Chapter 5
Use of Different PCR Primer-Based
Strategies for Characterization of
Natural Microbial Communities
James I. Prosser, Shahid Mahmood, and Thomas E. Freitag
5.1 INTRODUCTION
Ammonia oxidizers convert ammonia to nitrite in the first
step of nitrification, with subsequent oxidation of nitrite
to nitrate. The process is usually limited by ammonia
oxidation, and ammonia oxidizers therefore control an
essential step in the global nitrogen cycle. Ammonia
oxidizers exemplify, in many ways, the enormous benefits
of molecular techniques in microbial ecology. They grow
slowly under laboratory conditions, are difficult to purify,
and have few distinguishing phenotypic characteristics.
Community or population ecology of ammonia oxidizers
is therefore effectively impossible using traditional
techniques alone. Quantification of abundance relied
on the most probable number technique, which com-
pounds many of the well-accepted limitations of dilution
plate counting, including selective media and growth
conditions, lack of enumeration of dormant organisms,
increases in abundance with incubation time, and addi-
tional statistical variability introduced through application
of probability distributions. As a consequence, despite
their enormous importance in the global nitrogen cycle,
traditional techniques provided little reliable information
on the ecology, community structure, diversity, and
abundance of ammonia oxidizers.
The introduction of 16S rRNA gene-based tech-
niques for analysis of communities of ammonia-oxidizing
bacteria (AOB) [Stephen et al., 1996] demonstrated, as
for other microbial groups, the enormous diversity within
Handbook of Molecular Microbial Ecology, Volume I: Metagenomics and Complementary Approaches, First Edition. Edited by Frans J. de Bruijn.
© 2011 Wiley-Blackwell. Published 2011 by John Wiley & Sons, Inc.
natural communities, links between community composi-
tion and environmental conditions, and indications of their
activity, through RNA-targeted studies. These studies
were complemented by analysis of a key functional gene,
amoA, encoding subunit A of the ammonia monooxy-
genase enzyme. This enabled analysis of community
structure and transcriptional activity and quantification
of 16S rRNA, and amoA genes enabled determination of
ammonia oxidizer abundance. The importance of molec-
ular techniques was further demonstrated, dramatically,
by the discovery of archaeal ammonia oxidizers [Treusch
et al., 2005; Venter et al., 2004; Könneke et al., 2005],
which are even more difficult to cultivate, leading to
research into the respective roles of bacterial and archaeal
ammonia oxidisers [Prosser and Nicol, 2008]. Distinction
of their activities in natural environments currently relies
solely on molecular techniques.
Ammonia oxidizers reach high abundance in wastew-
ater treatment systems, where use of fluorescence in situ
hybridization (FISH) of 16S rRNA probes is feasible. Use
of FISH is severely limited in soil, because of background
fluorescence and high levels of particulate matter; it is also
severely limited in both soil and natural aquatic envi-
ronments, because of low cell abundance and possibly
by lower transcriptional activity, reducing signal inten-
sity. FISH also does not capture the high diversity of
natural communities. The majority of molecular studies
of AOB ecology therefore involve amplification of 16S
rRNA or amoA genes or gene transcripts from extracted
41
42 Chapter 5 PCR-Primer Based Strategies for Characterization
environmental DNA or RNA, followed by quantification,
using competitive [Phillips et al., 2000] or real-time [e.g.,
Hermansson and Lindgren, 2001] qPCR, sequencing [e.g.,
Stephen et al., 1996], or fingerprint analysis [e.g., Stephen
et al., 1998].
Molecular analysis of ammonia oxidizers removes the
major disadvantages and biases of cultivation-based tech-
niques, associated with selective growth and difficulties
in cultivating representative organisms and identification
and classification of isolates. Molecular techniques do,
however, introduce other biases [Prosser et al., 2010], but
increasing acceptance of molecular techniques during the
last decade has reduced the degree to which bias is con-
sidered, assessed, and controlled for. One potential bias
is introduced by analysis of different genes and use of
different primers. With the exception of gammaproteobac-
terial Nitrosococcus strains, which are restricted to marine
environments, known AOB belong to the betaproteobacte-
ria. Phylogenetic analysis of betaproteobacterial ammonia
oxidizer 16S rRNA genes places them within two genera,
Nitrosospira and Nitrosomonas, each containing several
subgroups [Purkhold et al., 2000]. This phylogeny is gen-
erally congruent with that obtained by analysis of amoA
genes. Nevertheless, use of 16S rRNA and amoA genes
is likely to generate different views of bacterial ammonia
oxidizer diversity, even when using primers designed to
target all betaproteobacterial ammonia oxidizers.
