the prokaryotes || the family actinosynnemataceae

CHAPTER 1.1.10The Family Actinosynnemataceae

The Family Actinosynnemataceae

DAVID P. LABEDA

Introduction and Phylogenetic Position

The family Actinosynnemataceae (Labeda andKroppenstedt, 2000), as currently defined byphylogenetic studies based on the analysis of 16SrDNA sequences, contains the genera Actinoki-neospora (Hasegawa, 1988), Actinosynnema(Hasegawa et al., 1978), Lechevalieria (Labedaet al., 2001), Lentzea (Yassin et al., 1995; Labedaet al., 2001) and Saccharothrix (Labeda et al.,1984; Labeda and Lechevalier, 1989a). This fam-ily lies within the suborder Pseudonocardiniae inthe class Actinobacteria (Stackebrandt et al.,1997). Earlier phylogenetic studies (Embley etal., 1988; Warwick et al., 1994) proposed that thegenus Saccharothrix was associated with the fam-ily Pseudonocardiaceae, but this affiliation wasnot supported statistically in phylogenetic analy-ses, and diagnostic chemotaxonomic characteris-tics of Saccharothrix species were different fromthose of taxa whose placement in the Pseudono-cardiaceae was well supported.

The genera included in the family Actinosyn-nemataceae share similar chemotaxonomic char-acteristics and exhibit a diagnostic whole cellsugar pattern (i.e., galactose, rhamnose and man-nose are diagnostic sugars) distinct from the pat-tern observed for the taxa within the familyPseudonocardiaceae (i.e., arabinose and galac-tose). The family Actinosynnemataceae forms acoherent group (Fig. 1) within the actinobacte-rial phylogenetic tree calculated from ribosomalDNA sequences (Labeda and Kroppenstedt,2000), and on the basis of current data, forms alineage separate from, but phylogenetically clos-est to, the Pseudonocardiaceae. Moreover, adiagnostic nucleotide signature pattern of TA(823–975), GC (824–874) is observed in the 16SrDNA sequence for all taxa within the familyActinosynnemataceae.

Within the suborder, related genera (phyloge-netically not quite within this family, but whichshare some properties with family members)include Actinoalloteichus, Crossiella, Kutzneriaand Streptoalloteichus. These genera may well

represent another family within the suborderPseudonocardiniae, but insufficient taxa havebeen isolated and described to provide adequatedata for resolution of their actual phylogeneticposition.

The genera currently circumscribed by thefamily Actinosynnemataceae are all aerobic, cat-alase positive, non-acid-fast, and lysozyme resis-tant. Their common chemotaxonomic propertiesinclude type III cell wall composition (meso-diaminopimelic acid), whole cell sugar patternconsisting of galactose (in the absence of signifi-cant quantities of arabinose) along with varyingquantities of rhamnose and mannose, a PII phos-pholipid profile typically displaying a significantamount of phosphatidylethanolamine, andmenaquinones containing nine isoprenoid units(MK-9). The Actinosynnemataceae exhibit arange of morphological and physiological prop-erties, including spore motility (expressed byseveral genera). Representatives of the familyhave been primarily isolated from soil or plantmaterial, but their role in these environmentsis unclear. Some taxa have also been isolatedfrom human or animal sources, but their capacityto cause infection or disease has not beenconfirmed.

Description of Genera

The genus Actinokineospora was described byHasegawa (1988) to contain a single species,Actinokineospora riparia, which produces shortaerial mycelia that fragment into motilearthrospores, but exhibit no sporangialstructures. Subsequently, Tamura et al. (1995)isolated and described four additional species,Actinokineospora diospyrosa, Actinokineosporaglobicatena, Actinokineospora inagensis andActinokineospora terrae, from soil and fallenleaves collected in the Yamanashi Prefecture ofJapan; these species exhibit the same capacity toproduce motile arthrospores from fragmentationof the aerial mycelia.

Hasegawa et al. (1978) described the genusActinosynnema to contain actinomycetes that

Prokaryotes (2006) 3:654–668DOI: 10.1007/0-387-30743-5_25

CHAPTER 1.1.10 The Family Actinosynnemataceae 655

were observed to have unique morphologicalproperties. Strains of this genus were observedto produce synnemata or dome-like bodies onmost media, and the aerial mycelia producedon the synnemata eventually fragment intoperitrichously flagellated motile zoospores inaqueous environments. The genus currently con-tains Actinosynnema mirum, the type species,and Actinosynnema pretiosum and its subspeciesActinosynnema pretiosum subsp. auranticum(Hasegawa et al., 1983). All of the species of thisgenus formally described to date have been iso-lated from the surface of blades of grass.

The genus Lechevalieria was recentlydescribed by Labeda et al. (2001) and containsLechevalieria aerocolonigenes and Lechevalieriaflava; both species were transferred from Saccha-rothrix aerocolonigenes and Saccharothrix flava,respectively, when they were shown to be phylo-genetically distinct from either Saccharothrix orLentzea and exhibited chemotaxonomic differ-ences from these genera.

The genus Lentzea was first described (Yassinet al., 1995) on the basis of a single strainobtained from a tissue specimen from theabdominal cavity of a patient with peritonealcarcinoma. Phylogenetic analysis based on 16SrDNA sequences indicated that this isolate wasrelated to Actinosynnema and Saccharothrix,which have similar chemotaxonomic characteris-tics. The pathogenicity of this species was neverconfirmed, particularly since the description isbased on only one isolate. Recently, Lee et al.(2000) proposed that the type species Lentzeaalbidocapillata be transferred to the genusSaccharothrix as Saccharothrix albidocapillata,

effectively abolishing this genus. A subsequentphylogenetic study (Labeda et al., 2001)reported phylogenetic and chemotaxonomic evi-dence that supported the revival of this genusand addition of the new species Lentzea californ-iensis and Lentzea albida, which had been pro-posed as the taxon “Asiosporangium albidum”(Itoh et al., 1987; Runmao et al., 1995; Tamuraand Hatano, 1998). The transfer of Saccharothrixviolacea and Saccharothrix waywayandensis tothe genus Lentzea as Lentzea violacea andLentzea waywayandensis, respectively, addedtwo more species.

