Archaeal AmoA Gene Distribution and Relative Abundance in Nitrifying
Aquiculture Bioreactors
Meagan Mauter
Department of Chemical Engineering (Environmental Program), Yale University
[email protected]
Introduction
Nitrification is a key biological process in environmental systems of all scales.
On a global level, this rate limiting process in organic nitrogen mineralization determines
the rate of nitrogen turnover in the global nitrogen cycle. In small scale systems, such as
marine aquiculture water treatment facilities, the specific capacity of ammonia oxidizing
organisms determine the design of the nitrifying bioreactor, the upper limits on water
treatment, the percent makeup water required, and the environmental characteristics of
the receiving water body. The dramatically different scope of modeling large and small
scale processes in the environmental nitrogen cycle obscures the unifying biological role
in these two systems.
Until recently, the first stage of ammonia oxidation was described as
phylogenetically restricted to the alpha and beta classes of proteobacteria. These
chemoautotrophic organisms catalyze the oxidation of ammonia to nitrite via an ammonia
monooxygenase enzyme encoded by amoA.(1) This Recent discoveries of anammox
bacteria, planctomycetes with the capacity to directly oxidize ammonia to N2 using
nitrite,(2) and crenarchaeal organisms, the first identified ammonia oxidizing archaea,(3)
have significantly changed our conception of biological nitrification. 16S rRNA gene
surveys suggest that crenarchaea may inhabit all major mesophilic environments, with
their relative abundance exceeding 5% of the total microbial community of some marine
environments.(4) The extensive diversity uncovered in the biological processes and
phylogenetic origins of nitrifying communities suggests that nitrogen cycling is an open
ended research question on both the large earth science and basic engineering/aquiculture
scales.
The functional gene for ammonia oxidation, amoA, is conserved across all known
nitrification processes in ammonia oxidizing bacteria (AOB) and ammonia oxidizing
archaea (AOA). Archaeal and Bacterial amoA share 40% amino acid homology, but only
25% sequence identity. This suggests that they share a common, albeit distant,
evolutionary origin.(4) This distance allows for definitive phylogenetic distinction
between AOA and AOB populations, and is employed in this research to characterize the
phylogenetic lineage of ammonia oxidizing microbes in a small aquiculture system.
In this independent project I sought to assess the distribution and relative
abundance of ammonia oxidizing marine crenarchaea in nitrifying bioreactors. I
hypothesized that ammonia oxidizing archaea (AOA) would exhibit greater community
diversity and higher relative abundance in biofilters maintained at higher temperatures.
As a subset of this research project, I also hypothesized that environmental stressors, such
as oxidative stress, will impair the function and viability of nitrifying communities. This
question is relevant to the management of marine aquiculture systems that must balance
the risks of bacterial pathogens against the inhibitory effects of upstream ozonation.
MATERIALS AND METHODS
Site description. The Marine Resources Center (MRC) is the primary aquaculture
facility for the Marine Biological Laboratory in Woods Hole, MA. Its mission is to
maintain, culture, and provide aquatic organisms essential to advanced biological,
biomedical, and ecological research. The diversity of aquatic organisms under their care
extends from local indigenous species such as scallops and horseshoe crabs to highly
sensitive model organisms such as cuddlefish. The species-specific environmental
conditions required for aquaculture of these organisms requires separation of the water
treatment facilities into eight primary recirculation loops. Key environmental variables
include flow rate, temperature, pH, total organics, ammonia/nitrate/total N
concentrations, and the application of ozone pretreatment. The primary objective of
water treatment is nitrification of ammonia species prior to mixing with 20% make-up
water and recirculation through the aquaculture facility. Nitrifying reactors found in the
MRC are three-story trickling filters packed with plastic “bioballs” with an estimated
surface area of 20,000 m^2 per filter. The biofilms that grow on the surfaces of the balls
contain a complex heterotrophic microbial community.
Sample collection. No extensive characterization or consistent water quality
monitoring program is currently in place for the nitrifying reactors at the MRC. Water
samples were collected in 50mL Falcon tubes and immediately delivered to the
Ecosystems Center at MBL (Richard McHorney) for quantification of Total Organic
Carbon (TOC), total ammonia, and total nitrate concentrations. Mature biofilm samples
were collected by extraction of bioballs from the second floor of the MRC. Samples
were contained in plastic sampling bags with water from the filter loop where the sample
originated. An alternative biofilm growth substrate, plastic “macaroni”, was placed at the
inlet of filters 5 and 7 and allowed to incubate for one week prior to extraction. This
provided a secondary sampling point and an opportunity to evaluate variation in
microbial diversity under higher ammonia levels and increased oxidative stress.
DNA extraction, PCR amplification of AmoA genes, cloning, and sequencing.
DNA was extracted from the biofilms using a phenol-chloroform extraction protocol
from the lab of Lutgarde Raskin (University of Michigan). MRC loops 5 and 7 were
selected for clone library analysis on the basis of significant temperature variation.
