, hydroxy-PE, and lyso-PE. Inaddition to various other families, members ofthe family Streptomycetaceae contain the lipidsof phospholipid type II. Additional lipids (e.g.,phosphomonoester [PME] and OH-PE) and thepresence or absence of PI and PG allow furtherdifferentiation (Fig. 5).

Menaquinones Streptomycetes contain onlymenaquinones (Collins and Jones, 1981), andlike the majority of actinomycetes, they syn-thesize quinones that have a partly saturatedisoprenoid side chain at position 3 of the naph-thoquinone ring. Menaquinone composition isvery useful for differentiation of actinomycetesbecause of the different numbers of isopreneunits, the different degree of hydrogenation, andthe position of hydrogenated isoprene units(Table 2). These three variations are useful forclassification and identification. Streptomycetessynthesize menaquinones with a highly hydroge-nated isoprenoid chain. Three to four (rarelyfive) isoprene units are saturated. The actino-mycetes which belong to this type synthesizemenaquinones with the same chain length butdifferent degree of saturation (Fig. 6).

Fig. 5. Two-dimensional thin layer chromatograms of polar lipids of A) Streptomyces albus (DSM 40313) and B) Streptomycesrimosus (DSM 40260). Abbreviations: DPG, diphosphatidyglycerol; PI, phosphatidylinositol; PIM, phosphatidylinositol man-nosides; PE, phosphatidylethanolamine; OH-PE, hydroxy-phosphatidylethanolamine; and P, phospholipids of unknownstructure. (Courtesy of R. M. Kroppenstedt.)

40313 40260

a b

PDPG

PE

PI

P

PIPIM

OH-PEPE

DPG

P


Phenotypic Methods for Classification within the Genus Streptomyces

Phenotypic methods comprise all those thatare not directed towards DNA or RNA. Theyinclude also chemotaxonomic techniques.Between 1916 and 1943, most of the studies onstreptomyces were published by soil microbiolo-gists, who were mainly interested in ecologicalquestions. Only few species were described atthat time, mainly on the basis of morphologicalcriteria, pigmentation and ecological require-ments (Waksman and Curtis, 1916; Waksman,1919; Jensen, 1930).

The discovery of actinomycin from S. antibioti-cus (Waksman and Woodruf, 1940) was the start-ing point of the investigations of antibiotics andother bioactive substances produced by strepto-mycetes in the 1940s and this led to extensivescreening approaches for novel bioactive com-pounds in the following two decades.

The description of each producer of a novelnatural product as a new species (often pat-ented) led to an explosion of species descriptionsand resulted in an overclassification of the genus.In the 1970s, the number of species increased toover 3000 (Trejo, 1970).

Reduction in the number of species names wasfirst attempted in 1964 by the InternationalStreptomyces Project (ISP), which introducedstandard criteria for determining species(described in Shirling and Gottlieb, 1968a, Shirl-ing and Gottlieb, 1968b, Shirling and Gottlieb,1969, and Shirling and Gottlieb, 1972) to reducethe number of poorly described synonymousspecies. The major drawback of these descrip-tions was that they were based mainly on mor-phology (i.e., spore chain morphology, spore

surface ornamentation, color of spores, substratemycelium, soluble pigments, and production ofmelanin pigment), in addition to a few physio-logical properties, which were mainly restrictedto utilization tests of different carbon sources.

In these classical papers, more than 450 Strep-tomyces species were redescribed, and typestrains were deposited in internationally recog-nized culture collections. Although intended, theefforts of the ISP did not result in an applicableidentification scheme.

A first step in this direction was the develop-ment of numerical taxonomic methods in the1960s including the methods of numerical iden-tification. The first numerical taxonomic studiesof streptomycetes by Silvestri et al. (1962) foundconsiderable diversity within the genus but alsogroups that corresponded to the initial morpho-logical descriptions. These studies did not resultin nomenclatural changes, and despite the devel-opment of other small databases for identifica-tion of streptomycetes (Kurylowicz et al., 1975;Gyllenberg, 1976), these studies had no impacton streptomyces systematics (Table 3). Datafrom a large-scale numerical taxonomic study byWilliams et al. (1983a) of 475 strains (including394 Streptomyces type cultures from the ISP) for139 unit characters were analyzed with simplematching, the Jaccard coefficient, and the aver-age linkage algorithm. Consequently, the genusStreptomyces was subdivided into species groups.Streptomyces type strains (394) were clusteredaccording to similarities obtained from the phe-netic tests. At the 77 ± 5% simple matching coef-ficient (SSM) level, 19 major, 40 minor and 18single strain clusters were recovered. Many ofthe minor clusters consisted of less than fivestrains. Major clusters varied in size from 6 to 71strains. Each cluster was addressed as a single“species” despite the high diversity observed

Fig. 6. Menaquinone profile of Strep-tomyces griseus (DSM 40236). Theextent of hydrogenation of the iso-prene units is shown by the subscriptof the abbreviation. For instance,MK-9 (H8) is a menaquinone withfour hydrogenated isoprene units.(Courtesy of R. M. Kroppenstedt.)

DSM 40 236Streptomyces griseusRP - 18

MK - 9(H4)

MK - 9(H6)

MK - 9(H6)

MK - 10(H4)MK - 9(H8)

MK - 9(H8)inject.

5 10 15 20 25 30 35 min. 390


within some clusters, and these therefore wereaddressed as “species groups.” The largest spe-cies group is Streptomyces albidoflavus (cluster1), containing 71 strains, including 44 typestrains, 15 invalidly published species, and 12unnamed strains. This cluster is further subdi-vided into three clusters: cluster 1a, Streptomycesalbidoflavus subsp. albidoflavus (20 strains), clus-ter 1b, Streptomyces albidoflavus subsp. anulatus(38 strains), and cluster 1c, Streptomyces albid-oflavus subsp. halstedii (13 strains; Williams etal., 1989).

The high phenotypic diversity of this cluster isobvious from the different test pattern. Allstrains produced yellow gray colonies, producedsmooth spores in straight chains and no melanin,and exhibited resistance to a number of anti-biotics including penicillin, lincomycin andcephaloridine. Many of the strains showedalso antimicrobial activity; 39% produced com-pounds with antifungal activity, 32% producedcompounds active against Gram-positive micro-organisms and 10% against Gram-negativemicroorganisms (Williams et al., 1983b), showingthe large diversity within one cluster and exem-plifying clearly the problems with streptomycetesystematics (Anderson and Wellington, 2001).

Nevertheless, the comprehensive survey ofWilliams et al. (1983a) resulted subsequently ina reduction of the number of described Strep-tomyces species; however, the problem of over-speciation remained. Numerous species andsubspecies were described and many naturalisolates did not match the reference strainsused to construct the identification matrices(Goodfellow and Dickenson, 1985). Althoughprobability matrices for identification purposeswere published (Williams et al., 1983b; Langhamet al., 1989), these matrices were not widelyadopted by the scientific community.

This study was the basis of the taxonomicscheme for streptomyces presented in the 1989edition of Bergey’s Manual of Systematic Bacte-riology, in which 142 species are listed (Williamset al., 1989), in contrast to 463 species describedin the 1974 edition of Bergey’s Manual of Deter-minative Bacteriology (Pridham and Tresner,1974). A further numerical taxonomic analysisby Kämpfer et al. (1991b) included more strainsand more than one strain of each species whenavailable. A total of 821 strains were tested for329 physiological properties, and the resultingcluster analysis was compared with the data pub-lished by Williams et al. (1983a) in addition topublished genetic and chemotaxonomic data.

Many of the clusters defined by Williams et al.(1983a) were again recognized; for example theS. albidoflavus, S. anulatus, S. griseus, S. halstediigroup appeared as cluster 1 in both studies, inwhich 28 of the S. griseus strains were grouped.

Interestingly, most of the strains sharing thesame specific epithet were grouped together,indicating previous identification was reliable,but some exceptions were also observed. Forexample S. hygroscopicus strains were recoveredin cluster 1 but also in several other clusters andsubclusters.

On the basis of this study, a probability matrixwas constructed (Kämpfer and Kroppenstedt,1991a), but this matrix was likewise not widelyused by other reseach groups.

Parallel with the numerical taxonomic studies,additional chemotaxonomic and also molecularmethods were developed that are now often usedtogether with (often) few physiological tests tostudy streptomycetes; however, a clear speciesconcept is still pending. In Table 4, the clusterallocation of the species is given in comparison.

Other phenotypic methods include cell wallanalysis (Lechevalier and Lechevalier, 1970),fatty acid profiling (Hofheinz and Grisbach,1965; Lechevalier, 1977a; Saddler et al., 1986;Saddler et al., 1987; Kroppenstedt, 1992), rapidbiochemical assay for utilization of 4-methyl-umbelliferone-linked substrates (Goodfellow etal., 1987c), serological assay (Ridell et al., 1986),phage typing (Wellington and Williams, 1981a;Korn-Wendisch and Schneider, 1992b), and pro-tein profiling (Manchester et al., 1990; Goodfel-low and O’Donnell, 1993; Lanoot et al., 2002),including comparison of ribosomal protein pat-terns (Ochi, 1989; Ochi, 1992; Ochi, 1995).

Fatty acids

The initial studies on actinomycete fatty acidswere carried out by Hofheinz and Grisebach(1965) on Saccharopolyspora erythraeus (for-merly “Streptomyces erythraeus”) and Streptomy-ces halstedii to elucidate the biosynthetic pathwayof branched fatty acids. It was shown that strep-tomyces synthesize terminally branched fattyacids. Anteiso-branched fatty acids are synthe-sized from 2-methylbutyrate, leading to anteisofatty acids with an odd number of carbon atoms.In contrast, isovalerate and isobutyrate as startingcompounds lead to the formation of iso-branchedfatty acids with even and odd numbers of C-atoms, respectively. For this reason iso- andanteiso-branched fatty acids appear in pairs withodd numbers of C-atoms only.

In their early studies, Hofheinz and Grisebach(1965) separated the fatty acids as their methylesters by gas chromatography on different sta-tionary phases. Identification of the individualfatty acids was obtained by comparing the equiv-alent chain lengths of unknown fatty acids withthose of standard mixtures. The results were con-firmed by preparative gas-chromatography andby physical methods such as mass spectrometry


and nuclear magnetic resonance (NMR) spec-trometry. In both species, iso- and anteiso-branched fatty acids with chain lengths of 15 and17 carbon atoms were detected. High amounts of14-methyl pentadecanoic acid (iso-C16:0) werefound in addition, while minor amounts ofunbranched fatty acids, tuberculostearic acid andtheir homologues, could be detected in “S. eryth-raeus” (now Saccharopolyspora erythraea) butnot in Streptomyces halstedii. These results arecongruent with those of several other studies(Lechevalier et al., 1977a; Saddler et al., 1985;Saddler et al., 1987) in which 10-methylbranched fatty acids could not be detectedamong streptomycetes. Usually only smallamounts of hydroxy fatty acids are synthesizedby a limited number of streptomycetes underoptimal oxygen supply. The hydroxy fatty acidsare easily destroyed in a non-deactivated injec-tion port of capillary gas chromatographysystem. Therefore hydroxy fatty acids in strepto-mycetes often go unnoticed. If streptomyces aregrown under reproducible culture conditions, thehydroxy fatty acids they produce are highly diag-nostic for some streptomyces species. Hydroxyfatty acids were detected in all strains of S. coeli-color (30), S. rimosus (14), and S. violaceusniger(18) and in 20 of 27 S. hygroscopicus strains butnot in S. violaceoruber (16), S. lavendulae (18), S.griseus (22), S. fradiae (25), S. viridochromogenes(25), S. glaucescens (8) and S. albus (33; Krop-penstedt, 1992; R. M. Kroppenstedt, unpub-lished observation). Standardized growth andcultivation conditions are a general prerequisitefor the use of fatty acid patterns below the genuslevel (Saddler et al., 1986). Saddler et al. (1987)used fatty acid profiles to investigate the taxon-omy of Streptomyces cyaneus strains and soil iso-lates showing also blue spores. The S. cyaneuscluster harbors 13 of 19 blue-spored strains ofstreptomycetes (Hütter, 1962; Pridham andTresner, 1974; Korn et al., 1978). In the study ofSaddler et al. (1987), 8 of their 10 blue-sporedisolates clustered, while 17 of the 34 S. cyaneusstrains were assigned to a separate cluster. Theconclusions of the fatty acid study (Saddler et al.,1987) and Williams et al. (1983a) agree that con-ventional features like spore chain morphology,color and ornamentation of spores may be help-ful for presumptive identification but are notdefinitive for classification of streptomycetes.The same combination of features may be foundin different clusters, yet one cluster may havemembers with different features. The study ofSaddler et al. (1987), however, demonstratedalso the heterogeneity of the Streptomyces cya-neus taxon as defined by Williams et al. (1983a).Fatty acid patterns in general cannot delimitStreptomyces species (Phillips, 1992; R. M. Krop-penstedt, unpublished observation), but usingstandardized conditions, they are still of high

value for the rapid characterization (indepen-dent of the taxonomic status) of large numbersof wild-type streptomycetes isolated from theenvironment (Saddler et al., 1987). By using theautomated commercially available MIDI systemconsisting of a Hewlett-Packard model 5890 cap-illary gas chromatograph and a computer withspecific software (Microbial ID, Inc., Newark,DE), the fatty acids are automatically identifiedand quantified by the computer using fatty acidstandard mixtures for comparison. In their chap-ter on The Family Nocardiopsaceae in this Vol-ume, Kroppenstedt and Evtushenko give a tableof types and the fatty acids diagnostic for differ-ent genera of Actinomycetales, including Strep-tomyces. The comparison of different methodsrevealed that the use of numerical methods todetermine taxonomy lumped too many strainstogether into some clusters (Williams et al.,1983a; Kämpfer et al., 1991b).