The genus Saccharothrix was described byLabeda et al. (1984) to contain nocardioform act-inomycetes that morphologically resembledNocardiopsis in the ability of their aerial andsubstrate mycelia to fragment into ovoid, non-motile elements. Saccharothrix strains containthe meso-isomer of diaminopimelic acid in theircell walls, but are chemotaxonomically distinctfrom Nocardiopsis by containing rhamnose inaddition to galactose as a diagnostic whole cellsugar. When first described, the genus consistedof only one species, Saccharothrix australiensis,but currently it consists of four species represent-ing strains originally isolated from nature, i.e.,Saccharothrix australiensis, Saccharothrix espa-naensis (Labeda and Lechevalier, 1989), Saccha-rothrix texasensis (Labeda and Lechevalier,1989b) and Saccharothrix tangerinus (Kinoshitaet al., 1999), as well as 7 species and subspeciestransferred from other genera, i.e., Saccharothrixmutabilis subsp. capreolus (Grund and Krop-pentstedt, 1989), transferred from the genusNocardia; and Saccharothrix mutabilis subsp.

Fig. 1. Radial phylogenetic tree of the suborder Pseudonocardiniae calculated from 16S rDNA sequences using Kimura’sevolutionary distance methods (Kimura et al., 1980) and the neighbor-joining method of Saitou and Nei (1987). This treeillustrates the relationship of the family Actinosynnemataceae to the family Pseudonocardiaceae.

Saccharomonospora

Amycolatopsis

Kibdelosporangium aridum ATCC 39323T /X53191

Actinoalloteichus cyanogriseus IFO 14455T /AB006178

Streptoalloteichus hindustanus IFO 15115T /D85497

Crossiella equi NRRL B-24104T /AF245017

Crossiella cryophila NRRL B-16238T /AF114807

Kutzneria kofuensis NRRL B-16061T /AF114801

Arthrobacter globiformis DSM 20124T/M23411

Kutzneria viridogrisea DSM 43850T /X70429Actinokineospora

Actinosynnemataceae

Pseudonocardiaceae

Saccharothrix

Actinosynnema

0.10

LechevalieriaLenizea

Pseudonocardia

Saccharopolyspora

Thermocrispum

Prauserella rugosa DSM 43194T /AF051342

656 D.P. Labeda CHAPTER 1.1.10

mutabilis (Grund and Kroppentstedt, 1989;Labeda and Lechevalier, 1989), Saccharothrixcoeruleofusca (Grund and Kroppentstedt, 1989),Saccharothrix coeruleoviolacea (Grund andKroppentstedt, 1989), Saccharothrix longispora(Grund and Kroppentstedt, 1989) and Saccharo-thrix syringiae (Grund and Kroppentstedt, 1989),transferred from the genus Nocardiopsis.

Habitats of Each Genus

Actinokineospora

Strains of the species of Actinokineospora havebeen isolated from plant material or directlyfrom soil. More specifically, Actinokineosporariparia and Actinokineospora terrae have beenisolated from soil samples taken in the proximityof a pond (Hasegawa, 1988; Tamura et al., 1995),strains of Actinokineospora inagensis and Acti-nokineospora diospyrosa from fallen leaves(Tamura et al., 1995), and strains of Actinokine-ospora globicatena from both soil samples andfallen leaves collected adjacent to the shores oflakes and ponds (Tamura et al., 1995).

Actinosynnema

The strains of the described species of Actinosyn-nema have all been isolated directly from plantmaterial, primarily the surfaces of blades of grass(Hasegawa, 1978) and sedge (Carex species;Hasegawa et al., 1983). Hayakawa et al. (2000)recently reported the use of a new technique thatpermits the isolation of Actinosynnema strainsfrom several different soil samples.

Lechevalieria

The genus Lechevalieria, at present, consists ofLechevalieria aerocolonigenes and Lechevalieriaflava, originally isolated from soil samplesobtained in Japan and Russia, respectively.

Lentzea

The first described strain of Lentzea, the typespecies Lentzea albidocapillata, was isolatedfrom a tissue sample from the peritoneal cavityof a cancer patient (Yassin et al., 1995), but spe-cies subsequently added to this genus are fromsoil samples obtained in China, Korea, and theUnited States, suggesting that the distribution ofgenus members in soils is probably global.

Saccharothrix

The original strain of Saccharothrix australiensis,the type species of the genus, was isolated from

a soil sample from Australia. The genus appearsto be ubiquitous in soils and has a worldwidedistribution. Isolates described as members ofthis genus have come from soil samples collectedin the United States, Japan, Panama, Africa, andRussia.