Specific primer sequences for Bacterial and Archaeal AmoA gene fragments were
obtained from Francis et al. and Liesack et al. (5-7) and ordered from Integrated DNA
Technologies (IDT). Archaeal amoA gene (635bp): Arch-amoAF (5 STAATGGTCTGGCTTAGACG-3 ) and Arch-amoAR (5 GCGGCCATCCATCTGTATGT-3 ). Bacterial amoA gene (490bp): AmoA-1F; 59GGGGTTTCTACTGGTGGT and AmoA-2R; 59-CCCCTCKGSAAAGCCTTCT
TC [K 5 G or T; S 5 G or C]. PCR was conducted using a one minute extension time and
a 49 degree Celsius annealing temperature for 30 amplification cycles. Sufficient levels
of DNA amplification for AOB cloning were obtained from loops 5 and 7, while AOA
primer amplification efficiency was only successful in loop 5. PCR products from MRC
loops 5 and 7 were cloned using the TOPO-TA cloning kit (Invitrogen) and clones were
screened via kanamycin and B-gal blue/white screening. White colonies were transferred
to 96 well plates containing SOC and 50ug/L kanamycin and allowed to grow overnight
at 37* Celsius. Plasmid and insert sequencing was performed by core facilities at the
MBL.
Phylogenetic Analysis. Sequences from archaeal and bacterial AmoA gene clone
libraries were analyzed for sequence quality using 4Peaks, and vector sequence was
excised from the relevant AmoA region of interest. Thirty-two Archaeal amoA clones
from loop 5 and 80 Bacterial amoA clones from loops 5 and 7 were sequenced and
analyzed. Functional gene AmoA databases could not be located, and instead I
constructed discrete databases of Bacterial and Archaeal functional genes against which I
compared sequences from the clone libraries. Databases were constructed using a
combination of published GenBank accession numbers from AOA and AOB(5, 6), as
well as BLAST results from the nearest neighbors of sequences retrieved through my
clone library. Functional gene alignment was performed using ClustalW and
phylogenetic trees were constructed via neighbor joining techniques in ARB. Clone
libraries were analyzed for species diversity and richness using DOTER and LIBSHUFF.
QPCR. We performed QPCR on genomic DNA extracted from the mid-region of
the eight loops and the inlet regions of loops 5 and 7. Identical primers to those used in
PCR were also used in QPCR analysis. Standard curves were generated from serial
dilution of plasmids purified from AOA and AOB clone libraries in loops 5 and 7. As
confirmed through clone library sequencing, Archaeal amoA primers amplify a 635bp
region, while Bacterial amoA primers target a 490bp region. QPCR was performed on 48
well plates using the Applied Biosystems QPCR machine and StepOne software.
Technical duplicates were conducted for each sample, but time constraints prevented
biological replicates coupled to technical triplicates. In the instances where I observed
significant variation in threshold concentrations or copy number between my replicates,
the runs were repeated or the data was excluded from my analysis.
Community Resilience to Oxidative Stress. One significant difference between
MRC loops 5 and 7 is the application of ozone to the input waters. Though the oxidationreduction potential (ORP/Eh) of the feed water was identical at the sampling point
midway down the trickling filter, I sought to evaluate the resilience of nitrifying
communities to oxidative stressors. Paraquot (Methyl-violagen, Sigma-Aldrich) was
selected as an oxidative stress inducer. Biofilm samples from loop 5 were incubated in
.5mM and 4mM concentrations of Seawater Complete (SWC) media for 30 minute time
increments. Functional resilience of the nitrifying community was quantified using
change in dissolved oxygen concentrations as a surrogate for nitrification capacity.
Oxygen depletion rate was measured with a Unisense microelectrode respirometry
system and an initial ammonia concentration of 1mM. General community resilience
was assessed using a BactoLight Live Dead Staining kit from Invitrogen. This kit
contains Syto9 and Propidium Iodide for visualization of cell membrane disruption.
Biofilms on the MRC tricking filters were visualized using a Ziess Confocal Axion
Exciter microscope.
RESULTS AND DISCUSSION
The nitrification trickling filters at the Marine Resources Center were
characterized by eight standard water quality parameters. While the MRC aquaculture
system does not typically measure or record this type of data, we enlisted and are grateful
for the help of Richard McHorney in the Ecosystem Center for running these samples.
Based upon my stated hypothesis that AOA would exhibit greater relative abundance and
species diversity at higher growth temperatures, treatment loops 5 and 7 appeared to be
strong candidates for community analysis. These samples represented significant
variation in operation temperature, while ammonia (the critical substrate for the system)
concentrations were relatively close. The variation in ozonation was of concern, but the
Oxidation Reduction Potential (ORP/Eh) of the water systems was equal at the sampling
point on the second floor of the MRC. While I recognized the shortcomings in designing
and drawing conclusions from an experiment in which critical environmental variables
were uncontrolled, but given time constraints it was essential that I use a system with
mature biofilms held at a constant growth rate over the course of the experiments.
Table 1. Water quality characterization of the MRC trickling filters.
