changes in the community structure of ammonia-oxidizing bacteria during secondary succession of...

Changes in the community structure of ammonia-oxidizing bacteria during secondary succession ofcalcareous grasslands

George A. Kowalchuk,1* Atie W. Stienstra,1

G. Hans J. Heilig,1 John R. Stephen2 and

Jan W. Woldendorp1

1Netherlands Institute for Ecology, Centre for Terrestrial

Ecology, PO Box 40, 6666 ZG Heteren, The Netherlands.2Center for Environmental Biotechnology,

10515 Research Drive, Unit 300, Knoxville, TN 37923,

USA.

Summary

The community structure of b-subclass Proteobac-

teria ammonia-oxidizing bacteria was determined in

semi-natural chalk grassland soils at different stages

of secondary succession. Both culture-mediated (most

probable number; MPN) and direct nucleic acid-based

approaches targeting genes encoding 16S rRNA and

the AmoA subunit of ammonia monooxygenase were

used. Similar shifts were detected in the composition

of the ammonia oxidizer communities by both culture-

dependent and independent approaches. A predomi-

nance of Nitrosospira sequence cluster 3 in early suc-

cessional ®elds was replaced by Nitrosospira sequence

cluster 4 in late successional ®elds. The rate of this

shift differed between the two areas examined. This

shift occurred in a background of relative stability in

the dominant bacterial populations in the soil, as

determined by domain-level polymerase chain reaction±

denaturing gradient gel electrophoresis (PCR±DGGE).

Molecular analysis of enrichment cultures obtained

using different ammonia concentrations revealed

biases towards Nitrosospira sequence cluster 3 or

Nitrosospira sequence cluster 4 under high- or low-

ammonia conditions respectively. High-ammonia

MPNs suggested a decease in ammonia oxidizer num-

bers with succession, but low-ammonia MPNs and

competitive PCR targeting amoA failed to support

such a trend. Ammonia turnover rate, not speci®c

changes in plant diversity and species composition,

is implicated as the major determinant of ammonia

oxidizer community structure in successional chalk

grassland soils.

Introduction

High levels of nitrogen input negatively affect plant diver-

sity in grassland ecosystems (Tilman, 1987; Bobbink et al.,

1998), and this decrease in plant species diversity has

become a major point of concern for many nature conser-

vationists. Nutrient deprivation management has therefore

been introduced in recent decades to steer secondary

plant succession in abandoned ®elds towards the restora-

tion of species-rich grassland habitats (Bakker, 1989).

These impoverishment regimes usually include cessation

of fertilizer application, annual hay-making with litter

removal and irregular grazing.

The secondary succession that occurs after productive

grasslands have been taken out of fertilization is charac-

terized by an increase in plant diversity and decreases in

plant productivity, net nitrogen mineralization and potential

ammonia oxidation (Willems, 1980; Bobbink and Willems,

1987; Olff and Bakker, 1991; Stienstra et al., 1994; unpub-

lished). At advanced stages of secondary succession,

plant diversity is high, and nitrogen turnover is low. In

chalk grassland pastures, a shift in the plant community

structure occurs, in which the species-poor Arrhenatheretum

elatoris (nutrient-rich grassland) community of fertilized

®elds is replaced by a species-rich Mesobrometum erecti

(nutrient-poor chalk grassland) community at later succes-

sional stages (Willems, 1983). The calcareous region in

the south of The Netherlands contains several locations

where such ®eld conservation measures have been imple-

mented. In this region, the start of impoverishment man-

agement at various times in the past has provided an

opportunity for the study of successional nitrogen trans-

formation processes and the organisms responsible for

these nitrogen conversions.

In many soils, autotrophic ammonia-oxidizing bacteria

of the b-subclass of the Proteobacteria are the main con-

tributors to ammonia oxidation, which is often the rate-

limiting step in the nitri®cation process (Belser, 1979; De

Boer et al., 1990). The physical, chemical and biotic prop-

erties of grassland soils change during the process of

secondary succession (Stienstra et al., 1994). Thus, the

environmental conditions encountered by ammonia-oxidizing

Environmental Microbiology (2000) 2(1), 99±110

Q 2000 Blackwell Science Ltd

Received 24 August, 1999; revised 11 October, 1999; accepted 14October, 1999. *For correspondence. E-mail [email protected];Tel. (�31) 0 2647 91314; Fax (�31) 0 2647 23227.

bacteria vary between ®elds of different location, age with

respect to last fertilization and above-ground vegetation.

For instance, decreasing amounts of available ammonia

may be encountered as ®elds reach later successional

stages as a result of lower levels of nitrogen mineralization,

thus increasing the stress imposed on ammonia- oxidizing

bacteria. Although some nitrifying bacteria have been

shown to survive long periods of dormancy (Jones and Mor-

ita, 1985; Johnstone and Jones, 1988) during times of sub-

strate limitation (Batchelor et al., 1997; Stein et al., 1997;

Stein and Arp, 1998), it is not known to what extent such

adaptation might occur under ®eld conditions. Such adapta-

tion must include successful competition with macrophytes

and heterotrophic microorganisms for available ammonia

(Bodelier et al., 1998). Alternatively, certain lineages of

ammonia-oxidizing bacteria may be particularly well

adapted to the soil conditions of either early or late succes-

sional stages. Hence, the roles of physiological adaptation

and species selection are as yet poorly understood with

respect to ammonia oxidizer communities in successional

chalk grasslands. One of the underlying assumptions in stu-

dies addressing the effect of plant diversity on ecosystem

function is that it in¯uences soil properties and microbial

diversity. The increase in plant diversity encountered during

secondary succession of semi-natural grasslands might

serve to increase soil heterogeneity, and thus the number

of microniches in the soil (Marrs, 1993; Gross et al., 1995).

Studies on the distribution, diversity, competitive inter-

actions and population dynamics of ammonia-oxidizing

bacteria have proved notoriously dif®cult using conven-

tional pure culture methodologies (Schmidt and Belser,

1982). These dif®culties are often associated with the

low growth rate and low biomass yield of ammonia oxidizer

cultures and the unrepresentative nature of pure culture

isolation (Prosser, 1989). However, the monophyletic nat-

ure of the b-subclass of the ammonia-oxidizing bacteria

with regard to 16S rDNA sequences (Head et al., 1993;

Teske et al., 1994) has facilitated the development of molecu-

lar approaches that speci®cally target this group (Hiorns et al.,

1995; Kowalchuk et al., 1997). Additionally, the universal

possession of the gene amoA, encoding the active site of

ammonia monooxygenase (Hyman and Wood, 1985), has

provided a second marker for culture-independent analysis

of this group (Rotthauwe et al., 1997; Stephen et al., 1999).

Phylogenetic analysis of 16S rRNA gene sequences

divides the b-subclass ammonia-oxidizing bacteria into two

genera, Nitrosomonas and Nitrosospira, each containing at

least four phylogenetically supported sequence clusters

(Fig. 1; Stephen et al., 1996; Maidak et al., 1999). The distri-

bution of speci®c sequence clusters has been shown to be

affected by various environmental parameters, including

tillage, marine organic pollution and soil pH (Stephen

et al., 1996, 1998; Bruns et al., 1999; McCaig et al.,

1999; Kowalchuk et al., 2000).

