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Applied and Environmental Microbiology, July 2000, p. 2873-2881, Vol. 66, No. 7
Institute of Marine
Sciences1 and Biology
Department,3 University of California, Santa
Cruz, California, and Department of Oceanography, Texas A & M
University, College Station, Texas 778432
Received 11 October 1999/Accepted 2 May 2000
Ammonia-oxidizing bacteria were detected by PCR amplification of
DNA extracted from filtered water samples throughout the water column
of Mono Lake, California. Ammonia-oxidizing members of the Mono Lake is an alkaline,
hypersaline, closed-basin lake in central California just east of the
Sierra Nevada Mountains (38°N, 119°W). Exceptionally heavy rainfall
in 1982 and 1983 caused a freshening of the surface layer; the
resulting large-density difference between surface and deep waters
caused the lake to become ectogenically meromictic (15, 18).
The lake became stratified with a chemocline in the vicinity of 15 m (total depth, approximately 30 m) and did not turn over or mix
thoroughly until 1988. In the interval, the difference in salinity
between the deep water and surface layer gradually decreased
(15), due to evaporation and removal of surface water to
supply drinking water to southern California. After 1988, the lake
resumed its normal pattern of annual winter holomixis (15).
The seasonal density stratification leads to stratification in oxygen
and other parameters, such as nitrogenous nutrients (16),
and this stratification is expected to be reflected in the depth
distribution of microbial activities. Both ammonium and methane
accumulate in the deep layer (25); the major biological sinks for these compounds are thought to be oxidation by obligately aerobic chemolithotrophic bacteria in the surface layer and, for methane, anaerobic oxidation in the deep layer. The depth distributions of methane and ammonia oxidation, measured by radiotracer experiments, were previously reported (19).
Ammonia-oxidizing bacteria are responsible for the oxidation of ammonia
to nitrite, the first step in nitrification, in terrestrial and aquatic
habitats of all kinds and are found in the Considering Mono Lake's recent history of salinity variation, it was
not obvious what kinds of ammonia oxidizers might be responsible for
the nitrification rates observed in the lake (19). While the
lake is in a "normal" temperate phase, the usual freshwater bacteria, such as Nitrosomonas and Nitrosospira
strains in the Water samples.
Water column samples were collected near a
permanently moored buoy at a station near the middle of the lake (28-m
depth) in April and July 1995. Water samples from discrete depths were
collected with a 5-liter polyvinyl chloride sampling bottle
(25) and stored on ice in 1-liter brown polyethylene bottles
prior to filtration. A peristaltic pump was used to circulate water
samples (400 to 700 ml) through Sterivex (0.22-µm-pore-size) filter
heads (Millipore). Filters were stored frozen in 1.8 ml of lysis buffer
(40 mM EDTA, 50 mM Tris, 0.74 M sucrose [pH 8.3]) prior to DNA extraction.
Bacterial strains and DNA extraction.
Bacterial strains were
maintained in a pure culture as previously described (45),
using freshwater (33) or seawater (48) medium as
appropriate. High-molecular-weight, bacterial genomic DNA from various
strains in the culture collection (required for controls and for
standardization in the denaturing gradient gel electrophoresis [DGGE]
analysis) was obtained from cells harvested from 1-liter cultures and
purified as previously described (41).
0099-2240/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.
Analysis of Ammonia-Oxidizing Bacteria from
Hypersaline Mono Lake, California, on the Basis of 16S rRNA
Sequences
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ABSTRACT
Top
Abstract
Introduction
Materials and Methods
Results and Discussion
References
subdivision of the division Proteobacteria (-subdivision Proteobacteria) were detected using previously
characterized PCR primers; target sequences were detected by direct
amplification in both surface water and below the chemocline.