Isolation and Cultivation

Strains of Actinokineospora have been routinelyisolated on humic acid-vitamin agar (HV agar;Hayakawa and Nonomura, 1987), using a modi-fication of the procedure of Makkar and Cross(1982). Soil or leaf litter samples are air dried at28

°C for 7 days, 0.5 g of sample is mixed with50 ml of sterile tap water, and then the mixtureis incubated at 20

°C for 55 minutes with occa-sional shaking. Aliquots (0.1 ml) of dilutions ofthe supernatant are spread on the surface ofplates and incubated at 28

°C for 2–3 weeks.Hayakawa et al. (2000) recently described a

new technique for the isolation of actinomyceteshaving motile zoospores. In this technique, a0.5 g sample of air-dried soil or leaf litter isplaced in a conical beaker (46 mm

× 60 mm) andgently flooded with 50 ml of sterile 10 mMphosphate buffer (pH 7.0) containing 10% soilextract. The vessel is loosely covered and incu-bated statically at 30

°C for 90 minutes to permitliberation of motile zoospores. An 8-ml aliquotof the supernatant is transferred to a 16.5

×105 mm screw cap tube and centrifuged for 20minutes at 1,500 g at room temperature. The tubecontents are allowed to settle for 30 minutes, aportion of the supernatant is diluted serially withsterile tap water, and 0.2 ml aliquots are spreadonto the surface of HV medium (containingcycloheximide [50

µg/ml]) with and without theaddition of trimethoprim (20

µg/ml) and nalid-ixic acid (10

µg/ml). The plates are incubated for2–3 weeks at 30

°C and are then observed with amicroscope fitted with a high-dry long workingdistance objective for the tentative identificationof putative Actinokineospora based on morpho-logical criteria.

Humic Acid-Vitamin (HV) Agar (Hayakawa and Nonomura, 1987)

Humic acid 1.0 gNa2HPO4 0.5 gKCl 1.7 gMgSO4 · 7H 2O 50 mgFeSO4 · 7H 2O 10 mgCaCO3 10 mgAgar 15.0 g

Before it is added to the medium, the humic acid is dis-solved in 10 ml of 0.2 N NaOH. The medium componentsare dissolved in 1 liter of distilled water, and the mediumis adjusted to pH 7.2, then autoclaved, and finallyamended with 1 ml of the filter-sterilized vitamin stocksolution after tempering.


Vitamin Stock SolutionThiamine hydrochloride 0.5 mgRiboflavin 0.5 mgNiacin 0.5 mgPyridoxine hydrochloride 0.5 mgInositol 0.5 mgCalcium pantothenate 0.5 mgp-Aminobenzoic acid 0.5 mgBiotin 0.25 mg

These vitamins (per ml) are dissolved in distilled waterand the solution is filter sterilized.

Strains of the genus Actinosynnema have beenisolated from grass blades placed on yeastextract agar (0.02% yeast extract and 1.5% agarin distilled water). It has been reported that Acti-nosynnema mirum is not inhibited by the pre-sence of nystatin at 100 µg/ml and candicidin at50 µg/ml, so it might be possible to use theseantibiotics to suppress the growth of fungal com-petitors. The plates containing the grass bladesare incubated for 3 weeks at 28°C, after whichthe agar surface may be covered with varioustypes of growth, but small synnemata can beobserved on the grass blade itself using a stereo-scopic microscope. These synnemata can becarefully picked off using a sterile loop andtransferred to fresh media. The technique ofHayakawa et al. (2000) described above for Act-inokineospora has also been reported to permitthe isolation of Actinosynnema strains from soilsand plant material.

Strains of Lechevalieria, Lentzea and Saccha-rothrix have been isolated from soil samples byspreading serial soil dilutions onto the surface ofroutine selective media (such as 1.5% crude agarand 0.4% casein hydrolysate in tap water) usedfor the general isolation of actinomycetes. Theuse of antibiotics to selectively isolate membersof this group has also been reported (Shearer,1987).

Typical actinomycete isolation media, such asAV (arginine-vitamin) agar or starch-casein agar,amended with a combination of penicillin G (5–10 µg/ml) and nalidixic acid (15 µg/ml), havebeen used to isolate Saccharothrix strains selec-tively (Shearer, 1987).

AV Agar (Nonomura and Ohara, 1969)L-Arginine 0.3 gGlucose 1.0 gGlycerol 1.0 gK2HPO4 0.3 gMgSO4 · 7H 2O 0.2 gNaCl 0.3 gFe2(SO4)3 10 mgCuSO4 · 5H 2O 1.0 mgMnSO4 · H 2O 1.0 mgZnSO4 · 7H 2O 1.0 mgAgar 15 g

The pH of the medium is adjusted to 6.4 prior to auto-claving. A vitamin stock solution (1 ml; for composition,see above) is added to each liter of sterilized, temperedmedium just prior to dispensing.

Starch-Casein Agar (Kuster and Williams, 1964)Soluble starch 10.0 gKNO3 2.0 gCasein (vitamin-free) 0.3 gK2HPO4 2.0 gMgSO4 · 7H 2O 50 mgNaCl 2.0 gFeSO4 · 7H 2O 10 mgCaCO3 20 mgAgar 18.0 g

The pH of this medium is adjusted to 7.0 to 7.2 prior toautoclaving.

More recently Lee et al. (2000) reported theuse of tap water agar and oligotrophic medium(M5) for the isolation of Lentzea strains fromserial dilutions of soils from a gold mine inKorea.

Oligotrophic Medium M5 (Lee et al., 2000)Glucose 0.1 gK2HPO4 0.5 gNaH2PO4 0.7 gKNO3 0.1 gMgSO4 · 7H 2O 0.1 gNaCl 0.3 gCaCl2 · H 2O 20 mgFeSO4 · 7H 2O 200 mgCuSO4 · 5H 2O 90 µgMnSO4 · 4H 2O 20 µgZnSO4 · 7H 2O 180 µgCoSO4 · 7H 2O 10 µg(NH4)6Mo7O24 · 4H 2O 5 µgH3BO3 200 µgAgar 15 g

The ingredients are dissolved in one liter of tap water,adjusted to pH 7.2 and autoclaved.

Cultivation

Strains of the family Actinosynnemataceae arerather nonfastidious in their growth require-ments and can be cultivated on a range of stan-dard media typically used for actinomycetes,such as media described by Pridham et al. (1957)or used by the International StreptomycesProject (Shirling and Gottlieb, 1966): inorganicsalts-starch agar, yeast extract-malt extract agar,or glycerol-asparagine agar. Two excellent mediafor the routine cultivation of strains are N-Zamine with soluble starch and glucose medium(ATCC Medium No. 172; Cote et al., 1984) andBennett’s Agar (ATCC Medium No. 174; Coteet al., 1984).


