Blackwell Science, LtdOxford, UKEMIEnvironmental Microbiology 1462-2912Blackwell Publishing Ltd, 200571218831895Original ArticleMetagenomic analysis of freshwater bacteriaM. T. Cottrell, L. A.

Waidner, L. Yu and D. L. Kirchman

Environmental Microbiology (2005) 7(12), 18831895

doi:10.1111/j.1462-2920.2005.00762.x

Bacterial diversity of metagenomic and PCR libraries

from the Delaware River
Matthew T. Cottrell, Lisa A. Waidner, Liying Yu and
David L. Kirchman*
University of Delaware, College of Marine Studies, 700
Pilottown Road., Lewes, DE 19958, USA.
Summary
To determine whether metagenomic libraries sample
adequately the dominant bacteria in aquatic environments, we examined the phylogenetic make-up of a
large insert metagenomic library constructed with
bacterial DNA from the Delaware River, a polymerase
chain reaction (PCR) library of 16S rRNA genes, and
community structure determined by fluorescence in
situ hybridization (FISH). The composition of the
libraries and community structure determined by
FISH differed for the major bacterial groups in the
river, which included Actinobacteria, beta-proteobacteria and Cytophaga-like bacteria. Beta-proteobacteria were underrepresented in the metagenomic library
compared with the PCR library and FISH, while
Cytophaga-like bacteria were more abundant in the
metagenomic library than in the PCR library and in
the actual community according to FISH. The Delaware River libraries contained bacteria belonging to
several widespread freshwater clusters, including
clusters of Polynucleobacter necessarius, Rhodoferax sp. Bal47 and LD28 beta-proteobacteria, the
ACK-m1 and STA2-30 clusters of Actinobacteria, and
the PRD01a001B Cytophaga-like bacteria cluster.
Coverage of bacteria with >97% sequence identity
was 65% and 50% for the metagenomic and PCR
libraries respectively. Rarefaction analysis of replicate PCR libraries and of a library constructed with
re-conditioned amplicons indicated that heteroduplex
formation did not substantially impact the composition of the PCR library. This study suggests that
although it may miss some bacterial groups, the
metagenomic approach can sample other groups
(e.g. Cytophaga-like bacteria) that are potentially
underrepresented by other culture-independent
approaches.

Received 9 March, 2004; revised 26 July, 2004; accepted 27 July,

2004. *For correspondence. E-mail: Kirchman@cms.udel.edu; Tel.
(+1) 302 6454375; Fax (+1) 302 6454028.

2005 Society for Applied Microbiology and Blackwell Publishing Ltd

Introduction
Metagenomic clone libraries of environmental DNA
enable the exploration of the phylogenetic and metabolic
diversity of microbes in the environment without cultivation
or polymerase chain reaction (PCR). New aspects of
microbial metabolism in uncultured microbes have been
uncovered using this metagenomic approach, as first
shown in a study of marine Bacteria and Archaea (Stein
et al., 1996), and then later in soils (Rondon et al., 2000).
Intriguing examples include studies revealing different
types of phototrophy in the ocean (Beja et al., 2000a,
2001; Sabehi et al., 2003). Other studies have examined
heterotrophic metabolism, including one study that identified chitinase genes of uncultured marine microbes (Cottrell et al., 1999). In soils, different types of metabolism
have been targeted such as those involving alcohol oxidoreductases (Knietsch et al., 2003), lipases, amylases
and nucleases (Rondon et al., 2000). Soil clones have
also been obtained that produce broad-spectrum antibiotics (Gillespie et al., 2002) while other
have
se e naoclones
cultivavel, como
fazer para reobter os
hemolytic properties (Rondon et al., 2000).
The data on the metabolic diversity revealed by metagenomic libraries are most valuable when viewed in the
context of community structure. Phylogenetic information
is needed together with estimates of metabolic potential
in order to link specific members of the community to
biogeochemical processes. However, the phylogenetic
information present in metagenomic libraries has received
little attention (Beja et al., 2000b; Liles et al., 2003). One
approach to obtaining this phylogenetic information is to
screen metagenomic libraries for 16S rRNA genes in
order to identify clones that then can be used to explore
the metabolic potential of targeted bacterial groups (Beja
et al., 2001; Quaiser et al., 2002; Liles et al., 2003). It is
also important to compare the phylogenetic make-up of
metagenomic libraries to other measures of community
structure, such as 16S rDNA clone libraries and fluorescence in situ hybridization (FISH), in order to determine if
any of the dominant groups of bacteria are missing.
Finally, the phylogenetic information in metagenomic
libraries provides another view of community structure
without the biases of PCR-dependent approaches (von
Wintzingerode et al., 1997).
Only one study examined the similarity between the
phylogenetic composition of a metagenomic library and
published surveys of marine bacterial diversity (Beja et al.,

1884 M. T. Cottrell, L. A. Waidner, L. Yu and D. L. Kirchman

2000b). Phylogenetic analysis of metagenomic clones
bearing 16S rRNA genes from surface water of the Pacific
Ocean identified several groups of Archaea and Bacteria
that are common in marine bacterial communities, including a Euryarchaeota clone and clones related to SAR86,
SAR116 and SAR11 bacteria, Roseobacter spp. and
Cytophaga-like bacteria (Giovannoni and Rapp, 2000).
However, no study has directly compared the phylogenetic
composition of a metagenomic library and the microbial
community in situ. It may be especially important to examined this issue in freshwaters where Gram-positive bacteria can be abundant (Glckner et al., 2000; Zwart et al.,
2002). These bacteria could be underrepresented in
metagenomic libraries if DNA extraction from Gram-positive bacteria is less efficient.
Bacteria in rivers and other freshwater ecosystems
have not been examined previously using metagenomic
libraries. Bacteria in rivers play an important role in the
consumption of organic matter transported from the terrestrial environment to the ocean (Amon and Benner,
1996; Opsahl et al., 1999; Hernes and Benner, 2002),
and the make up of those communities likely has an
impact on organic matter consumption (Covert and
Moran, 2001). The diversity of bacteria in rivers may be
especially high as they may contain mixtures of aquatic
and terrestrial bacteria, including phylogenetically and
metabolically diverse soil taxa (Liles et al., 2003; Topp,
2003). Microbes from soil may be introduced into waterways by runoff (Zaitlin et al., 2003) and mix with freshwater bacteria (Zwart et al., 2002). Less is known about
riverine bacteria than their counterparts in other aquatic
systems. A GenBank search in November 2003 for riverine bacterial and archaeal 16S rRNA genes returned
approximately 1000 hits from studies in 14 rivers, compared with more than 17 000 sequences from marine
environments and lakes.