Sample
location
Loop 1
Loop 2
Loop 3
Loop 4
Loop 5
Loop 6
Loop 7
Loop 8
Flow
(gpm)
31
37
30
32
32
39
45
28
Temperature
(celsius)
16
17.5
10
13
27
15
16.5
16
pH
6.7
6.8
6.6
6.6
6.6
6.6
6.7
7.4
Ozonation
at inlet
N
N
N
N
Y
N
N
N
NH4
(uM)
0.52
4.28
1.34
3.78
1.25
1.35
1.21
4.14
NO3
(uM)
2.68
77.7
16.4
7.33
15
7.95
4.37
714.00
Total
Nitrogen
(uM)
15.98
97.62
29.40
23.89
24.80
20.79
15.09
893.00
TOC
(uM)
119.8
171.2
132.3
143.4
139.4
132.40
132.70
449.00
Biofilm samples were collected via extraction of the bioballs from the appropriate
loops and immediately sectioned for DNA extraction. While nanodrop analysis
suggested DNA concentrations on the order of 1-20 ng/uL and 260/280 readings between
1.8 and 2.0 for all DNA extractions, PCR amplification of the archeal amoA gene was
only successful in loop 5. Repeated attempts to obtain PCR product from loop 7 were
unsuccessful, despite varying levels of primer, template, and annealing temperature, and
repeated poison controls. While this prohibited the development of an archaeal clone
library for loop 7, I suspect that the failed PCR attempts reflects the low copy number of
archaea in the colder (16*C) system rather than an absence of AOA. This hypothesis that
I reached the threshold detection limit for PCR product on a standard 1% agarose gel is
positively supported by later QPCR experiments on the sample of genomic DNA. Other
hypotheses included the absence of archaea in the system or a primer sequence that did
not amplify the specific archaeal amoA genes found in archaea from loop 7. PCR using
bacterial amoA primers was successful, further suggesting that sample quality was not a
factor in failed PCR amplification.
81 clones were sequenced for Bacterial amoA and 32 were sequenced for
Archaeal amoA phylogenetic analysis. Functional gene trees were constructed using
GenBank accession numbers listed in appendix A. The Bacterial amoA sequences
exhibited distinct clustering between sequences from loop 5 and those from loop 7. All
of the sequences from loop 5 formed a monophyletic clade, where as the sequences from
loop 7 were nearest neighbors with each other but exhibited higher levels of OTU
diversity. Nearest Neighbor matricies were developed using ARB and input into ∫LIBSHUFF(8). ∫-LIBSHUFF confirmed the distinct community composition of
bacterial amoA from loops 5 and 7 with XY and YX p values uniformly equal to zero.
Figure 1. BACTERIAL amoA Functional Gene Tree
Figure 2. Archaeal amoA Functional Gene Tree
The archaeal amoA gene contains two distinct crenarchaeal amoA lineages
originating from soil environments and marine environments. (4, 6) The primer
sequences used in this study target the second group of marine crenarchaeota. These
amoA lineages exhibit high conservation of sequence similarity, with 73% homology on
the amino acid level and 66% homology on the DNA level.
An Archaeal amoA gene tree was also constructed from the 31 clones from loop
5. Many of these sequences shared a common ancestor with the marine crenarcheota
Nitrosopumilus maritimus isolated by Stahl et al. (3) Others showed strong sequence
homology with uncultured marine crenarchaeota, such as those isolated from the
Sargasso Sea by Venter et al. (9) or marine sediments by Francis et al. (5).
The significant diversity in nucleic acid sequence homology among the archaeal
amoA functional genes prompted further questioning about the degree of amoA diversity
in the loop 5 filter. I used DOTUR (10) to generate rarefaction curves to estimate the
number of archaeal amoA sequence clones that would be necessary to observe all OTU’s
with 1%, 3%, 8%, and 16% OTU discrepancy. A unique species is generally designated
at 3% sequence discrepancy.
Figure 3. Archaeal Rarefaction Curve
Archaeal Rarefaction Curve
30
Number of OTUs Observed
Archaeal amoA
25
Sample 1
Sample
2
20
Sample 3
15
Sample
4
Sample
5
10
Sample M5
5
Sample
6
Sample 7
0
Sample
M7 5
0
Sample 8
ng/uL Extracted
DNA
Copy Number
StDev
12.9
0.09737 5.26907
11
4.23493
7.3228
18.5
7.27565 1.61072
18.4
7.10257 6.75555
15.9
4635.01165
1.8876
0.7
12.35024 1.42144
20.3
5.20373 2.10252
20.7
24.97013 1.15923
42.5 20
6.17025
10
15
25 5.43081
30
35
13.7 of amoA 16.48497
2.95482
number
sequence clones
Copy Number per
100 ng DNA
0
0.754806 1.847954
38.49936 57.832111% difference
39.32784 451.7017
38.60092 105.13683% difference
29151.02 245550.5
1764.32 868.85418% difference
25.63414 247.4997
16%
120.6286 2154.027difference
12.77838
88.01604
40
45
50
120.3282
557.901
Rather than pursue higher definition of species diversity in loop 5 using larger
clone libraries, at this point I changed the focus of my research to investigate the presence
and relative abundance of AOA in the other nitrifying reactor loops at the MRC. I used
QPCR to quantify the copy number of archaeal and bacterial amoA genes per ng of total
genomic DNA extracted. Technical replicates were performed for all samples, though
QPCR results would have been more significant with biological replicates and technical
triplicates. Furthermore, it is difficult to make meaningful comparisons of AOA and
AOB copy number because the 48 well limit in the QPCR machine prohibited me from
running samples in the same QPCR amplification. Finally, the potential for PCR bias in
primer efficiency should be investigated before definitive comparisons between AOA and
AOB copy number are performed.
Despite the challenges of comparison, the tables below suggest that archaea are
present in all loops at low copy number, and comprise a large percentage of nitrifying
microorganisms in loop 5. The relative abundance [AOA/(AOA+AOB)] of AOA in loop
5 is estimated at 25.8%, while the relative abundance in loop 7 is estimated at .785%.