Denaturing gradient gel electrophoresis analysis indicated the presence
of at least four different -subdivision ammonia oxidizers in some
samples. Subsequent sequencing of amplified 16S rDNA fragments verified the presence of sequences very similar to those of cultured
Nitrosomonas strains. Two separate analyses, carried out
under different conditions (different reagents, locations, PCR
machines, sequencers, etc.), 2 years apart, detected similar ranges of
sequence diversity in these samples. It seems likely that the
physiological diversity of nitrifiers exceeds the diversity of their
ribosomal sequences and that these sequences represent members of the
Nitrosomonas europaea group that are acclimated to
alkaline, high-salinity environments. Primers specific for
Nitrosococcus oceanus, a marine ammonia-oxidizing bacterium
in the 
subdivision of the Proteobacteria, did not
amplify target from any samples.
INTRODUCTION
Top
Abstract
Introduction
Materials and Methods
Results and Discussion
References
and subdivisions of
the division Proteobacteria. The number of species of
ammonia oxidizers that have been described is relatively small (21), although a greater diversity of uncultured strains has been detected in various environments (10, 12, 22, 23, 36, 37, 41,
43).
subdivision, might be favored, while
Nitrosococcus oceanus strains, representatives of the
-subdivision ammonia oxidizers, might be favored in the lake's
current saline state. This question was investigated by using PCR and
sequence analysis focusing on the 16S rRNA gene in nitrifying bacteria.
MATERIALS AND METHODS
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Abstract
Introduction
Materials and Methods
Results and Discussion
References
PCR amplification, cloning, and sequencing.
The PCR primers
used here have been described previously, and their positions relative
to that of the E. coli 16 S ribosomal DNA (rDNA) are
listed in Table 1. General bacterial
primers (EUB1 and EUB2 [24]) were used to verify that
the sample DNA was of PCR amplification quality. The NitA and NitB
primers (41) were designed to be specific for all nine
described ammonia oxidizers within the subdivision of the
Proteobacteria (11, 50), and the NitD primer is
100% specific for Nitrosomonas europaea (47). The NOC1 and NOC2 primers (42) are specific for N. oceanus. Amplification conditions for all of these primer sets
were optimized according to the method of Cobb and Clarkson
(7). The primer sequences and optimal assay conditions
(slightly different from previously published conditions) are shown in
Table 1. In addition to those reagents listed in the table, the
reaction mixture contained DNA template (1 µl of purified DNA, from
either a culture or an extract of natural sample) and 2.5 U of
Taq polymerase in a total reaction volume of 100 µl. When
two-stage amplification was performed, 1 µl of the initial
(first-stage) reaction mixture was used as the template in the second
amplification without further purification.
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Hybridization.
To verify that the amplified sequences
represented rRNA genes, the amplified fragments were analyzed by
hybridization with the EUB amplification product of N. europaea DNA, which was previously verified as being of rDNA
origin (41). The PCR amplified DNA was purified (Geneclean;
BIO 101, San Diego, Calif.), labeled with digoxigenin by random priming
according to the Genius protocol (Boehringer Mannheim), purified again,
and used at a final concentration of 100 ng ml
1. The test
DNAs were bound to a nylon membrane via a slot blotting procedure
(46). The hybridization conditions and procedures for
posthybridization washes and visualization of the probe-target conjugate were those recommended by the manufacturer (Boehringer Mannheim).
DGGE. DGGE was performed using a Bio-Rad mini-Protean II gel system modified for buffer recirculation as described by Myers et al. (28) with a linear gradient with 8% (wt/vol) acrylamide stock solutions (acrylamide-N,N'-methylenebisacrylamide; 37:1) which contained 20% (or 35%) and 70% denaturant (100% denaturant = 7 M urea [Sigma] and 40% formamide [Fisher] deionized with AG501-X8 mixed-bed resin [Bio-Rad]). Electrophoresis was performed at a constant voltage of 105 V and at 60°C for 3 to 4 h. After electrophoresis, the gels were stained in ethidium bromide and visualized with UV transillumination.