N-Z Amine with Soluble Starch and Glucose (ATCC Medium No. 172)

Glucose 10.0 gSoluble starch 20.0 gYeast extract 5.0 gN-Z amine type A 5.0 gCaCO3 1.0 gAgar 15.0 gDistilled water 1.0 liter

Adjust pH to 7.3 prior to autoclaving. A good substitutefor N-Z amine type A is enzymatic hydrolyzate of casein(cat. no. C0626; Sigma Chemical Company, St. Louis,Missouri, USA).

Bennett’s Medium (ATCC Medium No. 174)Yeast extract 1.0 gBeef extract 1.0 gN-Z amine type A 2.0 gGlucose 10.0 gAgar 15.0 gDistilled water 1.0 liter

Adjust pH to 7.3 prior to autoclaving. A good substitutefor N-Z amine type A is enzymatic hydrolyzate of casein(cat. no. C0626; Sigma Chemical Company, St. Louis,Missouri, USA).

Preservation of Cultures

Members of the family Actinosynnemataceaecan generally be preserved as mycelia/spore sus-pensions or colony plugs prepared from youngplate cultures (7–14 days). Mycelia/spores har-vested from liquid grown cultures or agar plugsare suspended in sterile 20% (vol/vol) aqueousglycerol and stored at –40°C or colder. The via-bility of strains frozen in this manner is quitegood for considerable periods of time, but forlong-term archival storage, these preparationsshould be stored in vapor phase liquid nitrogenor the strains lyophilized using standard proce-dures and stored at 4°C.

Identification Procedures

Morphology, as is typical for actinomycetes ingeneral, plays an important role in the identifica-tion of the taxa classified within the family Acti-nosynnemataceae, particularly those formingdistinctive sporulation structures, such as Actino-synnema and Actinokineospora. The micromor-phological properties of the other genera withinthe family (i.e., Lechevalieria, Lentzea and Sac-charothrix) are quite similar and thus are not asuseful for generic differentiation. The morphol-ogy observed may vary with medium composi-tion, and specific media may be required toencourage the formation of sporulation struc-tures. Media used successfully to culture strainsfor this purpose are HV agar (Hayakawa and

Nonomura, 1987), tyrosine agar (Shirling andGottlieb, 1966), and 1.5% agar in tap water. Themicromorphology of colonies growing on agarplates can be observed using a long workingdistance objective (30–40X) and a standardbinocular microscope. Agar blocks containingsporulating colonies can also be cut from agarplates, fixed with osmium tetroxide vapor, criti-cal point dried, and mounted on a stub for coat-ing and observation in a scanning electronmicroscope.

Chemotaxonomic markers have generallybeen found to be extremely stable and usefulfeatures for the classification of actinomycetegenera and are likewise useful when applied tothe identification of members of the family Acti-nosynnemataceae. All of the genera within thefamily share some common properties, such asmeso-diaminopimelic as the diamino acid in thepeptidoglycan of the cell wall, a whole cell sugarpattern containing galactose, a phospholipidpattern including a significant amount ofphosphatidylethanolamine, and menaquinoneshaving nine isoprenoid units. Other chemotaxo-nomic characteristics used to differentiate thegenera of the family Actinosynnemataceae areshown in Table 1.

The chemotaxonomic properties of strains canbe determined following the detailed proceduresoutlined by Lechevalier and Lechevalier (1970,1980) and the thin-layer chromatography proce-dures suggested by Staneck and Roberts (1974)and Meyertons et al. (1988) for isomers of diami-nopimelic acid and whole-cell sugars, respec-tively. Sugar content within hydrolyzates has alsobeen determined by gas chromatography of aldi-tol acetate derivatives (Englyst and Cummings,1984; Saddler et al., 1991) and using high perfor-mance liquid chromatography (Tamura et al.,1994). Phospholipid content can be determinedby the protocol outlined by Minnikin et al.(1984). Menaquinone analyses can be performedusing the procedures outlined by Collins et al.(1977), Kroppenstedt (1982), and Tamaoka et al.(1983), and fatty acid profiles can be determinedby the method of Kroppenstedt (1985).

Physiological characterization is extremelyimportant in distinguishing between the specieswithin each genus, and it is best evaluated usingthe methods described by Gordon et al. (1974),Kurup and Schmitt (1973), and Goodfellow(1971).

DNA-DNA hybridization still serves as thedefinitive means of determining species related-ness in bacteria. Several methods have been usedto determine DNA relatedness in the familyActinosynnemataceae. DNA relatedness amongspecies of Lechevalieria, Lentzea and Saccharo-thrix (as shown in Table 2) has been determinedfrom Cot0.5 values (Cot is the initial DNA concen-


tration [Co] times time [t] and Cot1/2 = Cot at which1/2 of the DNA has reannealed) for renaturationin 5X standard saline citrate (SSC; 1X SSC is0.15 M NaCl and 0.015 M trisodium citrate) and20% dimethylsulfoxide at Tm – 25°C (e.g., 66°C)using the method of Seidler and Mandel (1971)and Seidler et al. (1975) as modified by Kurtz-man et al. (1980). More recent studies of DNArelatedness among the species of Actinokine-ospora have been performed fluorometricallyusing biotinylated DNA by the method of Ezakiet al. (1989).