Curie-point Pyrolysis Mass Spectrometry (PyMS)

This method has also been applied to the classi-fication and identification of actinomycetes (San-glier et al., 1992). Similar to fatty acid profiling,highly standardized conditions are necessary.Whole cells are subject to high temperatures andsubsequent nonoxidative thermal degradation.The resulting pyrolysate is then analyzed usingmass spectrometry, resulting in a fingerprint foreach organism.

Sanglier et al. (1992) applied this method tostrains belonging to the largest Streptomycesspecies group, Streptomyces albidoflavus. Inter-estingly, Streptomyces albidoflavus and Strepto-myces anulatus strains could be separated intodistinct groups. Three of the six Streptomyces hal-stedii strains investigated also clustered into adistinct group, whereas the remaining strainsclustered into two other groups. The study ofKämpfer et al. (1991b) also found that Strepto-myces albidoflavus strains and Streptomyces anu-latus strains grouped separately. Interestingly, itwas confirmed that Streptomyces anulatus ISP5361T, the strain used to name the Streptomycesanulatus cluster, formed also a single-membercluster (Table 4).

Serology

Few results using serological methods have beenpublished. Antisera against the mycelia fromstreptomycetes, streptoverticillia and Nocardio-psis species (Ridell et al., 1986) were used toconfirm the high similarity between Streptomyceslavendulae and the streptoverticillia (Witt andStackebrandt, 1990; Kämpfer et al., 1991b). Theantisera of Kirby and Rybick (1986) raisedagainst Streptomyces griseus (Streptomyces anu-


latus, cluster 1B of Williams et al., 1983a) and“Streptomyces cattleya” (cluster 47) were shownto be genus-specific and to a certain degree alsogroup-specific. The monoclonal antibody pro-duced by Wipat et al. (1994) to “Streptomyceslividans” 1326 was shown to be specific for“Streptomyces lividans” strain 1326. Interest-ingly, antigenic reaction was observed also forstrains grouped into cluster 21 of Williams et al.(1983a).

Phage Typing

Phage typing can be used for host identificationat the genus and the species level (Welsch et al.,1957; Kutzner, 1961a; Kutzner, 1961b; Korn et al.,1978; Wellington and Williams, 1981a). Manyactinophages (most of them virulent) for phagetyping have been described. Streptomycetephages can be either polyvalent (e.g., C31; Chateret al., 1986b) or species-specific (Anderson andWellington, 2001; Table 5). The specificity of act-inophages at the genus level (e.g., Wellington andWilliams, 1981a; Korn-Wendisch, 1982; Prauser,1984) was an additional feature that justified thetransfer of Actinopycnidium, Actinosporangium,Chainia, Elytrosporangium, Microellobosporia,Kitasatoa and Streptoverticillium to thegenus Streptomyces (Goodfellow et al., 1986b;Goodfellow et al., 1986c; Goodfellow et al.,1986d; Goodfellow et al., 1986e; Witt and Stack-ebrandt, 1990). Other transfers justified by phagespecificity include Actinoplanes armeniacus tothe genus Streptomyces (Kroppenstedt et al.,1981; Wellington et al., 1981b) and “S. eryth-raeus” to the genus Saccharopolyspora (Labeda,1987). Species or group identification of Strepto-myces using phage typing has been less success-ful, but there are a few exceptions (Table 5).

Phages are also useful in industrial microbiol-ogy studies (Carvajal, 1953; Ogata, 1980) and ingenetic studies (for review, see Chater [1986a]and chapter 12 of Kieser et al. [2000]). One ofthe best-investigated actinophages is C31, a tem-perate phage with a broad host range within thegenus Streptomyces (Lomovskaya et al., 1980).This phage has become the subject of extensivestudies and has been employed for many pur-poses (e.g., transfection, transduction, detectionof transposon-like elements of host DNA, andcloning). Details are given in chapter 12 ofKieser et al. (2000).

Protein Profiling

Polyacrylamide gel electrophoresis (PAGE) oftotal protein extracts generate more or lesscomplex banding patterns. These patterns can beused to differentiate species and subspecieswithin various bacterial genera. Protein patternscan be determined using one-dimensional (1-D)or two-dimensional (2-D) protein electrophore-sis. The protein profiles of streptomycetes werefirst analyzed by Manchester et al. (1990), whoinvestigated 37 Streptomyces strains (amongthem 5 streptoverticillia). Some taxonomic cor-relations were found between these profiles andthe phenotypic groupings observed by Williamset al. (1983a) and Kämpfer et al. (1991b) in addi-tion to some DNA hybridization groupings(Table 4). But Lanoot et al. (2002) confirmedonly a few of these correlations. For Streptomy-ces isolates that are the causal agent of commonpotato scab, Paradis et al. (1994) used bothPAGE and DNA-DNA hybridizations to eluci-date the taxonomy of their strain. Isolatesobtained from potato tubers were divided intotwo groups with a correlation coefficient of 0.75

Table 5. Species-specific actinophages of the genus Streptomyces (modified according to Anderson and Wellington, 2001).

Abbreviations: T, type strain; SMC, single-member cluster; DSM, Deutsche Sammlung von Mikroorganismen und Zellkul-turen; and ATCC, American Type Culture Collection.aAccording to Williams et al. (1983); SMC according to Williams et al. (1989).bAccording to Kämpfer et al. (1991).Modified from Anderson and Wellington (2001).

Phage HostHost species

group

Hostcluster

no.aCluster

no.b References

98 S. coelicolor Müller ATCC 23899T S. albidoflavus 1A 1-1 Wellington and Williams, 198114, 24, 233 S. coelicolor Müller ATCC 23899T S. albidoflavus 1A 1-1 Korn-Wendisch and Schneider, 199289, DP 9 S. griseus ATCC 23345T S. albidoflavus 1B 1-3 Wellington and Williams, 198190 S. griseinus ATCC 23915T S. albidoflavus 1B 1-3 Wellington and Williams, 198133 ‘S. scabies’ ATCC 23962 S. atroolivaceus 3 1-3 Wellington and Williams, 1981SV1, SV2 S. venezuelae ATCC 10712T S. violaceus 6 2 Stuttard, 198241 S. matensis ATCC 23935T S. rochei 12 6 Wellington and Williams, 1981S3 S. albus DSM 40313T S. albus 16 32 Korn-Wendisch and Schneider, 1992SAt1 S. azureus ATCC 14921T S. cyaneus 18 9 Ogata et al., 1985100 ‘S. caesius’ ATCC 19828 S. griseoruber 21 6 Wellington and Williams, 19814, 5a, 5b, 49 S. violaceoruber DSM 40049T S. violaceoruber SMC 69 Korn-Wendisch and Schneider, 1992


using sodium dodecylsulfate (SDS)-PAGE anal-ysis. The same two groups were resolved atapproximately 44% similarity using DNA-DNAhybridization analysis. The fatty acid analysisresults of the same study did not correlate withthe SDS-PAGE and the DNA-DNA hybridiza-tion groupings, which can be explained by theinfluence of growth conditions on the profilesobtained (Saddler et al., 1986; Saddler et al.,1987). Protein profiling was not able to differen-tiate pathogenic from nonpathogenic strains.

Other more specific patterns are obtained withmultilocus enzyme electrophoresis (MLEE) anddepend on the relative mobilities of cellularenzymes in a gel matrix. Oh et al. (1996) studied24 Streptomyces strains and demonstrated howMLEE could be used for both inter- andintraspecific characterization of streptomycetes,provided the appropriate enzymes were used.However, because only a restricted set of strainswas studied, no general recommendations can bemade for the usefulness of this method. In amore comprehensive study, 93 Streptomycesreference strains were investigated using SDS-PAGE of whole-cell proteins (Lanoot et al.,2002). Subsequent computer-assisted numericalanalysis revealed 24 clusters encompassingstrains with very similar protein profiles. Five ofthem included several type strains with visuallyidentical patterns. DNA-DNA hybridizationsrevealed similarities higher than 70% amongthese type strains. On the basis of these results,consideration of Streptomyces albosporeussubsp. albosporeus LMG 19403T as a subjectivesynonym of Streptomyces aurantiacus LMG19358T, Streptomyces aminophilus LMG 19319T

as a subjective synonym of Streptomyces cacaoisubsp. cacaoi LMG 19320T, Streptomyces niveusLMG 19395T and Streptomyces spheroides LMG19392T as subjective synonyms of Streptomycescaeruleus LMG 19399T, and Streptomyces viola-tus LMG 19397T as a subjective synonym ofStreptomyces violaceus LMG 19360T was pro-posed (Table 4).

Two-dimensional PAGE of the total cellularproteins allows a finer resolution of the individ-ual gene products. This technique results in verycomplex patterns and seems to be too sensitiveto investigate proteins with high rates of evolu-tion (Hori and Osawa, 1987). Mikulik et al.(1982) and later Ochi (1989) were the first toapply 2-D PAGE to determine the variability ofribosomal proteins for use in streptomycete tax-onomy. These studies were later extended byfocusing on AT-L30 proteins, which give genus-specific profiles (Ochi, 1992). In an even morespecific analysis, Ochi (1995) correlated the Ntermini sequences of the ribosomal AT-L30 pro-tein of 81 streptomycete strains from differenttaxonomic groups to phylogenetic groupings

within the genus and pointed out that on thisbasis the genus Streptomyces seems to be welldescribed. However, Ochi’s groupings did notcorrelate with those of Williams et al. (1983a)and Kämpfer et al. (1991b). For details of thesegroupings, see Table 4.

More detailed taxonomic studies of Strepto-myces have been performed by isolating andsequencing specific proteins. For example, Tagu-chi et al. (1996) used the Streptomyces subtilisininhibitor protein (SSI), which plays unidentifiedrole(s) in physiological or morphological regula-tion, to investigate the taxonomic status of theStreptomyces coelicolor strains. The amino acidsequence, of SSI from “Streptomyces lividans”66, Streptomyces coelicolor Müller ISP 5233T and“Streptomyces coelicolor”A3(2) were compared.The alignments supported ribosomal sequencecomparisons, indicating that “Streptomyces coeli-color” A3(2) is more closely related to “Strepto-myces lividans” 66 (cluster 21 of Williams et al.,1983a) than to the type strain, Streptomycescoelicolor Müller ISP 5233T (cluster 1).

Genotypic Methods for Classification within the Genus Streptomyces

Genotypic methods comprise all those that aredirected towards DNA or RNA molecules(Schleifer and Stackebrandt, 1983; Vandamme etal., 1996). The development of molecular meth-ods to analyze bacterial genomes has provided anew basis for studying bacterial taxonomy and insome cases phylogenetic relationships of theprokaryotes at the genus, species and subspecieslevel. These methods are widely used in moderntaxonomic studies. The general taxonomic valuesof different molecular techniques are given byVandamme et al. (1996) and in Prokaryote Char-acterization and Identification in Volume 1. Theirusefulness in taxonomic studies to delimit spe-cies within the genus Streptomyces is discussedbriefly in the following chapters and has alsobeen reviewed by Anderson and Wellington(2001).

Problems in assigning new strains to existingspecies on the basis of this molecule alone persistand doing this will not be possible in the future.As already pointed out by Stackebrandt andSchumann in Introduction to the Classificationof the Actinomyces in this Volume, comparativeanalysis of sequences of homologous and genet-ically stable semantides has demonstrated thatseveral classification systems based on morphol-ogy and physiology do not reflect the naturalrelationships among actinomycetes and relatedorganisms. In this respect, rRNA sequence com-


parison is a powerful tool in modern taxonomyand has revolutionized our insight in phyloge-netic lineages of major taxonomic groups. How-ever, the resolving power of 16S rRNA sequencesis not sufficient to delimit species. Nevertheless,rRNA sequence comparisons are important inthe taxonomy of Streptomyces and the resultshave also been used to answer questions abouthorizontal transfer of genes within the genus(Huddleston et al., 1997). Genes for 16S RNAsare highly conserved within bacteria. Within thegenus Streptomyces, three regions within thegene have enough sequence variation to be use-ful as genus-specific (a and b regions) and spe-cies-specific (c regions) probes (see Stackebrandtet al., 1991a; Stackebrandt et al., 1991b; Stacke-brandt et al., 1992; Anderson and Wellington,2001). Not only 16S rRNA genes, but also 23SrRNA and 5S rRNA genes (Mehling et al.,1995), 16S-23S rRNA internally transcribedspacer (ITS) sequences (Song et al., 2003), andribosomal protein sequences have also been usedto investigate species relationships within thegenus Streptomyces (Liao and Dennis, 1994;Ochi, 1995). Wenner et al. (2002) studied thenucleotide composition of the ITS sequences ofthe six rDNA operons of two Streptomycesambofaciens strains. Their findings suggested thatrecombination frequently occurs between therDNA loci, leading to the exchange of nucleotideblocks, and confirmed that a high degree of ITSvariability is a common characteristic amongStreptomyces spp. Note that rRNA sequencescannot be used alone because of the intraspecificvariation and intragenomic heterogeneity.