Analysis of amoA sequences reveals similar phylo-

genetic groupings as seen with 16S rDNA sequence infor-

mation at the genus level (Rotthauwe et al., 1997). However,

the level of phylogenetic congruence of these two molecu-

lar markers is not yet known for ®ne taxonomic levels, and

it remains to be seen whether 16S rDNA sequence clus-

ters predict similar clustering of amoA sequences. Never-

theless, a higher degree of sequence variation can be

observed within the amoA marker (Rotthauwe et al.,

1997), allowing for a greater level of discrimination of closely

related ecotypes (Stephen et al., 1999) and more sensitive

determinations of genetic diversity within the b-subclass

ammonia-oxidizing bacteria.

A variety of molecular and culture-based approaches

were implemented to monitor the relative abundance, diver-

sity and community structure of the b-subclass ammonia-

oxidizing bacteria inhabiting chalk grassland soils differing

in the period of time since the last application of fertilizer.

Approaches included b-subclass ammonia oxidizer-speci®c

polymerase chain reaction (PCR) followed by denaturing

gradient gel electrophoresis (DGGE) and hybridization or

sequence analysis (Kowalchuk et al., 1997; Stephen

et al., 1998), 16S rDNA and amoA analysis of enrichment

cultures derived from the highest positive dilutions of most

probable number (MPN) counts, semi-speci®c PCR fol-

lowed by cloning and sequence analysis (Stephen et al.,

1996) and competitive PCR targeting amoA to approxi-

mate gene targets in the soil (Stephen et al., 1999). We

were interested to see how changes in N availability during

vegetational succession might be re¯ected in the structure

and size of the ammonia oxidizer communities in these

pH-neutral soils. The ®elds that were studied included

two locations, the Gerendal and Wrakelberg, where a series

of successional stages are present because ®elds have

been taken out of fertilization at different times in the

past. Two other nearby ®elds that are under the same

regime of impoverishment management, Stokhem and

Kunderberg, were also included in the study. The results

not only provide a description of ammonia oxidizer com-

munities in the ®elds under study, but also allow for compar-

ison of molecular and culture-based analyses. Enrichment

culture experiments, designed to test the importance of

substrate concentration on ammonia oxidizer growth,

were carried out to investigate the role of substrate avail-

ability in the selection of speci®c ammonia oxidizer groups.

Results

PCR-based analyses of directly extracted DNA from

successional grassland soils

All ammonia oxidizer DGGE patterns revealed two to six

bands between 44.5% and 47.0% denaturant (Fig. 2).

Bandsoccurredasdoublets,whichwaspreviouslyattributed

Q 2000 Blackwell Science Ltd, Environmental Microbiology, 2, 99±110

100 G. A. Kowalchuk et al.

to an ambiguity in the reverse primer (Kowalchuk et al.,

1997). DGGE patterns were further interpreted by oligo-

nucleotide hybridization to reveal the community composi-

tion of the b-subclass of the ammonia-oxidizing bacteria in

the different grassland soils (Stephen et al., 1998). All

bands, except for the lowest two bands in the Gerendal

samples, hybridized with a Nitrosospira-speci®c oligonu-

cleotide probe (Nsp436; results not shown). Nitrosospira

bands could be further identi®ed as Nitrosospira clusters

3 and 4 (Fig. 2B and C), and no other ammonia oxidizer-

like sequences were detected. This result is consistent

with previous studies that detected a predominance of

Nitrosospira-like sequences among the b-subclass ammo-

nia oxidizers in other terrestrial environments (Stephen et al.,

1996; 1998; Hastings et al., 1997; Kowalchuk et al., 1997;

1998). DGGE bands were also excised, re-ampli®ed with

the CTO189f-GC/CTO654r primers and sequenced to

con®rm hybridization results. In all cases, DGGE band

sequence analysis revealed the phylogenetic af®liations

predicted by the hybridization results (Fig. 1). The sequence

of the lowest doublet of the Gerendal samples was basal to

the Nitrosospira/Nitrosomonas clade upon phylogenetic

analysis (Fig. 1). These bands also did not hybridize with

an all b-subclass ammonia oxidizer-speci®c probe (b-AO233;

Stephen et al., 1998). It is therefore parsimonious to

assume that these sequences werederived from b-subclass

Proteobacteria that, while containing the CTO primer

sites, do not possess the trait of chemolithotrophic ammo-

nia oxidation. These bands were therefore excluded from

further analyses.

Comparison of relative hybridization intensities using

Nitrosospira sequence cluster-speci®c probes revealed a

pronounced shift in community composition from Nitro-

sospira cluster 3 in young ®elds to Nitrosospira cluster 4

in older ®elds (Fig. 2B±D). Although all ®eld sites showed

the same shift towards Nitrosospira cluster 4 with increased

age of the ®eld, this effect was less pronounced in the

Wrakelberg sites than in the Gerendal sites (Fig. 2D). In


Fig. 1. Neighbour-joining tree of partial 16SrDNA sequences highlighting the b-subclassof the ammonia-oxidizing bacteria.Phylogenetic analysis and tree constructionwas performed as described in the text.Labels beginning with `pHH' correspond toclones recovered from Gerendal soils thatwere partially sequenced during the course ofthis study. For each clone, the plot name isgiven, followed by the DGGE migrationcategory. For example, pHHGIII.3 woulddesignate a clone recovered from theGerendal-III soil that fell within DGGEmigration class 3. Sequences obtained fromexcised environmental DGGE bands arelabelled `bHH' as described in the legend toFig. 2A. Sequences beginning with `pH' or`Env' refer to environmental clones recoveredfrom soil (of the pH indicated) and marinesediment, respectively, and the Enr-ZD5 andA1bM3 sequences were recovered from soiland marine enrichments respectively(Stephen et al., 1996). Only bootstrap values(> 70) relevant to the discussion are shown.

Ammonia-oxidizing populations in chalk grassland soils 101

the Gerendal sites, nearly 100% of the hybridization signal

was from Nitrosospira cluster 3 in the least mature ®elds

(G-0 and G-I; 0 and 5 years without fertilizer respectively),

whereas Nitrosospira cluster 4 accounted for nearly 100%

of the hybridization signal in the most mature ®eld (G-IV;

>50 years without fertilizer). A similar situation is seen in

the Wrakelberg sites, except that Nitrosospira cluster 4

only accounted for < 20% of the total Nitrosospira signal in

the 41-year-old ®eld (W-IV). The Stokhem and Kunderberg

sites appeared to follow the trend seen in the Gerendal site.

Bacterial 16S rDNA ®ngerprints, produced by DGGE

separation of PCR-ampli®ed DNA for all the grassland

soils under study (Fig. 3A), showed no correlation between

the number of DGGE bands detected and the period of

time since the stoppage of fertilization or the diversity of

the standing vegetation. All samples produced between

14 and 22 visible bands. Diversity indices, based upon

the number of bands and the relative band intensities

seen in the bacterial DGGE patterns, showed no trend

with respect to ®eld age (Fig. 3B). All ®elds produced

very similar bacterial DGGE patterns, and dendrogram

analysis of the community ®ngerprints revealed greatest

similarity between ®elds at a single location, rather than

between ®elds of similar age (Fig. 3B).