Figure 4. Archaeal QPCR Standard Curve
40
35
y = -1.5943Ln(x) + 37.083
30
2
Ct Mean
R = 0.9924
25
20
15
10
5
0
1
10
100
1000
10000
100000
1000000
10000000
Quantity
Table 2. Bacterial amoA QPCR
Sample Name
Sample 1
Sample 2
Sample 3
Sample 4
Sample 5
Sample M5
Sample 6
Sample 7
Sample M7
Sample 8
ng/uL Extracted DNA
12.9
11
18.5
18.4
1.59
0.7
20.3
20.7
42.5
13.7
Copy Number
StDev
Copy Number per 100 ng DNA
0.303649
2950.369
1797.687
10.58455
0.316785
3181.98
6.883049
31.14134
0.133499
1917.209
2.121832
0.031178
3967.727
3.069353
5.817822
1.641348
16034.61
113062.1
1512.079
1.560517
15371.89
16.19541
227.3091
Copy Number
0.09737
4.23493
7.27565
7.10257
4635.01165
12.35024
5.20373
24.97013
5.43081
16.48497
StDev
5.26907
7.3228
1.61072
6.75555
1.8876
1.42144
2.10252
1.15923
6.17025
2.95482
Copy Number per 100 ng DNA
0.754806202
38.49936364
39.32783784
38.60092391
29151.01667
1764.32
25.63413793
120.6286473
12.77837647
120.3282482
Table 3. Archaeal amoA QPCR
Sample 1
Sample 2
Sample 3
Sample 4
Sample 5
Sample M5
Sample 6
Sample 7
Sample M7
Sample 8
ng/uL Extracted DNA
12.9
11
18.5
18.4
15.9
0.7
20.3
20.7
42.5
13.7
PART II: Community Resilience to Oxidative Stress
Pre-ozonation of the feed water to the nitrifying filters prevents excessive
microbial growth in high temperature feed waters and limits the proliferation of
potentially harmful fish pathogens in the re-circulating aquiculture water. Despite the
constant ORP across the filter loops at our selected sampling point, pre-ozonation of the
feed water may exert oxidative stress on the microbial community in the nitrifying
reactor. In order to investigate this effect I evaluated both total community resilience and
functional resilience to oxidative stress induced by methyl viologen (paraquot).
In order to determine inhibitory concentrations of paraquot, I began a series of
“enrichments” using the MRC biofilm community on filter 5 as an innoculum and E.coli
K-12 as a control. I exposed each balsh tube to 1 atm pure oxygen over pressure and
increasing concentrations of paraquot. Cultures were allowed to grow shaking for 24
hours at 37*C prior to transfer. After the tertiary enrichment, no growth was observed in
the control for concentrations of .5mM and 4mM paraquot, while inhibited growth was
observed in the MRC sample at the same concentrations. Thus, I chose .5mM and 4mM
as the inhibitory concentrations for community growth to be used in subsequent
experiments.
Figure 5. Enrichment Cultures
Total community resilience to oxidative stress was assessed qualitatively using
Bactolight Live/Dead Stains and confocal microscopy on mature biofilms from MRC
loop 5. Membrane damage, as indicated by red, is observed in the biofilm samples
exposed to both .5mM and 4mM concentrations of paraquot. The red zone in the control
image may be attributed to the well documented effect of slow metabolic function in the
center of biofilms which inhibit their ability to efflux propidium iodide from their
otherwise healthy cell membranes.
Figure 6. Qualitative Visual Comparison of Cell Membrane Damage after Exposure to
Increasing Concentrations of Paraquot
While overall community resilience to oxidative stress is interesting, it does not
shed light on any inhibitory effect that oxidative stress might have on the functional
capacity of a nitrifying community. In order to quantitatively assess the degree of
functional inhibition that oxidative stress exerts on a nitrifying community, the relative
rates of ammonia oxidation were compared before and after incubation in 4mM paraquot
for 30 minutes. Oxygen depletion rates were selected as an appropriate surrogate for
substrate depletion rates according to the well known Monod model for non-inhibitory
substrate depletion. S represents substrate concentration, Y the biomass yield coefficient,
um the specific growth rate, and Ks the half reaction rate.
Equation 1. Monod Model
Thus, oxygen consumption can be used as a surrogate measure for substrate consumption
by employing the relationship below where So=Initial Substrate Conc.: 1mM NH4+ [=]
M COD and Y=Biomass yield coefficient: 0 for small time
Equation 2.
O2 = O2o – (1 – Y) (So-S)
Figure 7. Functional Resilience to Oxidative Stress
0.1
0.09
Initial Rate
0.08
Rate after 4mM
paraquot incubation
d[O2]/Dt
0.07
0.06
0.05
0.04
0.03
0.02
0.01
0
1
2
3
4
Normalized to Protein Content
Four identical sized samples were evaluated for rates of oxygen consumption in
the presence of 1mM ammonia. The relative rates were further normalized to total
protein content of the biofilms using the Brandford Assay technique for quantifying total
protein content in a system. An average 54% reduction in the initial rates of oxygen
consumption was observed after exposure to 4mM paraquot, suggesting that oxidative
stress does exhibit an inhibitory effect on the nitrifying capacity of the biofilm in MRC
trickling filter loop number 5.