DNA-DNA Hybridizations

The percent DNA-DNA hybridization and thedecrease in thermal stability of the hybrid are atpresent the “gold standard” methods of speciesdelineation in bacteriology (Wayne et al., 1987).An ad hoc committee for the re-evaluation of thespecies definition in bacteriology (Stackebrandtet al., 2003) has encouraged investigators toverify the species concept with other methods;however, DNA-DNA similarity and change inmelting temperature (∆Tm; Wayne et al., 1987)remain the acknowledged standard for definitionof species.

DNA-DNA hybridizations of total chromo-somal DNA have also been used within thegenus Streptomyces. In an initial study, Mordar-ski et al. (1986) compared the numerical andphenetic groupings of strains of the Streptomycesalbidoflavus cluster 1 of Williams et al. (1983a)with the results of DNA-DNA hybridizations(reassociation of labeled DNA on nitrocellulosefilters). In congruence with numerical studies,DNA-DNA hybridization studies showed this

cluster could be subdivided into three subclus-ters and confirmed the homogeneity of the Strep-tomyces albidoflavus subcluster albidoflavus.Two further subclusters obtained by DNA-DNAhybridizations were not congruent with thegroupings of S. anulatus or halstedii by Williamset al. (1983a); however, some correlations werefound with the groupings of Kämpfer et al.(1991b). Unification using DNA-DNA similarity(Witt and Stackebrandt, 1990) of Streptoverticil-lium strains with the genus Streptomyces (reasso-ciation of labeled DNA on filters) was confirmedby phenotypic data in the numerical study ofKämpfer et al. (1991b).

The most extensive application of DNA-DNAhybridizations (thermal renaturation method) tothe study of the major streptomycete pheneticgroups of Williams et al. (1983a) was by Labedaand coworkers. In studies using Streptomycescyaneus (Labeda and Lyons, 1991a), Streptomy-ces violaceusniger (Labeda and Lyons, 1991b),Streptomyces lavendulae (Labeda, 1993), the ver-ticil-forming streptomycetes (formerly Strep-toverticillium species; Labeda, 1996; Hatano etal., 2003), and S. fulvissimus and S. griseoviridisphenotypic clusters (Labeda, 1998), the degreeof DNA similarities was often not congruentwith the phenetic groupings of Williams et al.(1983a). But again, more correlations werefound with the groupings of Kämpfer et al.(Kämpfer et al., 1991b; Table 4). The usefulnessof the DNA-DNA hybridization technique todelineate species within the genus Streptomyceshas been questioned. In support of the usefulnessof this technique is the considerable geneticinstability of certain regions within the Strep-tomyces chromosome (Redenbach et al.,1993). The two complete Streptomyces genomesequences available at this time (Omura et al.,2001; Ikeda et al., 2003) indicate that the centralcore region contains mostly the essential house-keeping genes, while the chromosome armscomprise laterally acquired contingency genes.Another issue is the presence of large plasmidsin strains of Streptomyces, which can consider-ably influence the results of DNA-DNA hybrid-izations. General properties of Streptomycesplasmids and their use for gene cloning are givenin chapter 11 of Kieser et al. (2000).

Fingerprinting Techniques

Randomly Amplified Polymorphic DNA(RAPD) Polymerase Chain Reaction (PCR)RAPD-PCR is used as a rapid screening methodto detect similarity among streptomycete strains.Single primers with arbitrary nucleotidesequences to amplify DNA are used in additionto a low annealing temperature so that polymor-phisms can be detected. Stringent standardiza-


tion of the reaction parameters is required.These include primer sequence, annealing tem-peratures, buffer components, concentration andquality of template DNA. The resulting charac-teristic fingerprint of PCR products enablesdetection of chromosomal differences betweenindividual isolates without having any priorknowledge of the chromosomal sequence (Will-iams et al., 1990). Applying this technique,Mehling et al. (1995) could not detect any char-acteristic banding patterns for closely relatedspecies unless a highly specific actinomyceteprimer was used. As a major disadvantage, theresulting fingerprints contained only few bands(one to four), reducing the effectiveness of thismethod. Huddleston et al. (1995) reported simi-lar results. They evaluated the use of RAPD-PCR for the resolution of interspecific relation-ships among members of the Streptomyces albi-doflavus cluster of Williams et al. (1983a). Anzaiet al. (1994) demonstrated the variation in finger-prints obtained when a single base was substi-tuted on the arbitrary primer; 11 primers wereinvestigated and the number of bands rangedfrom zero to 20. The most significant differenceswere observed when the sequence at the 3′ endwas altered. Anzai et al. (1994) also investigatedthe relationship of Streptomyces virginiae strainsto Streptomyces lavendulae strains by RAPD-PCR. Williams et al. (1983a) found that Strepto-myces virginiae was a synonym of Streptomyceslavendulae. These strains were also grouped intothe same cluster in the study of Kämpfer et al.(1991b). In addition to RAPD-PCR, DNA-DNAhybridization, low-frequency restriction frag-ment analysis (LFRFA), and cultural and physi-ological tests were performed. Consistent resultswere obtained using all these methods afterRAPD PCR optimization. It was however notpossible to clarify the interspecific relationshipof Streptomyces lavendulae and Streptomycesvirginiae.

Restriction Digests of Total ChromosomalDNA Similar to the RAPD PCR techniques,low-frequency restriction fragment analysis(LFRFA) uses the entire bacterial chromosome,which is digested with restriction endonucleasesthat cut infrequently. Because streptomycetesbelong to bacteria with a high DNA G + C con-tent, rare AT cutters are used. The fragmentsobtained were separated by pulsed-field gel elec-trophoresis (PFGE). In a first study, Beyazovaand Lechevalier (1993) included 59 strains fromeight species groups and found the method use-ful for the clustering of related strains. However,some discrepancies were found, for example forstrains grouped into the Streptomyces cyaneuscluster of Williams et al. (1983a). Again thismethod seems to be useful for the detection of

very closely related strains, but similar to RAPD-PCR, it cannot resolve interspecific relation-ships. In addition, Rauland et al. (1995) foundthat large chromosomal amplifications or dele-tions may also lead to misinterpretations ofresulting banding patterns.

Nucleic Acid Sequence Comparisons of 16SrRNA and Other Genes The application of16S rRNA gene sequence analysis to the studyof the taxonomy of streptomycetes is reviewedby Stackebrandt et al. (1992), who highlightedthe importance of the region selected for com-parison. Sequence analysis of rRNA genes hasalready been applied at the genus, species andstrain level. The relationships obtained differedconsiderably depending on the variable region(a, b or c). Kataoka et al. (1997) subsequentlyinvestigated the c region from 89 streptomycetetype strains representing several clusters ofWilliams et al. (1983a). Though these variableregions were useful for resolving inter- andintraspecies relationships within the strepto-mycetes, they were too variable for determininggeneric relationships. Among the 89 strains stud-ied, 57 variants were detected and 42 strainswere found to have unique sequences. After thispublication, the c regions from 485 strepto-mycete strains were sequenced and deposited inGenBank by this group. At present, this is thelargest publically available set of streptomycete16S rDNA sequence data.

Anderson and Wellington (2001) published aphylogenetic tree based on comparison of the cregions from representatives of the major clustergroups defined by Williams et al. (1983a). Thetaxonomic status of the phenotypic groups wasconfirmed, although they did not clustertogether. Only Streptomyces olivaceoviridis andStreptomyces griseoruber strains (which arefound in clusters 20 and 21 of Williams et al.[1983a], but in a single cluster 9 of Kämpfer etal. [1991b]) had identical c regions. The Strepto-myces albidoflavus group, which was previouslydivided into three species groups by Williams etal. (1983a) and contained more than 60 strains(Williams et al., 1989), was now divided into sixgroups using sequence comparisons of the cregion (Kataoka et al., 1997). The three pheno-typic subgroups of Williams et al. (1983a) weremaintained but did not cluster together.

Hain et al. (1997) designed 16S rRNA oligo-nucleotide probes to determine intraspecificrelationships within the Streptomyces albidofla-vus group. As a result, sequence comparisonswere found to be useful for species delimitationbut of no value for strain differentiation.

Also the intergenic 16S-23S rRNA spacerregions were investigated in detail and they wereobviously more suitable for delineation of the


intraspecific relationships within that cluster.Genus specific probes based on 23S rRNA genesequences (Mehling et al., 1995) and 5S rRNAgene sequences (Park et al., 1991) have beendeveloped. The reclassification of the generaChainia, Elytrosporangium, Kitasatoa, Microel-lobosporia and Streptoverticillium into the genusStreptomyces (Park et al., 1991) was confirmedusing 5S rRNA gene sequence evaluation.

At present about 350 complete 16S rRNAsequences are available from public databases.Although 16S rRNA sequence analyses haveprovided a framework for prokaryotic classifica-tion, note that the current classification systembased on this molecule has not yet solved thetaxonomy within the genera (especially withinthe genus Streptomyces). Several studies haveattempted to use sequence data from variableregions of 16S rRNA to establish taxonomicstructure within the genus, but the variation istoo limited to help resolve problems of speciesdifferentiation (Witt and Stackebrandt, 1990;Stackebrandt et al., 1991b; Stackebrandt et al.,1992; Anderson and Wellington, 2001).

The situation is complicated by the fact thatStreptomyces species may harbor different 16SrRNA gene sets. For example, S. coelicolorA3(2), S. lividans and several Streptomyces spe-cies harbor six ribosomal rRNA gene sets. Eachset comprises one gene copy for 16S, 26S, and 5SrRNA (Van Wezel et al., 1991) and lacks tRNAgenes.

A comprehensive study of the phylogeneticrelationships between 64 whorl-forming strepto-mycetes using partial gyrB sequences (the struc-tural gene of the B subunit of the DNA gyrase)has been published by Hatano et al. (2003). Thestrains in this study consisted of 46 species andeight subspecies and in addition 13 species whosenames have not been validly published (includ-ing 10 strains examined by the InternationalStreptomyces Project [ISP]). Two major groupswere found. The typical whorl-forming species(59 strains) were further divided into six majorclusters of three or more species, seven minorclusters of two species, and five single-memberclusters on the basis of the threshold value of97% gyrB sequence similarity. Major clusterscontained Streptomyces abikoensis, Streptomycescinnamoneus, Streptomyces distallicus, Strepto-myces griseocarneus, Streptomyces hiroshimensisand Streptomyces netropsis. Phenotypically,members of each cluster resembled each otherclosely except for those in the S. distallicus clus-ter (which was divided phenotypically into the S.distallicus and Streptomyces stramineus subclus-ters) and the S. netropsis cluster (which wasdivided into the S. netropsis and Streptomyceseurocidicus subclusters). Strains in each minorcluster closely resembled each other phenotypi-

cally. At the conclusion, 59 strains of typicalwhorl-forming Streptomyces species were placedinto the following 18 species, including subjectivesynonym(s): S. abikoensis, Streptomyces ardus,Streptomyces blastmyceticus, S. cinnamoneus, S.eurocidicus, S. griseocarneus, S. hiroshimensis,Streptomyces lilacinus, “Streptomyces luteoreti-culi,” Streptomyces luteosporeus, Streptomycesmashuensis, Streptomyces mobaraensis, Strepto-myces morookaense, S. netropsis, Streptomycesorinoci, S. stramineus, Streptomyces thioluteusand Streptomyces viridiflavus (Table 4). Allstrains showing 98.5–100% gyrB sequence simi-larity also had a high DNA-DNA similarity (70–100%), showing better resolution with gyrBsequences than with 16S rRNA sequences.

Other genes known to be conserved betweenspecies, such as housekeeping genes (e.g., elon-gation factors and ATPase subunits), may beuseful as primary target genes for study (Lud-wig and Schleifer, 1994). Huddleston et al.(1997) used tryptophan synthase genes in addi-tion to 16S rRNA gene comparisons to deter-mine the phylogeny of streptomycin-producingstreptomycetes and provide evidence for thehorizontal transfer of antibiotic resistancegenes. Predictably, sequence information onother genes (i.e., other housekeeping genes) willlead to better insight into the intraspecific struc-ture of the genus Streptomyces (Stackebrandtet al., 2003).

Rapid Methods for Gene Analysis in Streptomycete Taxonomy

Several alternative methods have been describedthat do not involve sequencing, using eitherrestriction analysis (Clarke et al., 1993; Fulton etal., 1995) or specialized gel electrophoresis tech-niques to monitor the mobility of the product(Hain et al., 1997; Heuer et al., 1997). Restrictionfragment length polymorphism (RFLP) patternsof purified rRNA were used by Clarke et al.(1993) with strains from the Streptomyces albid-oflavus cluster (subgroups 1A and 1B of Will-iams et al., 1983a). The following combination ofenzymes was used: BglI, EcoRI, PstI and PvuII.The resulting RFLP profiles varied considerablybetween species groups but were found to enabledifferentiation of phenotypically similar strains.Fulton et al. (1995) performed ribosomal restric-tion analysis using MseI fingerprints of rRNAoperons (RiDiTS) to group 98 named strepto-mycete strains including members of clustergroup A (comprising clusters 1–41) and F (com-prising clusters 55–67) of Williams et al. (1983a)and other strains. The resulting RiDiTS belongedto 11 pattern types with varying degrees of sim-ilarity to the Williams subclusters. At low resolu-tion (70% similarity), cluster groups A and F


could be differentiated, but individual clusterscould not.