In this study, we determined the coverage and composition of metagenomic and PCR libraries from the Delaware River. We also contrasted the composition of these
libraries with community structure determined by FISH. In
order to identify why the composition of libraries might
differ, we tested the hypothesis that heteroduplex artefacts
of the PCR step lead to overestimates of bacterial
diversity (Thompson et al., 2002). We found that the
metagenomic analysis and PCR libraries identified
Actinobacteria, Cytophaga-like bacteria and betaproteobacteria, which are potentially important members
of the bacterial community in the Delaware River.

Results
Overview of the libraries
The metagenomic library was comprised of 4608 clones
with an average insert size of 40.5 3.1 kb (n = 15). The
library contained approximately 180 Mb of DNA, which is
equivalent to 90 bacterial genomes, assuming two Mbp
per bacterial genome (Button and Robertson, 2001).
Eighty clones tested positive for 16S rRNA genes, which
is consistent with the expected number of rRNA genes in
the library, assuming a single 16S rRNA gene per bacterial genome. Bacteria isolated from aquatic and low-nutrient environments typically have only one or two rRNA
operons (Button et al., 1998; Fegatella et al., 1998; Fogel
et al., 1999; Klappenbach et al., 2000).
The compositions of the metagenomic library and PCR
libraries were not significantly different at a high phylogenetic level based on analyses of coverage calculated
using the LIBSHUFF tool. BLAST analysis was consistent
with this result, revealing essentially the same major
groups of bacteria in the two libraries (Table 1). Cytophaga-like bacteria, beta-proteobacteria and Actinobacteria

Table 1. Phylogenetic composition of the metagenomic and PCR libraries and structure of the actual community determined by FISH.

Group
Alpha-proteobacteria
Beta-proteobacteria
Gamma-proteobacteria
Epsilon-proteobacteria
Cytophaga-like
Actinobacteria
Firmicutes
Bdellovibrio
Verrucomicrobiales
Spirochaetaceae
Total

% of metagenomic
clones

% of PCR clones

% of total prokaryotes
detected by FISH

3
17
4
0
54
14
3
0
3
1

100

12
50
0
2
13
16
0
4
2
0

100

62
25 5
32
ND
17 4
26 5
ND
ND
ND
ND

77

Composition of the metagenomic library is based on nucleotide sequences from 72 clones bearing 16S rRNA genes. Fifty-eight clones from the
PCR library were sequenced and 5001000 DAPI-positive prokaryotes were analysed with each FISH probe.
ND, not determined.
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Metagenomic analysis of freshwater bacteria 1885

comprised the largest fractions of the libraries while alphaproteobacteria, gamma-proteobacteria, Firmicutes, Verrucomicrobiales and Spirochaetaceae were minor members. However, closer examination of the relative
abundance of different bacterial groups revealed noteworthy differences between the metagenomic and PCR
libraries.
Cytophaga-like bacteria in clone libraries
The metagenomic and PCR libraries differed in the number and kinds of Cytophaga-like bacteria. One obvious
difference was the percentage of Cytophaga-like clones.
Only 13% of the clones in the PCR library belonged to the
Cytophaga-like group, compared with 54% for the
metagenomic library (Table 1). In addition, the variety of
Cytophaga-like bacteria was greater in the metagenomic
library than in the PCR library. More Cytophaga-like clusters contained fosmid sequences than PCR sequences in
the phylogenetic tree of Cytophaga-like bacteria (Fig. 1).
The metagenomic library but not the PCR library contained Cytophaga-like clones in the Chitinophaga and
Myroides groups (Fig. 1). One Cytophaga-like clone in the
PCR library (Sta297) was not similar to any of the
metagenomic clones.
Seventy percent of the Cytophaga-like bacteria were
associated with the Flavobacteriales group (Fig. 1) and
these clones could be grouped into three clusters (AC).
A fourth cluster (D) was in the Flectobacillus group
(Fig. 1). Clusters A and B were related to different kinds
of Flavobacterium spp. and included some cultured bacteria. Cluster A included an isolate from the Elbe River
and cluster B included Flavobacterium xinjiangensi. The
average sequence identity in these two clusters with cultured members was 96.6% 3.4% and 94.5% 6.1%
respectively. Clusters C and D were associated with the
Myroides and Flectobacillus groups, respectively, and
included bacteria with a higher degree of sequence similarity (98.5% 0.5% and 99.5% 0.4% respectively), but
these clusters had no cultured members.
Some of the Cytophaga-like bacteria were represented
in both libraries. Half of the Cytophaga-like clones in the
PCR library were highly similar to clones in the metagenomic library, and every Cytophaga-like group with clones
from the PCR library also had representatives from the
metagenomic library. For example, cluster D was comprised of three PCR clones and four metagenomic clones
(Fig. 2). Cluster A included three metagenomic clones
and a highly similar clone from the PCR library. However,
similarity between Cytophaga-like bacteria in the two
libraries was restricted to these two clusters.
Beta-proteobacteria in clone libraries. The metagenomic and PCR libraries also differed in the number and
kinds of beta-proteobacteria. The PCR library had more

than twice as many clones in this group than the metagenomic library (50% versus 17% respectively) (Table 1).
The beta-proteobacteria clones in the two libraries
belonged to five clusters (AE). Clusters D and E were
comprised exclusively of clones from the PCR library
(Fig. 2). The degree of relatedness in the clusters made
up of only PCR clones and in cluster A was low, ranging
from 89% to 95% sequence identity. In contrast, some of
the beta-proteobacteria sampled by the two libraries were
similar. Clusters B and C, which included clones from both
libraries, were comprised of very closely related bacteria
with similarities of 96.4% 1.6% and 97.5% 3.1%
respectively.
Other bacteria in the clone libraries
In contrast to the differences outlined above for the
Cytophaga-like and beta-proteobacteria groups, Actinobacteria in the metagenomic and PCR libraries were quite
similar. The libraries had equivalent percentages of Actinobacteria (about 15%), and there was considerable overlap in the types of Actinobacteria in the two libraries. Of
the three clusters of Actinobacteria clones (AC), each
included clones from both libraries (Fig. 3). Cluster B
included the most closely related clones with average
similarities of 96.4% 2.2%. The Actinobacteria clones in
clusters A and C were less closely related. Sequence
similarities in these clusters were 93.2% 4.1% and
92.9% 5.4% respectively.
Alpha-proteobacteria comprised a minor component of
the libraries, accounting for only 3% and 12% of the
clones in the metagenomic and PCR clones respectively
(Table 1). The alpha-proteobacteria clones in the two
libraries were closely related to each other and to some
cultured alpha-proteobacteria (results not shown). Three
clones from the PCR library and two from the metagenomic library belonged to a cluster that included cultured
Sphingomonas spp. One alpha-proteobacteria clone from
the PCR library belonged to the Rhodospirillales group.
The libraries also included clones belonging to five
groups of bacteria that can be present in aquatic systems but are typically minor members of the community.
These groups included Firmicutes and Spirochaetaceae
that occurred in only the metagenomic library and epsilon-proteobacteria and Bdellovibrio that were found
exclusively in the PCR library (Table 1). Clones belonging to the Verrucomicrobiales group occurred in both
libraries.
Library coverage and diversity
Library coverage increased with the genetic distance, as
expected. At an evolutionary distance of 0.03 coverages
of the metagenomic and PCR libraries were 65% and 51%