Identification of Individual Genera

Actinokineospora

The cultural characteristics of all Actinokine-ospora species are rather similar: yellow toorange colonies with no visible aerial mycelia.Observation of mature cultures grown on anappropriate medium, such as HV agar ortyrosine agar, reveals the presence of short aerialhyphae that fragment into chains of spores (Figs.2 and 3). These arthrospores are rod shaped and

Table 1. Chemotaxonomic characteristics of the family Actinosynnemataceae and related genera.a

aAll genera have meso-diaminopimelic acid (DAP) as the cell wall diamino acid, are of cell wall chemotype III, and containstraight chain, monounsaturated iso and anteiso fatty acids.bPhospholipid pattern type sensu Lechevalier etal., 1977.Abbreviations: DPG, diphosphatidylglycerol; PE, phosphatidylethanolamine; PG, phosphatidylglycerol; PG (v), phosphati-dylglycerol (variable presence); PI, phosphatidylinositol; PIM, phosphatidylinositol mannosides; PME, phosphatidylmethyl-ethanolamine; HO-PE, phosphatidyl-ethanolamine containing hydroxylated fatty acids; and MK-9 and -10, menaquinonescontaining 9 and 10 isoprenoid units, respectively.

Taxon Whole-cell sugar patternPhospholipidb

type PhospholipidsPredominant

menaquinones

Actinoalloteichus Galactose, mannose, andribose

PII PE, DPG, PMI, PG, PIM MK-9(H4)

Actinokineospora Galactose, mannose, andrhamnose

PII PE, HO-PE MK-9(H4)

Actinosynnema Galactose and mannose PII PE, HO-PE, PI, PIM, DPG MK-9(H4) MK-9(H6)Crossiella Galactose, mannose,

rhamnose, and ribosePII PE, DPG, PI, PIM, PME MK-9(H4)

Kutzneria Galactose, trace rhamnose PII PE, HO-PE, PI, DPG MK-9(H4)Lechevalieria Galactose, mannose, and

rhamnosePII PE MK-9(H4)

Lentzea Galactose, mannose, andribose

PII PE, DPG, PG, PI MK-9(H4)

Saccharothrix Galactose, rhamnose, andmannose (trace)

PII, PIV PE, HO-PE, PI, PIM, DPG,PG (v)

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

Streptoalloteichus Galactose, mannose, andribose

PII PE MK-10(H4) MK-10(H6)

Table 2. DNA relatedness among species currently and formerly classified within the genus Saccharothrix.

aNRRL 11239T is the type strain of Saccharothrix australiensis.

Strain

% DNA relatedness to strain

NRRL11239Ta

NRRL15764T

NRRLB-16077T

NRRLB-16107T

NRRLB-16238T

NRRLB-3289T

Saccharothrix espanaensis 25NRRL 15764T

Saccharothrix mutabilis 18 0subsp. mutabilis NRRL B-16077T

Saccharothrix texasensis 21 17 12NRRL B-16107T

Crossiella cryophila 6 9 9 4NRRL B-16238T

Lechevalieria aerocolonigenes 10 2 17 0 11NRRL B-3298T

Lentzea waywayandensis 3 12 4 7 4 44NRRL B-16159


become actively motile when suspended in ster-ile water.

Actinokineospora strains have galactose as thepredominant sugar present in whole cell hydro-lyzates, but smaller quantities of rhamnose,mannose and arabinose are also observed. Phos-phatidylethanolamine and phosphatidylethano-lamine containing 2-hydroxy-fatty acids wereobserved as the diagnostic phospholipids. Themajor menaquinone found to be present wasMK-9(H4).

The described species of Actinokineospora canbe distinguished from each other on the basis ofdifferential physiological properties as shown inTable 3.

Actinosynnema

The cultural characteristics of Actinosynnemastrains differ between the described species andsubspecies, with A. mirum producing a pale yel-

low to yellowish brown substrate mycelium onmost media, with or without white to yellowishwhite sparse aerial mycelia. Actinosynnema pre-tiosum subsp. pretiosum produces yellow to paleorange-yellow substrate mycelia, while A. pretio-sum subsp. auranticum produces distinctivelyyellow to orange substrate mycelia. A sparsewhite to yellowish-white aerial mycelium, whereproduced, is observed for all species. The moststriking diagnostic characteristic of members ofthe genus Actinosynnema is the production ofsynnemata (also called “coremia”), or com-pacted groups of erect hyphae originating fromthe substrate mycelium which give rise to chainsof conidia (Fig. 4). These synnemata can be large(50–200 µm × 200–1,500 µm), and the chains ofconidia on these synnemata give rise to peritric-hously flagellated rod-shaped to ellipsoidalspores which are motile for about 30 minutesafter being introduced into a liquid medium (Fig.5). Synnemata appear to form in greatest num-bers on water agar, tyrosine agar, Bennett’s agar,yeast dextrose agar, and TPC agar (Higgins et al.,1967).

Thin Potato-Carrot (TPC) Agar (Higgins et al., 1967)Potatoes 30.0 gCarrots 2.5 g

Boil potatoes and carrots in 1 liter of distilled water, filter,and make up to 1 liter with distilled water. Add 15 gramsof agar per liter and autoclave.

Actinosynnema strains have galactose as thepredominant whole cell sugar, but also containmannose. The phospholipid content observedincludes phosphatidylethanolamine, phosphati-dylethanolamine containing 2-hydroxy-fattyacids, phosphatidylinositol, phosphatidylinositolmannosides, and diphosphatidylglycerol. Thepredominant menaquinones observed are MK-9(H4) and MK-9(H6).

Fig. 2. Light micrograph of Actinokineospora riparia.Photograph graciously provided by Dr. Kazunori Hatano,Institute for Fermentation, Osaka. It is from the Atlas ofActinomycetes and is used with the permission of the editor,Shinji Miyadoh, on behalf of the Society for Actinomycetes,Japan.

Fig. 3. Scanning electron micrograph of 21-day growth ofActinokineospora riparia.