Further studies are based on genotypic varia-tion monitored using denaturing gradient gelelectrophoresis (DGGE; Muyzer et al., 1993)with or without DNA-binding agents (Hainet al., 1997). Anderson and Wellington (2001)recommended DGGE in combination with othertechniques. Using the variable 16S rRNAregions, this method enables delimitation ofgenus groups and species-groups. Huddlestonet al. (1995) allocated isolates ASB33, ASB37and ASSF22 to Streptomyces albidoflavus,Streptomyces griseoruber and Streptomycesalbidoflavus, respectively, using a combination oftechniques including numerical taxonomy,PFGE and sequence comparisons (Huddlestonet al., 1997).

Identification of Streptomycetaceae at the Genus Level

Sequencing of 16S rRNA genes and comparisonof these sequences after careful alignment withpublished sequences is currently the most reli-able method for assigning unknown organisms tothe different genera. The calculation of phyloge-netic trees at the subgeneric level should be donevery carefully and may lead to misinterpreta-tions. Notably, phylogenetic analysis on the basisof 16S rRNA comparisons does not allow speciesdelineation.

Colony morphology (color of the aerial myce-lium, color of the substrate mycelium, and solu-ble pigment) is (especially in the case of thegenus Streptomyces) very useful (Tables 6 and 7;Figs. 3 and 4). Here the traditional methodsextensively described by Korn-Wendisch andKutzner (1992a) are highly recommended.

A microscopic characterization (particularlythe morphologies of the aerial mycelium,arthrospores and vegetative mycelium) is ofhigh value (Figs. 7–11). See Korn-Wendisch andKutzner (1992a) and chapter 3 of Kieser et al.(2000) for details of the methods on microscopy.

Furthermore, the detection of LL-A2pm in cellwall or whole-cell hydrolysates, the lack ofmycolic acids, the predominance of mainly iso-and anteiso-methyl branched fatty acids, and the16S rRNA sequence are well suited for genusidentification (Table 1).

Identification of Species

Kitasatospora Novel isolates can be readilyidentified as members of the genus by 16S rRNAgene and 16S-23S rRNA gene spacer analyses.The presence of meso-A2pm in whole cell

hydrolysates is an important chemotaxonomiccharacter for differentiation from Streptomyces(Table 1). Additional chemotaxonomic investi-gations (polar lipids, fatty acid patterns, andmenaquinone type) are helpful for allocation ofan unknown isolate to the genus. The meso-A2pm content is 49–89% in Kitasatospora strainsand 1–16% in Streptomyces strains (Zhang et al.,1997). Strains belonging to the genus Strepta-cidophilus contain (like Streptomyces) LL-diaminopimelic acid as predominant diaminoacid (Kim et al., 2003). For further species iden-tification, the characters shown in Table 8 arehelpful.

Streptacidiphilus Novel isolates can be readilyidentified as members of the genus by 16S rRNAgene analysis. The presence of LL-A2pm inwhole cell hydrolysates and other chemotaxo-nomic characters (e.g., polar lipids, fatty acidpatterns, and menaquinone type; see familydescription) are shared by members of the genusStreptomyces. The three species of the genusStreptacidophilus can be differentiated on thebasis of some phenotypic properties (Table 9).

Streptomyces The identification of species posessevere problems. Because of the high number ofvalidly published species (Table 4), most ofwhich are based on a single strain description, itis at present not possible to recommend a singlemethod or even a set of methods for identifica-tion at the species level. Because a clear speciesconcept within the genus Streptomyces is stillpending, investigators should be very carefulwith species allocations on the basis of the resultsof one or few methods. The ICSP Subcommitteeon the Systematics of Streptomycetaceae(Kämpfer and Labeda, 2003) has recommendedthat before a species concept of the genus Strep-tomyces is formulated more genomic informa-tion should be evaluated, and it was agreed “thatthe proposal of new species should only beaccepted on the basis of very careful studies donewith sufficient practice and considering all otherspecies.” In recent years, only a few species havebeen proposed on the basis of 16S rRNAsequence analysis and phenotypic characteriza-tion (most often restricted to those speciesclosely related by phylogenetic analysis [e.g.,Bouchek-Mechine et al., 2000; Kim et al., 2000;Kim and Goodfellow, 2002a, b; Li et al., 2002a;Li et al., 2002b; Meyers et al., 2003; Petrosyan etal., 2003; Zhang et al., 2003]). The ICSP Subcom-mittee on the Systematics of Streptomycetaceae(Kämpfer and Labeda, 2003) recommended acareful look at synonymy as a first step to reducethe number of “species” within the genus.

At present two complete Streptomycesgenomes are available (Omura et al., 2001; Ikeda


Table 6. Spore colors for the grouping of streptomycetes and representatives of each color (according to Korn-Wendisch andKutzner 1992).

Abbreviations: DSM, Deutsche Sammlung von Mikroorganismen und Zellkulturen; and ISP, International StreptomycesProject.aDSM no. 40XXX = ISP no. 5XXX; e.g., 40236 = ISP 5236.bFigures 3a–j show the aerial mycelia of the strain after cultivation on three different media for 21 days (left: starch-casein-nitrate agar, middle: GYM agar, right: oatmeal agar; for compositions, see Tables 10 and 12).From Korn-Wendisch and Kutzner (1992).

Representative species (DSM no.)a Figure (strain no.)b Color of aerial mycelium

S. griseus (40236); S. coelicolor (40233) 3a (40236) Yellow-gray: “griseus”S. fradiae (40063); S. toxytricini (40178) 3b (40178) Pink/light violetS. lavendulae (40069); S. flavotricini (40152) 3c (40069) Gray-pink/lavender: “cinnamomeus”S. eurythermus (40014); S. fragilis (40044) 3d (40014) Brown (plus gray or red)S. viridochromogenes (40110); S. cyaneus (40108) 3e (40108) Blue: “azureus”S. glaucescens (40155) 3f (40155) Blue-green: “glaucus”S. prasinus (40099); S. hirsutus (40095) 3g (40099) Green: “prasinus”S. violaceoruber (40049); S. echinatus (40013) 3h (40049) Gray: “cinereus”S. albus (40313); S. longisporus (40166) 3i (40166) White: “niveus”S. alboniger (40043); S. rimosus (40260) 3j (40043) Not definable: white plus various light-colored

shades

Table 7. Colors of substrate mycelium and soluble pigment occurring in streptomycetes.

Abbreviations: DSM, Deutsche Sammlung von Mikroorganismen und Zellkulturen; and ISP, International StreptomycesProject.aDSM no. 40XXX = ISP no. 5XXX.bFigures 4a–f show the substrate mycelia of the strain after cultivation on three different media for 7 days; left: starch-casein-nitrate agar, middle: GYM agar, right: oatmeal agar; for compositions, see Tables 10 and 12).From Korn-Wendisch and Kutzner (1992).

Representative species (DSM no.)a Figure (strain no.)b Color

S. aurantiacus (40412); S. griseoruber (40275) 4a (40412) Orange to dark redS. longispororuber (40599); S. spectabilis (40512) (mainly endopigment)S. californicus (40058); S. cinereoruber (40012) 4b (40058) Red to blue/violetS. violaceus (40082); S. purpurascens (40310) (mainly endopigment)S. coelicolor (40233); S. cyaneus (40108) 4c (40163) Red-violet to blueS. violaceoruber (40049); S. lateritius (40163) (endo- or exopigment or both)S. atroolivaceus (40137); S. canarius (40528) 4d (40089) Yellow-orange/greenish-yellowS. galbus (40089); S. tendae (40101) (endo- and exopigment)S. flavoviridis (40210); S. olivoviridis (40211) 4e (40071) Green to gray-oliveS. viridochromogenes (40110); S. nigrifaciens (40071) (endo- and exopigment)“S. malachiticus” (40167); “S. malachitorectus” (40333) Green (endopigment)S. badius (40139); S. eurythermus (40014) 4f (40100) Red-brown to dark-brownS. phaeochromogenes (40073); S. ramulosus (40100) (endo- and exopigment)S. alboniger (40043); S. hygroscopicus (40578) Gray-brown to blackS. purpeofuscus (40283); S. mirabilis (40553) (mainly endopigment)

et al., 2003). Bentley et al. (2002) reported thefirst complete genome sequence of S. coelicolorand described a model for the evolution of thelarge linear chromosome, where the central coreregion contains mostly the essential housekeep-ing genes and the “arms” contain laterallyacquired contingency genes. A comparison of theS. averilitils and S. coelicolor genomes supportsthis model (Bentley et al., 2003). Most of thesecondary metabolism gene clusters are locatedin the arms. The detailed study of genomes ofdifferent streptomycetes and housekeeping genesmay provide a more reliable basis for an intrage-neric subdivision (Stackebrandt et al., 2003).

Ecophysiology and Habitat

The following sections are largely based on theinformation summarized by Korn-Wendisch andKutzner (1992a). Members of the family Strep-tomycetaceae are ubiquitous in nature. Membersof the genus Kitasatospora have been predomi-nantly isolated from soils (Zhang et al., 1997),and Streptacidiphilus species have been isolatedfrom acidic soils and litter (Kim et al., 2003).Streptomycetes can be isolated in highnumbers in soil, which is their natural habitat.Most streptomycetes can degrade complex and


Fig. 7. Aerial mycelium of the fertile (left) and the sterile (right) strain of two streptomycetes. First and third lines, lightmicroscopy (@ 250); second and fourth lines, electron microscopy (×15,000). (From Kutzner [1956], with permission.)


Fig. 8. Morphology of the aerial mycelium of some strains of Streptomyces (×250). (From Flaig and Kutzner [1960b], withpermission.)


Fig. 10. Scanning electron microscopyof arthrospore chains. (A) Streptomy-ces torulosus (knobby); (B) S. bluensis(spiny); (C) S. antimycoticus (rugose);and (D) “S. karnatakensis” (hairy).(Courtesy of A. Dietz.)

A

2µm

B

5,000x2µm

5,000x

Fig. 9. Electron micrographs of four types of arthrospores of streptomycetes: smooth, warty, hairy and spiny. The spores areabout 1 m long. (From Kutzner [1956], with permission.)


Fig. 11. Morphology of the aerial mycelium of some species of Streptoverticillium. (A)–(E) Light microscopy (×250).(Courtesy of C. Mütze.) (A) Sv. netropsis (DSM 40259). (B) “Sv. reticulum” (DSM 40893). (C) “Sv. cinnamomeum subsp.azacolutum” (DSM 40646). (D) Sv. septatum (DSM 40577). (E) Sv. mobaraense (DSM 40847). (F) Scanning electronmicroscopy: a Streptoverticillium species (×6,200).

A B

C D

Fig. 10. ContinuedC

2µm

D

5,000x1µm

10,000x


Table 8. Differentiation of Kitasatospora species (according to Tajima et al., 2001).a

Symbols and abbreviations: +, positive; -, negative; ND, not determined; T, type strain; and IFO, Institute for Fermentation,Osaka, Japan.aTaxa are identified as: 1) strain SK-3255T; 2) strain SK-3406T; 3) K. setae KM-6054T; 4) K. phosalacinea KA-338T; 5) K.griseola AM-9660T; 6) K. chochleata IFO 14768T; 7) K. cystarginea IFO 14836T; and 8) K. paracochleata IFO 14769T. All strainsare positive for peptonization of milk, hydrolysis of starch, and utilization of D-glucose. All strains are negative for liquefac-tion of gelatin and utilization of inositol and d-mannitol.Data were from Tajima et al. (2001) for strains SK-3255T and SK-3406T, mura et al. (1982) for K. setae KM-6054T, Takahashiet al. (1984b) for K. phosalacinea KA-338T and K. griseola AM-9660T, Nakagaito et al. (1992a) for K. cochleata IFO 14768T

and K. paracochleata IFO 14769T and Nakagaito et al. (1992b) for K. cystarginea IFO 14836T. From Tajima et al. (2001).

Characteristic 1 2 3 4 5 6 7 8

Fermentation of melanoid pigment - - - - - + - +Reduction of nitrate - + - + - - - +Coagulation of milk - - + - + - - -Utilization of

l-Arabinose + + + + + + - -d-Xylose + + + + + - - -Raffinose + - - + + - - -Melibiose + - - - - ND ND NDd-Fructose - - - + - - - +d-Rhamnose + - - + - - - -Sucrose - - - + - - - -Cellulose - - - - - ND ND ND

Temperature for growth (∞C) 15-37 15-41 15-37 15-42 15-37 13-38 17-40 11-38DNA G+C content (mol %) 73.7 73.5 73.1 73.4 73.1 72.4 70.6 73.1

O

Fig. 11. Continued

E F

recalcitrant plant and animal materials, oftenpolymeric residues including polysaccharides(e.g., starch, pectin, cellulose and chitin), pro-teins (e.g., keratin and elastin), lignocellulose,and aromatic compounds.