16S rDNA and amoA analyses of highest positive

dilutions from MPN counts and amoA-targeted

competitive PCR

MPN counts conducted under the high- and low-ammonia

conditions produced contrasting results. The high-ammonia

conditions suggested a decrease in ammonia oxidizer

numbers from between 4.5 and 5.0 log10 gÿ1 dry soil for

the youngest ®elds to 3.4±3.8 log10 gÿ1 dry soil for the

most mature ®elds (Table 1). This trend was not observed

in the Gerendal sites using low-ammonia culture conditions,

with all ®elds giving counts between 4.1 and 4.3 log10 gÿ1

dry soil. The latter trend was in agreement with results

based upon competitive PCR targeting the amoA gene

from soil DNA extracts (Table 1). The calculated number

of amoA targets in the soil detected by the competitive

PCR assay was 1.5 and two orders of magnitude higher

than that observed with the MPN. This increased level of

detection is consistent with previous reports comparing

results of MPN and competitive PCR targeting amoA

(Kowalchuk et al., 1999).

For the Gerendal plots, the screening of highest positive

dilution MPN cultures by 16S rDNA-directed PCR, DGGE

and speci®c hybridization revealed the same shift from

Nitrosospira cluster 3 sequence recovery in young ®elds

to an increased relative recovery of Nitrosospira cluster

4 in later successional ®elds (Fig. 2D). This trend was

observed for both high- and low-ammonia culture conditions,

although high-ammonia conditions consistently detected a


Fig. 2. A. DGGE gel of 16S rDNA fragments from chalk grasslandsoils. Numbers above lanes indicate the number of years that thegiven ®eld has been without fertilizer. Band labels correspond tosequences shown in Fig. 1 and are as follows: A, bHH-4.1; B,bHH-3.1; C, bHH-4.2; D, bHH-3.2; E, bHH4.3; F, bHH4.4; G,bHH-X.1. Labels shown with a prime (8) have a single basedifference from their namesake band, which was introduced by anambiguous position in the CTO654r primer.B. Oligonucleotide hybridization of DGGE gel with a probe speci®cfor Nitrosospira cluster 3 (NspCl3-454) and (C) Nitrosospira cluster4 (NspCl4-446).D. Relative proportion of Nitrosospira clusters 3 and 4 detected inchalk grasslands. Open circles and squares indicate the relativeproportion of Nitrosospira clusters 3 and 4 for samples from theGerendal and Wrakelberg sites, respectively, as judged by speci®chybridization of the DGGE gel shown in (A). The circled `S' and `K'points indicate the hybridization results for the Stokhem andKunderberg sites respectively. Closed and open diamonds denotethe proportion of enrichment cultures obtained from Gerendalsoils in which the given sequence clusters were detected forhigh-ammonia and low-ammonia culture media respectively.


higher proportion ofNitrosospiracluster 3,and low-ammonia

conditions a higher proportion of Nitrosospira cluster 4

(Fig. 2D).

Results similar to those observed for direct hybridiza-

tion analysis were also seen for the other study locations,

where high-ammonia MPN culture conditions revealed the

following distributions of Nitrosospira clusters 3 and 4: W-I,

100:0; W-II, 100:0; W-III, 89:11; W-IV, 80:20; St, 63:37;

Ku, 10:90. As observed using the direct PCR±DGGE

and hybridization approach, the Wrakelberg sites showed

a retarded shift between the two detected sequence clus-

ters, and the Stokhem and Kunderberg sites againappeared

to follow the trend observed in the Gerendal ®elds.

For the assessment of the genetic variability of MPN-

derived enrichment cultures, the amoA gene was targeted

by PCR, and diversity was expressed as a Shannon±

Weaver (S±W) index based upon MspI restriction classes

(Table 1). A total of 11 different MspI restriction classes

could be detected, with six patterns being derived from

Nitrosospira cluster 3-containing enrichments and ®ve

from Nitrosospira cluster 4-containing enrichments (Fig. 4).


Fig. 3. Analysis of bacterial DGGE ®ngerprints of grassland soils.A. DGGE gel of 16S rDNA fragments recovered bybacteria-speci®c PCR using the primers 968f-GC and 1401r(NuÈbel et al., 1996).B. Dendrogram comparison of bacterial DGGE ®ngerprints. Fielddesignations are as in Table 1, and the numbers in parenthesesindicate the Shannon±Weaver indices calculated from the numberof bands and the relative intensity of bands (see text). Onlybootstrap values (> 70) relevant to the discussion are shown.

Tab

le1.

Pote

ntia

lnitr

i®catio

n,

net

Nm

inera

lizatio

n,

com

petit

ive

PC

Rand

MP

Ncultu

rere

sults

from

soil

sam

ple

scolle

cte

din

August

1996.

MP

Ncount

MP

Ncount

Tim

esin

ce

Pote

ntia

lnitr

ificatio

nN

et

Nm

inera

lizatio

nb

5m

Mam

monia

0.3

mM

am

monia

Estim

ate

dam

oA

targ

ets

S±

Win

dex

cof

am

oA

Fie

ldfe

rtili

zer

use

aactiv

ity

(mM

Ngÿ

1dry

soil)

(mM

Ngÿ

1dry

soil)

(log

10

gÿ

1dry

soil)

(log

10

gÿ

1dry

soil)

(log

10

no.

targ

ets

gÿ

1dry

soil)

Msp1

dig

ests

G-0

0197.8

16

4.7

35.1

96

0.5

64.2

60.4

ND

5.8

60.3

1.3

1G

-I5

226.4

56

2.5

62.4

96

0.3

14.5

60.1

4.2

60.4

6.3

60.5

1.3

0G

-II

10

253.4

16

1.7

33.1

86

0.0

94.7

60.1

4.3

60.3

5.9

60.4

1.7

6d

G-I

II24

64.7

76

0.4

60.5

56

0.0

14.2

60.2

4.2

60.3

5.7

60.3

1.5

3G

-IV

>50

7.8

06

1.8

90.1

66

0.0

33.7

60.1

*4.1

60.4

5.6

60.4

0.7

5e

W-I

5607.7

26

2.4

66.3

86

1.3

05.1

60.6

ND

6.0

60.2

1.3

6W

-II

11

288.6

76

2.3

11.4

06

0.1

74.5

60.4

ND

5.7

60.3

1.2

7W

-III

21

156.5

26

1.5

51.4

66

0.0

84.2

60.1

ND

5.8

60.3

1.7

1W

-IV

41

48.8

66

5.8

30.1

16

0.0

43.8

60.2

*N

D5.6

60.5

1.3

7S

t15

133.3

36

0.1

11.6

56

0.0

64.3

60.1

ND

5.8

60.2

1.9

7d

Ku

>50

8.3

36

0.2

80.2

06

0.0

33.4

60.2

ND

5.6

60.5

0.8

2e

Fie

ldlo

catio

ns:

G,

Gere

ndal;

W,

Wra

kelb

erg

;S

t,S

tokhem

;K

u,

Kunderb

erg

.a.

Fie

ldage

isgiv

en

rela

tive

toth

etim

eof

sam

plin

g.

b.

Dete

rmin

ed

aft

er

100

days

of

incubatio

nat

21

8C.

c.

S±

Win

dex,

Shannon

±W

eaver

index.

d.

Sta

tistic

ally

gre

ate

rth

an

(P<

0.0

1)

expecte

d.

e.

Sta

tistic

ally

less

than

(P<

0.0

01)

expecte

d.

Aste

risks

indic

ate

asta

tistic

ally

low

er

num

ber

than

oth

er

sam

ple

s.