Accumulation of 16S rRNA gene sequences from
pure and enrichment cultures and from sequences
amplified directly from environmental nucleic acids
promoted the design of RNA-targeted probes, specific
to all bacterial ammonia oxidizers and subgroups, for
FISH studies. A similar range of primers were designed
for PCR amplification of bacterial ammonia oxidizer
16S rRNA genes. Purkhold et al. [2000] concluded that
βAMO primers [McCaig et al., 1994] were most inclusive
of available AOB database sequences, but that specificity
was incomplete. The numbers of nontarget sequences
amplified by βAMO primers depend on the environment
under study and AOB abundance, but their use requires
screening of amplicons to eliminate non-AOB sequences.
The greatest specificity was found for CTO primers
[Kowalchuk et al., 1997], but these did not target several
members of the Nitrosomonas oligotropha cluster and
have some mismatches with the Nitrosomonas communis
cluster. In addition, in environments with very low
AOB abundance, CTO primers amplify many non-AOB
sequences [Schmidt et al., 2007].
Fingerprinting analysis of AOB communities
has been carried out using denaturing gradient gel
electrophoresis (DGGE) [Kowalchuk et al., 1997],
temperature gradient gel electrophoresis (TGGE)
[Fouratt et al., 2003], terminal-restriction fragment
length polymorphism (T-RFLP) [Mintie et al., 2003],
and single-strand conformation polymorphism (SSCP)
[Backman et al., 2003]. These approaches have, in
turn, utilized a range of PCR strategies most frequently
amplifying AOB 16S rRNA genes using βAMO and/or
CTO primers, sometimes in combination with primers
designed to amplify 16S rRNA genes of all bacteria.
Each primer set generates products of different size,
preventing direct comparison of community structure
obtained with different primer strategies and assessment
of primer bias. Mahmood et al. [2006] addressed this
problem by analysis of natural AOB communities using
several commonly used primer strategies, with both direct
and nested PCR amplification and with a common final
PCR step to enable direct comparison.
5.2 METHODS
Both soil and marine sediment samples were analysed and
details of sites and of all methods are provided in Mah-
mood et al. [2006]. The soil was a managed grassland,
subject to fertilizer addition, and marine samples were
obtained from sediment of a marine loch (Loch Duich,
Scotland). AOB had been characterized previously in sev-
eral studies of these sites [Webster et al., 2005; Freitag and
Prosser, 2003]. DNA was extracted from soil and sediment
samples using standard techniques; then it was purified,
and 16S rRNA genes were amplified by PCR using five
strategies, illustrated in Figure 5.1.
a. Direct amplification with βAMO primers
(βAMO143f–βAMO1315r [Stephen et al., 1996]).
b. Direct amplification with AOB-specific CTO primers
(CTO189f–CTO654r [Kowalchuk et al., 1997]).
c. Nested PCR amplification with first-round amplifi-
cation using βAMO143f-βAMO1315r and secondary
amplification with CTO189f–CTO654r.
d. Nested PCR amplification with first-round amplifica-
tion using βAMO143f–βAMO1315r and secondary
amplification with CTO189f in conjunction with the
general bacterial primer Pf1053r [Edwards et al.,
1989].
e. Nested PCR amplification with first-round amplifi-
cation using universal bacterial primers 27f–1492r
[Lane, 1991] and secondary amplification with
CTO189f–CTO654r.
Amplicons generated by each approach were ampli-
fied further using 357f–GC–518r primers [Muyzer et al.,
1993], which are considered to be universal bacterial
 5.3 Results
Figure 5.1 Five PCR strategies (a–e) used to amplify ammonia
oxidising bacterial 16S rRNA genes from DNA extracted from soil
and marine sediment samples. Strategies a and b involve direct
amplification with βAMO primers (βAMO143f and βAMO1315r
[Stephen et al., 1996]) or CTO primers (CTO189f-CTO654r
[Kowalchuk et al., 1997]), which are designed to target
ammonia-oxidizing bacteria. Strategies c, d, and e involved nested
PCR with primary amplification with either βAMO primers (c and d)
or 27f–1492r primers [Lane, 1991], which target all bacteria.
Secondary amplification involves either CTO primers (c, e) or a
combination of CTO189f [Kowalchuk et al., 1997] and Pf1053r
[Edwards et al., 1989], which targets all bacteria. Products from each
strategy were amplified using the universal bacterial primer
357f-GC-518r, to generate products of the same size for DGGE
analysis. (Modified from Mahmood et al. [2006] with permission.)
primers. They were not selective for AOB and served
only to generate products of equal size from each of the
five strategies, which could then be characterized and
compared using DGGE analysis.