Members of the genus Streptomyces areinvolved in the biodegradation of various poly-mers abundant in soil owing to their ability toproduce extracellular enzymes. The biodegra-datve activities of actinomycetes in general werereviewed in the 1980s by Lechevalier (Lecheva-lier, 1981a; Lechevalier, 1988), Crawford (1988),and Peczynska-Czoch and Mordarski (1988).Streptomycetes are among the very few bacteriaable to degrade lignin which occurs in nature

closely associated with cellulose and xylan (hemi-cellulose), i.e., in the lignocellulose complex.Although fungi play the more important role inlignin decomposition (Crawford, 1981; Crawford,1988; Janshekar and Fiechter, 1983; Kirk andFarell, 1987), evidence from experiments using14C-labeled lignin now shows that streptomycetes(Crawford, 1978; Antai and Crawford, 1981) andalso several other genera of actinomycetes areinvolved in this process (McCarthy and Broda,1984a; McCarthy et al., 1984b; McCarthy et al.,1986). As a constituent of the lignocellulose com-plex, cellulose can be degraded by the few ligni-nolytic streptomycetes. More details are given byRamachandra et al. (1988), Wang et al. (1990),


Crawford et al. (1993), Chamberlain and Craw-ford (2000), Kormanec et al. (2001), Gottschalket al. (2003), and Kaneko et al. (2003).

In addition, multicomponent cellulases consist-ing of several endoglucanases and exoglucanaseshave been found in thermophilic and mesophilicstreptomycetes (Enger and Sleeper, 1965; Craw-ford and McCoy, 1972; MacKenzie et al., 1984;Schrempf and Walter, 1995; Harchand and Singh,1997; Marri et al., 1997; Ulrich and Wirth, 1999;Wirth and Ulrich, 2002). Also, xylanases involvedin the decomposition of the lignocellulose com-plex have been found in streptomycetes(Kluepfel and Ishaque, 1982; Kluepfel et al., 1986;Deobald and Crawford, 1987; Godden et al.,1989; Schäfer et al., 1996; Morosoli et al., 1999).Again, xylanases seem to be more widespreadamong thermophilic actinomycetes (McCarthy etal., 1985). Pectinolytic streptomycetes have beenreported (Sato and Kaji, 1975; Sato and Kaji,1977; Sato and Kaji, 1980a; Sato and Kaji, 1980b)and chitinolytic complexes consisting of chitinaseand chitobiase have been isolated from variousstreptomycetes: Streptomyces griseus (Bergerand Reynolds, 1958), Streptomyces antibioticus(Jeuniaux, 1966), and othe streptomycetes (Beyerand Diekmann, 1985). For more details, see TheFamily Streptomycetaceae, Part II: MolecularBiology in this Volume).

The ability to decompose starch (the primarymaterial for the textile, paper and food industry)is widespread among fungi and bacteria. Theinvolved enzymes, amylases, have been detectedin various streptomycetes (Mordarski et al.,1970; Suganuma et al., 1980; Fairbairn et al.,1986; McKillop et al., 1986).

In addition to degrading polymeric com-pounds (as described above), streptomycetes can

play an important role in the destruction of otherorganic materials used by humans for diversepurposes, among them cotton and plant fibers(Khan et al., 1978; Lacey and Lacey, 1987), wool(Noval and Nickerson, 1959), hydrocarbons injet fuel and emulsions (Genner and Hill, 1981),and rubber (Cundell and Mulcock, 1975; Hutch-inson et al., 1975). The biodeterioration of natu-ral and synthetic substances has been reviewedin detail by Lacey (1988) and Behal (2000). Moredetails are given in The Family Streptomyceta-ceae, Part II: Molecular Biology in this Volume.

Most isolated streptomycetes are nonfastidi-ous; they do not require organic nitrogen sourcesor vitamins and other growth factors. Soil as ahabitat gives support to their mycelial growth.Furthermore, spore formation enables strepto-mycetes to adapt to various physical conditionsin soil (such as shifts in aeration, moisture ten-sion, and pH), periods of drought, frost, hydro-static pressure, and anaerobic conditions whichmay change dramatically and quickly.

The spores can be regarded as a semi-dormantstage in the life cycle that facilitates survival insoil for long periods (Mayfield et al., 1972;Ensign, 1978). Morita (1985) reported viable cul-tures from 70-year-old soil samples. A relativelyhigh number of streptomycetes in soil is almostalways present as inactive spores. The very lowgermination efficiencies often obtained may becaused by competition with other indigenousmicroorganisms, but pre-germinated spores arefound to grow for a short time and then re-sporulate (Lloyd, 1969a). Germination maydepend on the presence of special signalingfactors, and there is evidence that exogenousnutrients, water and Ca2+ are required (Ensign,1978). Furthermore the nutrient status of the

Table 9. Differentiation of Streptacidiphilus species (according to Kim et al., 2003) using phenotypic properties.

Symbols: +, positive; -, negative; and v, variable.From Kim et al. (2003).aThe number of species.bThe numbers in parentheses are the concentrations of the compounds in mg per ml.

Characteristic No. of strains

Streptacidiphilusalbus

Streptacidiphilusneutrinimicus

Streptacidiphiluscarbonis

7a 5a 6a

Growth at pH 6.0 + - +Growth on sole carbon sources (1%, w/v)

d-Gluconic acid - - +d-Glucosamine hydrochloride + + -meso-Inositol - - +Inulin - - +l-Rhamnose v - +d-Ribose + - -

Growth in presence of (mg·ml -1)Cadmium acetate (50)b + - -Lead acetate (100) + + -Cephaloridine hydrochloride (2) + + -Penicillin-G (16) + + -Streptomycin sulfate (16) + + -


germination site influences the extent of hyphalgrowth and the time to differentiation into aerialhyphae. Many other “habitats” may come intocontact with soil owing to human or other activ-ities. As listed by Korn-Wendisch and Kutzner(1992a), these are 1) fodders and other organicmaterial and 2) freshwater and marine habitatsas well as potable water systems. Mesophilicand especially thermophilic streptomycetes areinvolved in the degradation of many natural sub-strates (e.g., hay, fodder, grain, and wood) andcan degrade synthetic products (e.g., cotton tex-tiles, fabric, paper, rubber, plastics and plasticiz-ers). Drainage after heavy rainfalls causes creeksand rivers to become contaminated with soilstreptomycetes that find their way into the sedi-ments of freshwater lakes and even to marinebiotopes. According to some reports, drinkingwater supplies may also become contaminatedwith streptomycetes; some of them produceodorous compounds leading to the spoilage ofthe water. Streptomycetes play only a minor roleas plant pathogens, and although very few strep-tomycetes have been isolated from pathologicalmaterial so far, their role as agents of infectiousdiseases cannot be ignored (more details aregiven below).

Soil as Habitat

A number of biotic and abiotic properties char-acterize any habitat and determine the currentcomposition of the community and also the num-bers of microorganisms. These are: 1) vegetationand content and kind of organic matter, 2) soiltype, 3) season and climate, 4) temperature, 5)circulation of water and air, and 6) pH. As alreadypointed out by Korn-Wendisch and Kutzner(1992a), the reports published on the occurrenceof streptomycetes in soil are numerous and tooextensive to be treated quantitatively. The readeris referred to reviews by Lechevalier (Lecheva-lier, 1981a; Lechevalier, 1988), Williams (1982a),Goodfellow and Williams (1983), Williams et al.(1984b), Goodfellow and Simpson (1987a), Korn-Wendisch and Kutzner (1992a) and to a series ofpapers by Williams and coworkers dealing spe-cifically with the ecology of actinomycetes in var-ious soils and under specific conditions: Daviesand Williams (1970), Williams and Mayfield(1971b), Williams et al. (Williams et al., 1971c;Williams et al., 1972; Williams et al., 1977), May-field et al., (1972), Ruddick and Williams (1972),Watson and Williams (1974), Khan and Williams(1975), Flowers and Williams (1977), Williamsand Robinson (1981). By direct observation ofthe soil microflora, several authors have shownthat streptomycetes perform their typical lifecycle in this natural habitat. For details of this lifecycle and its genetic control, the reader is referred

to Kieser et al. (2000). As summarized by Korn-Wendisch and Kutzner (1992a), in most soils, 104

to 107 colony forming units (CFU) per g can beexpected, accounting for about 1–20% of the totalviable count; in some soils however strepto-mycetes dominate. The number of strepto-mycetes and also the number of subgroups varyunder different conditions. Details can be foundin the studies of Flaig and Kutzner (1960a), Mis-iek (1955), Szabó and Marton (1964), and Küster(1976).

Note that the detection and localization of dif-ferent Streptomyces species or types in theirnatural habitat are based mainly on cultivationdependent techniques. In addition, the difficul-ties in the intrageneric classification of the genusStreptomyces often do not allow a comparison ofthese ecophysiological studies. With respect tomoisture, it has been shown by Williams et al.(1972) that streptomycetes resist desiccationbecause they form arthrospores. In addition theyneed a lower water tension for growth than otherbacteria need, but they may be very sensitive towater-logged conditions.

Most streptomycetes prefer neutral to alkalinesoils as a natural habitat (e.g., Flaig and Kutzner,1960a). But several studies of acidic soils employ-ing media adjusted to acid pH and supplementedwith antifungal agents found numerous acido-philic as well as acido-tolerant streptomycetes.Khan and Williams (1975), Hagedorn (1976), andWilliams et al. (1977) showed in detail that acidicforest soils and other acidic habitats containeddifferent groups or species of streptomycetes.These streptomycetes also have unusual proper-ties when compared with neutrophilic strains,e.g., production of specific amylases (Williamsand Flowers, 1978b) and chitinases (Williams andRobinson, 1981). Only a few reports of alkalo-philic, acid-sensitive actinomycetes have beenpublished (Taber, 1959; Taber, 1960; Mikami etal., 1982; Mikami et al., 1985).

Streptomycetes, like other soil bacteria, mayalso be found in the intestinal tract of earth-worms (Brüsewitz, 1959; Parle, 1963b; Parle,1963a), the gut of arthropods (Szabó et al., 1967;Bignell et al., 1980; Bignell et al., 1981; Bignell,1984), and the pellets produced by millipedesand woodlice (Márialigeti et al., 1984).

Studies on the occurence of streptomycetes inthe rhizosphere have been published by variousauthors (for a review, see Goodfellow andWilliams, 1983). The competitive advantage ofantibiotic-producing organisms over nonproduc-ing microbes in soil has been suggested since thetime these compounds were first discovered.However, evidence for the in situ production ofantibiotics in soil is still not clear (Williams,1982a). This may be due to 1) the instability andlow concentrations in soil (Brian, 1957; Williams,


1982a) and 2) possible adsorption to soil colloidsin combination with inadequate and insensitivedetection methods (Williams, 1982a) and 3) theephemeral growth of producers in responseto nutrient shortage (Williams and Khan, 1974;Williams, 1982a).

However, Rothrock and Gottlieb (1984)reported antibiotic production occurs in steril-ized soil supplemented with nutrients andinoculated with a potent producer. Many soilmicrobiologists support the assumption thatstreptomycetes play an important role in thecontrol of fungal root pathogens (Williams,1978a; Williams, 1982a; Sing and Mehrotra, 1980;Rothrock and Gottlieb, 1981). In addition manystreptomycetes are often successful in competi-tion with other rhizosphere bacteria such aspseudomonads and bacilli, especially in rela-tively dry soil.

Thermophilic Streptomycetes

The genus Streptomyces contains mainly meso-philic species in addition to some thermotolerant(growing up to 45°C) and a few thermophilicspecies. A detailed taxonomic study of thermo-philic streptomycetes has been published by Kimet al. (1999). Additional thermophilic species (S.thermocoprophilus and S. thermospinisporus)have been described by Kim et al. (2000) andKim and Goodfellow (2002b). The thermophilicstreptomycetes described so far grow at 28–55°C,and several grow at even higher temperatures.

Thermophilic streptomycetes go through aninteresting cycle in nature in regard to their dis-persal: active growth takes place at sites of hightemperature such as in compost, manure, and self-heating hay or grain. The vegetative phase endswith the formation of a large number of spores.These are returned with the compost or manureto the fields and pastures where they infect plantmaterial and hay directly or via soil dust (Korn-Wendisch and Kutzner, 1992a). Not surprisingly,the majority of actinomycetes isolated frombioaerosols in the surroundings of compostingfacilities belong to the genus Streptomyces (P.Kämpfer et al., unpublished observation). Forthis reason thermophilic actinomycetes are wide-spread and can be isolated from various sourceslike soils (Tendler and Burkholder, 1961; Craveriand Pagani, 1962), pig feces (Ohta and Ikeda,1978), sewage-sludge compost (Millner, 1982),and freshwater habitats (Cross, 1981).

Freshwater Environments, Water Supplies and Marine Environments

Actinomycetes can easily be isolated from freshwater and especially from sediments of rivers

and lakes. Cross (1981) stated, however, thatmost of these organisms may not be active atthese sites. Nevertheless, these wash-in forms(“aliens”) from surrounding terrestrial environ-ments can survive as dormant spores in aquatichabitats for a long time (Al-Diwany and Cross,1978), and especially rivers carry a load ofvarious actinomycetes, among them also strep-tomycetes. In a study on the occurence ofactinomycetes in the river Thames, Burman(1973) found 59–200 streptomycetes per ml and10–20 micromonosporae per ml. These organ-isms were found to grow on decaying vegetationon riverbanks and mud flats at low water or onfloating mats of decaying algae or other vegeta-tion. Under these conditions odorous substancesare produced, and subsequent increase in riverlevels washes them into the water, thus givingrise to “earthy taste” complaints. Of these odor-ous compounds, geosmin and methyl iso-borneolare most often detected (Gerber, 1979a; Gerber,1979b). As summarized by Wood et al. (1983),the prevention of earthy tastes in reservoirs andwater supply systems depends on locating theproduction sites and determining the patterns ofdistribution of these compounds (Silvey andRoach, 1975; Lechevalier et al., 1980).

