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1.  Vibrio diversity and dynamics in the Monterey Bay upwelling region 
The Vibrionaceae (Vibrio) are a ubiquitous group of metabolically flexible marine bacteria that play important roles in biogeochemical cycling in the ocean. Despite this versatility, little is known about Vibrio diversity and abundances in upwelling regions. The seasonal dynamics of Vibrio populations was examined by analysis of 16S rRNA genes in Monterey Bay (MB), California from April 2006–April 2008 at two long term monitoring stations, C1 and M2. Vibrio phylotypes within MB were diverse, with subpopulations clustering with several different cultured representatives including Allivibrio spp., Vibrio penaecida, and Vibrio splendidus as well as with many unidentified marine environmental bacterial 16S rRNA gene sequences. Total Vibrio population abundances, as well as abundances of a Vibrio sp. subpopulation (MBAY Vib7) and an Allivibrio sp. subpopulation (MBAY Vib4) were examined in the context of environmental parameters from mooring station and CTD cast data. Total Vibrio populations showed some seasonal variability but greater variability was observed within the two subpopulations. MBAY Vib4 was negatively associated with MB upwelling indices and positively correlated with oceanic season conditions, when upwelling winds relax and warmer surface waters are present in MB. MBAY Vib7 was also negatively associated with upwelling indices and represented a deeper Vibrio sp. population. Correlation patterns suggest that larger oceanographic conditions affect the dynamics of the populations in MB, rather than specific environmental factors. This study is the first to target and describe the diversity and dynamics of these natural populations in MB and demonstrates that these populations shift seasonally within the region.
doi:10.3389/fmicb.2014.00048
PMCID: PMC3921578  PMID: 24575086
Vibrio; upwelling; Monterey Bay; seasonal variability; 16S rRNA gene diversity
2.  Aphotic N2 Fixation in the Eastern Tropical South Pacific Ocean 
PLoS ONE  2013;8(12):e81265.
We examined rates of N2 fixation from the surface to 2000 m depth in the Eastern Tropical South Pacific (ETSP) during El Niño (2010) and La Niña (2011). Replicated vertical profiles performed under oxygen-free conditions show that N2 fixation takes place both in euphotic and aphotic waters, with rates reaching 155 to 509 µmol N m−2 d−1 in 2010 and 24±14 to 118±87 µmol N m−2 d−1 in 2011. In the aphotic layers, volumetric N2 fixation rates were relatively low (<1.00 nmol N L−1 d−1), but when integrated over the whole aphotic layer, they accounted for 87–90% of total rates (euphotic+aphotic) for the two cruises. Phylogenetic studies performed in microcosms experiments confirm the presence of diazotrophs in the deep waters of the Oxygen Minimum Zone (OMZ), which were comprised of non-cyanobacterial diazotrophs affiliated with nifH clusters 1K (predominantly comprised of α-proteobacteria), 1G (predominantly comprised of γ-proteobacteria), and 3 (sulfate reducing genera of the δ-proteobacteria and Clostridium spp., Vibrio spp.). Organic and inorganic nutrient addition bioassays revealed that amino acids significantly stimulated N2 fixation in the core of the OMZ at all stations tested and as did simple carbohydrates at stations located nearest the coast of Peru/Chile. The episodic supply of these substrates from upper layers are hypothesized to explain the observed variability of N2 fixation in the ETSP.