With respect to impoverishment management, the diver-

sity of amoA genes within enrichment cultures was lowest

for cultures derived from the oldest successional ®elds,

Gerendal-IV and Kunderberg. Enrichment cultures from

®elds of intermediate age gave the highest amoA diversity,

with the Gerendal-II, Wrakelberg-III and Stokhem ®elds yield-

ing S±W indicessigni®cantly greater than expected,given the

total distribution of amoA restriction pattern classes.

Screening and sequencing of cloned 16S

rDNA fragments

Direct cloning of PCR products using a different primer set,

b-AMOf and b-AMOr (McCaigetal., 1994), also revealed the

presence of Nitrosospira-like 16S rDNA sequences in the

Gerendal I±IV ®elds. Owing to the semi-speci®c nature of

these primers, a variable proportion of recovered clones

was expected to be derived from b-subclass Proteobac-

teria falling outside the Nitrosospira/Nitrosomonas radiation

(Stephen et al., 1996). Recovered clones were therefore

screened using the CTO189f-GC and CTO654r primers

to check for ammonia oxidizer-like inserts (Kowalchuk

et al., 1997). The percentage of ammonia oxidizer-like

clones varied between 37.5% for the G-IV sample and

55.0% for the G-I sample (Table 2). In agreement with

results reported by Kowalchuk et al. (1997), all clones

that gave positive PCR results with the CTO189f-GC and

CTO654r primers contained 16S rDNA sequences showing

af®nity with the b-subclass ammonia oxidizer clade. Ten

clones that were negative for this assay were also chosen

at random for partial sequence determination to check the

®delity of the PCR screening procedure used. All 10

sequences fell outside the Nitrosomonas/Nitrosospira radia-

tion (results not shown). The PCR products produced dur-

ing the screening procedure were subjected to DGGE and

revealed a total of four DGGE mobility classes, with

between one and three migration classes being present

per sample (Table 2). The phylogenetic placement of

recovered ammonia oxidizer-like clones followed the

same trend as seen by DGGE and hybridization. Namely,

clones (migrating to 46% denaturant) recovered from late

successional grassland soils (G-III and G-IV) clustered

with Nitrosospira cluster 4, whereas all clones recovered

from early successional ®elds (G-I and G-II) clustered

with Nitrosospira cluster 3 (Fig. 1). Identical clones were

recovered from multiple ®eld sites in some cases, and

some clone sequences were identical to DGGE band

sequences for the 16S rDNA region analysed (Fig. 1).

Discussion

Recovery of ammonia oxidizer-like 16S rDNA from

grassland soils

Using speci®c PCR primers, it was possible to detect b-

subclass ammonia oxidizer-like 16S rDNA directly in all


Fig. 4. Bar graph showing distributionof Msp1 restriction fragment lengthpolymorphism (RFLP) patterns of amoA genefragments from successional chalk grasslandsoils. Msp1 restriction analysis was ofPCR-ampli®ed amoA gene fragments thatwere recovered by PCR from enrichmentcultures derived from the highest positivedilutions of MPNs using high-ammonia cultureconditions (5 mM NH3/NH4

�).

Table 2. Screening of clones recovered aftersemi-speci®c PCR with bAMO primers. % of clones giving positive No. of clones per DGGE migration category

Field PCR with CTO primer 1 (44.5%) 2 (45.0%) 3 (46.0) 4 (46.5)

G-I 55.0 2 20 0 0G-II 50.0 0 20 0 0G-III 42.5 0 2 14 1G-IV 37.5 0 0 13 2

Forty insert-containing clones, recovered from each of the G-I to G-IV fields, were screened forthe presence of ammonia oxidizer-like 16S rDNA fragments by PCR using CTO189f-GC andCTO654r primers. Products from all positive PCRs were subjected to DGGE analysis, andthe number of clones falling into the different DGGE migration categories is given. The percen-tage of denaturing chemicals is given with respect to the lower band of each doublet producedin the DGGE screening procedure.


the successional ®elds examined. All ammonia oxidizer-like

sequences detected by the various PCR-based approaches

used in this study showed an af®liation with the genus

Nitrosospira. It is therefore unlikely that the failure to

recover Nitrosomonas sequences was a procedural arti-

fact. That the CTO primers can co-amplify some 16S rDNA

sequences basal to the Nitrosospira/Nitrosomonas radia-

tion has been observed previously (Kowalchuk et al.,

1998), illustrating the importance of hybridization or

sequence analysis in the interpretation of DGGE results.

A shift from Nitrosospira cluster 3 dominance in early

successional ®elds to an increase in Nitrosospira cluster

4 in later successional ®elds was detected using two different

primer sets, with both DGGE and cloning-based approaches.

Interestingly, this shift in dominant ammonia oxidizer popula-

tions occurred in a background of general stability in the domi-

nant bacterial populations, as determined by domain-level

PCR±DGGE community pro®ling. These analyses only

reveal trends in the most numerically dominant bacterial

ribotypes (Muyzer et al., 1993; Stephen et al., 1999).

Although it is dif®cult to ascertain the ®delity with which

such molecular strategies represent actual community

structure, the agreement between the different approaches

used suggests that the observed community shifts are

real. Although competitive PCR experiments using mixed

templates of ammonia oxidizer-like clones did not reveal

any evidence of preferential PCR ampli®cation (Kowalchuk

et al., 1997), this possibility cannot be fully discounted,

and caution must be exercised when extrapolating relative

PCR product abundance to cell numbers in the environ-

ment (Suzuki and Giovannoni, 1996). The sequencing of

only one clone of each DGGE `type' per sample in the clon-

ing experiment may have missed some variation within the

created clone libraries, given that previous studies have

shown DGGE band mobility to be an unreliable indicator

of nucleotide sequence identity (Kowalchuk et al., 1997).

However, there is no reason to believe that such minor

variability would affect the phylogenetic tree topology

(Fig. 1) or the sequence cluster placement of recovered

clones. No sequences showed evidence of a chimeric

nature, although such detection would be dif®cult consid-

ering the high level of 16S rDNA sequence identity within

the b-subclass ammonia oxidizers (Larsen, 1997).

Comparison of direct extraction and enrichment

culture results

The 16S rDNA analysis of enrichment cultures, derived

from the highest positive dilutions of MPN counts, showed

the same shift from Nitrosospira cluster 3 in early succes-

sional ®elds to Nitrosospira cluster 4 in later successional

stages. The relative recovery of these two sequence clus-

ters was dependent on ammonia concentration in the

medium. High-ammonia conditions enhanced recovery

of Nitrosospira cluster 3 strains, whereas low-ammonia

conditions enhanced Nitrosospira cluster 4 recovery.

The media and conditions currently used for the isolation

and enumeration of ammonia-oxidizing bacteria are

thought to favour certain strains (Belser and Schmidt,

1978) and may explain the over-representation of Nitroso-

monas cluster 7 (Nitrosomonas europaea-like strains) and

Nitrosospira cluster 3 (Nitrosospira briensis-like strains) in

culture collections (Schmidt and Belser, 1982). The MPN

data presented here suggest that ammonia availability

may be a major factor in the observed shift of Nitrosospira

cluster 3 to Nitrosospira cluster 4 with succession. The fact

that both Nitrosospira clusters 3 and 4 were detected in the

highest positive dilutions of MPN counts demonstrates that

both groups are amenable to the culture conditions used.

Speci®c enrichment for these ammonia oxidizer groups

may facilitate experiments designed to test the hypoth-

eses generated here.