Details of all PCR conditions and DGGE analysis are
given in Mahmood et al. [2006]. Gels were silver-stained
and scanned, and band intensity was quantified. In addi-
tion, DNA from a number of excised DGGE bands was
extracted, reamplified, purified, and sequenced to check
for bias between gels, to ensure that only AOB sequences
had been amplified and to check that bands migrating to
43
the same position had identical sequences. Sequences were
obtained for the majority of bands with high relative inten-
sity and for some with low relative intensity.
5.3 RESULTS
5.3.1 Analysis of Soil Ammonia
Oxidizer Communities
DGGE patterns from soil samples contained 15 distinct
bands, and reproducibility between replicate samples was
high, with no detectable difference for any particular
PCR strategy. The influence of different PCR strategies
can be seen in Figure 5.2, with quantitative analysis of
DGGE gels in Table 5.1. Strategies a and d gave similar
profiles that were dominated by three bands consisting
of non-AOB sequences related to Variovorax . Primary
PCR amplification with these two strategies employed
βAMO143f–βAMO1315r primers, which are known to
amplify non-AOB sequences in some environments, and
CTO primers, which amplify non-AOB sequences at low
AOB abundance. Although it is not relevant to analysis
of AOB communities in this case, the relative intensities
of some bands (S5–S8, S15) was greater for strategy d,
and this difference may be applicable to communities
where AOB sequences only are amplified. Although
preferentially amplifying non-AOB sequences, a and d
were the only strategies that detected Nitrosospira-bands
S6 and S7, which were not amplified by CTO-primer-
based strategies. Strategy a did not detect a Nitrosospira
briensis-like band (S15), possibly because of lower
abundance than Variovorax -like sequences.
Strategies b, c, and e generated similar DGGE profiles
with a relatively high number of common bands, most of
which consisted of Nitrosospira-related sequences, and
these were the only strategies to amplify Nitrosospira-like
bands S9, S10, S13, and S14. Strategies b, c, and e did
not amplify two of the Variovorax -like bands (S1 and S2)
but amplified Variovorax -like band S3, although at lower
relative intensity than a and d. Strategies c and e gen-
erated similar DGGE profiles. Both involved secondary
amplification with CTO189f–CTO654r primers, follow-
ing amplification with either βAMO143f–βAMO1315r or
bacterial 27f–1492r primers. All CTO-based approaches
led to high relative intensity of Nitrosospira-like band
S8, which was less intense in strategy a. This may
have resulted from relatively high abundance within the
AOB community, but lower abundance than Variovo-
rax -like sequences. Band S15 (Nitrosospira briensis-like
sequence) was absent in approach a, probably due to lower
copy numbers than Variovorax -like sequences types.
44 Chapter 5 PCR-Primer Based Strategies for Characterization

Table 5.1 Numbers of Bands and Similarity Coefficients (in parentheses) for DGGE Profiles of Ammonia Oxidizer 16S
rRNA Genes Amplified from (a) Soil and (b) Marine Sediment Samples Using PCR Strategies a–ea
PCR Strategy a b c d e
(a) Soil
a 5 (1.00)
b 2 (0.40) 8 (1.00)
c 2 (0.40) 8 (1.00) 8 (1.00)
d 2 (0.44) 4 (0.73) 4 (0.73) 7 (1.00)
e 2 (0.40) 8 (1.00) 8 (1.00) 4 (0.67) 8 (1.00)
(b) Marine Sediments
a 8 (1.00)
b 8 (0.94) 9 (1.00)
c 7 (0.82) 8 (0.89) 10 (1.00)
d 8 (0.89) 8 (0.89) 9 (0.95) 10 (1.00)
e 7 (0.82) 8 (0.89) 10 (1.00) 9 (0.95) 10 (1.00)
a
Analysis was restricted to bands that were confirmed, by sequence analysis, to be from ammonia oxidizing bacteria. Values presented are the
number of sequenced bands found in each approach (values on diagonal) or shared among different approaches

5.3.2 Analysis of Marine Ammonia
Oxidizer Communities
DGGE patterns from marine sediment samples contained
11 distinct bands. Again, reproducibility between repli-
cate samples was high, with no detectable difference for
any particular PCR strategy, and the influence of different
PCR strategies can be seen in Figure 5.3, with quantita-
tive analysis in Table 5.1. All PCR strategies generated
similar DGGE profiles for marine sediment samples, and
very few non-AOB sequences were amplified. The major-
ity of sequences were related to uncultured Nitrosospira
and Nitrosomonas sequences, in addition to some uniden-
tified betaproteobacterial AOB, and most sequences were
closely related to those obtained in cloning studies of sedi-
ments from the same site using βAMO143f–βAMO1315r
primers [Freitag and Prosser, 2003; Mortimer et al., 2004].