doi:10.1371/journal.pone.0081265
PMCID: PMC3861260  PMID: 24349048
3.  Two Strains of Crocosphaera watsonii with Highly Conserved Genomes are Distinguished by Strain-Specific Features 
Unicellular nitrogen-fixing cyanobacteria are important components of marine phytoplankton. Although non-nitrogen-fixing marine phytoplankton generally exhibit high gene sequence and genomic diversity, gene sequences of natural populations and isolated strains of Crocosphaera watsonii, one of the two most abundant open ocean unicellular cyanobacteria groups, have been shown to be 98–100% identical. The low sequence diversity in Crocosphaera is a dramatic contrast to sympatric species of Prochlorococcus and Synechococcus, and raises the question of how genome differences can explain observed phenotypic diversity among Crocosphaera strains. Here we show, through whole genome comparisons of two phenotypically different strains, that there are strain-specific sequences in each genome, and numerous genome rearrangements, despite exceptionally low sequence diversity in shared genomic regions. Some of the strain-specific sequences encode functions that explain observed phenotypic differences, such as exopolysaccharide biosynthesis. The pattern of strain-specific sequences distributed throughout the genomes, along with rearrangements in shared sequences is evidence of significant genetic mobility that may be attributed to the hundreds of transposase genes found in both strains. Furthermore, such genetic mobility appears to be the main mechanism of strain divergence in Crocosphaera which do not accumulate DNA microheterogeneity over the vast majority of their genomes. The strain-specific sequences found in this study provide tools for future physiological studies, as well as genetic markers to help determine the relative abundance of phenotypes in natural populations.
doi:10.3389/fmicb.2011.00261
PMCID: PMC3247675  PMID: 22232617
comparative genomics; Crocosphaera; exopolysaccharide biosynthesis; genome conservation; mobile genetic elements; nitrogen fixation
4.  Analogous nutrient limitations in unicellular diazotrophs and Prochlorococcus in the South Pacific Ocean 
The ISME Journal  2011;6(4):733-744.
Growth limitation of phytoplankton and unicellular nitrogen (N2) fixers (diazotrophs) were investigated in the oligotrophic Western South Pacific Ocean. Based on change in abundances of nifH or 23S rRNA gene copies during nutrient-enrichment experiments, the factors limiting net growth of the unicellular diazotrophs UCYN-A (Group A), Crocosphaera watsonii, γ-Proteobacterium 24774A11, and the non-diazotrophic picocyanobacterium Prochlorococcus, varied within the region. At the westernmost stations, numbers were enhanced by organic carbon added as simple sugars, a combination of iron and an organic chelator, or iron added with phosphate. At stations nearest the equator, the nutrient-limiting growth was not apparent. Maximum net growth rates for UCYN-A, C. watsonii and γ-24774A11 were 0.19, 0.61 and 0.52 d−1, respectively, which are the first known empirical growth rates reported for the uncultivated UCYN-A and the γ-24774A11. The addition of N enhanced total phytoplankton biomass up to 5-fold, and the non-N2-fixing Synechococcus was among the groups that responded favorably to N addition. Nitrogen was the major nutrient-limiting phytoplankton biomass in the Western South Pacific Ocean, while availability of organic carbon or iron and organic chelator appear to limit abundances of unicellular diazotrophs. Lack of phytoplankton response to nutrient additions in the Pacific warm pool waters suggests diazotroph growth in this area is controlled by different factors than in the higher latitudes, which may partially explain previously observed variability in community composition in the region.
doi:10.1038/ismej.2011.152
PMCID: PMC3309360  PMID: 22094348
Crocosphaera; cyanobacteria; group A; nitrogen fixation; qPCR; UCYN-A
5.  Seasonal Synechococcus and Thaumarchaeal population dynamics examined with high resolution with remote in situ instrumentation 
The ISME Journal  2011;6(3):513-523.
Monterey Bay, CA is an Eastern boundary upwelling system that is nitrogen limited much of the year. In order to resolve population dynamics of microorganisms important for nutrient cycling in this region, we deployed the Environmental Sample Processor with quantitative PCR assays targeting both ribosomal RNA genes and functional genes for subclades of cyanobacteria (Synechococcus) and ammonia-oxidizing Archaea (Thaumarchaeota) populations. Results showed a strong correlation between Thaumarchaea abundances and nitrate during the spring upwelling but not the fall sampling period. In relatively stratified fall waters, the Thaumarchaeota community reached higher numbers than in the spring, and an unexpected positive correlation with chlorophyll concentration was observed. Further, we detected drops in Synechococcus abundance that occurred on short (that is, daily) time scales. Upwelling intensity and blooms of eukaryotic phytoplankton strongly influenced Synechococcus distributions in the spring and fall, revealing what appear to be the environmental limitations of Synechococcus populations in this region. Each of these findings has implications for Monterey Bay biogeochemistry. High-resolution sampling provides a better-resolved framework within which to observe changes in the plankton community. We conclude that controls on these ecosystems change on smaller scales than are routinely assessed, and that more predictable trends will be uncovered if they are evaluated within seasonal (monthly), rather than on annual or interannual scales.