Ammonia oxidizer community differences between

®elds of different location and successional stage

Ammonia availability is a critical regulatory factor in nitri-

®cation activity and ammonia oxidizer growth (Prosser,

1989), and potential nitri®cation activity has been shown

to correlate well with the decreasing amount of net N

mineralization in semi-natural chalk grasslands (Table 1;

Stienstra et al., 1994). This study suggests that nitrogen

turnover may be a key factor affecting the structure of

the ammonia oxidizer community in these soils. Nitrosospira

cluster 3 cultured strains are known to grow well in high-

ammonia culture media (see above), and this cluster

appears to dominate the ammonia oxidizer community in

early successional ®elds, where N mineralization levels

are high. Corresponding with the decrease in the amount

of ammonia released via mineralization, the relative abun-

dance Nitrosospira cluster 4 increased in later succes-

sional soils. This result agrees with the suggested

dominance of Nitrosospira cluster 4 in never-tilled soils

of neutral pH (Bruns et al., 1999).

Thus, although ammonia-oxidizing bacteria have the

ability to adapt to changes inammoniaavailability (Laanbroek

and Woldendorp, 1995), prolonged exposure to lowered

ammonia availability may select for strains af®liated with

Nitrosospira cluster 4. That such strains might outcompete

other ammonia-oxidizing bacteria seems reasonable, con-

sidering the observed differences between cultured strains

with respect to metabolic activity, Km for ammonia, Vmax

for ammonia oxidation and recovery after periods of star-

vation (Belser and Schmidt, 1978; Batchelor et al., 1997;

Stein and Arp, 1998). It may be hypothesized that strains

within Nitrosospira clusters 3 and 4 exhibit signi®cant dif-

ferences in these properties relevant to growth and survival

in successional soils. The combination of culture techniques



and molecular monitoring strategies, as used in this study,

should prove helpful in the further isolation and physiologi-

cal characterization of these bacteria.

Plant diversity and species composition also change

during the secondary succession of chalk grasslands,

and alterations in the plant community may have both

direct and indirect effects on ammonia oxidizer commu-

nities. In addition to changes in competition for ammonia

and the rate of plant decomposition, the production of allelo-

chemicals may in¯uence nitri®cation and the structure of

nitri®er communities. Plants typical of late successional

stages may produce increased amounts of allelochemicals

(Lodhi and Killingbeck, 1980; Rice, 1984), avariety of tannins,

polyphenols and alkaloids. Additional experiments, perhaps

using model systems and the enrichment and monitoring

strategies employed in this study, are necessary to assess

the importance of allelochemicals to microbial diversity.

Both the Gerendal and Wrakelberg sites showed the

same shift towards Nitrosospira cluster 4 in later succes-

sional ®elds, although this shift with respect to ®eld age

was less pronounced for the latter site. The latter location

was used until the mid-1950s for crop production, with

heavy reliance on manure fertilizers, whereas the Gerendal

location was less intensively farmed. It may be that the his-

tory of these grasslands before deprivation management

in¯uences the rate of the observed shift in ammonia oxidizer

community structure. The results obtained from the

Stokhem ®eld, however, do not support this conclusion.

The Stokhem ®eld was also a productive arable ®eld

before impoverishment management, but changes in the

ammonia oxidizercommunity resembled the trend observed

in the Gerendal soils. Thus, although differences in soil treat-

ment (disturbances and fertilization) before impoverishment

management may be important, more recent biotic and/or

abiotic factors may also have in¯uenced the structure of

the ammonia oxidizer communities in these ®elds.

The data obtained in this study from bacterial and

ammonia oxidizer DGGE pro®les do not support the notion

that increased plant diversity necessarily stimulates

microbial diversity in the soil. The high-diversity grassland

situation may represent a more narrowly selective below-

ground environment than that of less diverse early succes-

sional ®elds. Increases in above-ground diversity do not

necessarily result in increases in biodiversity in the below-

ground compartment. Such uncoupling means that caution

must be taken not to examine microbial biodiversity

through a `plant window'.

Experimental procedures

Sample sites and sampling procedure

The chalk grasslands examined, located in the Limburg regionin the south of The Netherlands (508 518 N; 58 548 E), were

purchased by the government for conversion into species-rich semi-natural habitats. These ®elds are mown once peryear, and above-ground biomass is removed. The Gerendaland Wrakelberg sites include ®elds that have been takenout of fertilization for different periods of time and are at differ-ent stages of secondary plant succession (Table 1). The Geren-dal ®elds were previously used as highly productive pastures,whereas the Wrakelberg plots were originally used as arable®elds (> 50 years ago), which were converted to productivepastures before the application of fertilizer was stopped. Atthe outset of impoverishment management, the vegetationconsisted of an Arrhenatheretum elatoris community, typicalof fertilized ®elds. Dominant plant species included Heracleumsphondylium, Festuca pratensis, Knautia arvensis and Daucuscarota. Secondary succession leads to the development of aMesobrometum erecti community, typical of chalk grasslandsof low productivity. This species-rich plant community is char-acterized by Brachypodium pinnatum, Koeleria macrantha,Koeleria pyramidata, Avenula pratensis, Scabiosa columbaria,Sanquisorba minor, Gentianella germanica, Thymus pule-gioides and Ophrys apifera (Willems, 1980; 1982; 1983; Willemsand Bobbink, 1990). A strong inverse correlation exists betweenplant diversity and ®eld productivity (Willems, 1980). TheStokhem and Kunderberg plots are subjected to the samemanagement strategies and have not been fertilized for 15and at least 50 years respectively. Net nitrogen mineraliza-tion, potential nitri®cation activity and culturable nitrifyingbacterial numbers (5 mM ammonia) all decreased with theperiod of time that a ®eld had been without fertilization(Table 1; A. W. Stienstra et al., unpublished results).

To facilitate sample collection, a 200 m2 area was selectedin the centre of each ®eld. A 10 m ´ 20 m grid was used toselect 15 equidistant (5 m apart) points within each plot. Threesoil cores (30 mm diameter, 100 mm depth) were collected ateach sampling point. Each group of three cores was sievedand analysed for moisture content. The 15 samples per sitewere pooled using an equal amount of each three-core sub-sample, based upon dry weight. All samples were taken inAugust 1996. Samples were stored at 48C for no more than5 days before MPN enumeration and subsequently stored atÿ808C. Soil analyses (Table 1) were conducted as describedby Stienstra et al. (1994).

16S rDNA analyses of DNA extracted directly

from soil

DNA was extracted directly from soil by mechanical disruption(Stephen et al., 1996), followed by composite agarose gel-mediated DNA puri®cation (Kowalchuk et al., 1997). PCR,using DNA extracted from the soil, was performed with theCTO189f-GC and CTO654r primers that are described speci-®cally to target 16S rDNA b-subclass ammonia oxidizers, withthe addition of a 40 bp GC clamp to facilitate DGGE analyses(Shef®eld et al., 1989; Kowalchuk et al., 1997). Reactionsused 50 ng of template DNA in a total volume of 50 ml usingthe PCR conditions described by Kowalchuk et al. (1997).PCR product concentrations were estimated by comparisonwith known standards after agarose gel electrophoresis(1.5% agarose, 0.5 ´ TBE; 1 ´ TBE� 90 mM Tris borate, 2 mMEDTA, pH 8.3) and ethidium bromide staining. Approximately1 mg of PCR product per sample was subjected to DGGE,



according to the protocol of Muyzer et al. (1998) as adaptedfor the study of ammonia-oxidizing bacteria [Kowalchuk et al.,1997; 8% polyacrylamide, 1.5 mm thickness, 0.5 ´ TAE;37:1 acrylamide:bisacrylamide; 38±50% denaturant (100%denaturant� 7 M urea� 40% v/v formamide); 200 mm ´200 mm]. DNA was transferred to nylon membranes (Muyzeret al., 1998), and hybridization conditions as well as quanti®-cation of hybridization signals were according to Stephen et al.(1998). Hybridization intensities were calibrated using thecontrol lanes, which contained known amounts of DNA fromeach of the ammonia oxidizer sequence clusters targeted inthe probing. The sum of sequence cluster-speci®c hybridiza-tions could explain in excess of between 95% and 103% ofthe total b-subclass ammonia oxidizer hybridization signal inall cases. All values were transformed to 100% for graphicalpresentation (Fig. 2D).