Most major bands were found with all five strategies,
except for Nitrosospira-like bands M6 and M9, which
were only detected by strategies involving the complete
CTO primer set (c, e) or, with lower relative intensity,
using the CTO189f-primer (strategy d). Some other bands
(M1, M4, and M5) detected using strategies a, b, and d
were less evident when using strategies c and e. Strate-
gies a and d and c and e generated similar profiles, and
UPGMA analysis indicated greater similarities between
amplification strategies than in analyzing soils samples.
To assess the influence of the final, tertiary amplifi-
cation step, both soil and marine sediment samples were
analyzed using only primary and secondary amplification
steps of strategies c and e. This generated fewer distinct
bands (4–5), several of which did not comigrate with
AOB cluster controls.
5.4 DISCUSSION
The study of Mahmood et al. [2006] provides impor-
tant information for those using PCR-based analysis of
AOB and also generic information of relevance to any
PCR-based study. It does not, however, determine which
method is “best,” because the exact composition of the
soil and marine sediment studies was not known. Deter-
mination of the accuracy of analysis would require use
of model communities, inoculation of sterile environmen-
tal samples with known proportions of different sequence
types, or additional, independent techniques that provide
greater confidence. The practical difficulties in achieving
this, while maintaining relevance to complex soil and sed-
iment communities, preclude such analysis.
The study confirmed the disadvantages of using
βAMO143f-βAMO1315r primers for analysis of AOB
communities, particularly in the soil. Soil DGGE profiles
generated using strategies a and d were dominated by
non-AOB (presumably), Variovorax sequence types.
These organisms were likely more abundant than AOB
in this soil and were amplified through close sequence
similarity, with only one mismatch against the forward
and reverse βAMO primers [Mahmood et al., 2006].
Similar problems have been reported for other soil AOB
studies. For example, Stephen et al. [1996] reported
amplification of betaproteobacterial sequences related to
 5.4 Discussion 45

a b c d e a b c d e
M 1 2 1 2 1 2 1 2 1 2 M M 1 2 1 2 1 2 1 2 1 2 M

VII VII VII S1 VII

S2
VI VI VI VI
M1
S3
V M2 V V V
S4
M3 S5
S6
S7
M4
M5 IV S8 IV
IV IV
M6 S9
I M7 I I S10 I
II II II S11 II
M8 S12
S13
M9 S14
III M10 III III S15 III
M11
(A) (A)

2 2
e e
1 1
2 2
c c
1 1
2 2
b b
1 1
2 d 2
d
1 1
2 2
a a
1 1

(B) 0.80 0.85 0.90 0.95
Figure 5.2 DGGE analysis of soil ammonia-oxidizing bacteria
(AOB) using PCR strategies a–e to amplify AOB 16S rRNA genes
from extracted DNA. (a) DGGE profiles of genes amplified from
duplicate samples (lanes 1 and 2 for each strategy). White
arrowheads indicate major bands whose presence, absence, or
relative intensity differed between PCR-amplification strategies.
Black arrowheads indicate bands that were PCR artifacts from which
a sequence could not be generated. Bands S1–S15 were excised and
sequenced.The band above band S8 is an artifact, possibly arising
from excessive loading of strong PCR amplification products.
Asterisks indicate non-AOB 16S rRNA gene sequences. Markers in
lane M consisted of seven AOB cluster controls I–VII: I, EnvB1-8
(Nitrosospira); II, pH4.2A/27 (Nitrosospira); III, pH4.2A/4
(Nitrosospira); IV, pH7B/C3 (Nitrosospira); V, EnvA1-21
(Nitrosomonas); VI, EnvC1-19 (Nitrosomonas); VII, N. europaea
(Nitrosomonas). (b) UPGMA (unweighted pairwise grouping method
with mathematical averages) dendrogram showing similarities
between DGGE banding profiles based on presence or absence of all
bands in each lane. (From Mahmood et al. [2006] with permission.)
1.00 0.50 0.60 0.70 0.80 0.90 1.00
(B)
Figure 5.3 DGGE analysis of ammonia oxidising bacteria (AOB)
from marine sediment samples using PCR strategies a–e to amplify
AOB 16S rRNA genes from extracted DNA. (a) DGGE profiles of
genes amplified from duplicate samples (lanes 1 and 2 for each
strategy). Bands M1–M11 were excised and sequenced, and other
information is as described in the legend for Figure 5.2. (b) UPGMA
(unweighted pairwise grouping method with mathematical averages)
dendrogram showing similarities between DGGE banding profiles
based on presence or absence of all bands in each lane. (From
Mahmood et al. [2006] with permission.)