doi:10.1038/ismej.2011.127
PMCID: PMC3280143  PMID: 21975596
Crenarchaeota; Environmental Sample Processor; Monterey Bay; Synechococcus; Thaumarchaeota; time series
6.  Nitrogenase (nifH) gene expression in diazotrophic cyanobacteria in the Tropical North Atlantic in response to nutrient amendments 
The Tropical North Atlantic (TNAtl) plays a critical role in the marine nitrogen cycle, as it supports high rates of biological nitrogen (N2) fixation, yet it is unclear whether this process is limited by the availability of iron (Fe), phosphate (P) or is co-limited by both. In order to investigate the impact of nutrient limitation on the N2-fixing microorganisms (diazotrophs) in the TNAtl, trace metal clean nutrient amendment experiments were conducted, and the expression of nitrogenase (nifH) in cyanobacterial diazotrophs in response to the addition of Fe, P, or Fe+P was measured using quantitative PCR. To provide context, N2 fixation rates associated with the <10 μm community and diel nifH expression in natural cyanobacterial populations were measured. In the western TNAtl, nifH expression in Crocosphaera, Trichodesmium, and Richelia was stimulated by Fe and Fe+P additions, but not by P, implying that diazotrophs may be Fe-limited in this region. In the eastern TNAtl, nifH expression in unicellular cyanobacteria UCYN-A and Crocosphaera was stimulated by P, implying P-limitation. In equatorial waters, nifH expression in Trichodesmium was highest in Fe+P treatments, implying co-limitation in this region. Nutrient additions did not measurably stimulate N2 fixation rates in the <10 μm fraction in most of the experiments, even when upregulation of nifH expression was evident. These results demonstrate the utility of using gene expression to investigate the physiological state of natural populations of microorganisms, while underscoring the complexity of nutrient limitation on diazotrophy, and providing evidence that diazotroph populations are slow to respond to the addition of limiting nutrients and may be limited by different nutrients on basin-wide spatial scales. This has important implications for our current understanding of controls on N2 fixation in the TNAtl and may partially explain why it appears to be intermittently limited by Fe, P, or both.
doi:10.3389/fmicb.2012.00386
PMCID: PMC3487379  PMID: 23130017
nitrogenase; P-limitation; Fe-limitation; UCYN-A; UCYN-B; Trichodesmium; diazotrophs; nitrogen fixation
7.  Nitrogen fixation and nitrogenase (nifH) expression in tropical waters of the eastern North Atlantic 
The ISME Journal  2011;5(7):1201-1212.
Expression of nifH in 28 surface water samples collected during fall 2007 from six stations in the vicinity of the Cape Verde Islands (north-east Atlantic) was examined using reverse transcription-polymerase chain reaction (RT-PCR)-based clone libraries and quantitative RT-PCR (RT-qPCR) analysis of seven diazotrophic phylotypes. Biological nitrogen fixation (BNF) rates and nutrient concentrations were determined for these stations, which were selected based on a range in surface chlorophyll concentrations to target a gradient of primary productivity. BNF rates greater than 6 nmolN l−1 h−1 were measured at two of the near-shore stations where high concentrations of Fe and PO43− were also measured. Six hundred and five nifH transcripts were amplified by RT-PCR, of which 76% are described by six operational taxonomic units, including Trichodesmium and the uncultivated UCYN-A, and four non-cyanobacterial diazotrophs that clustered with uncultivated Proteobacteria. Although all five cyanobacterial phylotypes quantified in RT-qPCR assays were detected at different stations in this study, UCYN-A contributed most significantly to the pool of nifH transcripts in both coastal and oligotrophic waters. A comparison of results from RT-PCR clone libraries and RT-qPCR indicated that a γ-proteobacterial phylotype was preferentially amplified in clone libraries, which underscores the need to use caution interpreting clone-library-based nifH studies, especially when considering the importance of uncultivated proteobacterial diazotrophs.
doi:10.1038/ismej.2010.205
PMCID: PMC3146282  PMID: 21228888
nitrogen fixation; nifH; nitrogenase; molecular; Cape Verde; Atlantic
8.  Global distribution patterns of distinct clades of the photosynthetic picoeukaryote Ostreococcus 
The ISME Journal  2011;5(7):1095-1107.