Burman (1973) studied the fate of actino-mycetes of river water in the course of produc-tion of drinking water. Filtration processesreduce the number of streptomycetes consider-ably. In the distribution system, a new typenamed “aquatic strains of Streptomyces” hasappeared, which can be enriched (for details, seeBurman, 1973).

Okazaki and Okami (1976), Cross (1981), Wey-land (1981b), Weyland (1981a), Weyland andHelmke (1988), and Goodfellow and Haynes(1984) have reviewed the occurrence of strepto-mycetes in marine habitats including sediments.Two localities can be distinguished: 1) the littoraland inshore zone and 2) deep-sea sediments. Fromboth localities streptomycetes can be isolated;however, similarly to streptomycetes in freshwa-ter environments, most of these organisms are“survivors” rather than constituents of theautochthonous microflora. From the littoral zone,streptomycetes have been isolated both from sed-iments (Roach and Silvey, 1959) and from decay-ing seaweed (Siebert and Schwartz, 1956). Theseisolates were able to grow on polymeric sub-stances, e.g., agar and chitin (Humm and Shepard,1946), alginate and laminarin (Chesters et al.,1956), and cellulose (Chandramohan et al., 1972),characteristic for these microsites.

In sediments, both the depth and location ofsample sites play important roles in determiningthe ratio of different taxa of actinomycetes(Weyland, 1981b; Weyland and Helmke, 1988).Sites in the open sea generally contain only low


numbers of actinomycetes (viable counts wereabout 100 CFU per ml of wet sediment). Thedistribution (horizontal as well as vertical) isassumed to correlate with the physiologicalproperties of the three taxa Streptomycetes,Micromonosporae and Rhodococci barotoler-ance (Helmke, 1981), halotolerance, and psy-chrophilism (Weyland, 1981a). Goodfellow andHaynes (1984), however, were not able to find acorrelation between salinity, pH, or depth andthe number of actinomycetes recovered frommarine sediments. In this study numerous iso-lates are described in detail, and from a total of732 organisms, 250 belonged to Streptomyces,250 to Micromonospora, 140 to Rhodococcus,and 92 to Thermoactinomyces. A selected num-ber of isolates belonging to the genus Strep-tomyces was subsequently identified using 41diagnostic tests and applying the MATIDENprogram and the Streptomyces probability matrix(Williams et al., 1983b). Around 50% of thesestreptomycetes were similar to those groupedinto cluster 1 of Williams et al. (1983a).

Okami and Okazaki (1978) found strepto-mycetes (300–1270 colonies per cm3) mainlyin the sediments of shallow seas (70–520 mdeep), whereas in samples 700–1600 m deep,Micromonospora dominated. No actinomyceteswere obtained from depths of 2800 and 5000 min the Pacific Ocean. A higher salt tolerance ofmarine streptomycetes than of their terrestrialcounterparts was observed. However, alreadyTresner et al. (1968) found that salt toleranceamong streptomycetes is widespread and thisfeature may be due to selection of the more tol-erant organisms in marine habitats. Few strepto-mycetes isolated from marine environmentswere found to be obligate halophiles (Okazakiand Okami, 1976).

Notably, marine habitats are essential forscreening programs in the search of new antimi-crobial and anti-insecticidal compound produc-ers. In early studies, Nissen (1963) found a highpercentage of antibiotic-producing strepto-mycetes from decaying seaweed, and subsequentinvestigations confirmed these findings (Okamiand Okazaki, 1972; Okami et al., 1976; Hotta etal., 1980).

Isolation

The procedures of isolation of streptomycetes(extensively summarized by Korn-Wendisch andKutzner, 1992a) are partly summarized in thenext paragraphs. Additional information aboutisolation for special purposes, growth of strepto-mycetes, and preservation of streptomycetes canbe obtained from the excellent textbook Practi-cal Streptomyces Genetics (Kieser et al., 2000).

Generally, all procedures for the isolation ofmicroorganisms are influenced by the natureof the microorganism and the number ofpropagules relative to the number of othermicrobes within the habitat (Stolp and Starr,1981). If the organism to be isolated is bestadapted to the selected isolation conditions, thendirect plating of a serial dilution on a nutrientagar medium can readily lead to a pure culture.This procedure does not work well for isolationof streptomycetes. These actinomycetes are iso-lated usually by enrichment or use of selectivemedia and specific isolation conditions or both.

As already pointed out by Korn-Wendisch andKutzner (1992a), Streptomycetaceae memberscan be isolated using general selective isolationprocedures (Williams and Wellington, 1982b;Williams and Wellington, 1982c; Williams et al.,1984a). These procedures require 1) choice ofthe material containing the selected microorgan-isms, 2) pretreatment of the sample, and in somecases, enrichment of the chosen microbialgroups, 3) use of selective media or selectiveincubation conditions or both, and 4) colonyselection on the basis of colony morphology andpurification.

Streptomycetes can be isolated from a widevariety of habitats, and most isolation proce-dures involve extraction from soil or anotherenvironmental sample followed by dilution ofthe cells (cell aggregates) to allow cultivation onsolid media.

Isolation and Enrichment from Soil

Because the vegetative mycelium and sporechains are often closely associated with the min-eral and organic particles of the soil, vigorousshaking of the sample with the diluent is oftenneeded to suspend the spores of mycelial frag-ments. Use of glass beads and agitation on ashaker may aid suspension. In the literature,methods involving mechanical devices such asthe Ultrasonics sonicator-disrupter, Ultra-Turraxhomogenizer, Turmix blender, Waring blender,or a mortar and pestle are described, but adetailed comparison of the dilution efficiency ofthese pretreatments is still missing. Chemical dis-ruption methods are also reported in literature.Gently shaking soil samples with an ion-exchange resin Chelex-100 (Biorad) followed bydifferential centrifugation and filtration was usedto separate the mycelium from the spores(Herron and Wellington, 1990).

A subsequent treatment of samples (i.e., pre-paring dilutions and plating) differs little fromgeneral bacteriological practice. Most often thecoarse particles of the soil suspension areallowed to settle before dilutions are made. Butsoil particles may also be used directly for incu-


bation of “soil plates” (Warcup, 1950), which arealso used to isolate fungi. The addition of lime tosoil can enrich for streptomycetes (see chapter 2of Kieser et al. [2000] and references therein).Isolation plates may be surface-inoculated witha sterile glass rod (or Drigalski spatula).

Spread of motile bacteria via water films canbe avoided by drying the plates at 45°C beforeincubation or mixing the soil suspension withthe molten agar, which is highly recommended(Korn-Wendisch and Kutzner, 1992a). The addi-tion of CaCO3 to air-dried soil samples (10:1 w/w) and subsequent incubation at 26°C for 7–9days in a water-saturated atmosphere can lead toa 100-fold increase of streptomycete colonies onisolation plates (Tsao et al., 1960; El-Nakeeb andLechevalier, 1963).

The enrichment of streptomycetes by soilamendment with keratin was first carried out byJensen (1930). Later the addition of chitin to soilwas found to stimulate growth of actinomycetes(Williams and Mayfield, 1971b). Williams andRobinson (1981) similarly obtained the enrich-ment of acidophilic and neutrophilic strepto-mycetes in acidic soil and litter containing pureand fungal chitin. Another isolation strategyusing chitin in the form of insect wings has beenused as a baiting method (Veldkamp, 1955; Jag-now, 1957; Okafor, 1966). Porter and Wilhelm(1961) studied isolation using various otherorganic materials, like salmon viscera meal, pea-nut meal, cottonseed meal, and dried blood flour(15 mg/g of soil). They found that incubation ofthe enrichment cultures under moist conditionsled to an up to 1000-fold increase in the numberof streptomycetes.

Besides the frequently used arginine glycerolagar (El-Nakeeb and Lechevalier, 1963), thefollowing media (details below) are also oftenapplied for selective isolation of streptomycetes:HV agar (Hayakawa and Nonamura, 1987a,1987b), colloidal chitin agar (Hsu and Lockwood,1975), and reduced arginine starch salts agar.

Several physical, chemical and biologicalmethods have been studied to reduce or inhibitother microbes (for review, see Goodfellow andWilliams, 1986a). Nüesch (1965) centrifuged soilsuspensions for 20 min at 1600 × g to separatethe spores of streptomycetes (in the supernatant)from other bacteria and spores of fungi (in thesediment); however, the method has not beenvery successful. Using a similar approach, El-Nakeeb and Lechevalier (1963) obtained a sig-nificantly smaller number of streptomycete colo-nies as compared with the control. Voelskow(1988/1989) described a simple sedimentationmethod in which 1 g of soil was suspended in15 ml of salt solution, vigorously shaken, andthen treated with ultrasonic vibrations. After 1,2, and 4 h of sedimentation, samples were taken

from different levels of this solution, furtherdiluted, and plated on agar surfaces.

Initial drying and heating procedures wereapplied because arthrospores have a relativelyhigh resistance to low moisture tension. Dryingof the sample or prolonged storage at ambienttemperatures for mesophiles and at 50–60°C forthermophiles led to a relative increase in strep-tomycete concentrations. Williams et al. (1972)showed that heat treatment of soil (40–50°C, 2–16 h) leads to a significant reduction of the veg-etative bacterial proportion without affecting thecolony counts of streptomycetes.

Membrane filtration has been mainly used forthe enrichment of streptomycetes from watersamples (Burman et al., 1969) and from seawaterand mud (Okami and Okazaki, 1972), but it hasalso been a first step in the isolation of strepto-mycetes from soil (Trolldenier, 1966). Theseauthors used 1 ml of a series of 10-fold dilutions,which were membrane-filtered (0.3-µm poresize). This filter was then placed upside down ona suitable agar medium, which was supple-mented with 10% compost soil. Coloniesdeveloped between the agar surface and themembrane filter, and the streptomycetes (but notother bacteria or fungi) were able to growthrough the pores. Using this method, the selec-tive effects of the physical barrier (the mem-brane) and the soil lead to a 3–5-fold increase inthe number of streptomycete colonies in com-parison with poured plates without soil.

Hirsch and Christensen (1983) introduced theuse of cellulose ester membrane filters (poresize 0.01–3.0 µm), which were placed ontonutrient agar containing antifungal antibiotics(cycloheximide and candicidin). These plateswere inoculated with different samples fromsoil, water and vegetable materials. After 4days, the hyphae of actinomycetes penetratedthe filter pores and grew on the underlying agarmedium, whereas the growth of the other bac-teria was restricted to the surface of the filter.To allow further development of actinomycetecolonies, the membrane filter was removed andthe plates were reincubated. Filters (0.22–0.45 µm) were also found to be suitable for theexclusive recovery of actinomycetes. Polsinelliand Mazza (1984) and Hanka et al. (1985) usedthis approach independently.

Several authors have added chemicals toimprove the isolation efficiency. Phenol treat-ment of a dense soil suspension (1.4% for10 min) was recommended to eliminate bacteriaand fungi, but El-Nakeeb and Lechevalier (1963)obtained less favorable results with this method.Chloramine, ammonia, and sodium hypochloritewere mainly employed for the treatment ofwater samples, because it has been found thatstreptomycetes and other actinomycetes are


slightly more resistant to these agents than otherbacteria are (Burman et al., 1969).

Isolation of Airborne Spores

For the isolation of Streptomyces spores fromself-heating material such as hay or compost,samples can be agitated in a wind channel (Laceyand Dutkiewicz, 1976b) or sedimentation cham-ber (see below; Lacey and Dutkiewicz, 1976a).Plates are then inoculated with the aerosol usingan Andersen sampler (Goodfellow and Williams,1986a). This method, widely employed for theisolation of thermophilic actinomycetes, mayalso be used for the isolation of mesophilic strep-tomycetes from soil.

In addition, other devices like filtration sam-plers (e.g., Sartorius MD 8) are suitable for thesampling of airborne streptomycetes.

Use of Selective Media and Incubation

Selective media always play an important role inthe isolation of the desired microorganisms.A number of factors can be varied: 1) nutrientcomposition and concentration of the isolationmedium, i.e., choice of carbon and nitrogensources preferred by the organisms; 2) additionof chemical substances to inhibit selectively theaccompanying flora of the natural habitat or thosewhich are stimulating the desired organisms; 3)pH, for acidophilic, neutrophilic, and alkalophilicorganisms; and 4) temperature, e.g., for the iso-lation of thermophiles or psychrophiles.

Many different media have been formulatedempirically and proposed for the isolation ofstreptomycetes. Selected carbon and nitrogencompounds listed in Table 10 are especially suit-able for the isolation of these organisms. The

most frequently used media with their formulasare listed in Tables 11 and 12. Alternatively,streptomycetes can be grown on very poor mediasuch as water agar.

Different Carbon and Nitrogen Sources forEnrichment Because it was early recognizedthat streptomycetes can degrade chitin (Veld-kamp, 1955; Jagnow, 1957), a chitin medium wasdevised by Lingappa and Lockwood (1962) forselective isolation. However, the authors andlater also El-Nakeeb and Lechevalier (1963)found that their chitin agar was only a little bet-ter than water agar. Hsu and Lockwood (1975)added mineral salts to this medium (Table 11),which was shown to be useful for the isolationof actinomycetes (Streptomyces, Nocardia andMicromonospora) from water samples but hadlittle effect when isolating actinomycetes fromsoil. Note that chitinolytic activity is not a genusspecific feature for streptomyces. Williams et al.(1983a) found that only 25% of over 300 strainswere strongly chitinolytic, so this widely usedmedium selects the chitinolytic streptomycetestrains, which may not be the most abundantstrains in soil. Starch is degraded by the vastmajority of streptomycetes and can thereforebe used as a selective carbon source. An earlyfinding was that the combination of starch withnitrate, which is utilized by many streptomycetes(in contrast to other bacteria) as nitrogen source,is very useful for the selective isolation of strep-tomycetes (Flaig and Kutzner, 1960a). Küsterand Williams (1964), who improved this medium,stated: “The three best media, allowing gooddevelopment of streptomycetes while suppress-ing bacterial growth, were those containingstarch or glycerol as the carbon source withcasein, arginine or nitrate as the nitrogensource.”