The described species of Actinosynnema canbe distinguished from each other on the basis ofdifferential physiological properties as shown inTable 4.

Saccharothrix

All strains of Saccharothrix are quite similar inmorphological appearance. Both substrate andaerial hyphae are approximately 0.5–0.7 µm indiameter and fragment into ovoid bacillary unitstypical of nocardioform actinomycetes (Fig. 6).The color of the substrate mycelium of Saccha-rothrix species ranges from yellow to yellowish-brown, and that of the aerial mycelium tends tobe white to yellowish-white or gray, except for S.coeruleofusca and S. longispora, which produceblue aerial mycelia on glycerol-nitrate agar. Sac-charothrix mutabilis is photochromogenic andtends to produce orange-yellow aerial myceliaon many media when cultivated in the light,

Table 3. Differential physiological properties of Actinokineospora species.

Data from Tamura et al. (1995).

Characteristic

ActinokineosporadiospyrosaIFO 15665T

ActinokineosporaglobicatenaIFO 15664T

Actinokineosporainagensis

IFO 15663T

Actinokineosporariparia

IFO 14541T

Actinokineosporaterrae

IFO 15668T

Hydrolysis of:Gelatin + + - - +Starch + + - - +Peptonization of milk + - - - +

Decomposition ofCalcium malate - + - - +

Utilization ofD-Mannose + + - + ±Sucrose + + - + -Reduction of nitrate + + + + -

Growth in NaCl (%) <3 <2 <4 <4 <4Formation of clusters - + - - -

Fig. 4. Synnemata or coremia of Actinosynnema mirumgrowing on tyrosine agar (ISP-7). Photograph graciously pro-vided by Dr. Kazunori Hatano, Institute for Fermentation,Osaka. It is from the Atlas of Actinomycetes and is used withthe permission of the editor, Shinji Miyadoh, on behalf of theSociety for Actinomycetes, Japan.

Fig. 5. Peritrichously flagellated motile zoospore from Acti-nosynnema mirum. Photograph graciously provided by Dr.Kazunori Hatano, Institute for Fermentation, Osaka. It isfrom the Atlas of Actinomycetes and is used with thepermission of the editor, Shinji Miyadoh, on behalf of theSociety for Actinomycetes, Japan.


while the aerial mycelia are white when culturedin the dark. Soluble pigments are produced byvery few species (e.g., S. australiensis and S. syrin-gae). Aerial hyphae often display the “zig-zag”morphology, which is also typical of the genusNocardiopsis (Fig. 7), thus making the differen-tiation between these genera rather difficultbased on morphological criteria. Aerial myce-lium is best observed on minimal media such as1.5% crude agar in tap water or Czapek’s agar(Pridham and Lyons, 1980).

Saccharothrix strains have galactose as thepredominant sugar in their whole cell sugar pat-tern, but also contain rhamnose and a trace ofmannose. The predominant phospholipids arephosphatidylethanolamine, phosphatidylethano-lamine containing 2-hydroxy fatty acids, phos-phatidylinositol and phosphatidylinositolmannoside, and diphosphatidylglycerol. Glu-cosamine-containing phospholipids of unknowncomposition have been observed in Saccharo-thrix mutabilis subsp. mutabilis and Saccharo-thrix espanaensis. The major menaquinoneobserved in all species is MK-9(H4), but MK-10(H4) is observed in Saccharothrix australiensis,Saccharothrix coeruleofusca and Saccharothrixtangerinus, while MK-9(H2) is observed in Sac-charothrix longispora.

Diagnostic nucleotide signature patterns inthe16S rDNA gene sequence, CACG (607-610),GTGG (617–620) and GTC (843–845), areextremely useful for differentiating Saccharo-thrix species from Lentzea and Lechevalieria(Fig. 8). Saccharothrix tangerinus is the most

Table 4. Differential physiological properties of Actinosynnema species.

Data from Hasegawa et al. (1989).

Characteristic

Actinosynnemamirum

IFO 14064T

Actinosynnemapretiosum subsp.

auranticumIFO 15620T

Actinosynnemapretiosum subsp.

pretiosumIFO 15621T

Growth at10∞C + - -38∞C - + +

Utilization ofMelibiose - + +Raffinose - + +

Fragmentation of substrate hyphae in liquid media - + +

Fig. 6. Scanning electron micrograph of vegetative hyphaeof a 28-day culture of Saccharothrix australiensis on ATCCMedium No. 172. Note fragmentation of the mycelium intococcoid elements.

Fig. 7. Scanning electron micrograph of aerial hyphae of a14-day culture of Saccharothrix australiensis on ATCCMedium No. 172. The aerial mycelium has fragmented intoovoid arthrospores and demonstrates typical “zig-zag” mor-phology. Bar = 1 µm.


recently described and validated species of thegenus and has the appropriate chemotaxonomicproperties. In the phylogenetic tree shown in Fig.9, this species appears to be the most phylo-genetically distinct member of the genus, and thesignature patterns within the 16S rDNA gene areslightly different than those of “authentic”Saccharothrix. Additional sequence determina-tion will be necessary to establish whether thepublished sequences are real or in error.

The described species of Saccharothrix can bebest distinguished from each other on the basisof differential physiological properties as shownin Table 5.

Lentzea

Lentzea strains are quite similar in gross mor-phology to members of the genera Saccharothrixand Lechevalieria, producing aerial mycelium,sometimes exhibiting “zig-zag” morphology,and fragmenting into rod-shaped elements.The members of this genus are observed tolack phosphatidylethanolamine containing 2-hydroxy-fatty acids, although they contain phos-phatidylinositol and diphosphatidylglycerol,which differentiates them from Saccharothrix.They also appear to have galactose, mannose andsmall quantities of ribose, but no rhamnose in

Fig. 8. Signatures in 16S rDNA sequence for genera in the family Actinosynnemataceae.