Ostreococcus is a marine picophytoeukaryote for which culture studies indicate there are ‘high-light' and ‘low-light' adapted ecotypes. Representatives of these ecotypes fall within two to three 18S ribosomal DNA (rDNA) clades for the former and one for the latter. However, clade distributions and relationships to this form of niche partitioning are unknown in nature. We developed two quantitative PCR primer-probe sets and enumerated the proposed ecotypes in the Pacific Ocean as well as the subtropical and tropical North Atlantic. Statistical differences in factors such as salinity, temperature and NO3 indicated the ecophysiological parameters behind clade distributions are more complex than irradiance alone. Clade OII, containing the putatively low-light adapted strains, was detected at warm oligotrophic sites. In contrast, Clade OI, containing high-light adapted strains, was present in cooler mesotrophic and coastal waters. Maximal OI abundance (19 555±37 18S rDNA copies per ml) was detected in mesotrophic waters at 40 m depth, approaching the nutricline. OII was often more abundant at the deep chlorophyll maximum, when nutrient concentrations were significantly higher than at the surface (stratified euphotic zone waters). However, in mixed euphotic-zone water columns, relatively high numbers (for example, 891±107 18S rDNA copies per ml, Sargasso Sea, springtime) were detected at the surface. Both Clades OI and OII were found at multiple euphotic zone depths, but co-occurrence at the same geographical location appeared rare and was detected only in continental slope waters. In situ growth rate estimates using these primer-probes and better comprehension of physiology will enhance ecological understanding of Ostreococcus Clades OII and OI which appear to be oceanic and coastal clades, respectively.
doi:10.1038/ismej.2010.209
PMCID: PMC3146286  PMID: 21289652
picoeukaryotes; Ostreococcus; quantitative PCR; mamiellales; prasinophytes; niche differentiation
9.  Nitrogen fixation and transfer in open ocean diatom–cyanobacterial symbioses 
The ISME Journal  2011;5(9):1484-1493.
Many diatoms that inhabit low-nutrient waters of the open ocean live in close association with cyanobacteria. Some of these associations are believed to be mutualistic, where N2-fixing cyanobacterial symbionts provide N for the diatoms. Rates of N2 fixation by symbiotic cyanobacteria and the N transfer to their diatom partners were measured using a high-resolution nanometer scale secondary ion mass spectrometry approach in natural populations. Cell-specific rates of N2 fixation (1.15–71.5 fmol N per cell h−1) were similar amongst the symbioses and rapid transfer (within 30 min) of fixed N was also measured. Similar growth rates for the diatoms and their symbionts were determined and the symbiotic growth rates were higher than those estimated for free-living cells. The N2 fixation rates estimated for Richelia and Calothrix symbionts were 171–420 times higher when the cells were symbiotic compared with the rates estimated for the cells living freely. When combined, the latter two results suggest that the diatom partners influence the growth and metabolism of their cyanobacterial symbionts. We estimated that Richelia fix 81–744% more N than needed for their own growth and up to 97.3% of the fixed N is transferred to the diatom partners. This study provides new information on the mechanisms controlling N input into the open ocean by symbiotic microorganisms, which are widespread and important for oceanic primary production. Further, this is the first demonstration of N transfer from an N2 fixer to a unicellular partner. These symbioses are important models for molecular regulation and nutrient exchange in symbiotic systems.
doi:10.1038/ismej.2011.26
PMCID: PMC3160684  PMID: 21451586
nanoSIMS; symbioses; cyanobiont; diatoms; N2 fixation
10.  Misannotations of rRNA can now generate 90% false positive protein matches in metatranscriptomic studies 
Nucleic Acids Research  2011;39(20):8792-8802.