To con®rm the results obtained using the CTO189f-GC andCTO654r primers, a cloning-based analysis was also con-ducted with a different primer set. This second primer set,designated b-AMOf and b-AMOr (McCaig et al., 1994), hasbeen shown previously to provide a semi-speci®c ampli®ca-tion of 16Sr DNA from environmental samples. This was per-formed only for Gerendal samples I±IV. 16S rDNA fragmentswere recovered by PCR and cloned in E. coli as described byStephen et al. (1996). Potentially positive colonies (white)were subjected to colony PCR, using the vector-encoded SP6and T7 primers. Forty colonies, whose PCR products indicatedinsertsof thepropersize (1.1 kb),werescreened further byPCRfor the presence of b-subclass ammonia oxidizer-like 16S rDNAinserts using the CTO189f-GC and CTO654r primers (Kowal-chuk et al., 1997). PCR products, where recovered, were sub-jected to DGGE as above. One clone of each unique DGGEpattern was selected for double-stranded sequence analysis.

Sequencing of clones and DGGE bands

For cloned material, double-stranded cycle sequencing wasperformed using the CTO189f (no GC clamp) and CTO654rprimers and 1 mg of plasmid DNA per reaction. Plasmid isola-tions were performed using the Qiaquick mini-plasmid prepara-tion kit according to the manufacturer's speci®cations (Qiagen).Sequencing reactions were performed using an ABI PRISMDye Terminator Cycle Sequence ready reaction kit accordingto the manufacturer's instructions (Perkin-Elmer), and theproducts were analysed using an Applied Biosystemsautomatic sequencer (model 373 with a `Stretch' adapter;Department of Biotechnology, Wageningen, The Nether-lands). Sequences were assembled using the SEQUENCE NAVIGA-

TOR program (version 1.0, release 3.0.1; Applied Biosystems).Extraction of DNA from DGGE gels and subsequent re-

ampli®cation were performed as described by Kowalchuket al. (1997). Excess primers and unincorporated nucleotideswere removed from the PCR products with the QiaQuick PCRpuri®cation kit (Qiagen) before direct double-stranded sequenc-ing, using 1 mg of puri®ed PCR product per reaction, asdescribed above.

Phylogenetic analysis

DNA sequence manipulations were performed using the SEQAPP

program, version 1.9a169 (Gilbert, 1993), and phylogenetic

analyses were implemented through PHYLIP 5.57 (Felsenstein,1993). Distance matrix analyses were performed according tothe method of Jukes and Cantor (1969) with a masking func-tion to exclude ambiguous data. Phylogenetic tree constructionwas by neighbour joining (Saitou and Nei, 1987). Phylogeneticanalysis was performed for the 287 nucleotide positionsthat could be unambiguously aligned for all sequencesused. Bootstrapping was conducted with 100 replicatesusing the program SEQBOOT (Felsenstein, 1993) (Fig. 1).

Competitive PCR targeting the amoA gene

The estimation of amoA gene targets was performed accord-ing to Stephen et al. (1999), using the same DNA isolationsused for the 16S rDNA analysis. As suggested by theseauthors, competitive PCR used the amoA deletion construct,p428-NAB_8_23, and the size difference conversion factor1.11. PCR signals were quanti®ed from digitized gel images(The Imager System; Ampligene) obtained after ethidium bro-mide staining, using the IMAGEMASTER ELITE software package,version 3.0 (Amersham-Pharmacia Biotech).

Enrichment conditions and molecular analyses of

DNA extracted from enrichment cultures

Ammonia-oxidizing bacteria were enumerated using the MPNmethod of Alexander (1982). The experimental procedure hasbeen described by Stienstra et al. (1994), except that 132 mg(high-ammonia conditions) or 15 mg (low-ammonia conditions)of (NH4)2SO4 was added to the sterile incubation medium.Incubation was at 278C for 12 weeks in the dark without shak-ing. Nitri®cation activity was determined by the presence ofnitrate and/or nitrite (> 0.1 mM NO3

ÿ�NO2

ÿ), as detectedusing a Technicon Traacs 800 autoanalyser (TechniconInstruments). Of the highest MPN dilution tubes showing posi-tive nitri®cation for the high-ammonia conditions, 0.5 ml wasused to inoculate 9.5 ml of the same medium, but with theaddition of 0.04% bromthymol blue as pH indicator. Enrich-ment cultures were grown in the dark at 278C for 60 days with-out shaking. Positive cultures (< 95%) were centrifuged at14 000 g for 15 min. All but 0.4 ml of the supernatant was de-canted, and cell pellets were resuspended in the remainingmedium and transferred to 0.5 ml microcentrifuge tubes. Con-centrated cell suspensions were incubated for 10 min at1008C, frozen for 30 min at ÿ208C and heated once more tolyse cells. Lysates were stored at ÿ208C until further use.16S rDNA-directed PCR analyses used 1 ml of lysate astemplate source, and DGGE and hybridization were used tocharacterize ammonia oxidizers within enrichment culturesto the sequence cluster level, as described above. SelectedPCR products were chosen for partial sequence analysis,and sequence results supported the cluster placements sug-gested by the hybridization results in all cases (not shown). Allbut two of the enrichment cultures produced DGGE patternscontaining a single doublet, whose hybridization suggestedthe presence of only a single ammonia oxidizer sequencecluster. In the remaining two cultures, two separate doubletscould be detected by the DGGE and hybridization, andeach detected ribotype was treated separately in the analysisof sequence cluster distribution (Fig. 2D).



The amoA genotypes of MPN-derived enrichment culturesfrom the high-ammonia enrichment conditions (5 mM NH3/NH4

�) were assessed by MspI restriction analysis of PCR-ampli®ed amoA gene fragments. PCR ampli®cation was carriedout according to the protocol of (Stephen et al., 1999) andused 1 ml of lysate as template. Approximately 0.5 mg ofPCR product was digested with MspI as speci®ed by the man-ufacturer (Boehringer Mannheim), examined on 2.0% agarose,0.5 ´ TBE gels, and DNA was visualized after standard ethidiumbromide staining. A Shannon±Weaver index was determinedfor the enrichment cultures derived from each ®eld using thenumber of amoA classes detected per sample and their rela-tive frequency as variable parameters. The statistical signi®-cance of differences in the diversity of recovered amoAgene fragments was based on the comparison of the actualdiversity index with the range of values obtained in 1000 randompermutations of the total data set (Potvin and Roff, 1993).