CONCLUSIONS
From this study I was able to conclude that ammonia oxidizing archaea are present in
nitrifying biofilms at the MBL MRC. Amongst the loops, the warmest temperature loop
exhibited the highest relative abundance of AOA amoA genes, though the unique
environmental conditions of each loop prevent the deduction of deterministic variables
impacting the relative abundance of these organisms. Community analysis and
LIBSHUFF allowed me to conclude that the bacterial communities in loops 5 and 7 are
significantly different (p-value of zero) despite the close relationship they share on the
amoA functional gene tree. Finally, in the last portion of this independent project, I was
able to demonstrate that exposure to moderate oxidative stress damages cell membranes
and reduces nitrifying capacity in loop 5 biofilms.
ACKNOWLEDGEMENTS
Much thanks to the course instructors, TA’s, and class mates for fostering such an
open and enthusiastic learning environment. Scott Lindell and Reese at the MRC were
invaluable in their assistance and willingness to open their facilities. Thanks also to
Richard McHorney for analyzing water quality samples. Finally, thanks to my advisor
Meny Elimelech at Yale for giving me the intellectual freedom to explore something
entirely outside his area of expertise.
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Appendix A.
GenBank Accession Numbers for Archaeal amoA Database
1: DQ085098
Candidatus Nitrosopumilus maritimus putative archaeal ammonia monooxygenase
subunit A gene, partial cds
gi|71668108|gb|DQ085098.1|[71668108]
2: DQ148898
Uncultured crenarchaeote clone ES-VM-15 putative ammonia monooxygenase subunit A
(amoA) gene, partial cds
gi|73697451|gb|DQ148898.1|[73697451]
3: DQ148895
Uncultured crenarchaeote clone ES-VM-12 putative ammonia monooxygenase subunit A
(amoA) gene, partial cds
gi|73697445|gb|DQ148895.1|[73697445]
4: DQ148893
Uncultured crenarchaeote clone ES-VM-10 putative ammonia monooxygenase subunit A
(amoA) gene, partial cds
gi|73697441|gb|DQ148893.1|[73697441]
5: DQ148880
Uncultured crenarchaeote clone OKR-C-20 putative ammonia monooxygenase subunit A
(amoA) gene, partial cds
gi|73697415|gb|DQ148880.1|[73697415]
6: DQ148873
Uncultured crenarchaeote clone OKR-C-10 putative ammonia monooxygenase subunit A
(amoA) gene, partial cds
gi|73697401|gb|DQ148873.1|[73697401]
7: DQ148869
Uncultured crenarchaeote clone OKR-C-5 putative ammonia monooxygenase subunit A
(amoA) gene, partial cds
gi|73697393|gb|DQ148869.1|[73697393]
8: DQ148865
Uncultured crenarchaeote clone OKR-C-1 putative ammonia monooxygenase subunit A
(amoA) gene, partial cds
gi|73697385|gb|DQ148865.1|[73697385]
9: DQ148839
Uncultured crenarchaeote clone MB_C130m_21 putative ammonia monooxygenase
subunit A (amoA) gene, partial cds
gi|73697333|gb|DQ148839.1|[73697333]
10: DQ148827
Uncultured crenarchaeote clone MB_C130m_9 putative ammonia monooxygenase
subunit A (amoA) gene, partial cds
gi|73697309|gb|DQ148827.1|[73697309]
11: DQ148819
Uncultured crenarchaeote clone MB_C130m_1 putative ammonia monooxygenase
subunit A (amoA) gene, partial cds
gi|73697293|gb|DQ148819.1|[73697293]
12: DQ148816
Uncultured crenarchaeote clone ES_HL_21 putative ammonia monooxygenase subunit A
(amoA) gene, partial cds
gi|73697289|gb|DQ148816.1|[73697289]
13: DQ148814
Uncultured crenarchaeote clone ES_HL_19 putative ammonia monooxygenase subunit A
(amoA) gene, partial cds
gi|73697285|gb|DQ148814.1|[73697285]
14: DQ148811
Uncultured crenarchaeote clone ES_HL_15 putative ammonia monooxygenase subunit A
(amoA) gene, partial cds
gi|73697279|gb|DQ148811.1|[73697279]
15: DQ148808
Uncultured crenarchaeote clone ES_HL_12 putative ammonia monooxygenase subunit A
(amoA) gene, partial cds
gi|73697273|gb|DQ148808.1|[73697273]
16: DQ148796
Uncultured crenarchaeote clone ES_HI_29 putative ammonia monooxygenase subunit A
(amoA) gene, partial cds
gi|73697249|gb|DQ148796.1|[73697249]
17: DQ148774
Uncultured crenarchaeote clone ES_HI_5 putative ammonia monooxygenase subunit A
(amoA) gene, partial cds
gi|73697205|gb|DQ148774.1|[73697205]
18: DQ148771
Uncultured crenarchaeote clone ES_HI_2 putative ammonia monooxygenase subunit A
(amoA) gene, partial cds
gi|73697199|gb|DQ148771.1|[73697199]
19: DQ148769
Uncultured crenarchaeote clone ETNP_26 putative ammonia monooxygenase subunit A
(amoA) gene, partial cds