In the course of analyzing 9 522 746 pyrosequencing reads from 23 stations in the Southwestern Pacific and equatorial Atlantic oceans, it came to our attention that misannotations of rRNA as proteins is now so widespread that false positive matching of rRNA pyrosequencing reads to the National Center for Biotechnology Information (NCBI) non-redundant protein database approaches 90%. One conserved portion of 23S rRNA was consistently misannotated often enough to prompt curators at Pfam to create a spurious protein family. Detailed examination of the annotation history of each seed sequence in the spurious Pfam protein family (PF10695, ‘Cw-hydrolase’) uncovered issues in the standard operating procedures and quality assurance programs of major sequencing centers, and other issues relating to the curation practices of those managing public databases such as GenBank and SwissProt. We offer recommendations for all these issues, and recommend as well that workers in the field of metatranscriptomics take extra care to avoid including false positive matches in their datasets.
doi:10.1093/nar/gkr576
PMCID: PMC3203614  PMID: 21771858
11.  Differential Distributions of Synechococcus Subgroups Across the California Current System 
Synechococcus is an abundant marine cyanobacterial genus composed of different populations that vary physiologically. Synechococcus narB gene sequences (encoding for nitrate reductase in cyanobacteria) obtained previously from isolates and the environment (e.g., North Pacific Gyre Station ALOHA, Hawaii or Monterey Bay, CA, USA) were used to develop quantitative PCR (qPCR) assays. These qPCR assays were used to quantify populations from specific narB phylogenetic clades across the California Current System (CCS), a region composed of dynamic zones between a coastal-upwelling zone and the oligotrophic Pacific Ocean. Targeted populations (narB subgroups) had different biogeographic patterns across the CCS, which appear to be driven by environmental conditions. Subgroups C_C1, D_C1, and D_C2 were abundant in coastal-upwelling to coastal-transition zone waters with relatively high to intermediate ammonium, nitrate, and chl. a concentrations. Subgroups A_C1 and F_C1 were most abundant in coastal-transition zone waters with intermediate nutrient concentrations. E_O1 and G_O1 were most abundant at different depths of oligotrophic open-ocean waters (either in the upper mixed layer or just below). E_O1, A_C1, and F_C1 distributions differed from other narB subgroups and likely possess unique ecologies enabling them to be most abundant in waters between coastal and open-ocean waters. Different CCS zones possessed distinct Synechococcus communities. Core California current water possessed low numbers of narB subgroups relative to counted Synechococcus cells, and coastal-transition waters contained high abundances of Synechococcus cells and total number of narB subgroups. The presented biogeographic data provides insight on the distributions and ecologies of Synechococcus present in an eastern boundary current system.
doi:10.3389/fmicb.2011.00059
PMCID: PMC3153035  PMID: 21833315
Synechococcus; picocyanobacteria; biogeography; CCS; eastern-Pacific; qPCR; narB
12.  Hydrogen Cycling by the Unicellular Marine Diazotroph Crocosphaera watsonii Strain WH8501 ▿  
Applied and Environmental Microbiology  2010;76(20):6797-6803.
The hydrogen (H2) cycle associated with the dinitrogen (N2) fixation process was studied in laboratory cultures of the marine cyanobacterium Crocosphaera watsonii. The rates of H2 production and acetylene (C2H2) reduction were continuously measured over the diel cycle with simultaneous measurements of fast repetition rate fluorometry and dissolved oxygen. The maximum rate of H2 production was coincident with the maximum rates of C2H2 reduction. Theoretical stoichiometry for N2 fixation predicts an equimolar ratio of H2 produced to N2 fixed. However, the maximum rate of net H2 production observed was 0.09 nmol H2 μg chlorophyll a (chl a)−1 h−1 compared to the N2 fixation rate of 5.5 nmol N2 μg chl a−1 h−1, with an H2 production/N2 fixation ratio of 0.02. The 50-fold discrepancy between expected and observed rates of H2 production was hypothesized to be a result of H2 reassimilation by uptake hydrogenase. This was confirmed by the addition of carbon monoxide (CO), a potent inhibitor of hydrogenase, which increased net H2 production rates ∼40-fold to a maximum rate of 3.5 nmol H2 μg chl a−1 h−1. We conclude that the reassimilation of H2 by C. watsonii is highly efficient (>98%) and hypothesize that the tight coupling between H2 production and consumption is a consequence of fixing N2 at nighttime using a finite pool of respiratory carbon and electrons acquired from daytime solar energy capture. The H2 cycle provides unique insight into N2 fixation and associated metabolic processes in C. watsonii.