DGGE analysis of bacterial 16S rDNA

Ampli®cation of bacterial 16S rDNA fragments was performedusing the 968f-GC and 1401r primers (NuÈbel et al., 1996).DGGE (6% acrylamide gel; 45±65% denaturant) was run at80 V for 16 h at 608C. DGGE banding patterns were comparedwith respect to Rf values, which were scaled between 0 and 1,within the IMAGEMASTER ELITE software package (version 3.01).Total pro®le similarity was calculated as a Pearson's coef®-cient, thus taking both band presence and band intensityinto account, and dendrogram construction was by neighbourjoining performed with the IMAGEMASTER ELITE DATABASE program(version 2.0). Bootstrap values were based upon 100 repli-cates. DGGE band intensity for diversity index calculationswas determined by integration of the area under a givenpeak after background subtraction, which used a rolling circlealgorithm (r�30 pixels). Relative band evenness was calcu-lated by dividing the intensity of each band by the intensityof the summed bands for a given lane after background sub-traction, and band diversity was based upon band numberand relative intensity of bands expressed as a Shannon±Weaver index, which was calculated manually.

Nucleotide sequence accession numbers

Novel partial 16S rDNA sequences have been deposited inthe EMBL databank with the accession numbers AJ131802±AJ131810.

Acknowledgements

We thank David Wardle, Ken Giller, Peter van Tienderen,Wim van der Putten, Wietse de Boer and Hans van Veenfor helpful support and comments. This work was supportedby the Netherlands Royal Academy of Arts and Sciences, agrant from the Netherlands Science Foundation (NOW) anda COBASE grant from the US National Research Council.

References

Alexander, M. (1982) Most probable number method formicrobial populations. In Methods of Soil Analysis, 2nd

edn. Page, A.L., Miller, R.H., and Keeney, D.R. (eds).Madison, WI: American Society for Agronomy, pp. 815±820.

Bakker, J.P. (1989) Geobotany 14: Nature Management byGrazing and Cutting. Dordrecht, The Netherlands: Kluwer.

Batchelor, S.E., Cooper, M., Chabra, S.R., Glover, L.A.,Stewart, G.S.A.B., Williams, P., et al. (1997) Cell density-regulated recovery of starved bio®lm populations ofammonium-oxidising bacteria. Appl Environ Microbiol 63:2281±2286.

Belser, L.W. (1979) Population ecology of nitrifying bacteria.Annu Rev Microbiol 33: 309±333.

Belser, L.W., and Schmidt, E.L. (1978) Diversity in theammonium-oxidizing nitri®er population of a soil. ApplEnviron Microbiol 36: 584±588.

Bobbink, R., Hornung, M., and Roelofs, J.G.M. (1998) Theeffects of air-borne nitrogen pollutants on species diversityin natural and semi-natural European vegetation. J Ecol86: 717±738.

Bobbink, R., and Willems, J.H. (1987) Increasing dominanceof Brachypodium pinnatum (L.) Beauv. in chalk grasslands:a threat to a species-rich ecosystem. Biol Conserv 40:301±314.

Bodelier, P.L.E., Duyts, H., Blom, C.W.P.M., and Laanbroek,H.J. (1998) Interactions between nitrifying and denitrifyingbacteria in gnotobiotic microcosms planted with the emer-gent macrophyte Glyceria maxima. FEMS Microbiol Ecol25: 63±78.

Bruns, M.A., Stephen, J.R., Kowalchuk, G.A., Prosser, J.I.,and Paul, E.A. (1999) Comparative diversity of ammoniaoxidiser 16S rRNA gene sequences in native, tilled, andsuccessional soils. Appl Environ Microbiol 65: 2994±3000.

De Boer, W., Klein Gunnewiek, P.J.A., and Troelstra, S.R.(1990) Nitri®cation in Dutch heathland soils. II. Character-istics of nitrate production. Plant Soil 127: 193±200.

Felsenstein, J. (1993) PHYLIP: Phylogeny Inference Package.Seattle.

Gilbert, D.G. (1993) SEQAPP Sequence Alignment Editor.Bloomington, IN. Available from author by ftp (ftp.bio.indiana.edu).

Gross, K.L., Pregitzer, K.S., and Burton, A.J. (1995) Spatialvariation in nitrogen availability in three successionalplant communities. J Ecol 83: 357±367.

Hastings, R.C., Ceccherini, M.T., Miclaus, N., Saunders, J.R.,Bazzicalupo, M., and McCarthy, A.J. (1997) Direct molecu-lar biological analysis of ammonia oxidising bacteria popu-lations in cultivated soil plots treated with swine manure.FEMS Microbiol Ecol 23: 45±54.

Head, I.M., Hiorns, W.D., Embley, T.M., McCarthy, A.J., andSaunders, J.R. (1993) The phylogeny of autotrophicammonia-oxidising bacteria as determined by analysis of16S ribosomal RNA gene sequences. J Gen Microbiol139: 1147±1153.

Hiorns, W.D., Hastings, R.C., Head, I.M., McCarthy, A.J.,Saunders, J.R., Pickup, R.W., et al. (1995) Ampli®cationof 16S ribosomal RNA genes of autotrophic ammonia-oxidising bacteria. Microbiology 141: 2793±2800.

Hyman, R.K., and Wood, P.M. (1985) Suicidal inactivationand labeling of ammonia mono-oxygenase by acetylene.Biochem J 27: 719±725.

Johnstone, B.H., and Jones, R.D. (1988) Physiologicaleffects or long-term energy-source deprivation on the



survival of a marine chemolithotrophic ammonium-oxidisingbacterium. Mar Ecol Prog Ser 49: 295±303.

Jones, R.D., and Morita, R.Y. (1985) Survival of a marineammonium oxidiser under energy-source deprivation.Mar Ecol Prog Ser 26: 175±179.

Jukes, T.H., and Cantor, C.R. (1969) Evolution of proteinmolecules. In Mammalian Protein Metabolism. Munro,H.N. (ed.). New York: Academic Press, pp. 21±132.

Kowalchuk, G.A., Stephen, J.R., De Boer, W., Prosser, J.I.,Embley, T.M., and Woldendorp, J.W. (1997) Analysis ofb-Proteobacteria ammonia-oxidising bacteria in coastalsand dunes using denaturing gradient gel electrophoresisand sequencing of PCR ampli®ed 16S rDNA fragments.Appl Environ Microbiol 63: 1489±1497.

Kowalchuk,G.A., Bodelier, P.L.E., Heilig, G.H.J., Stephen, J.R.,and Laanbroek,H.J. (1998) Community analysisofammonia-oxidising bacteria, in relation to oxygen-availability in soilsand root-oxygenated sediments, using PCR, DGGE andoligonucleotide probe hybridisation. FEMS Microbiol Ecol27: 339±350.

Kowalchuk, G.A., Naoumenko, Z.S., Derikx, P.J.L., Felske, A.,Stephen, J.R., and Arkhipchenko, I.A. (1999) Molecularanalysis of ammonia-oxidising bacteria of the b-subdivisionof the class Proteobacteria in compost and compostingmaterials. Appl Environ Microbiol 65: 396±405.

Kowalchuk, G.A., Stienstra, A.W., Heilig, G.H.J., Stephen, J.R.,and Woldendorp, J.W. (2000) Composition of communitiesof ammonium-oxidising bacteria in wet, slightly acid grass-land soils using 16S rDNA-analysis. FEMS Microbiol Ecol(in press).