Table 10. Nutritional substances and selective agents for isolation of streptomycetes from soil (according to Korn-Wendischand Kutzner, 1992).

From Korn-Wendisch and Kutzner (1992).

Preferred C and N sourceSelective agents in the medium

Antibiotic(s) Others References

Starch and KNO3 None None Flaig and Kutzner, 1960bStarch, casein, and KNO3 None None Küster and Williams, 1964Chitin None None Lingappa and Lockwood, 1962Glycerol and arginine None None El-Nakeeb and Lechevalier, 1963Glycerol, casein, and KNO3 None None Küster and Williams, 1964Raffinose, histidine None None Vickers et al., 1984Starch, casein, and KNO3 Rifampicin None Vickers et al., 1984Starch, casein, and KNO3 Cycloheximide, nystatin, penicillin, and

polymyxinNone Williams and Davies, 1965

Glycerol and arginine Cycloheximide, pimaricin, and nystatin None Porter et al., 1960Dextrose and asparagine Cycloheximide None Corke and Chase, 1956Asparagine None Propionate Crook et al., 1950Starch, casein, and KNO3 Cycloheximide Rose bengal Ottow, 1972


Tabl

e 11

.So

me

med

ia u

sefu

l for

the

sel

ecti

ve is

olat

ion

of s

trep

tom

ycet

es.

a Ref

eren

ces:

Küs

ter

and

Will

iam

s, 19

64; E

l-N

akee

b an

d L

eche

valie

r, 19

63; H

su a

nd L

ockw

ood,

197

5; a

nd V

icke

rs e

t al

., 19

48; D

ifco

Lab

orat

orie

s.b A

lter

nati

vely

, gly

cero

l at

10g/

liter

can

be

used

.c N

ot c

onta

ined

in t

he d

ehyd

rate

d m

ediu

m; a

dded

at

the

tim

e of

pre

para

tion

.d T

he d

iffe

rent

am

ount

s of

the

aga

r ar

e du

e to

the

var

ying

qua

lity

used

by

the

indi

vidu

al a

utho

rs.

Ref

eren

cesa

12

34

5In

gred

ient

s (g

/lite

r)St

arch

-cas

ein-

KN

O3

agar

Gly

cero

l-ar

gini

ne a

gar

Act

inom

yces

isol

atio

n ag

arC

hiti

n ag

arR

affin

ose-

hist

idin

e ag

ar

Chi

tin

(col

loid

al)

——

—4.

0—

Star

ch10

.0b

——

——

Gly

cero

l—

12.5

5.0c

——

Raf

finos

e—

——

—10

.0So

dium

pro

pion

ate

——

4.0

——

KN

O3

2.0

——

——

Cas

ein

0.3

——

——

Sodi

um c

asei

nate

——

2.0

——

Asp

arag

ine

——

0.1

——

Arg

inin

e—

1.0

——

—H

isti

din e

——

——

1.0

NaC

l2.

01.

0—

——

KH

2PO

4—

——

0.3

—K

2HP

O4

2.0

1.0

0.5

0.7

1.0

MgS

O4 ·

7H2O

0.05

0.5

0.1

0.5

0.5

CaC

O3

0.0 2

——

——

Fe2(

SO4)

3 · 6H

2O—

0.01

——

—Fe

SO4 ·

7H2O

0.01

—0.

001

0.01

0.01

CuS

O4 ·

5H2O

—0.

001

——

—Z

nSO

4 · 7H

2O—

0.00

1—

0.00

1—

MnS

O4 ·

H2O

—0.

001

——

—M

nCl 2

· 4H

2O—

——

0.00

1—

Aga

rd18

.015

.015

.020

.012

.0pH

Adj

uste

d to

7.0

–7.5

or

low

eror

hig

her

depe

ndin

g on

the

flora

to

be is

olat

ed.

Adj

uste

d to

7.0

–7.5

or

low

eror

hig

her

depe

ndin

g on

the

flora

to

be is

olat

ed.

Adj

uste

d to

7.0

–7.5

or

low

eror

hig

her

depe

ndin

g on

the

flora

to

be is

olat

ed.

Adj

uste

d to

7.0

–7.5

or

low

eror

hig

her

depe

ndin

g on

the

flora

to

be is

olat

ed.

Adj

uste

d to

7.0

–7.5

or

low

eror

hig

her

depe

ndin

g on

the

flora

to

be is

olat

ed.


Benedict et al. (1955) were the first to reportthat the combination of glycerol and argininefavored streptomycete isolation. El-Nakeeb andLechevalier (1963) found that this medium(Tables 11 and 12) was superior to nine othermedia, resulting in higher number and propor-tion of streptomycete colonies.

Other compounds (e.g., pectin [Wieringa,1955], poly-ß-hydroxybutyrate [Delafield et al.,1965], rubber [Nette et al., 1959], cholesterol[Brown and Peterson, 1966], elemental sulfur[Wieringa, 1966], and natural and artificial humicacids [Hayakawa and Nonomura, 1987a, 1987b])have been used successfully, and most of them

Table 12. Composition of some media suitable for the cultivation of streptomycetes.a

aRecipes 1–5 are from Shirling and Gottlieb (1966) and recipe 6 from Voelskow (1988/89).From Korn-Wendisch and Kutzner (1992).

Ingredients Comments

1. Glucose-yeast extract-malt extract (GYM) agarGlucose 4.0g Addition of CaCO3 (2.0g/liter) is advantageous for the growth of many

streptomycetes. Adjust pH to 7.2.Yeast extract 4.0gMalt extract 10.0gAgar 12.0gDistilled water 1 liter

2. Oatmeal agarOatmeal 20.0g Cook 20.0g of oatmeal in 1 liter of distilled water for 20min. Filter through

cheesecloth. Add distilled water to restore the volume of the filtrate to 1 liter, then add trace salts solution and agar. Adjust pH to 7.2.

Agar 12.0gTrace salts solution (see no. 5) 1.0mlDistilled water 1 liter

3. Inorganic salts-starch agarStarch (soluble) 10.0g Make a paste of the starch with a small amount of cold distilled water and

bring to a volume of 1 liter; then add the other ingredients. Adjust pH (if necessary) to 7.0–7.4.

(NH4)2SO4 2.0gK2HPO4 (anhydrous basis) 1.0gMgSO4·7H 2O 1.0gNaCl 1.0gCaCO3 2.0gTrace salts solution (see no. 5) 1.0mlAgar 12.0gDistilled water 1 liter

4. Glycerol-asparagine agarGlycerol 10.0g The pH should be about 7.0–7.4. Do not adjust if it is within this range.l-Asparagine (anhydrous) 1.0gK2HPO4 1.0gTrace salts solution (see no. 5) 1.0mlAgar 12.0gDistilled water 1 liter

5. Trace salts solutionFeSO4·7H 2O 0.1gMnCl2·4H 2O 0.1gZnSO4·7H 2O 0.1gDistilled water 100.0ml

6. Trace elements solution SPV-4CaCl2·2H 2O 4.0g SPV-4 is used as an alternative to (5). Five ml of this stock solution is added

to 1 liter of medium.Fe (III) citrate 1.0gMnSO4 0.2gZnCl2 0.1gCuSO4·5H 2O 0.04gCoCl2 0.022gNa2MoO4·2H 2O 0.025gNa2B4O7·10H 2O 0.1gDistilled water 1 liter


strongly select certain organisms producingvisible zones of clearing or other changes in themedium.

The use of compounds with antifungal activity(antibiotics) as supplements to isolation mediahas also been widely used to suppress fungalgrowth (Table 10). The most frequently usedcompound is cycloheximide (actidione, 50–100 µg/ml) by Williams and Davies (1965). Pima-ricin and nystatin (each 10–50 µg/ml) were foundto be even more effective (Williams and Davies,1965).

The use of compounds with antibacterial activ-ity is more restricted because actinomycetes areoften also sensitive to them. Williams and Davies(1965) found polymyxin (5 µg/ml) and penicillin(1 µg/ml) suppressed bacterial flora; however,they also inhibited streptomycetes. Preobrazhen-skaya et al. (1978) showed that the genera ofActinomycetales may differ significantly in theirsensitivity to antibacterial antibiotics and thatstreptomycetes were the most sensitive group.Thus, the use of antibacterial compounds may bemore helpful in isolating other genera of thisorder (Cross, 1982).

In contrast, some antibiotics may facilitate theisolation of certain species or groups of Strepto-myces. For example, the selective isolation ofmembers of the Streptomyces diastaticus clustersensu Williams et al. (1983a) was achieved onstarch casein medium containing rifampicin(50 µg/ml; Vickers et al., 1984). Wellington et al.(1987) found a similar effect with several mediacontaining different C and N sources as well aswith media supplemented with inhibitors.

Hanka et al. (1985) described a selectiveisolation medium for streptoverticil-producingStreptomyces species containing cycloheximideand nystatin (each 50 g/ml, to control fungalgrowth) and oxytetracycline (25 µg/ml, to sup-press other actinomycete genera and other Strep-tomyces groups). Hanka and Schaadt (1988)enhanced selectivity of this medium by addinglysozyme (1000 µg/ml). Also, sodium propionatecan suppress the competing fungi (Crook et al.,1950; Table 11), and Rose Bengal (35 mg/liter)in starch casein nitrate agar (Ottow, 1972) cansuppress most of the bacteria and inhibit thespreading growth of fungi.

pH of the Isolation Medium and IncubationTemperature Most streptomycetes grow opti-mally at neutral pH values, i.e., are neutrophilic.Therefore, the pH of most isolation media is 7.0–7.5. However, for the isolation of acidophilicstreptomycetes, the medium pH is 4.5 (Khan andWilliams, 1975), and for alkalophilic strains, itis 10–11 (Mikami et al., 1982). Most strepto-mycetes isolated from soils are mesophilic, andtherefore plates are most often incubated at 22–

37°C (mostly at 28°C). Psychrophilic strains (e.g.,from marine environments) grow at 15–20°C. Incontrast, thermotolerant and thermophilic repre-sentatives can be isolated at higher temperatures(40, 45, 50, or 55°C). Note that thermophilesoften form colonies after a short period of incu-bation (within 2–5 days), and mesophilic mem-bers produce visible colonies within 7–14 days.However, marine and other psychrophilic organ-isms often need several weeks (up to 10) for theformation of visible colonies.

Colonies of Streptomyces are in most casesreadily recognized by their macroscopic andmicroscopic appearance. In most cases, Strepto-myces are easily purified by picking colonies andtransfer to a nonselective medium. According toWilliams and Wellington (1982b), purificationis “undoubtedly the most time-consuming andoften the most frustrating stage of the isolationprocedure.” Acidiphilic members of the Strepto-mycetaceae can be isolated on acidified starchcasein agar containing cycloheximide and nysta-tin (Kim et al., 2003).

Isolation of Antibiotic Producers and Strains for Genetic Studies

The isolation of antibiotic producing strepto-mycetes follows the same procedures as givenabove. Most often, the activity is tested after iso-lation of pure cultures, but this can also be com-bined with the isolation procedure. Thus, strainsexhibiting antibiotic activity can be recognizedeven on the initial dilution plates if they aretreated with an appropriate test organism, eitherby flooding or by spraying. Zones of inhibitioncan be detected after further incubation (Lind-ner and Wallhäusser, 1955; Wilde, 1964). Asan alternative, a simple replication procedureallows the examination of the antibiotic activityof the colonies against selected sensitive organ-isms (Lechevalier and Corke, 1953). Selectivetechniques for isolation and screening of actino-mycetes that produce antibiotics and other sec-ondary metabolites of clinical relevance havebeen summarized by Nolan and Cross (1988). Inaddition, Kieser et al. (2000) have provided aset of protocols for selective isolation of strepto-mycetes, generating spore suspensions, and sev-eral other more sophisticated procedures.

Isolation of Thermophilic Streptomycetes

As already stated, thermophilic streptomycetesand other thermophilic actinomycetes are oftenisolated from samples derived from high tem-perature environments (e.g., compost materials,manure heaps, and fodders). The high tempera-tures (45–60°C) can serve as a selective condition


that favors enrichment of the desired organisms(Festenstein et al., 1965).

As pointed out by Greiner-Mai et al. (1987), avery important requisite for the isolation of ther-mophilic streptomycetes is a humid atmosphere,which can be achieved by incubating plates inlarge jars with water in the bottom. Alternatively,the sealing of Petri dishes with masking tape isalso effective.

Interestingly, the media recommended for theisolation of thermophilic actinomycetes, includ-ing streptomycetes, contain higher nutrientconcentrations than those used for mesophilicstrains. In addition, antifungal agents andantibacterial agents are sometimes added as sup-plements (Lacey and Dutkiewicz, 1976b; Good-fellow et al., 1987c). For some special isolationtechniques, the reader is referred to the papersof Uridil and Tetrault (1959), Fergus (1964), Gre-gory and Lacey (1963), and Cross (1968). Addi-tional information can be obtained from thepublications of Kim et al. (Kim et al., 1996; Kimet al., 1998; Kim et al., 2000).