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1886 M. T. Cottrell, L. A. Waidner, L. Yu and D. L. Kirchman

Fig. 1. Phylogenetic relationships of selected Cytophaga-like bacteria as depicted in the ARB database tree constructed of sequences longer
than 1000 bp (June 2002). Delaware River Cytophaga-like bacteria were added using the ARB parsimony quick add tool. Clones in the library
of 16S rDNA amplicons are labelled PCR and 16S rDNA-bearing metagenomic DNA clones are labelled Fosmid. Values on the trapezoids indicate
the number of sequences in the group. Labels A, B, C and D refer to clusters of closely related bacteria that are discussed in the text. The scale
bar represents 0.1 substitutions per site.
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Metagenomic analysis of freshwater bacteria 1887

Fig. 2. Phylogenetic relationships of selected beta-proteobacteria as depicted in the ARB database tree constructed of sequences longer than
1000 bp (June 2002). Delaware River beta-proteobacteria were added using the ARB parsimony quick add tool. Clones in the library of 16S
rDNA amplicons are labelled PCR and 16S rDNA-bearing metagenomic DNA clones are labelled Fosmid. Values on the trapezoids indicate the
number of sequences in the group. Labels A, B, C, D and E refer to clusters of closely related bacteria that are discussed in the text. The scale
bar represents 0.1 substitutions per site.
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1888 M. T. Cottrell, L. A. Waidner, L. Yu and D. L. Kirchman

Fig. 3. Phylogenetic relationships of selected Actinobacteria as depicted in the ARB database tree constructed of sequences longer than 1000 bp
(June 2002). Delaware River Actinobacteria were added using the ARB parsimony quick add tool. Clones in the library of 16S rDNA amplicons
are labelled PCR and 16S rDNA-bearing metagenomic DNA clones are labelled Fosmid. Values on the trapezoids indicate the number of
sequences in the group. Labels A, B and C refer to clusters of closely related bacteria that are discussed in the text. The scale bar represents
0.1 substitutions per site.
2005 Society for Applied Microbiology and Blackwell Publishing Ltd, Environmental Microbiology, 7, 18831895

Metagenomic analysis of freshwater bacteria 1889

Fig. 5. Rarefaction curves of 16S rDNA clone libraries constructed

with and without re-conditioned PCR. PCR 1 and PCR 2 are clone
libraries constructed from replicate PCR reactions without re-conditioning. The error bars are one standard deviation.

Fig. 4. Coverage of the metagenomic and 16S rDNA libraries calculated using the LIBSHUFF tool.

respectively (Fig. 4). This distance is approximately equal

to 97% sequence identity, which is one approximation of
species-level identity (Stackebrandt and Goebel, 1994).
Coverage of the metagenomic library was higher than
coverage of the PCR library at all levels of genetic distance with the greatest differences occurring at small
genetic distance. Coverage decreased rapidly with
decreasing genetic distance, and at a genetic distance of
0.01 the coverage of the metagenomic library was 46%
while the coverage of the PCR library decreased even
more to just 16%.
Indices of diversity were higher for the metagenomic
library than the PCR library (Table 2). The Simpson
index was 13.4 for the metagenomic library compared
with 8.7 for the PCR library. The difference was not as
substantial for the ShannonWeaver index (Table 2).
Similarly, measures of evenness were higher for the
metagenomic library than the PCR library. Evenness calculated using the ShannonWeaver index was 0.73 and
0.66 for the metagenomic library and PCR library
respectively, while the Simpson index of evenness was
0.26 and 0.17 for the metagenomic and PCR libraries
respectively.

We examined the possibility that diversity based on

PCR clone library composition is overestimated because
of artefacts of the PCR. Re-conditioned PCR was used to
test for potential artefacts introduced when heterologous
PCR products re-anneal and form heteroduplexes that are
then subjected to mismatch repair upon cloning into
E. coli (Thompson et al., 2002). Re-conditioning of the
PCR amplicons reduced the number of RFLP types
expected in a library of 80 clones from 50 in the standard
PCR library to 40 in the library produced with re-conditioned PCR (Fig. 5). There was no difference between
rarefaction curves of replicate PCR libraries produced with
standard PCR (Fig. 5).

Comparison of FISH and clone libraries

Fluorescence in situ hybridization was used to assess the
correspondence between clone library composition and
bacterial community structure. Microscopic enumeration
of bacteria hybridized with fluorescently labelled 16S
rRNA directed probes revealed a community dominated
by beta-proteobacteria, Actinobacteria and Cytophagalike bacteria (Table 1). As expected for a freshwater system, Actinobacteria and beta-proteobacteria were the
most abundant groups, making up about 25% of the community. Cytophaga-like bacteria were also prominent,

Table 2. Diversity of 16S rRNA genes in metagenomic and PCR clone libraries from the Delaware River.
ShannonWeaver

Simpson

Library

Diversity

Evenness

Diversity

Evenness

Coverage

Metagenomic
PCR

2.9
2.6

0.73
0.66

13.4
8.7

0.26
0.17

0.65
0.52

Diversity indices were calculated using equations taken from Dunbar and colleagues (1999).
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1890 M. T. Cottrell, L. A. Waidner, L. Yu and D. L. Kirchman