gi|73697195|gb|DQ148769.1|[73697195]
20: DQ148759
Uncultured crenarchaeote clone ETNP_16 putative ammonia monooxygenase subunit A
(amoA) gene, partial cds
gi|73697175|gb|DQ148759.1|[73697175]
21: DQ148750
Uncultured crenarchaeote clone ETNP_7 putative ammonia monooxygenase subunit A
(amoA) gene, partial cds
gi|73697157|gb|DQ148750.1|[73697157]
22: DQ148743
Uncultured crenarchaeote clone BS15.9_23 putative ammonia monooxygenase subunit A
(amoA) gene, partial cds
gi|73697143|gb|DQ148743.1|[73697143]
23: DQ148706
Uncultured crenarchaeote clone BS15.8_7 putative ammonia monooxygenase subunit A
(amoA) gene, partial cds
gi|73697069|gb|DQ148706.1|[73697069]
24: DQ148695
Uncultured crenarchaeote clone BS15.7_18 putative ammonia monooxygenase subunit A
(amoA) gene, partial cds
gi|73697047|gb|DQ148695.1|[73697047]
25: DQ148672
Uncultured crenarchaeote clone SF_CB20_20 putative ammonia monooxygenase subunit
A (amoA) gene, partial cds
gi|73697001|gb|DQ148672.1|[73697001]
26: DQ148664
Uncultured crenarchaeote clone SF_CB20_9 putative ammonia monooxygenase subunit
A (amoA) gene, partial cds
gi|73696985|gb|DQ148664.1|[73696985]
27: DQ148658
Uncultured crenarchaeote clone SF_CB20_3 putative ammonia monooxygenase subunit
A (amoA) gene, partial cds
gi|73696973|gb|DQ148658.1|[73696973]
28: DQ148654
Uncultured crenarchaeote clone SF_NB1_22 putative ammonia monooxygenase subunit
A (amoA) gene, partial cds
gi|73696965|gb|DQ148654.1|[73696965]
29: DQ148645
Uncultured crenarchaeote clone SF_NB1_13 putative ammonia monooxygenase subunit
A (amoA) gene, partial cds
gi|73696947|gb|DQ148645.1|[73696947]
30: DQ148644
Uncultured crenarchaeote clone SF_NB1_12 putative ammonia monooxygenase subunit
A (amoA) gene, partial cds
gi|73696945|gb|DQ148644.1|[73696945]
31: DQ148633
Uncultured crenarchaeote clone SF_NB1_1 putative ammonia monooxygenase subunit A
(amoA) gene, partial cds
gi|73696923|gb|DQ148633.1|[73696923]
32: DQ148632
Uncultured crenarchaeote clone MX_6_32 putative ammonia monooxygenase subunit A
(amoA) gene, partial cds
gi|73696921|gb|DQ148632.1|[73696921]
33: DQ148626
Uncultured crenarchaeote clone MX_6_26 putative ammonia monooxygenase subunit A
(amoA) gene, partial cds
gi|73696909|gb|DQ148626.1|[73696909]
34: DQ148589
Uncultured crenarchaeote clone MX_4_17 putative ammonia monooxygenase subunit A
(amoA) gene, partial cds
gi|73696835|gb|DQ148589.1|[73696835]
35: DQ148585
Uncultured crenarchaeote clone MX_4_13 putative ammonia monooxygenase subunit A
(amoA) gene, partial cds
gi|73696827|gb|DQ148585.1|[73696827]
36: DQ148584
Uncultured crenarchaeote clone MX_4_12 putative ammonia monooxygenase subunit A
(amoA) gene, partial cds
gi|73696825|gb|DQ148584.1|[73696825]
37: DQ148546
Uncultured crenarchaeote clone HB_A_22 putative ammonia monooxygenase subunit A
(amoA) gene, partial cds
gi|73696797|gb|DQ148546.1|[73696797]
38: DQ148541
Uncultured crenarchaeote clone HB_A_17 putative ammonia monooxygenase subunit A
(amoA) gene, partial cds
gi|73696787|gb|DQ148541.1|[73696787]
39: DQ148536
Uncultured crenarchaeote clone HB_A_12 putative ammonia monooxygenase subunit A
(amoA) gene, partial cds
gi|73696777|gb|DQ148536.1|[73696777]
40: DQ148534
Uncultured crenarchaeote clone HB_A_10 putative ammonia monooxygenase subunit A
(amoA) gene, partial cds
gi|73696773|gb|DQ148534.1|[73696773]
BLAST Results for Closest Matches to Archaeal Sequences
F09
LOCUS
EF382494
585 bp DNA linear ENV 07-MAY-2007
DEFINITION Uncultured crenarchaeote clone DS2-27 putative ammonia
monooxygenase subunit A (amoA) gene, partial cds.
ACCESSION EF382494
VERSION EF382494.1 GI:146147027
KEYWORDS ENV.
SOURCE
uncultured crenarchaeote
E01
LOCUS
DQ085098
603 bp DNA linear BCT 15-FEB-2006
DEFINITION Candidatus Nitrosopumilus maritimus putative archaeal ammonia
monooxygenase subunit A gene, partial cds.
ACCESSION DQ085098
VERSION DQ085098.1 GI:71668108
KEYWORDS .
SOURCE
Candidatus Nitrosopumilus maritimus SCM1
E10
LOCUS
AM295171
620 bp DNA linear ENV 01-NOV-2006
DEFINITION Uncultured archaeon partial amoA gene for putative ammonia
monooxygenase, clone T4.
ACCESSION AM295171
VERSION AM295171.1 GI:117306978
KEYWORDS ENV; ammonia monooxygenase; amoA gene.
SOURCE
uncultured archaeon
A01
LOCUS
EF382609
585 bp DNA linear ENV 07-MAY-2007
DEFINITION Uncultured crenarchaeote clone PA6-15 putative ammonia
monooxygenase subunit A (amoA) gene, partial cds.