Phylogenetic analyses. Sequences were initially compared to the available databases by using the BLAST network service (1) to determine phylogenetic affiliation. Sequences were then compiled in Sequence Navigator (Perkin-Elmer Applied Biosystems) and aligned with representative nitrifier sequences from the Ribosomal Database Project. Evolutionary distance, parsimony, and maximum-likelihood analyses were performed on the aligned sequences with programs from the Phylogenetic Inference package (PHYLIP, version 3.5 [9]).
Nucleotide sequence accession numbers. The rDNA sequences of the 41 unique Mono Lake clones have been deposited in GenBank (accession numbers AF266805 to AF266845).
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RESULTS AND DISCUSSION |
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Top
Abstract
Introduction
Materials and Methods
Results and Discussion
References
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The density and chemical distributions of Mono Lake were
determined at the time of both sample collections. The oxic-anoxic interface was at a 12 to 13 m depth in April 1995 and at 13 to 17 m in July 1995. The characteristics of the water column above and below the oxycline are summarized in Table
2 and described in more detail in the
work of Joye et al. (19). Salinity was not measured at the
time of sampling but can be estimated from subsequent measurements
(17). It ranged from approximately 74 g
kg1 in the surface water to 88 g kg1
in the bottom water. This gradient coincided with a strong thermal gradient, which together were sufficient to maintain stratification beyond the normal winter mixing season (17). Ammonia
oxidation was detected in the surface layer down to 12 m in both
July and April and also at 15 and 17 m in July (19).
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Detection of -subdivision ammonia-oxidizing sequences.
DNA
was successfully amplified with the EUB primers from all samples,
yielding a single band of the correct size (Table
3). Extracts from 6 of the 11 depths from
April and from 8 of the 12 depths from July yielded the correct
(1,080-bp) fragment upon direct amplification with the NitA and NitB
primers. Target sequences were detected both above and below the
chemocline in both months, indicating the presence of nitrifiers, even
in anoxic water. In several cases (at depths of 1 and 10 m in
April and 1, 5, and 10 m in July), only one or two of the two or
three samples collected from each depth amplified directly with the
NitA and NitB primers, and these results were completely reproducible.
Only three and one sample(s) from April and July, respectively,
amplified directly with the NitD and NitB primers, suggesting that the
N. europaea target was relatively more abundant in those
samples.
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-subdivision ammonia-oxidizer target sequences were present at all depths in the
lake, and N. europaea-like sequences were present at most of
those depths. In one case, two-stage amplification with NitD and NitB
was positive, but neither single-stage nor two-stage amplification with
NitA and NitB was positive. This NitD-NitB fragment was not studied
further; it may reflect the greater selectivity of the specific NitD
primer for a rare target that was enriched by EUB amplification
sufficiently to detect with NitD but not with NitA.
The NitA and NitB primers were among the first to be developed for the
-subdivision ammonia oxidizers (41), and a few of the
more recently developed primers have slightly improved specificity (40). The NitA and NitB primers contain slight mismatches in order to amplify all nine known strains. Because of the potential ambiguity resulting from nonspecific amplification of the possible targets for the NitA and NitB primers, successful amplification with
these primers was interpreted as preliminary evidence that -subdivision ammonia oxidizers were present in the samples.
The NitD primer has 100% homology with N. europaea; the
next-closest sequence in the database, at 83% similarity, is
Nitrosomonas eutropha. The analysis by Utaker and Nes
(40) concluded that NitD is absolutely specific for N. europaea, based on the published sequences. Therefore, both
single- and two-stage amplification with the NitD and NitB primers is
strong evidence that -subdivision ammonia oxidizers are present,
specifically N. europaea.
NitA and NitB PCR products that were analyzed by hybridization reacted
strongly with the rRNA probe (results not shown), indicating that the
amplified sequences represented ribosomal sequences from the
environment. These fragments were then further characterized by
more-specific techniques. Because it is possible that nonnitrifier fragments of the right size could be amplified from the complex milieux
of the natural samples, PCR amplification alone is not proof that
-subdivision nitrifiers were present at all depths where
amplification was obtained. Because it seemed unlikely that the
familiar freshwater-terrestrial nitrifier, N. europaea,
would be present in the highly alkaline Mono Lake environment, the
NitD-NitB amplification seemed most suspect. Therefore, we focused on
the depths where NitD-NitB amplification was obtained to evaluate more
specifically whether nitrifier sequences were indeed present. Validation of these sequences as belonging to nitrifiers would tend to
validate the NitA-NitB amplifications, whereas the opposite was not true.