doi:10.1128/AEM.01202-10
PMCID: PMC2953037  PMID: 20709832
13.  Microbes in Earth's Aqueous Environments 
doi:10.3389/fmicb.2010.00004
PMCID: PMC3128466  PMID: 21772828
14.  Distribution and activity of diazotrophs in the Eastern Equatorial Atlantic 
Environmental Microbiology  2009;11(4):741-750.
The gene abundance and gene expression of six diazotroph populations from the Eastern Equatorial Atlantic in June 2007 were examined using nifH gene quantitative polymerase chain reaction (q PCR) methods. Of all the diazotrophs, Trichodesmium spp. was the most abundant with the highest number of gene copies in the Gulf of Guinea. Trichodesmium also had the highest nitrogenase gene transcript abundance overall with the maximum in samples collected at the equator and in waters influenced by the Congo River plume (> 105 cDNA nifH copies l−1). Both cyanobacterial unicellular groups (A and B) were detected, where group A was the second most abundant in surface samples, in particular at the stations along the equator. Transcript abundance for group A, however, was at the detection limit and suggests that it was not actively fixing N2. Trichodesmium and group B nifH gene abundances co-varied (P < 0.0001). Richelia associated with Hemiaulus hauckii diatoms were detected in 9 of 10 surface samples and the highest abundances (> 104nifH copies l−1) were found north-west of the Congo River plume. In contrast, the Calothrix symbionts (het-3) of Chaetoceros had low abundances at the surface, but were present at 3.7 × 104nifH copies l−1 at 40 m depth in the equatorial upwelling. This is the first report of the Calothrix symbiont in the Atlantic Ocean. This is also the first report of nifH gene copy and transcript abundance in an Equatorial upwelling zone. Although the number of gene copies for Richelia associated with Rhizosolenia were the lowest, the transcript abundance were high (9.4 × 101−1.8 × 104 cDNA nifH copies l−1) and similar to that of Trichodesmium. The distribution of the diazotroph groups, especially the three strains of symbiotic cyanobacteria, was different, and appeared largely controlled by riverine inputs and upwelling.
doi:10.1111/j.1462-2920.2008.01796.x
PMCID: PMC2702491  PMID: 19175790
15.  Temporal Patterns of Nitrogenase Gene (nifH) Expression in the Oligotrophic North Pacific Ocean 
Dinitrogen (N2)-fixing microorganisms (diazotrophs) play important roles in ocean biogeochemistry and plankton productivity. In this study, we examined the presence and expression of specific planktonic nitrogenase genes (nifH) in the upper ocean (0 to 175 m) at Station ALOHA in the oligotrophic North Pacific Ocean. Clone libraries constructed from reverse-transcribed PCR-amplified mRNA revealed six unique phylotypes. Five of the nifH phylotypes grouped with sequences from unicellular and filamentous cyanobacteria, and one of the phylotypes clustered with γ-proteobacteria. The cyanobacterial nifH phylotypes retrieved included two sequence types that phylogenetically grouped with unicellular cyanobacteria (termed groups A and B), several sequences closely related (97 to 99%) to Trichodesmium spp. and Katagnymene spiralis, and two previously unreported phylotypes clustering with heterocyst-forming nifH cyanobacteria. Temporal patterns of nifH expression were evaluated using reverse-transcribed quantitative PCR amplification of nifH gene transcripts. The filamentous and presumed unicellular group A cyanobacterial phylotypes exhibited elevated nifH transcription during the day, while members of the group B (closely related to Crocosphaera watsonii) unicellular phylotype displayed greater nifH transcription at night. In situ nifH expression by all of the cyanobacterial phylotypes exhibited pronounced diel periodicity. The γ-proteobacterial phylotype had low transcript abundance and did not exhibit a clear diurnal periodicity in nifH expression. The temporal separation of nifH expression by the various phylotypes suggests that open ocean diazotrophic cyanobacteria have unique in situ physiological responses to daily fluctuations of light in the upper ocean.