ACCESSION EF382609
VERSION EF382609.1 GI:146147257
KEYWORDS ENV.
SOURCE
uncultured crenarchaeote
A08
LOCUS
EF382485
585 bp DNA linear ENV 07-MAY-2007
DEFINITION Uncultured crenarchaeote clone DS2-18 putative ammonia
monooxygenase subunit A (amoA) gene, partial cds.
ACCESSION EF382485
VERSION EF382485.1 GI:146147009
KEYWORDS ENV.
SOURCE
uncultured crenarchaeote
GenBank Accession Numbers for Bacterial amoA Database
1: AY958704
Nitrosomonas sp. NL7 ammonia monooxygenase A (amoA) gene, partial cds
gi|62083007|gb|AY958704.1|[62083007]
2: AB234590
Uncultured bacterium amoA-like gene for ammmonia monooxygenase, oartial cds, clone:
A-Sep-clone44
gi|75992683|dbj|AB234590.1|[75992683]
3: AY702596
Uncultured ammonia-oxidizing bacterium clone P14-169 ammonia monooxygenase
(amoA) gene, partial cds
gi|51599231|gb|AY702596.1|[51599231]
4: AY702586
Uncultured ammonia-oxidizing bacterium clone P22-14 ammonia monooxygenase
(amoA) gene, partial cds
gi|51599211|gb|AY702586.1|[51599211]
5: AY702577
Uncultured ammonia-oxidizing bacterium clone P14-26 ammonia monooxygenase
(amoA) gene, partial cds
gi|51599193|gb|AY702577.1|[51599193]
6: AY616020
Uncultured bacterium clone AN 14 ammonia monooxygenase (amoA) gene, partial cds
gi|47680269|gb|AY616020.1|[47680269]
7: AY356421
Uncultured bacterium clone Marshall-66W ammonia monooxygenase (amoA) gene,
partial cds
gi|38706972|gb|AY356421.1|[38706972]
8: AY356418
Uncultured bacterium clone Marshall-60S ammonia monooxygenase (amoA) gene,
partial cds
gi|38706966|gb|AY356418.1|[38706966]
9: AF489639
Uncultured bacterium clone K_10 ammonia monooxygenase (amoA) gene, partial cds
gi|20136226|gb|AF489639.1|[20136226]
10: AY353050
Uncultured ammonia-oxidizing beta proteobacterium clone CT2-28 ammonia
monooxygenase subunit A (amoA) gene, partial cds
gi|37993378|gb|AY353050.1|[37993378]
11: AY353045
Uncultured ammonia-oxidizing beta proteobacterium clone CT2-23 ammonia
monooxygenase subunit A (amoA) gene, partial cds
gi|37993368|gb|AY353045.1|[37993368]
12: AY353033
Uncultured ammonia-oxidizing beta proteobacterium clone CT2-11 ammonia
monooxygenase subunit A (amoA) gene, partial cds
gi|37993344|gb|AY353033.1|[37993344]
13: AY353015
Uncultured ammonia-oxidizing beta proteobacterium clone CT1-25 ammonia
monooxygenase subunit A (amoA) gene, partial cds
gi|37993308|gb|AY353015.1|[37993308]
14: AY352993
Uncultured ammonia-oxidizing beta proteobacterium clone CT1-1 ammonia
monooxygenase subunit A (amoA) gene, partial cds
gi|37993264|gb|AY352993.1|[37993264]
15: AY352986
Uncultured ammonia-oxidizing beta proteobacterium clone CB3-26 ammonia
monooxygenase subunit A (amoA) gene, partial cds
gi|37993250|gb|AY352986.1|[37993250]
16: AY352985
Uncultured ammonia-oxidizing beta proteobacterium clone CB3-25 ammonia
monooxygenase subunit A (amoA) gene, partial cds
gi|37993248|gb|AY352985.1|[37993248]
17: AY352971
Uncultured ammonia-oxidizing beta proteobacterium clone CB3-10 ammonia
monooxygenase subunit A (amoA) gene, partial cds
gi|37993220|gb|AY352971.1|[37993220]
18: AY352969
Uncultured ammonia-oxidizing beta proteobacterium clone CB3-8 ammonia
monooxygenase subunit A (amoA) gene, partial cds
gi|37993216|gb|AY352969.1|[37993216]
19: AY352967
Uncultured ammonia-oxidizing beta proteobacterium clone CB3-6 ammonia
monooxygenase subunit A (amoA) gene, partial cds
gi|37993212|gb|AY352967.1|[37993212]
20: AY352964
Uncultured ammonia-oxidizing beta proteobacterium clone CB3-3 ammonia
monooxygenase subunit A (amoA) gene, partial cds
gi|37993206|gb|AY352964.1|[37993206]
21: AY352940
Uncultured ammonia-oxidizing beta proteobacterium clone CB2-12 ammonia
monooxygenase subunit A (amoA) gene, partial cds
gi|37993158|gb|AY352940.1|[37993158]
22: AY352932
Uncultured ammonia-oxidizing beta proteobacterium clone CB2-2 ammonia
monooxygenase subunit A (amoA) gene, partial cds
gi|37993142|gb|AY352932.1|[37993142]
23: AY352930
Uncultured ammonia-oxidizing beta proteobacterium clone CB1-33 ammonia
monooxygenase subunit A (amoA) gene, partial cds