Isolation from Aquatic Habitats

For the isolation of streptomycetes from water,the media listed in Table 11 can be used. In acomparative study on the suitability of media,Hsu and Lockwood (1975) found that chitin-agarwas superior to the other four (egg albumin,glycerol arginine, starch casein, and Actinomycesisolation agar; see also Table 11).

Water samples can be directly streaked ontothe solid medium after dilution. When low num-bers are expected, the samples can be concen-trated by membrane filtration (for details, seeBurman et al. [1969] or Trolldenier [1967]).

Streptomycetes from marine habitats weresuccessfully isolated on media containing 25or 75% seawater (Weyland, 1981a; Weyland,1981b), artificial seawater (Goodfellow andHaynes, 1984), or deionized water supplementedwith 3.0% NaCl (Okami and Okazaki, 1978). Forfurther details, see also Weyland (1981b) andGoodfellow and Haynes (1984).

Isolation from Infected Plants

For the isolation of streptomycetes from dis-eased plant tissue (i.e., from scabby potato orbeet surface layers), three general steps havebeen recommended (see also Korn-Wendischand Kutzner, 1992a): 1) sterilization of the sur-faces of tubers, beets, or roots; 2) maceration ofthe plant tissues; and 3) use of appropriate mediafor plating.

Methods for isolating Streptomyces scabiesfrom potatoes have been described in detail byseveral authors (Taylor, 1936; KenKnight and

Munzie, 1939; Menzies and Dade, 1959; Adamsand Lapwood, 1978; Archuleta and Easton,1981).

Cultivation

Nutritional Requirements and Media for Sporulation

The vast majority of streptomycetes are nonfas-tidious organisms, having a chemoorganotrophicmetabolism. The nutritional requirements are(in most cases) restricted to an organic carbonsource (e.g., starch, glucose, glycerol and lactate)and an inorganic nitrogen source (NH4

+ or NO3–),

in addition to the essential mineral salts forgrowth. The necessity to amend media withspecific trace elements has not been studied indetail. Many of the early used media (even the“synthetic” media) were not supplemented withtrace elements, although the positive effect oftrace elements in soil on the growth of strepto-mycetes has been reported (Spicher, 1955).Other authors used quite different recipes(Tables 11 and 12), each containing only a selectnumber of metal ions. A rather complete mixture(SPV-4; Table 12) has been found to be optimalfor actinomycetes and other bacteria (Voelskow,1988/1989).

Because no specific requirement for vitaminsor organic growth factors has been described,“synthetic media” can be used for their cultiva-tion. Notably, complex organic substrates (e.g.,oatmeal, yeast extract, or malt extract) are wellutilized and enhance growth rates and biomassproduction. A combination of a complex organiccarbon source with a single amino acid as nitro-gen source (e.g., glutamic acid, arginine or aspar-agine) is also suitable.

Several authors have proposed “generalmedia” for streptomycetes that allow the com-pletion of the streptomyces’ life cycle, i.e. germi-nation of spores, growth of substrate and aerialmycelium, and formation of spores (visiblebecause of the typical color of the spores). Someof these media have been used in the Interna-tional Streptomyces Project (ISP). Of the greatnumber of useful media (Pridham et al., 1956/1957), four are of particular value and most oftenused (Table 12). Additional media are listed byWaksman (1961) and by Williams and Cross(1971a). CaCO3 added to some media not onlysupplies Ca2+ for growth but also neutralizesacids produced by many streptomycetes. Thesemedia also allow good sporulation. Becausemacroscopically heavy aerial mycelia may con-tain very few spores and aerial mycelia hardlydetectable by the naked eye may be a goodsource of spores, it is advisable to check thesecultures microscopically. Specialized media,


especially for genetic studies, are given by Kieseret al. (2000).

Media Containing Soil, Clay, Minerals and Calcium Humate

Addition of soil to isolation media increases thenumber of colonies as it promotes growth, sporu-lation and pigmentation (Trolldenier, 1966).Martin et al. (1976) observed the stimulation ofboth growth and metabolic activity of some act-inomycetes when montmorillonite or Ca-humatewas added to a liquid medium. A similar effectwas observed for clay in dialysis tubes after ashort lag period, which was explained by a pos-sible adsorption of one or more inhibitory sub-stances produced during growth. Martin et al.(1976) also reported a positive effect of theseadsorbing materials on the genetic stability ofother bacteria and on fungi.

Temperature and Oxygen

As pointed out above, most streptomycetes aremesophilic organisms; however, psychrophilic aswell as thermotolerant and thermophilic speciesare also known. Note that in many instances, theoptimum temperature for fast growth or maxi-mal yield may not be the best choice for studyingthe production of secondary metabolites (e.g.,antibiotics and pigments). Most streptomyceteshave an obligately aerobic metabolism, but manystreptomycetes are able to reduce nitrate tonitrite under strictly anaerobic conditions.

In semisolid agar with a nutrient medium, theygrow at the surface of the agar column; however,in semisolid agars with a poor medium or non-utilizable carbon source, they grow microaero-philically. A stationary liquid culture grows as apellicle at the surface, and the medium remainscompletely clear.

Cultivation and Preparation of Inoculum

For subcultivation and maintenance, and formost diagnostic tests, streptomycetes are prefer-ably cultivated on solid media in dishes or slants.On solid media many strains produce aerialmycelia and spores when the entire surface iscovered by confluent growth. Since Streptomycescolonies, in contrast to most molds, spread overa limited distance, a point inoculation will oftennot lead to a confluent growth.

Some strains show sporulation only when theplates are cross-hatched inoculated, i.e., by amethod which leaves empty spaces betweenthe streaks. Sporulation generally occurs betterunder dry conditions. For this reason, slantsshould be incubated horizontally for the first twodays to allow the liquid to soak into the surface

of the agar (Hopwood et al., 1985). The startingmaterial should be a suspension of inoculum inliquid (Kieser et al., 2000).

The propagation of cultures by successiverounds of mass culture should be avoided, butinstead a single colony should be picked andstreaked to start the next culture. Especially ingenetic studies, this reduces the accumulation ofrevertants or the gradual loss of selected plas-mids or both (Kieser et al., 2000).

Note that morphological heterogeneity isoften observed when streptomycetes are culti-vated on solid media (for more details, seeKieser et al., 2000).

Because it may be difficult to produce a homo-geneous suspension from the grown colonies (aprerequisite for inoculation of some diagnostictests; Kämpfer et al., 1991b), precultivation inliquid media is sometimes useful. This is the casefor certain physiological studies (e.g., degrada-tion tests), for the provision of cell material forbiochemical analysis, and for the productionof secondary metabolites (e.g., antibiotics) orenzymes. For these purposes, streptomycetes canbe cultivated in liquid medium with agitation.

The use of liquid cultures started from an inoc-ulum of spores is also recommended for manydetailed studies, e.g., for preparation of proto-plasts for fusion, transformation or transfection.

Notably, the multicellular lifestyle of strepto-mycetes complicates the study of metabolicproperties, where all cells of the initial suspen-sion should be in the same physiological condi-tion. In general, streptomycetes grow by mycelialelongation and branching. But when centralparts of the colony become nutrient limited,physiological homogeneity cannot be sustained.To overcome these problems, spore germlingsare used for physiological studies, even though alarge amount of spores is needed. Other solu-tions include liquid cultures supplemented withdispersants, like sucrose, polyethylene glycol,Junlon®, starch, agar and carboxymethylcellu-lose. Chapter 2 of Kieser et al. (2000) summa-rizes the advantages and disadvantages. Sincestreptomycetes are highly aerobic, the culturesneed to be shaken during incubation. Use ofErlenmeyer flasks with indentations or stainlesssteel springs is recommended, but for smallquantities (3–5 ml being enough for some phys-iological tests), tubes in a slanted position on ashaker or roller also allow excellent supply ofoxygen. Note that some secondary metabolites(e.g., antibiotics and pigments, which are pro-duced on solid media) may fail to be synthesizedunder these conditions.

Korn-Wendisch and Kutzner (1992a) recom-mend two media that have been widely used forthe submerged cultivation of streptomycetes (g/liter): 1) GPYB broth (glucose, 10.0; peptonefrom casein, 5.0; yeast extract, 5.0; beef extract,


5.0; CaCl2 · 2H 2O, 0.74; pH 7.2) and 2) soybeanmeal-mannitol nutrient medium (soybean meal,20.0; mannitol, 20.0; pH 7.2). Two kinds of noc-ulation material can be employed for subcultur-ing streptomycetes: 1) arthrospores and 2)vegetative mycelium, occasionally including“submerged spores” (Wilkin and Rhodes,1955). Kieser et al. (2000) recommend similarprocedures.

Spore suspensions can be used over a periodof several weeks when stored at 4°C, but sincethe spores tend to settle and clump, the additionof a few glass beads to the screw-cap tube helpsto resuspend the spores before use. Chapters 8and 9 of Kieser et al. (2000) describe the prepa-ration of mycelia for detailed DNA or RNAstudies.

Preservation

Several different procedures have beenemployed for the short- and long-term preserva-tion of microorganisms (Kirsop and Snell, 1984).

Three short-term preservation methods havebeen described by Korn-Wendisch and Kutzner(1992a). First, agar slope cultures may be storedat 4°C for few months. Second, spore suspen-sions can be mixed with soft water agar and keptat 4°C (Kutzner, 1972). And third, glycerol canbe added to spore suspensions (final concentra-tion, 10%, v/v) and stored at –20°C (Wellingtonand Williams, 1978); these cultures (after thaw-ing) can serve as inoculum for most diagnostictests except carbon utilization (Williams et al.,1983a). Kieser et al. (2000) recommended forlong-term preservation the preparation of aspore suspension in 20% glycerol and freezingat –20°C. Another procedure is the growth ofstrains in complex media (like trypticase soybroth [TSB] agar), addition of 20% glycerol plus10% lactose, and storage in the vapor phase ofliquid nitrogen. In addition, drying on unglazedporcelain beads (Lange and Boyd, 1968), fol-lowed by soil culture (Pridham et al., 1973), andlyophilization (Hopwood and Ferguson, 1969)are used. For longer preservation (see The Fam-ily Nocardiopsaceae in this Volume), spore sus-pensions or homogenized mycelia are mixedwith glycerol to a final concentration of 25% andkept at 25°C (Wellington and Williams, 1978).Alternatively, spores and mycelia suspended in10% skim milk are lyophilized. A very simple,reliable, and time-saving method is liquid nitro-gen cryopreservation of living cells in small poly-vinyl chloride (PVC) tubes (“straws”) at –196°C.The procedure has been tested for various acti-nomycetes. The strains are harvested from well-sporulated cultures grown on suitable agarmedia in Petri dishes. A 2 × 25-mm piece of ster-ile PVC tubing is pressed into the mycelial mat

and agar and carefully raised to excise the agarplug. This is repeated until the tube is filled withagar. The filled tubes are placed in a sterile cry-ovial (the screw cap marked with the strainaccession number). A 1.8-ml vial will hold up to13 tubes. Two vials are prepared for each strainand then fixed to a metal clamp for freezing inthe gas phase of a liquid nitrogen container.After 10–15 min, when temperature falls below–130°C, the clamp can be immersed in the liquidphase at –196°C. A container with a capacity of250 liters will hold at least 8000 vials or 4000strains. For viability testing, one tube is removedfrom the vial within the nitrogen gas atmosphereof the container and placed directly and thawedon a suitable agar medium. After a few daysincubation, the mycelium will be visible. Forthose strains that do not produce an abundantmycelium, the plugs may be pushed out of thetubes by a sterile needle.

Detection of Streptomyces Using Cultivation-independent Methods

Microorganisms in the environment can beidentified without cultivation by retrieving andsequencing macromolecules and using oligonu-cleotide probes (largely based on small subunitrRNA; Amann et al., 1995; Rappé and Giovan-noni, 2003). Stackebrandt et al. (1991b) devel-oped 16S rRNA-targeted oligonucleotide probesspecific for certain Streptomyces species andsubsequently studied bacterial diversity in a soilsample from a subtropical Australian environ-ment (Stackebrandt et al., 1993). They found thatmost sequences were from alpha subclass Pro-teobacteria and only a few from streptomycetes.Hahn et al. (1992), using the in situ hybridizationapproach, were unable to analyze bacterial pop-ulations in soil without prior activation by addingnutrients. Growing cells, e.g., Streptomyces sca-bies hyphae, were easily detected. The use ofspecific primers in connection with environmen-tal clone libraries is a powerful approach forstudy of the microbial diversity in soil (Felske etal., 1997; Rheims et al., 1999; Rintala et al., 2001;Courtois et al., 2003) and discovery of novel bio-active metabolites (Donadio et al., 2002). Moredetails are given in The Family Streptomyceta-ceae, Part II: Molecular Biology in this Volume.

Acknowledgments. The basis of the sections onecophysiology, isolation and habitats has beenthe excellent and comprehensive treatise ofKorn-Wendisch and Kutzner (1992a) from thesecond edition of The Prokaryotes, which is stillrecommended for deeper study of classicalapproaches in Streptomyces biology.


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the prokaryotes || the family streptomycetaceae, part i: taxonomy

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