accounting for 17% 4% of the community. In contrast,
alpha-proteobacteria and gamma-proteobacteria were
considerably less abundant (less than 10%). Seventyseven percent of all prokaryotes were identified with a
group probe, and 69% 11% were detected with the general bacterial probe Eub338.
Bacterial community structure determined by FISH and
inferred from the PCR library was surprisingly similar. The
abundance of Cytophaga-like bacteria in the PCR library
was within 25% of the abundance determined by FISH,
the abundance of gamma-proteobacteria differed by 3%
or less and Actinobacteria differed by 38%, according to
the two analyses (Table 1). In contrast, the abundance of
beta-proteobacteria clones in the PCR library was twice
the abundance of bacteria detected with the beta-proteobacteria FISH probe (Table 1). Three types of bacteria,
including epsilon-proteobacteria, Bdellovibrio and bacteria belonging to the candidate division TM6, were present
in the PCR library and would not have been detected with
any of the FISH probes used in this study. Although we
have no estimate of the abundance of bacteria in these
groups, they likely accounted for some of the 23% of
bacteria not detected by FISH (Table 1).
Discussion
It is necessary to examine whether the phylogenic composition of metagenomic libraries reflects that of the original microbial composition in order to effectively link
community structure and function revealed by metagenomic analysis. If metagenomic libraries represent a
biased sample of microbial diversity, they will not yield an
accurate assessment of metabolic diversity. Assuming
they are unbiased, large insert metagenomic libraries may
be particularly powerful in examining the relationship
between community structure and metabolic capacities.
Large inserts increase the likelihood of obtaining a clone
containing both functional genes (coding for metabolic
function) and genes with phylogenetic information, e.g.
16S rRNA genes. Therefore, it is necessary to know how
the diversity of 16S rRNA genes sampled by large insert
metagenomic libraries corresponds to actual community
structure. Our main finding was that the composition of
the metagenomic library at a high phylogenetic level was
not statistically different from the PCR library, but closer
examination revealed noteworthy differences between the
libraries and the community structure determined by
FISH. The metagenomic library sampled a broader diversity of Cytophaga-like bacteria than the PCR library, but it
missed some groups of potentially important beta-proteobacteria. The metagenomic and PCR libraries sampled
the same groups of Actinobacteria, but FISH analysis
suggested that both types of libraries may have failed to
adequately sample Actinobacteria.

Metagenomic analysis of Delaware River bacteria provided a view of Cytophaga-like bacterial diversity that was
not possible with the PCR library and FISH analyses. The
number of clones representing Cytophaga-like bacteria in
the metagenomic library was threefold higher than the
abundance determined by FISH and fourfold higher than
in the PCR library. It was not surprising that the metagenomic library sampled a larger diversity of Cytophaga-like
bacteria than the PCR library because previous studies
had suggested that Cytophaga-like bacteria are underrepresented in 16S rDNA PCR libraries relative to the abundance of these bacteria detected by FISH (Cottrell and
Kirchman, 2000; Kirchman et al., 2003). It was also not
surprising that the metagenomic library had more clones
from Cytophaga-like bacteria than would have been
expected based on the FISH counts because the general
FISH probe CF319a for Cytophaga-like bacteria does not
recognize all Cytophaga-like bacteria (Weller et al., 2000).
The abundance of Cytophaga-like bacteria according to
the general FISH probe CFB560 for Cytophaga-like bacteria (OSullivan et al., 2002) was only 15% higher than
that determined with CF319a. The denaturing gradient
electrophoresis (DGGE) PCR primers used in this study
appear to adequately sample Cytophaga-like bacteria, at
least compared with FISH analysis (Castle and Kirchman,
2004), and these primers apparently recognize a broader
diversity of Cytophaga-like bacteria than the EubA and
EubB primers, as some Cytophaga-like bacteria detected
by DGGE in the metagenomic library were not sampled
by the PCR library.
The Cytophaga-like bacterial group includes a highly
diverse collection of bacteria but not all of this diversity
was present in our Delaware River sample. In fact, most
of the Cytophaga-like bacteria were most closely related
to just two genera of Flavobacteriales (Flavobacterium
and Myroides) and a cluster in the Flectobacillus group.
This latter group is distinct from the CytophagaFlavobacterium cluster, which is synonymous with the Flavobacteriales (Kirchman, 2002). The Delaware River bacteria in
the Flectobacillus group belong to the proposed
PRD01a001B cluster of Cytophaga-like bacteria identified
by Zwart and colleagues (2002). Nine of the 12 Delaware
River sequences in the Flectobacillus group were greater
than 98% similar to the PRD01a001B clone from the
Parker
River,
Massachusetts.
The
proposed
PRD01a001B cluster, which is currently comprised solely
of uncultivated bacteria, has also been found in Adirondack lakes, Lake Baikal and other freshwater environments (Zwart et al., 2002). However, this group of
Cytophaga-like bacteria has not been detected in all
freshwater environments; for example, it was not found in
the Columbia River (Zwart et al., 2002).
The Delaware River beta-proteobacteria belonged to
clusters widely distributed in freshwater systems. Three

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Metagenomic analysis of freshwater bacteria 1891

groups of Delaware River beta-proteobacteria clones (B,
D and E) were highly similar to bacteria in the freshwater
beta-Proteobacteria clusters proposed by Zwart and colleagues (2002). Sequences in Delaware River beta-proteobacteria group B were 9599% similar to the
Rodoferax sp. in the Rodoferax sp. BAL47 cluster. Group
D of Delaware Bay beta-proteobacteria corresponded to
the Polynucleobacter necessarius cluster (8899%
sequence similarity to P. necessarius) and sequences in
group E probably belong to the proposed LD28 cluster.
Sequences in group D were 8997% similarity to the
LD28 clone from Lake Loosdrech in the Netherlands
(Zwart et al., 1998).
The clusters of freshwater beta-proteobacteria proposed by Zwart and colleagues (2002) were more prevalent in the PCR library than in the metagenomic library,
suggesting that they may represent bacteria that are preferentially sampled by PCR libraries. Delaware River betaproteobacteria group B included clones from both the
metagenomic library and the PCR library, but Delaware
River groups D and E were comprised of only clones from
the PCR library. Different scenarios could lead to the
overrepresentation of bacterial groups in PCR libraries
compared with libraries constructed without a PCR step,
including selective PCR amplification with general bacterial primers, differences in rRNA gene copy number or
variation in DNA extraction efficiency (von Wintzingerode
et al., 1997). Fluorescence in situ hybridization probes are
needed to determine the abundance of these beta-proteobacteria, which appear to be in a variety of freshwater
environments.
There was a high degree of similarity in the Actinobacteria sampled by the metagenomic and PCR libraries. All
three groups of Actinobacteria identified in this study
included clones from the metagenomic and PCR libraries.
Our results indicate that the presumed thicker cell walls
of these G+ bacteria do not interfere with the extraction of
high molecular weight DNA from these bacteria. However,
the overall efficiency of DNA extraction may be low as
Actinobacteria were underrepresented in the libraries
compared with their abundance determined by FISH.
Although the libraries may be missing some Actinobacteria, they did recover clusters that are apparently
widespread in freshwater systems. Delaware River Actinobacteria groups B and C, which correspond to the ACKMI and STA-30 clusters identified by Zwart and colleagues
(2002), were identified in the metagenomic and PCR
libraries. Clones in cluster B were 9299% similar to the
ACK-M1 clone and clones in cluster C were 9297%
similar to the STA-30 clone. Delaware River Actinobacteria group A, which included clones from both libraries, did
not overlap with previously described clusters of freshwater Actinobacteria. None of the G+ bacterial groups we
detected represented a terrestrial bacterial signal