gi|37993138|gb|AY352930.1|[37993138]
24: AY352923
Uncultured ammonia-oxidizing beta proteobacterium clone CB1-26 ammonia
monooxygenase subunit A (amoA) gene, partial cds
gi|37993124|gb|AY352923.1|[37993124]
25: AY352916
Uncultured ammonia-oxidizing beta proteobacterium clone CB1-19 ammonia
monooxygenase subunit A (amoA) gene, partial cds
gi|37993110|gb|AY352916.1|[37993110]
26: AY352912
Uncultured ammonia-oxidizing beta proteobacterium clone CB1-15 ammonia
monooxygenase subunit A (amoA) gene, partial cds
gi|37993102|gb|AY352912.1|[37993102]
27: AY352905
Uncultured ammonia-oxidizing beta proteobacterium clone CB1-7 ammonia
monooxygenase subunit A (amoA) gene, partial cds
gi|37993088|gb|AY352905.1|[37993088]
28: AY352904
Uncultured ammonia-oxidizing beta proteobacterium clone CB1-6 ammonia
monooxygenase subunit A (amoA) gene, partial cds
gi|37993086|gb|AY352904.1|[37993086]
29: AY352902
Uncultured ammonia-oxidizing beta proteobacterium clone CB1-4 ammonia
monooxygenase subunit A (amoA) gene, partial cds
gi|37993082|gb|AY352902.1|[37993082]
30: AY123821
Nitrosospira briensis ammonia monooxygenase (amoA) gene, partial cds
gi|24474376|gb|AY123821.1|[24474376]
31: AY123819
Nitrosomonas sp. Nm86 ammonia monooxygenase (amoA) gene, partial cds
gi|24474372|gb|AY123819.1|[24474372]
32: AY123816
Nitrosomonas sp. Nm143 ammonia monooxygenase (amoA) gene, partial cds
gi|24474366|gb|AY123816.1|[24474366]
33: AF047705
Nitrosococcus oceanus ammonia monooxygenase subunit A (amoA) and ammonia
monooxygenase subunit B (amoB) genes, complete cds; and unknown gene
gi|3282844|gb|AF047705.1|[3282844]
34: AF042171
Nitrosolobus multiformis ammonia monooxygenase subunit AmoA (amoA) gene,
complete cds
gi|3282756|gb|AF042171.1|[3282756]
35: AF032438
Nitrosospira sp. NpAV ammonia monooxygenase operon copy 1: ammonia
monooxygenase 1 subunit C (amoC1) gene, partial cds, and ammonia monooxygenase
subunits A (amoA1) and B (amoB1) genes, complete cds
gi|2641609|gb|AF032438.1|[2641609]
36: AF006692
Nitrosospira sp. Np39-19 ammonia monooxygenase subunit A3 (amoA3) gene, complete
cds
gi|2266997|gb|AF006692.1|[2266997]
37: U92432
Nitrosospira sp. NpAV ammonia monooxygenase operon copy 3: ammonia
monooxygenase 3 subunits C (amoC3), A (amoA3) and B (amoB3) genes, complete cds
gi|2062745|gb|U92432.1|NSU92432[2062745]
38: AF272406
Nitrosomonas oligotropha ammonia monooxygenase (amoA) gene, partial cds
gi|11545302|gb|AF272406.1|AF272406[11545302]
39: AF272405
Nitrosomonas marina ammonia monooxygenase (amoA) gene, partial cds
gi|11545300|gb|AF272405.1|AF272405[11545300]
40: AF272404
Nitrosomonas nitrosa ammonia monooxygenase (amoA) gene, partial cds
gi|11545298|gb|AF272404.1|AF272404[11545298]
41: AF272403
Nitrosomonas ureae ammonia monooxygenase (amoA) gene, partial cds
gi|11545296|gb|AF272403.1|AF272403[11545296]
42: AF272402
Nitrosomonas cryotolerans ammonia monooxygenase (amoA) gene, partial cds
gi|11545294|gb|AF272402.1|AF272402[11545294]
43: AF272400
Nitrosomonas aestuarii ammonia monooxygenase (amoA) gene, partial cds
gi|11545290|gb|AF272400.1|AF272400[11545290]
BLAST Results for Closest Matches to Archaeal Sequences
A09
LOCUS
AY702615
489 bp DNA linear ENV 30-AUG-2005
DEFINITION Uncultured ammonia-oxidizing bacterium clone P14-7 ammonia
monooxygenase (amoA) gene, partial cds.
ACCESSION AY702615
VERSION AY702615.1 GI:51599269
KEYWORDS ENV.
SOURCE
uncultured ammonia-oxidizing bacterium
G12
LOCUS
EF615105
789 bp DNA linear ENV 05-JUL-2007
DEFINITION Uncultured ammonia-oxidizing beta proteobacterium clone Bsedi-
31
ammonia monooxxygenase subunit A (amoA) gene, partial cds.
ACCESSION EF615105
VERSION EF615105.1 GI:149782729
KEYWORDS ENV.
SOURCE
uncultured ammonia-oxidizing beta proteobacterium
C07
LOCUS
AB261613
491 bp DNA linear ENV 25-JUL-2007
DEFINITION Uncultured beta proteobacterium amoA gene for ammonia
monooxygenase
subunit A, partial cds, clone: OAU3.
ACCESSION AB261613
VERSION AB261613.1 GI:106363045
KEYWORDS ENV.
SOURCE
uncultured beta proteobacterium