Laanbroek, H.J., and Woldendorp, J.W. (1995) Activity ofchemolithotrophic nitrifying bacteria under stress in naturalsoils. Adv Microb Ecol 1: 275±304.

Larsen, N. (1997) Check_Chimera program of the RDP(Ribosomal Database Project: http: //www.cme.msu.edu/rdp).

Lodhi, M.A.K., and Killingbeck, K.T. (1980) Allelopathic inhibi-tion of nitri®cation and nitrifying bacteria in a ponderosapine (Pinus ponderosa Dougl.) community. Am J Bot 67:1423±1429.

McCaig, A.E., Embley, T.M., and Prosser, J.I. (1994) Molecularanalysis of enrichment cultures of marine ammonia oxidi-sers. FEMS Microbiol Lett 120: 363±368.

McCaig, A.E., Phillips, C.J., Stephen, J.R., Kowalchuk, G.A.,Harvey, M., Herbert, R.A., et al. (1999) Nitrogen cyclingand community structure of b-subgroup ammonia oxidisingbacteria within polluted, marine ®sh-farm sediments. ApplEnviron Microbiol 65: 213±220.

Maidak, B.L., Cole, J.R., Parker, Jr, C.T., Garrity, G.M.,Larsen, N., Li, B., et al. (1999) A new version of theRDP. Nucleic Acids Res 27: 171±173.

Marrs, R.H. (1993) Soil fertility and nature conservation inEurope: theoretical considerations and practical manage-ment solutions. In Advances in Ecological Research, Vol.24. Begon, M., and Fitter, A.H. (eds). London: AcademicPress, pp. 242±292.

Muyzer, G., De Waal, E.C., and Uitterlinden, A.G. (1993) Pro-®ling complex microbial populations by denaturing gradient gelelectrophoresis analysis of polymerase chain reaction-ampli®ed genes coding for 16S rRNA. Appl Environ Micro-biol 59: 695±700.

Muyzer, G., Hottentrager, S., Teske, A., and Wawer, C.(1998) Denaturing gradient gel electrophoresis (DGGE)in microbial ecology. In Molecular Microbial Ecology Manual.Akkermans, A.D.L., van Elsas, J.D., and de Bruijn, F.J.(eds). Dordrecht: Kluwer, pp. 1±27.

NuÈbel, U., Engelen, B., Felske, A., Snaidr, J., Weishuber, A.,Amann, R.I., et al. (1996) Sequence heterogeneities ofgenes encoding 16S rRNAs in Paenibacillus polymyxadetected by temperature gradient gel electrophoresis.J Bacteriol 178: 5636±5643.

Olff, H., and Bakker, J.P. (1991) Long-term dynamics ofstanding crop and species composition after cessation offertiliser application to mown grassland. J Appl Ecol 28:1040±1052.

Potvin, C., and Roff, D.A. (1993) Distribution±free and robuststatistical methods ± viable alternatives to parametric statis-tics. Ecology 74: 1617±1628.

Prosser, J.I. (1989) Autotrophic nitri®cation in bacteria. AdvMicrobiol Physiol 30: 125±181.

Rice, E.L. (1984) Natural ecosystems: Allelopathy and old-®eld or urban succession. In Allelopathy, 2nd edn. Rice,E.L. (ed.). Series Physiological Ecology. New York:Academic Press, pp. 206±231.

Rotthauwe, J.-H., Witzel, K.-P., and Liesack, W. (1997) Theammonia monooxygenase structural gene amoA as a func-tional marker: molecular ®ne-scale analysis of naturalammonia-oxidising populations. Appl Environ Microbiol63: 4704±4712.

Saitou, N., and Nei, M. (1987) The neighbor-joining method:a new method for reconstructing phylogenetic trees. MolBiol Evol 4: 406±425.

Schmidt, E.L., and Belser, L.W. (1982) Nitrifying bacteria. InMethods of Soil Analysis, 2nd edn. Page, A.L., Miller, R.H.,and Keeney, D.R. (eds). Agronomy 9, Part 2. Madison, WI:American Society of Agronomy.

Shef®eld, V.C., Cox, D.R., Lerman, L.S., and Myers, R.M.(1989) Attachment of a 40-base pair G�C-rich sequence(GC-clamp) to genomic DNA fragments by the polymerasechain reaction results in improved detection of single-basechanges. Proc Natl Acad Sci USA 86: 232±236.

Stein, L.Y., and Arp, D.J. (1998) Ammonium limitation resultsin the loss of ammonium-oxidising activity in Nitrosomonaseuropaea. Appl Environ Microbiol 64: 1514±1521.

Stein, L.Y., Arp, D.J., and Hyman, R. (1997) Regulation ofthe synthesis and activity of ammonium monooxygenasein Nitrosomonas europaea by altering pH to affect NH3

availability. Appl Environ Microbiol 63: 4588±4592.Stephen, J.R., McCaig, A.E., Smith, Z., Prosser, J.I., and

Embley, T.M. (1996) Molecular diversity of soil and marine16S rDNA sequences related to b-subgroup ammonia-oxidising bacteria. Appl Environ Microbiol 62: 4147±4154.

Stephen, J.R., Kowalchuk, G.A., Bruns, M.A., McCaig, A.E.,Phillips, C.J., Embley, T.M., et al. (1998) Analysis of b-subgroup ammonia oxidiser populations in soils by DGGEanalysis and hierarchical phylogenetic probing. Appl EnvironMicrobiol 64: 2958±2965.

Stephen, J.R., Chang, Y.-J., Macnaughton, S.J., Kowalchuk,G.A., Leung, K.T., Flemming, C.A., et al. (1999) Effect oftoxic metals on the indigenous b-subgroup ammonia oxidisercommunity structure and protection by inoculated metalresistant bacteria. Appl Environ Microbiol 65: 95±101.



Stienstra, A.W., Klein-Gunnewiek, P., and Laanbroek, H.J.(1994) Repression of nitri®cation in soils under a climaxgrassland vegetation. FEMS Microbiol Ecol 14: 45±52.

Suzuki, M.T., and Giovannoni, S.J. (1996) Bias causedby template annealing in the ampli®cation of mixtures of16S rRNA genes by PCR. Appl Environ Microbiol 62:625±630.

Teske, A., Elm, E., Regan, J.M., Toze, S., Rittmann, B.E.,and Stahl, D.A. (1994) Evolutionary relationships amongammonia- and nitrite-oxidising bacteria. J Bacteriol 176:6623±6630.

Tilman, D. (1987) Secondary succession and the pattern ofplant dominance along experimental nitrogen gradients.Ecol Monogr 57: 189±214.

Willems, J.H. (1980) An experimental approach to the

study of species diversity and above-ground biomass inchalk grasslands. Proc Kon Ned Akad Wet Ser C 83:279±306.

Willems, J.H. (1982) Phytosociological and geographical surveyof Mesobromion communities in western Europe. Vegeta-tio 48: 227±240.

Willems, J.H. (1983) Species composition and above groundphytomass in chalk grassland with different management.Vegetatio 52: 171±180.

Willems, J.H., and Bobbink, R. (1990) Spatial processes inthe succession of chalk grassland on old ®elds in TheNetherlands. In Spatial Processes in Plant Communities.Krahulec, F., Agnew, A.D.Q., Agnew, S., and Willems, J.H.(eds). The Hague: SPB Academic Publishing, pp. 237±249.



changes in the community structure of ammonia-oxidizing bacteria during secondary succession of...

Documents