because of transport of soils into the river. Zwart and

colleagues (2002) also did not find evidence for an important contribution of soil bacteria to bacterial communities
in rivers.
Coverage in the metagenomic library was consistently
higher than in the PCR library over a broad range of
evolutionary distances, although substantially higher coverage by the metagenomic library was restricted to evolutionary distances less than 0.05. Differences in
coverage were not the result of different sequence
lengths; we analysed 1400 bp for the PCR clones, but only
the highly variable V3 region of the 16S rRNA gene for
the metagenomic clones. LIBSHUFF analysis conducted
with the V3 region alone also revealed higher coverage by
the metagenomic library (data not shown). Differences in
coverage may reflect more comprehensive sampling of
bacterial diversity by the metagenomic library.
Lower coverage by the PCR library was consistent with
our hypothesis that PCR artefacts lead to overestimates
of diversity. Polymerase chain reaction re-conditioning flattened the rarefaction curve, but the effect was only 20%,
not as pronounced as would be expected if heteroduplexes were a substantial source of diversity in the PCR
libraries. The higher diversity in the metagenomic library
compared with the PCR library is also consistent with the
idea that the PCR libraries do not overestimate bacterial
diversity. In fact, they probably underestimate diversity,
because they miss certain groups, such as Cytophagalike bacteria (Cottrell and Kirchman, 2000). Additional factors such as cloning biases, variation in genome size
(Fogel et al., 1999), rRNA gene copy number (Klappenbach et al., 2000) and ribosome content (Fegatella et al.,
1998), could have resulted in larger differences than we
observed between the two library approaches and FISH.
Given the potential sources of differences the degree of
similarity between the different library and FISH
approaches was higher than expected.
These results and other suggest that the identities of
prokaryotes in aquatic environments are no longer completely hidden in the microbial black box. With the aid of
various culture-independent approaches, it is now clear
that various proteobacterial groups, Cytophaga-like bacteria and Actinobacteria are the dominant groups of bacteria in surface waters of aquatic systems (Giovannoni
and Rapp, 2000; Rapp and Giovannoni, 2003).
Archaea make a substantial contribution to prokaryotic
diversity in the ocean depths well below the euphotic zone
(Massana et al., 1998; Karner et al., 2001). Although
potentially these groups could contain many subgroups,
at least in marine waters, it appears that only a few clades
dominate, including alpha-proteobacteria belonging to the
SAR11 group (Morris et al., 2002; Rapp et al., 2002) and
Roseobacter spp. (Gonzalez and Moran, 1997), Cytophaga-like bacteria in Delaware cluster 2 (Kirchman et al.,

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1892 M. T. Cottrell, L. A. Waidner, L. Yu and D. L. Kirchman

2003), the marine Actinobacteria group (Rapp et al.,
1999) and Archaea in group I and group II (Fuhrman et al.,
1992; Massana et al., 2000; Karner et al., 2001).
Dominant groups in freshwater are also becoming
apparent. Our study revealed several widespread clusters
of freshwater bacteria in the Delaware River that were
identified in the metagenomic and PCR libraries. These
clusters are obvious candidates for further investigation,
including additional metagenomic analysis to explore
their metabolic potential. It is unclear if clusters such as
the P. necessarius and LD28 beta-proteobacteria clusters, which occurred in the PCR library but not the
metagenomic library, are selectively amplified by the PCR
or for some reason are missed by the metagenomic cloning. Additional examination by FISH could help resolve
the numerical importance of these freshwater bacteria.
Studies such as these will further advance our understanding of aquatic bacterial diversity as it evolves from a
view of apparently intractable diversity towards what
appears to be a manageable number of dominant bacterial groups.
Experimental procedures
Sample collection
A water sample was collected from a depth of 1 m in the
Delaware River near Trenton, New Jersey (407.7-N,
7449.3-W) in December 2001 and subsampled for the analyses performed in this study.

Fluorescence in situ hybridization

The sample for FISH analysis was fixed with 2% paraformaldehyde at 4C for 1218 h. Bacteria were then collected on
0.2 mm polycarbonate filters, rinsed twice with deionized
water and the filters were stored at -20C. Bacteria were
detected with FISH probe Eub338 (Amann et al., 1990). We
also tried a mixture of general probes for Bacteria (Daims
et al., 1999) and found that it detected 70 5% of DAPI
positive bacteria compared with 69 11% for Eub338 alone.
The FISH probes for alpha-, beta- and gamma-proteobacteria were Alf968 (Neef, 1997; Glckner et al., 1999), Bet42a
and Gam42a (Manz et al., 1992) respectively. Cytophaga-like
bacteria were assayed with probe CF319a (Manz et al.,
1996) and CFB560 (OSullivan et al., 2002), and Actinobacteria were enumerated using probe HGC69a (Roller et al.,
1994). Non-specific binding was assessed using a negative
control probe (Karner and Fuhrman, 1997). Hybridization of
Cy3-labelled oligonucleotide probes was performed with portions of polycarbonate filters incubated in a hybridization
buffer containing formamide to control the stringency of
hybridization (Cottrell and Kirchman, 2000). Unbound probe
was removed by washing the sample with a wash buffer at
48C. The filter piece was then rinsed in water and 70%
ethanol. The air-dried sample was mounted on a glass slide
with a coverslip using an antifade mountant containing
2 mg ml-1 DAPI. Probe-positive bacteria (5001000 bacteria

for each FISH probe) were enumerated using semiautomated

fluorescence microscopy (Cottrell and Kirchman, 2003).

Preparation of the bacterial size fraction

Environmental DNA was extracted from the bacterial size
fraction obtained by pumping river water (500 l) sequentially
through a 1 mm nominal pore-size polypropylene stringwound filter (Cole Parmer) and a 0.8 mm polycarbonate filter
(Nuclepore). Bacteria were collected from the filtrate by tangential flow filtration using a 0.1 mm hollow fibre filter (A/G
Technology) and an Amicon DC10 gear pump. The sample
was concentrated to 2 l, rinsed with a buffer (0.5 M NaCl,
0.1 M EDTA, 10 mM Tris pH 8.0), and stored frozen.