C

Lechevalieria aerocolori genes NRRL. B-3298T AAACTTGGGG. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

. . . . . .

. . . . . . .. . .

. . . . . .

. . . . . ... . .

. . . . . .

. . . . . . . . .

. . . . . .

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. . . . . . . .

. . . . . .

. . . . . . . . .

. . . . . . .

. . . . . . .

. . . . . . ..

. . . . . . ..

. .

. . .

. .. . . .

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. . . . . . .

. . . . . . .

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. . . . . . .

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. . . . . . . . .

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. . . . . . . . . .

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. . . . . . . . . .. . . . . . . . .. . . . . . .. . . . . . .. . . . . . .. . . . . . .. . . . . . . . . . .

. ..

. . . . . . .

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. . . . . . .

.

.

.

.

.

.

601 611 621 841 1001

CTTAACCCCG

T A

T AT A

T A

T

T

T CT

T

T

T

CC

CCCC

CC

CCC

CC

C

CC

CC

C

C

CACCACCACCAC

CACCAC

CAC

CAC

GTGGTGGTGGTG

GTGNGTG

GTGN

GTG

A

A

A

TCCT

A

TA

TT

T

T

CC

CC

CC

C

T A

TT

T

T

ATCG

G

TC

TC

A

AGCCTGCGGT ACGTTCTCCG GAAACCGGTALechevalieria flava NRRL. B-16131T

Lentzea albida IFO 16102T

Lentzea albidocapillata DSM 44073T

Lentzea californiensis NRRL. B-16137T

Lentzea violacea IMSNU 50388T

Lentzea waywayandensis NRRL. B-16159T

Actinosynnema mirum DSM 43827T

Actinosynnema pretiosum subsp. pretiosum NRRL B-16060T

Saccharothrix australiensis NRRL. 11239T

Saccharothrix coeruleofusca NRRL. B-16115T

Saccharothrix espanaensis NRRL. 15764T

Saccharothrix longispora NRRL. B-16116T

Saccharothrix mutabilis subsp. capreolus DSM 40225T

Saccharothrix mutabilis subsp. mutabilis DSM 43853T

Saccharothrix syringae NRRL. B-16468T

Saccharothrix texasensis NRRL. B-16134T

Fig. 9. Phylogenetic dendrogramreconstructed from evolutionary dis-tances (Kimura, 1980) by the neigh-bor-joining method (Saitou and Nei,1987) with stability of the groupingsestimated by bootstrap analysis(Felsenstein, 1985), indicating theposition of species within the familyActinosynnemataceae. Species inblue are within the family Actinosyn-nemataceae, and species in red arerepresentatives of the family Pseud-onocardiaceae. Scale bar represents0.1 nucleotide substitutions per site.

Lentzea violaceaLentzea albidocapillataLentzea californiensis

Lentzea albida‘Lentzea staureosporea’

Lentzea waywayandensisLechevalieria aerocolonigenesLechevalieria flava

Actinosynnema pretiosum subsp.Actinosynnema mirum

Saccharothrix mutabilis subsp. capreolusSaccharothrix mutabilis subsp. mutabilisSaccharothrix espanaensisSaccharothrix syringae

Saccharothrix coeruleofuscaSaccharothrix australiensis

Saccharothrix texasensisSaccharothrix longispora

Actinokineospora ripariaActinokineospora inagensis

Actinokineospora diospyrosaActinokineospora globicatenaKutzneria kofuensis

Crossiella cryophilaStreptoalloteichus hindustanus

Actinoalloteichus cyanogriseusAmycolatopsis orientalis

Saccharomonospora viridisSaccharopolyspora hirsuta

Kibdelosporangium aridumArthrobacter globiformis

8997

74

8974

98

85

82

83

0.10


Tabl

e 5.

Dif

fere

ntia

l pro

pert

ies

of S

acch

arot

hrix

spe

cies

.

Sym

bols

: +,p

osit

ive

in a

ll st

rain

s; -

,neg

ativ

e in

all

stra

ins;

w, w

eak

posi

tive

rea

ctio

n; v

, var

iabl

e re

acti

on; a

nd n

d, n

ot d

eter

min

ed.

Cha

ract

eris

tic

S.au

stra

liens

isN

RR

L11

239T

S.co

erul

eofu

scus

DSM

436

79T

S.co

erul

eovi

olac

eaD

SM 4

3935

T

S.es

pana

ensi

sN

RR

L 1

5764

T

S.lo

ngis

pora

DSM

437

49T

S.m

utab

ilis

subs

p . m

utab

ilis

NR

RL

B-1

6077

T

S.m

utab

ilis

subs

p.ca

preo

lus

DSM

402

25T

S.sy

ring

aeD

SM43

886T

Sct.