Characterization of nitrifier sequences.
DGGE results
represent another line of evidence strongly indicating the presence of
N. europaea-like sequences among the Mono Lake ammonia
oxidizers. The nine strains of -subdivision ammonia oxidizers each
yielded unique DGGE fragments in gradient gels run under the conditions
described here (22). Bands obtained from Mono Lake samples
appeared most similar to bands obtained from N. europaea and
Nitrosomonas marina; therefore, a mixed standard containing
both N. europaea and N. marina DNA was routinely
run on DGGE gels containing Mono Lake samples (Fig.
1A, lane 3). In most samples, a band that
migrated essentially identically with the band from pure cultures of
N. europaea was observed (Fig. 1). Several samples also
yielded additional bands, indicating the presence of multiple species
of -subdivision ammonia oxidizers in the sample (Fig. 1B).
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-subdivision ammonia oxidizers only. Sequencing
of amplification products indicated the presence of only 1 NitD-NitB
sequence per band.
Six of the seven Mono Lake sequences from the Laboratory A analysis
(all except 4.18.8, obtained at 23 m in April 1995) were very
similar to each other and to the -subdivision ammonia-oxidizer sequences in the database, especially those of N. europaea
and N. eutropha (Table 4).
Sequence 4.18.8, which was derived from the same NitA-NitB
amplification reaction as 4.18.4, was an outlier; its genetic distance
from the other Mono Lake sequences and from the known N. europaea sequences was about 0.10. Nevertheless, 4.18.8 is clearly
a nitrifier sequence, as it is more similar to the nitrifiers than to
any other sequence in the database.
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4 for
Taq polymerase, the expected error rate (or difference) per base in the amplified 582-bp fragment is computed to be 0.00117 per
base. The smallest genetic distance between our environmental sequences
(4.5PCR and 4.8PCR) (Table 4) and the published N. europaea
sequences was about 0.069 per base. Thus, these differences likely
represent real variability of naturally occurring sequences.
In the Laboratory B analysis, 10 clones each were sequenced from
samples 4.5 and 4.8 and 15 each from samples 4.18, 7.15, and 7.18, to
yield a total of 65 sequences. Of those clones, 5, 4, 9, 10, and 6 sequences, respectively (a total of 34), were unique. Multiple clones
of the same sequence were obtained from a single sample, and the same
sequences were obtained multiple times from various depths, indicating
the presence of the same strains at many depths. Although 10 or 15 clones is not an exhaustive number, the fact that the same sequences
were obtained more than once indicates that the degree of diversity in
these samples is well represented by the unique sequences reported.
The 34 sequences obtained from the Laboratory B analysis are included
in the phylogenetic analyses using 582 bp (an approximately 550-bp
internal fragment plus primers) that resulted from the GM5F-DS907R
amplification. This fragment includes the V6 and V7 variable regions
(4), and most of the variability lies in the V6 region. The
V6 region was not included in the NitD-NitB fragment, on which the
Laboratory A sequence analysis was based. The seven Laboratory A
sequences could not be included in the phylogenetic analysis because
they do not include the 180 bp upstream of the NitD site. A combined
analysis of the NitD-NitB fragments and GM5F-DS907R fragments would
have reduced the common region to only 400 bp and would have included
only the V7 region, which is the least variable of the variable
regions. Thus, we considered that analysis was not informative and do
not present results from it here.