Library construction using a fosmid vector

Bacteria were collected from thawed bacterial concentrate by
centrifugation in an SS34 rotor at 16 000 r.p.m. for 30 min.
The bacteria were resuspended in 1 ml of the supernatant
and then mixed with 1% Sea Plaque agarose in deionized
water at 55C (Stein et al., 1996). The molten mixture was
drawn into a 1 ml syringe and allowed to solidify. The sample
was equilibrated with buffer (10 mM Tris pH 8, 50 mM NaCl,
0.1 M EDTA, 1% Sarkosyl) and digested with 1 mg ml-1
lysozyme at 37C for 4 h. Buffer and lysozyme were replaced
with 1% Sarkosyl in 0.5 M EDTA and the sample was
digested with 1 mg ml-1 proteinase K at 55C for 18 h. Following the enzyme treatments, the sample was electrophoresed for 8 h at 23 V through 0.3% SeaKem Gold agarose in
TAE buffer. DNA entering the gel, but larger than the 48 kb
size standard was recovered from the gel by electroelution
and concentrated by ethanol precipitation. The DNA was then
sheared to 40 kb by 150 passages through a 200 ml pipette
tip. The DNA was prepared for ligation using a blunt end
repair kit (Epicentre Technologies) following the manufacturers protocol. The insert DNA was then size selected by
electrophoresis on 1% Sea Plaque GTG agarose. DNA was
recovered from the gel following treatment with Gelase
enzyme (Epicentre Technologies) digestion and ethanol precipitation. The DNA was then ligated to the pCC1FOS fosmid
vector (Epicentre Technologies) and packaged using phage
protein extract following the manufacturers recommended
procedure. EPI300 host E. coli infected with recombinant
phage were plated on LB media containing chloramphenicol.
Clones were picked and sorted into 96-well microtitre plates.

PCR library construction

Three PCR libraries were constructed with the DNA isolated
from the Delaware River. Two replicate libraries were constructed by cloning amplicons generated from separate PCR
reactions using the general bacterial primers EubA and EubB
(Lane, 1991). A third library was constructed using re-conditioned amplicons from the PCR reaction used to generate the
first library. Re-conditioning was performed with three rounds
of thermal cycling applied to amplicons diluted 10-fold in
fresh PCR reagents (Thompson et al., 2002). The 25 ml PCR
reactions included 0.5 ml, 1.2 ml and 2 ml additions of 10 mM
deoxynucloside triphosphate, 25 mM MgCl2, 10 mM primer

2005 Society for Applied Microbiology and Blackwell Publishing Ltd, Environmental Microbiology, 7, 18831895

Metagenomic analysis of freshwater bacteria 1893

stocks respectively. Bovine serum albumin was added to a
final concentration of 0.2 mg ml-1. Two and a half units of Taq
polymerase (Promega) and a one-tenth volume of 10 buffer
were added to each reaction. The thermal cycling conditions
consisted of a touchdown series in which the annealing temperature decreased from 65C to 55C by 1C per cycle,
followed by 15 cycles at 55C, each for 1 min. Each cycle
included a 1 min denaturation step at 95C and an extension
step of 2.5 min at 72C. Amplicons were cloned with a TOPOTA cloning kit with pCR2.1-TOPO vector (Invitrogen) according to the manufacturers instructions.

Screening PCR and metagenomic libraries

Clones were screened for insert size using M13 forward and
reverse PCR primers, which flank the cloned insert, and
those with full-length inserts were screened by restriction
fragment length polymorphism (RFLP) analysis by digesting
the M13 PCR amplicons with a mixture of HhaI and RsaI
(New England Biolabs). Restriction patterns on 2% Metaphore agarose (FMC) agarose gels were compared manually.
Clones with identical restriction patterns were grouped
together into RFLP types. The 16S rRNA genes from one
representative of each RFLP type in the first library were
completely sequenced.
Pools of 96 fosmid clones were screened for 16S rRNA
genes by DGGE of PCR amplicons generated with primers
GC358F and 517R (Muyzer et al., 1995). Selected bands
resolved on an 8% polyacrylamide gel containing a gradient
of 2555% denaturant (13.822% formamide and 10.523%
urea) were re-amplified and sequenced. Phylogenetic classification was determined using BLAST and the ARB sequence
analysis tool as described below. Bands with equal electrophoretic mobility were assigned the same phylogenetic
classification.

Sequence and library analysis

Nucleotide sequences were analysed using BLAST [version
2.1; National Center for Biotechnology Information (http://
www.ncbi.nlm.nih.gov/BLAST/) and the ARB package (Ludwig et al., 2004)]. Sequences were aligned with the ARB Fast
Aligner version 1.03, adjusted manually with secondarystructure criteria. Phylogenetic relationships were determined using the Quick Add Parsimony tool and the
1000_pub_may02 phylogenetic tree in the ARB database
(June 2002 release).
Sequence similarities were determined using the tools in
ARB and rarefaction curves were calculated using Analytic
Rarefaction 1.3 (http://www.uga.edu/strata/software/). Libraries were compared using the LIBSHUFF analysis tool
(http://www.arches.uga.edu/~whitman/libshuff.html) (Singleton et al., 2001). Diversity indices were calculated using
equations taken from Dunbar and colleagues (1999).

Statistical analysis
Libraries were compared using LIBSHUFF analysis (Singleton et al., 2001). This approach compares libraries based on
their coverage and the number of sequences in one library

that are not found in the second library. This so-called heterologous coverage is calculated over a range of evolutionary distances to obtain a coverage curve. The difference
between the heterologous coverage curve and the coverage
curve of the first library is calculated using the Cramr-von
Mises test statistic (Pettit, 1982) and compared with the coverage curve calculated after shuffling sequences between the
two libraries.

Nucleotide sequence accession numbers

The nucleotide sequence data reported in this work are
deposited in GenBank under accession numbers AY562245
AY562366.

Acknowledgements
This work was supported by the US Department of Energy
Microbial Genomes Program and the National Science
Foundation.

References
Amann, R.I., Binder, B.J., Olson, R.J., Chisholm, S.W.,
Devereux, R., and Stahl, D.A. (1990) Combination of 16S
ribosomal-RNA-targeted oligonucleotide probes with flow
cytometry for analyzing mixed microbial populations. Appl
Environ Microbiol 56: 19191925.
Amon, R.M.W., and Benner, R. (1996) Photochemical and
microbial consumption of dissolved organic carbon and
dissolved oxygen in the Amazon River system. Geochim
Cosmochim Ac 60: 17831792.
Beja, O., Aravind, L., Koonin, E.V., Suzuki, M.T., Hadd, A.,
Nguyen, L.P., et al. (2000a) Bacterial rhodopsin: evidence
for a new type of phototrophy in the sea. Science 289:
19021906.
Beja, O., Suzuki, M.T., Koonin, E.V., Aravind, L., Hadd, A.,
Nguyen, L.P., et al. (2000b) Construction and analysis of
bacterial artificial chromosome libraries from a marine
microbial assemblage. Environ Microbiol 2: 516529.
Beja, O., Spudich, E.N., Spudich, J.L., Leclerc, M., and
DeLong, E.F. (2001) Proteorhodopsin phototrophy in the
ocean. Nature 411: 786789.
Button, D.K., and Robertson, B.R. (2001) Determination of
DNA content of aquatic bacteria by flow cytometry. Appl
Environ Microbiol 67: 16361645.
Button, D.K., Robertson, B.R., Lepp, P.W., and Schmidt,
T.M. (1998) A small, dilute-cytoplasm, high-affinity, novel
bacterium isolated by extinction culture and having kinetic
constants compatible with growth at ambient concentrations of dissolved nutrients in seawater. Appl Environ
Microbiol 64: 44674476.
Castle, D., and Kirchman, D.L. (2004) Composition of estuarine bacterial communities assessed by denaturing gradient gel electrophoresis and fluorescence in situ
hybridization. Limnol Oceanogr 2: 303314.
Cottrell, M.T., and Kirchman, D.L. (2000) Community composition of marine bacterioplankton determined by 16S rRNA
gene clone libraries and fluorescence in situ hybridization.
Appl Environ Microbiol 66: 51165122.