tang

erin

usJC

M10

302T

S.te

xase

nsis

NR

RL

B-1

6134

T

Dec

ompo

siti

on o

fA

deni

ne-

--

-+

--

--

-H

ippu

rate

--

-+

-+

++

v+

Hyp

oxan

thin

e-

--

+-

++

-+

+St

arch

-+

--

++

++

++

Tyro

sine

+-

--

++

++

++

Ure

a-

--

-+

--

--

vP

rodu

ctio

n of

Solu

ble

pigm

ents

+-

+-

--

-+

--

Nit

rate

red

ucta

se+

-+

w+

+-

--

+A

ssim

ilati

on o

fC

itra

te-

-+

v+

+-

-+

-L

acta

tew

-nd

++

+-

-nd

+M

alat

e+

-nd

++

++

+nd

+A

cid

from

Ara

bino

se-

++

-+

++

++

+D

extr

in+

++

-+

++

++

+In

osit

ol-

--

--

++

-+

+L

acto

se-

++

-+

+-

++

+M

elib

iose

--

+-

-+

++

++

Raf

finos

e-

+-

--

+-

++

-R

ham

nose

-+

+-

+-

-+

++

Salic

in-

+-

-w

++

ww

+So

rbit

ol+

+-

--

--

-+

-Su

cros

e-

+-

++

+-

++

+X

ylos

e-

++

v+

++

++

+a-

Met

hyl-

d-g

luco

side

-+

w-

-+

--

w+

Gro

wth

in p

rese

nce

of4%

NaC

l+

+-

++

-+

++

-5%

NaC

l-

+-

-+

-+

+w

-G

row

th a

t37

∞C-

++

++

++

+-

+45

∞C+

++

--

++

+-

-


their whole cell sugar profiles, which differenti-ates them from the genera Saccharothrix andLechevalieria.

Lentzea species can also be differentiatedfrom Saccharothrix and Lechevalieria species byvirtue of the genus diagnostic nucleotide signa-ture TCCA (617–620) and GCC (843–845)regions in their 16S rDNA gene sequence, as canbe seen in Fig. 8.

The substrate mycelium is yellow to yellow-brown in Lentzea albidocapillata and Lentzeacaliforniensis, yellowish-orange in Lentzeaalbida, violet in Lentzea violacea, and pale todark yellow in Lentzea waywayandensis. Whiteaerial mycelium is produced by all species. Areddish-brown soluble pigment is produced onsome media by Lentzea violacea, while Lentzeacaliforniensis produces orange soluble pigments,particularly on Czapek’s agar.

The described species of Lentzea can bedistinguished from each other on the basis ofcolonial morphology and the differential physio-logical characteristics shown in Table 6.

Lechevalieria

The species within the genus Lechevalieria havehad a checkered taxonomic history, with bothspecies having spent some time in at least

four different genera, including Saccharothrix.Lechevalieria aerocolonigenes was originallydescribed as Streptomyces aerocolonigenes, whileLechevalieria flava was originally described asActinomadura flava. The members of this genushave morphology quite similar to that of Saccha-rothrix and Lentzea, i.e., branching vegetativemycelium and rudimentary aerial mycelium thatfragments into coccoidal elements. The wholecell sugar pattern consists of galactose and man-nose, with traces of rhamnose. The phospholipidpattern consists of significant quantities of phos-phatidylethanolamine lacking hydroxylated fattyacids. The sequence of the 16rRNA gene con-tains genus-specific diagnostic nucleotidesignature patterns of TT (844–845) and GGT(1107–1109; Fig. 8).

Both species of Lechevalieria exhibit similargross morphology when growing in culture, withsubstrate mycelium of yellow shades and onlytraces of white aerial mycelia, and can be bestdistinguished through the use of the differentialphysiological characteristics shown in Table 7.

Applications

New strains of genera within the family Actino-synnemataceae isolated from nature in the

Table 6. Differential physiological properties of Lentzea species.

Symbols: see footnote in Table 5.Data from Lee et al. (2000).

Characteristic

Lentzeaalbidocapillata

NRRL B-24057TLentzea albida

NRRL B-24073T

Lentzeacaliforniensis

NRRL B-16137TLentzea violaceaIMSNU 50388Tb

LentzeawaywayandensisNRRL B-16159T

Hydrolysis ofUrea w w + + +

Production ofNitrate reductase - - + - +

Assimilation ofAcetate - + + + +Citrate - + + - +Lactate - - - + +Malate + + + - +

Acid fromAdonitol + + - - +Cellobiose + + + - +Inositol + + + - +Maltose + + + - +Mannitol + + + - +Raffinose w - + + +Rhamnose + + + - +Sucrose + + + - +Trehalose + + + - +Xylose + + + - +

Growth at10∞C + - + + +37∞C + + + + w42∞C - + - - -45∞C - + - - -


future should have significant biotechnologicalpotential as sources of novel and useful second-ary metabolites in natural products discoveryresearch. The biosynthetic diversity of membersof this family is illustrated by the list of antibiot-ics produced by Actinokineospora, Actinosyn-nema, Lechevalieria, Lentzea and Saccharothrixspecies (Table 8).

Literature Cited

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Table 7. Differential physiological properties of Lechevalieria species.

Symbols: see footnote in Table 5.

CharacteristicLechevalieria flava

NRRL B-3298TLechevalieria flavaNRRL B-16131T

Growth in presence of4% NaCl + -5% NaCl + -

Utilization ofLactate + -

Acid fromAdonitol + waSalicin + -

Growth at45∞C - +

Table 8. Antibiotics produced by species within the family Actinosynnemataceae.

Species Strain no. Antibiotic Reference

Actinosynnema pretiosum subsp. auranticum ATCC 31309 Ansamitocins Higashide et al., 1977Actinosynnema pretiosum subsp. pretiosum ATCC 31281 Ansamitocins Higashide et al., 1977Lechevalieria aerocolonigenes ATCC 39243 Rebeccamycin Bush et al., 1987Lechevalieria flava INA 2171 Madumycin Gauze et al., 1974Saccharothrix australiensis NRRL 11239 LL-BM782 complex Tresner et al., 1980Saccharothrix espanaensis NRRL 15764 LL-C19004 Kirby et al., 1987Saccharothrix mutabilis subsp. capreolus NRRL 2773 Capreomycin Stark et al., 1967Saccharothrix mutabilis subsp. mutabilis ATCC 31520 Polynitroxin Jain et al., 1982Saccharothrix syringae INA 2240 Nocamycin Gauze et al., 1977Saccharothrix tangerinus JCM 10302 Formamicin Kinoshita et al., 1999


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the prokaryotes || the family actinosynnemataceae

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