The distance tree (Fig. 2) shows that the
greatest similarities among Laboratory B sequences were found between
sequences from the same depth (e.g., 7.15-B and 7.15-H), but sequences
from different depths were interspersed among clusters in the tree. This implies that the assemblage at 5 m was very similar to that at 20 or 23 m and that the assemblage present in April was very similar to that present in July. Greater differences might have been
expected between the shallow oxic habitat and the deep anoxic habitat,
but viability and persistence under these conditions complicate the
comparison.
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75%) by
parsimony and distance analyses are indicated by solid circles.
-subdivision Nitrosomonas-type nitrifiers and was nearly identical in an overlapping 400 bp with one of the sequences (4.8-B) obtained in the Laboratory B analysis. The outlier from the Laboratory B analysis (4.18-A, obtained at 23 m), based on 582 bp of
unambiguous sequence, is not derived from a nitrifier, being most
similar (95%) to a Thiomicrospira thyasirae strain. The
environmental sequence was not a perfect match for the
Thiomicrospira in the database, and the NitA and NitB primer
sequences with which it was amplified are not present in the published sequence.
Significance of N. europaea and N. eutropha sequences in a hypersaline environment. The finding that organisms like N. europaea and N. eutropha are present in the ammonia-oxidizer assemblage in Mono Lake has ecological significance, firstly because N. europaea is the most commonly isolated terrestrial ammonia oxidizer; for that reason it has been the subject of intense physiological and biochemical study of the pathways and enzymes of nitrification. If this strain is present in the environment, then it may be valid to extrapolate from laboratory studies to the environment in terms of the kinetics, biochemistry, and ecology of nitrification. Secondly, finding N. europaea in the environment suggests that at least some organisms in culture are in fact representative of those in nature, although the quantitative contribution of N. europaea to the natural assemblage cannot be determined.
One important caveat prevents this interpretation, however. This analysis is based entirely on ribosomal genes, which are the genes most commonly used to distinguish phylogenetic groups. However, the alkaline and saline conditions of Mono Lake are not particularly conducive to the growth of the type strains of N. europaea, which do not have a salt requirement, grow in distilled-water medium, and are inhibited by high-salt and -ammonium concentrations (20). The highest salt tolerance attributed to various genospecies of N. europaea by Koops et al. (20) was less than 500 mM NaCl (29 g liter1). Hunik et al. (14)
found that N. europaea activity (in terms of oxygen
consumption) was 90% inhibited by the presence of 500 mM NaCl but that
several different ions cause a similar degree of inhibition. Stehr et
al. (35) obtained N. eutropha and N. europaea isolates from the Elbe River estuary that exhibited
higher levels of salt tolerances than the Nitrosospira
isolates from the same location. The highest level of salt tolerance
tested was 500 mM NaCl (29 g liter1). The total salt
content of Mono Lake at present is on the order of 74 to 88 g
liter1, varying with depth and stratification
(17); sodium and chloride are the major ions. The surface
water at 74 g liter1 is less salty than the deep
water but has still more than twice the salinity of seawater. The
presence of ammonia oxidizers in the deep water cannot therefore result
simply from inoculation of the deep layer with normal, i.e.,
freshwater, Nitrosomonas strains from the upper layer. It
also therefore seems unlikely that the nitrifiers represented by these
ribosomal sequences are physiologically identical to the cultured
N. europaea or N. eutropha. Koops et al.
(20) identified a subgroup of the genus
Nitrosomonas which was composed of halotolerant
"genospecies," including some N. europaea strains and
Nitrosomonas halophila. These strains had salt requirements
but had optimal salt concentrations of 300 mM NaCl, much less than the
salt concentration of Mono Lake waters. The sequences described here
may represent a new more-halophilic group of Nitrosomonas species.