2005 Society for Applied Microbiology and Blackwell Publishing Ltd, Environmental Microbiology, 7, 18831895

1894 M. T. Cottrell, L. A. Waidner, L. Yu and D. L. Kirchman

Cottrell, M.T., and Kirchman, D.L. (2003) Contribution of
major bacterial groups to bacterial biomass production
(thymidine and leucine incorporation) in the Delaware estuary. Limnol Oceanogr 48: 168178.
Cottrell, M.T., Moore, J.A., and Kirchman, D.L. (1999) Chitinases from uncultured marine microorganisms. Appl Environ Microbiol 65: 25532557.
Covert, J.S., and Moran, M.A. (2001) Molecular characterization of estuarine bacterial communities that use high- and
low-molecular weight fractions of dissolved organic carbon.
Aquat Microb Ecol 25: 127139.
Daims, H., Bruhl, A., Amann, R., Schleifer, K.H., and Wagner,
M. (1999) The domain-specific probe EUB338 is insufficient for the detection of all Bacteria: Development and
evaluation of a more comprehensive probe set. Syst Appl
Microbiol 22: 434444.
Dunbar, J., Takala, S., Barns, S.M., Davis, J.A., and Kuske,
C.R. (1999) Levels of bacterial community diversity in four
arid soils compared by cultivation and 16S rRNA gene
cloning. Appl Environ Microbiol 65: 16621669.
Fegatella, F., Lim, J., Kjelleberg, S., and Cavicchioli, R.
(1998) Implications of rRNA operon copy number and ribosome content in the marine oligotrophic ultramicrobacterium Sphingomonas sp. strain RB2256. Appl Environ
Microbiol 64: 44334438.
Fogel, G.B., Collins, C.R., Li, J., and Brunk, C.F. (1999)
Prokaryotic genome size and SSU rDNA copy number:
Estimation of microbial relative abundance from a mixed
population. Microb Ecol 38: 93113.
Fuhrman, J.A., McCallum, K., and Davis, A.A. (1992) Novel
major Archaebacterial group from marine plankton. Nature
356: 148149.
Gillespie, D.E., Brady, S.F., Bettermann, A.D., Cianciotto,
N.P., Liles, M.R., Rondon, M.R., et al. (2002) Isolation of
antibiotics turbomycin A and B from a metagenomic library
of soil microbial DNA. Appl Environ Microbiol 68: 4301
4306.
Giovannoni, S.J., and Rapp, M.S. (2000) Evolution, diversity
and molecular ecology of marine prokaryotes. In Microbial
Ecology of the Oceans. Kirchman, D.L. (ed.). New York,
USA: Wiley-Liss, pp. 4784.
Glckner, F.O., Fuchs, B.M., and Amann, R. (1999) Bacterioplankton compositions of lakes and oceans: a first comparison based on fluorescence in situ hybridization. Appl
Environ Microbiol 65: 37213726.
Glckner, F.O., Zaichikov, E., Belkova, N., Denissova, L.,
Pernthaler, J., Pernthaler, A., and Amann, R. (2000) Comparative 16S rRNA analysis of lake bacterioplankton
reveals globally distributed phylogenetic clusters including
an abundant group of actinobacteria. Appl Environ Microbiol 66: 50535065.
Gonzalez, J.M., and Moran, M.A. (1997) Numerical dominance of a group of marine bacteria in the alpha- subclass
of the class Proteobacteria in coastal seawater. Appl Environ Microbiol 63: 42374242.
Hernes, P.J., and Benner, R. (2002) Transport and diagenesis of dissolved and particulate terrigenous organic matter
in the North Pacific Ocean. Deep-Sea Res Part II Top
Stud Oceanogr 49: 21192132.
Karner, M., and Fuhrman, J.A. (1997) Determination of active
marine bacterioplankton: a comparison of universal 16S

rRNA probes, autoradiography, and nucleoid staining. Appl

Environ Microbiol 63: 12081213.
Karner, M.B., DeLong, E.F., and Karl, D.M. (2001) Archaeal
dominance in the mesopelagic zone of the Pacific Ocean.
Nature 409: 507510.
Kirchman, D.L. (2002) The ecology of CytophagaFlavobacteria in aquatic environments. FEMS Microbiol Ecol 39:
91100.
Kirchman, D.L., Yu, L.Y., and Cottrell, M.T. (2003) Diversity
and abundance of uncultured Cytophaga-like bacteria in
the Delaware Estuary. Appl Environ Microbiol 69: 6587
6596.
Klappenbach, J.A., Dunbar, J.M., and Schmidt, T.M. (2000)
rRNA operon copy number reflects ecological strategies of
bacteria. Appl Environ Microbiol 66: 13281333.
Knietsch, A., Waschkowitz, T., Bowien, S., Henne, A., and
Daniel, R. (2003) Metagenomes of complex microbial consortia derived from different soils as sources for novel
genes conferring formation of carbonyls from short-chain
polyols on Escherichia coli. J Mol Microbiol Biotechnol 5:
4656.
Lane, D.J. (1991) 16S/23S rRNA sequencing. In Nucleic Acid
Techniques in Bacterial Systematics. Stackebrandt, E. and
Goodfellow, M. (eds). Chichester, UK: Wiley and Sons,
pp. 115175.
Liles, M.R., Manske, B.F., Bintrim, S.B., Handelsman, J., and
Goodman, R.M. (2003) A census of rRNA genes and linked
genomic sequences within a soil metagenomic library.
Appl Environ Microbiol 69: 26842691.
Ludwig, W., Strunk, O., Westram, R., Richter, L., Meier, H.,
Yadhukumar, et al. (2004) ARB: a software environment
for sequence data. Nucleic Acids Res 32: 13631371.
Manz, W., Amann, R., Ludwig, W., Wagner, M., and Schleifer, K.H. (1992) Phylogenetic oligodeoxynucleotide probes
for the major subclasses of Proteobacteria problems and
solutions. Syst Appl Microbiol 15: 593600.
Manz, W., Amann, R., Ludwig, W., Vancanneyt, M., and
Schleifer, K.H. (1996) Application of a suite of 16S rRNAspecific oligonucleotide probes designed to investigate
bacteria of the phylum CytophagaFlavobacterBacteroides in the natural environment. Microbiology 142: 1097
1106.
Massana, R., Taylor, L.J., Murray, A.E., Wu, K.Y., Jeffrey,
W.H., and DeLong, E.F. (1998) Vertical distribution and
temporal variation of marine planktonic archaea in the
Gerlache Strait, Antarctica, during early spring. Limnol
Oceanogr 43: 607617.
Massana, R., DeLong, E.F., and Pedros-Alio, C. (2000) A few
cosmopolitan phylotypes dominate planktonic archaeal
assemblages in widely different oceanic provinces. Appl
Environ Microbiol 66: 17771787.
Morris, R.M., Rapp, M.S., Connon, S.A., Vergin, K.L., Siebold, W.A., Carlson, C.A., and Giovannoni, S.J. (2002)
SAR11 clade dominates ocean surface bacterioplankton
communities. Nature 420: 806810.
Muyzer, G., Teske, A., Wirsen, C.O., and Jannasch, H.W.
(1995) Phylogenetic-relationships of Thiomicrospira species and their identification in deep-sea hydrothermal vent
samples by denaturing gradient gel-electrophoresis of 16S
rDNA fragments. Arch Microbiol 164: 165172.
Neef, A. (1997) Anwendung der in situ-Einzelzell-