Comamonas testosteroni and Rubrivivax gelatinosus,
particularly in soil clone libraries, but also in marine
sediments, and Bruns et al. [1999] found nonspecific
amplification products in clone libraries generated from
several soils. This problem appears to be less important
for marine environments [Freitag and Prosser, 2004;
46 Chapter 5 PCR-Primer Based Strategies for Characterization
Mahmood et al., 2006], but use of βAMO primers for
characterization of AOB communities requires screen-
ing of clones to ensure that non-AOB sequences are
eliminated from analysis.
CTO primers provided greater specificity, but are
less inclusive than βAMO primers and can amplify
non-AOB sequences when these are present at low
abundance [Schmidt et al., 2007]. This was not the
case in the soil and sediment samples investigated by
Mahmood et al. [2006] and CTO-primer-based primary
amplification strategies generated similar numbers of
bands in DGGE profiles. As indicated above, the number
of bands generated is not necessarily an indicator of
the quality or accuracy of a particular strategy. Many
phylotypes will be below the detection limit, and the
number of bands detected will depend on evenness
rather than richness. Band number will increase with
evenness and decrease with dominance, and “accurate”
detection of bands requires independent, alternative, and
reliable techniques or analysis of model communities
with known relative abundances of different phylotypes.
It also requires acceptance of relative band intensity
as a quantitative measure of relative abundance, which
remains a source of debate. Confidence in analysis
methods is increased when different strategies generate
similar results, reducing concerns about PCR or primer
bias. In this respect, marine sediment profiles showed
greater consistency with different strategies than soil, but
this is mainly a reflection of the increased number of
non-AOB sequences amplified from soil.
Nested PCR-based approaches (c, e) are frequently
employed, and necessary, for amplification of organisms
present at low abundance, particularly in the presence
of naturally occurring PCR inhibitors. These are often
criticized with respect to their ability to quantify relative
abundance (through relative band intensity) because of the
increased potential to introduce Taq-polymerase reading
errors [Barnes, 1992; Tindal and Kunkel, 1988]. However,
these criticisms are rarely supported by strong evidence,
and the reproducible DGGE banding patterns reported by
Mahmood et al. [2006] and high similarities of excised
bands sequences and clone library sequences suggest that
such errors were not significant. There may, however, be
carryover of highly abundant, nontarget sequences from
primary to secondary amplifications, possibly explaining
detection of Variovorax -related sequences (band S3) in
strategy c, despite use of AOB-specific CTO primers for
secondary amplification. Choice of PCR amplification
strategy is also influenced by the length of product
generated, and a balance is required between sequence
length required for phylogenetic analysis and sequences
with melting characteristics suitable for DGGE analysis
[Wu et al., 1998]. Thus, βAMO primers amplify 1.1-bp
products, which are suitable for phylogenetic analysis
but not DGGE. In contrast, CTO189f-CTO654r products
are insufficient for phylogenetic analysis, but better for
DGGE.
5.5 CONCLUSION
Design and choice of PCR primer strategies when target-
ing complex communities is often limited by the avail-
ability of sequences that are specific for target sequences,
that exclude nontarget sequences, that demonstrate effi-
cient PCR reaction kinetics, and that generate products
of the required length for sequencing or fingerprinting.
The potential for comparison of different primers strate-
gies is therefore limited by lack of alternative primers, but
their availability for AOB enabled comparison by Mah-
mood et al. [2006] of DGGE profiles obtained by different
strategies for two environments, facilitated by use of a
common primer set for a final amplification stage. Major
differences resulted from lack of specificity of one primer
set, which was more evident for analysis of soil samples,
but otherwise different primers strategies generated sim-
ilar DGGE profiles and amplified similar AOB sequence
types. Potential lack of specificity and potential constraint
to known target groups, even if not previously estab-
lished, requires caution, particularly when initiating study
of previously uncharacterised environments. Confidence
in results is increased by similarities between sequences of
excised DGGE bands, after two or three rounds of amplifi-
cation, and those obtained from clone libraries employing
a single PCR step, emphasizing the desirability of using
more than one technique for community analysis.
Acknowledgments
This work was carried out as part of a Marie Curie Indi-
vidual Fellowship (contract No. QLK3-CT-2000-52161)
awarded by the European Commission.
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