The genus Nitrosomonas includes marine members (e.g.,
N. marina and N. halophila) with documented salt
requirements. However, the Mono Lake sequences presented here were more
similar to N. europaea and N. eutropha than to
either the N. halophila (not shown) or N. marina
sequences (Table 4; Fig. 2). DGGE analysis did detect bands in some of
the samples that appeared to migrate identically with bands from the
N. marina standard DNA, but these bands were not analyzed
further. The positive NitA-NitB amplifications at most of the depths
also imply the presence of -subdivision nitrifiers in addition to
N. europaea- or N. eutropha-like organisms, and
further analysis of those fragments could yield information about the
broader ammonia-oxidizer assemblage. This study focused on the N. europaea-N. eutropha group because of our initial surprise at
finding sequences so close to the cultured strains in this extreme
environment. The sequence and DGGE results show that a wider diversity
of -subdivision ammonia oxidizers is also present.
The physiological diversity deriving from selective pressures in the
environment probably exceeds the phylogenetic diversity of
microorganisms reflected in their rRNA sequences. Environmental parameters such as salinity and temperature probably exert greater selection on functional genes (e.g., genes which encode enzymes with
different salt tolerances or membrane proteins) than on ribosomal genes. Thus, detecting an organism identified as nearly identical to
N. europaea, according to its ribosomal sequence in Mono
Lake, does not necessarily imply that a nitrifier with the
physiological traits of N. europaea in culture could reside
in Mono Lake. Future work in this area could fruitfully focus on genes
for ammonia monooxygenase, the enzyme responsible for the first step in
the oxidation of ammonia, which is more variable among nitrifying strains than is the ribosomal sequence of the same strains
(31).
These results suggest that N. europaea- and N. eutropha-like bacteria are present and perhaps dominant members of
the ammonia-oxidizer assemblage of Mono Lake. Hiorns et al.
(12) and Hastings et al. (10) obtained quite
different results from English lakes: they were unable to detect
N. europaea by using specific PCR amplification and
oligonucleotide hybridization probes but did detect
Nitrosospira-like sequences in several freshwater and
sediment environments. Enrichment cultures yielded strains in the
Nitrosomonas lineage, however (10). Sequences of
the amplified fragments were not reported, so the actual degree of
identity or lack thereof in those samples cannot be compared to the
data reported here. Nevertheless, the results suggest important
differences in species composition of ammonia oxidizers among different
kinds of aquatic environments. Subsequently, Whitby et al.
(49) used the Hastings et al. (10) primers to
retrieve two groups of sequences from aquatic sediments that were very
similar to N. europaea and N. eutropha, while
Nitrosospira still dominated sequences retrieved from the
water column of a freshwater lake.
Using a different set of nitrifier-specific primers, Stephen et al.
(36, 37) detected a preponderance of Nitrosospira sequences in soils and marine sediments. Again, enrichment cultures contained Nitrosomonas-like sequences. Kowalchuk et al.
(22) used the same primers and also reported detecting a
preponderance of Nitrosospira-like sequences in coastal sand
dune environments, and Nitrosomonas-like sequences were
detected only in the seawardmost dune site of their study. Compost
material and manure also contained mainly Nitrosospira
sequences (23). Relatives of the most easily culturable
Nitrosomonas types were detected only in enrichment cultures
from these materials. Both Phillips et al. (29), working with seawater, and Prin
i et al. (30), working
with wastewater, detected N. eutropha-like sequences in
natural assemblages. Hovanec and DeLong (13) found that
N. europaea-like targets were more common in seawater
aquarium filters than were Nitrosospira-like targets,
providing more evidence for salt-tolerant N. europaea strains. It appears from this summary of recent work that direct detection of strains or species by PCR amplification of extracted DNA
detects different strains than does culturing of the same samples.
Strains amenable to enrichment may be those which tolerate or require
high-substrate concentrations, while those which predominate in the
environment may be a less uniform group with substrate preferences that
vary depending on the environment. That might explain the prevalence of
N. europaea in culture and would predict that high-ammonium
environments might select for N. europaea. While we did not
attempt any isolations, our results differ from most of the reports
summarized above in detecting sequences in the environment that are
very similar to those of the easily cultured strains. This may be in
part an artifact associated with selectivity of different PCR primers,
which could be investigated by comparing results from other 16S rRNA
nitrifier primers on the same samples.