2005 Society for Applied Microbiology and Blackwell Publishing Ltd, Environmental Microbiology, 7, 18831895

Metagenomic analysis of freshwater bacteria 1895

Identifizierung von Bakterien zur Populationsanalyse in
Komplexen Mikrobiellen Biozonosen. Munich, Germany:
Technische Universitat Munchen.
Opsahl, S., Benner, R., and Amon, R.M.W. (1999) Major flux
of terrigenous dissolved organic matter through the Arctic
Ocean. Limnol Oceanogr 44: 20172023.
OSullivan, L.A., Weightman, A.J., and Fry, J.C. (2002) New
degenerate Cytophaga-Flexibacter-Bacteroides-specific
16S ribosomal DNA-targeted oligonucleotide probes reveal
high bacterial diversity in River Taff epilithon. Appl Environ
Microbiol 68: 201210.
Pettit, A.N. (1982) Cramer-von Misess statistic. In Encyclopedia of Statistical Sciences. Kotz, S., Johnson, N.L., and
Read, C.B. (eds). New York, USA: Wiley, pp. 220221.
Quaiser, A., Ochsenreiter, T., Klenk, H.P., Kletzin, A.,
Treusch, A.H., Meurer, G., et al. (2002) First insight into
the genome of an uncultivated crenarchaeote from soil.
Environ Microbiol 4: 603611.
Rapp, M.S., and Giovannoni, S.J. (2003) The uncultured
microbial majority. Annu Rev Microbiol 57: 369394.
Rapp, M.S., Gordon, D.A., Vergin, K.L., and Giovannoni,
S.J. (1999) Phylogeny of Actinobacteria small subunit
(SSU) rRNA gene clones recovered from marine bacterioplankton. Syst Appl Microbiol 22: 106112.
Rapp, M.S., Connon, S.A., Vergin, K.L., and Giovannoni,
S.J. (2002) Cultivation of the ubiquitous SAR11 marine
bacterioplankton clade. Nature 418: 630633.
Roller, C., Wagner, M., Amann, R., Ludwig, W., and Schleifer,
K.H. (1994) In-situ probing of Gram-positive bacteria with
high DNA G+C content using 23S-ribosomal-RNA-targeted
oligonucleotides. Microbiology 140: 28492858.
Rondon, M.R., August, P.R., Bettermann, A.D., Brady, S.F.,
Grossman, T.H., Liles, M.R., et al. (2000) Cloning the soil
metagenome: a strategy for accessing the genetic and
functional diversity of uncultured microorganisms. Appl
Environ Microbiol 66: 25412547.
Sabehi, G., Massana, R., Bielawski, J.P., Rosenberg, M.,
Delong, E.F., and Beja, O. (2003) Novel proteorhodopsin
variants from the Mediterranean and Red Seas. Environ
Microbiol 5: 842849.

Singleton, D.R., Furlong, M.A., Rathbun, S.L., and Whitman,

W.B. (2001) Quantitative comparisons of 16S rRNA gene
sequence libraries from environmental samples. Appl Environ Microbiol 67: 43744376.
Stackebrandt, E., and Goebel, B.M. (1994) A place for DNA
DNA reassociation and 16S ribosomal-RNA sequenceanalysis in the present species definition in bacteriology.
Int J Syst Bacteriol 44: 846849.
Stein, J.L., Marsh, T.L., Wu, K.Y., Shizuya, H., and DeLong,
E.F. (1996) Characterization of uncultivated prokaryotes:
Isolation and analysis of a 40-kilobase-pair genome fragment from a planktonic marine archaeon. J Bacteriol 178:
591599.
Thompson, J.R., Marcelino, L.A., and Polz, M.F. (2002) Heteroduplexes in mixed-template amplifications: formation,
consequence and elimination by reconditioning PCR.
Nucleic Acids Res 30: 20832088.
Topp, E. (2003) Bacteria in agricultural soils: Diversity, role
and future perspectives. Can J Soil Sci 83: 303309.
Weller, R., Glckner, F.O., and Amann, R. (2000) 16S rRNAtargeted oligonucleotide probes for the in situ detection
of members of the phylum Cytophaga-FlavobacteriumBacteroides. Syst Appl Microbiol 23: 107114.
von Wintzingerode, F., Gobel, U.B., and Stackebrandt, E.
(1997) Determination of microbial diversity in environmental samples: pitfalls of PCR-based rRNA analysis. FEMS
Microbiol Rev 21: 213229.
Zaitlin, B., Watson, S.B., Dixon, J., and Steel, D. (2003)
Actinomycetes in the Elbow River Basin, Alberta. Can
Water Quality Res J Can 38: 115125.
Zwart, G., Hiorns, W.D., Methe, B.A., Van Agterveld, M.P.,
Huismans, R., Nold, S.C., et al. (1998) Nearly identical 16S
rRNA sequences recovered from lakes in North America
and Europe indicate the existence of clades of globally
distributed freshwater bacteria. Syst Appl Microbiol 21:
546556.
Zwart, G., Crump, B.C., Agterveld, M., Hagen, F., and Han,
S.K. (2002) Typical freshwater bacteria: an analysis of
available 16S rRNA gene sequences from plankton of
lakes and rivers. Aquat Microb Ecol 28: 141155.

2005 Society for Applied Microbiology and Blackwell Publishing Ltd, Environmental Microbiology, 7, 18831895