Several of the -subdivision ammonia-oxidizer sequences reported here
were detected in samples (at 23 m in April 1995 and at 20 m
in July 1995) collected from below the oxic-anoxic interface (12 to
13 m in April and 13 to 17 m in July) of the stratified lake.
The present analysis cannot evaluate whether the target DNA derived
from intact cells or whether cells were active in these samples. Joye
et al. (19) estimated ammonium oxidation rates in these same
samples using methyl fluoride-sensitive bicarbonate fixation rates
(25). Significant rates were found at 15 and 17 m in
July, and amplification with NitA-NitB and NitD-NitB primers was
detected in both of these samples (Table 3). The sample obtained at
20 m in July had evidence of ammonium oxidation activity, but the
time course of bicarbonate fixation was not linear. This 20-m sample
was the source of the sequences identified by the 7.18 prefix. The
amplification results and retrieval of nitrifier sequences, in
combination with the detection of methyl fluoride-sensitive bicarbonate
fixation at these depths, imply the presence of viable and even active
ammonia oxidizers of the -subdivision type in this environment.
Depths below the oxycline were not sampled by Joye et al.
(19) in April, so we cannot make a direct comparison with
those samples. Detection of ammonia oxidizers in the anoxic hypolimnion
of lakes has been reported previously, using the NitA and NitB primers
in the analysis of the water column of a lake in northern Germany
(47) and by immunofluorescence of lake sediments (of a lake
in the U.S. midwest) and enrichment samples derived from anoxic
sediments (32). In the latter case, the organisms clearly
remained viable and could be retrieved by enrichment culturing, even in
very unsuitable environmental conditions. Without culturing the
assemblage in Mono Lake, it cannot be verified that the retrieved sequences actually represent viable organisms. However, multiple Nitrosomonas sequences were detected below the chemocline in
both April and July 1995, and activity was detected in July. Thus, it
is possible that these sequences do represent viable, if not actively
growing, cells. The combined approach of assaying presence, diversity,
and activity in the same samples has potential for understanding
regulation of nitrification in natural environments.
It is also of ecological significance that N. oceanus was
not detected in Mono Lake samples. None of the samples were amplified in a two-stage amplification procedure with the NOC1 and NOC2 primers.
A direct amplification with these primers was not attempted because the
higher-sensitivity two-stage amplification failed to obtain successful
amplification. It is conceivable that reliance on the two-stage
amplification procedure might have missed N. oceanus if it
were rare and if it were selected against by the EUB primers in the
initial amplification. The results of direct and two-stage
amplification with the NitAB primers (Table 3) suggest the opposite
effect, however, but we cannot rule out the presence of N. oceanus at some low level. N. oceanus is the only known
ammonia oxidizer in the subdivision of the
Proteobacteria, and it has been detected using the NOC1 and
NOC2 primers in seawater from the Southern California Bight
(42) and in several permanently ice-covered, saline lakes in
Antarctica (43). Immunofluorescence with specific antisera
(44) has also detected N. oceanus in these two
environments (43, 45). That N. oceanus was not
readily detectable in Mono Lake suggests that some factor other than
salinity or temperature limits its distribution.
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ACKNOWLEDGMENTS |
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T. J. Hollibaugh, C. Culbertson, L. Miller, and T. Connell assisted with the field work. Mandy Mazzotta extracted the DNA which was used in these experiments.
This research was supported by NSF grants to B.B.W. and S.B.J.
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FOOTNOTES |
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* Corresponding author. Mailing address: Geosciences Department, Princeton University, Princeton, NJ 08544. Phone: (609) 258-5150. Fax: (609) 258-1274. E-mail: bbw{at}princeton.edu.
Present address: Geosciences Department, Princeton University,
Princeton, NJ 08544.
Present address: Department of Marine Sciences, University of
Georgia, Athens, GA 30602.
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Top
Abstract
Introduction
Materials and Methods
Results and Discussion
References
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