doi:10.1128/AEM.71.9.5362-5370.2005
PMCID: PMC1214674  PMID: 16151126
16.  Spatial and Temporal Distribution of Two Diazotrophic Bacteria in the Chesapeake Bay 
The aim of this study was to initiate autecological studies on uncultivated natural populations of diazotrophic bacteria by examining the distribution of specific diazotrophs in the Chesapeake Bay. By use of quantitative PCR, the abundance of two nifH sequences (907h22 and 912h4) was quantified in water samples collected along a transect from the head to the mouth of the Chesapeake Bay during cruises in April and October 2001 and 2002. Standard curves for the quantitative PCR assays demonstrated that the relationship between gene copies and cycle threshold was linear and highly reproducible from 1 to 107 gene copies. The maximum number of 907h22 gene copies detected was approximately 140 ml−1 and the maximum number of 912h4 gene copies detected was approximately 340 ml−1. Sequence 912h4 was most abundant at the mouth of the Chesapeake Bay, and in general, its abundance increased with increasing salinity, with the highest abundances observed in April 2002. Overall, the 907h22 phylotype was most abundant at the mid-bay station. Additionally, 907h22 was most abundant in the April samples from the mid-bay and mouth of the Chesapeake Bay. Despite the fact that the Chesapeake Bay is rarely nitrogen limited, our results show that individual nitrogen-fixing bacteria have distinct nonrandom spatial and seasonal distributions in the Chesapeake Bay and are either distributed by specific physical processes or adapted to different environmental niches.
doi:10.1128/AEM.70.4.2186-2192.2004
PMCID: PMC383158  PMID: 15066812
17.  Determination of Nitrogen-Fixing Phylotypes in Lyngbya sp. and Microcoleus chthonoplastes Cyanobacterial Mats from Guerrero Negro, Baja California, Mexico 
In many environments, biological nitrogen fixation can alleviate nitrogen limitation. The high rates of N2 fixation often observed in cyanobacterial mats suggest that N2 fixation may be an important source of N. In this study, organisms expressing nifH were identified in a Lyngbya sp.- and two Microcoleus chthonoplastes-dominated cyanobacterial mats. The pattern of nitrogenase activity was determined for the Lyngbya sp. mat and a Microcoleus chthonoplastes mat sampled directly in Guerrero Negro, Mexico. Their maximum rates were 23 and 15 μmol of C2H4 m−2 h−1, respectively. The second Microcoleus mat, which was maintained in a greenhouse facility, had a maximum rate of 40 μmol of C2H4 m−2 h−1. The overall diel pattern of nitrogenase activity in the three mats was similar, with the highest rates of activity occurring during the dark period. Analysis of nifH transcripts by reverse transcription-PCR revealed that several different organisms were expressing nifH during the dark period. nifH phylotypes recovered from these mats were similar to sequences from the unicellular cyanobacterial genera Halothece, Myxosarcina, and Synechocystis, the filamentous cyanobacterial genera Plectonema and Phormidium, and several bacterial nifH groups. The results of this study indicate that several different organisms, some of which were not previously known to fix nitrogen, are likely to be responsible for the observed dark-period nitrogenase activity in these cyanobacterial mats.
doi:10.1128/AEM.70.4.2119-2128.2004
PMCID: PMC383108  PMID: 15066804
18.  Fingerprinting Diazotroph Communities in the Chesapeake Bay by Using a DNA Macroarray 
Investigations of the distribution and diversity of nitrogen-fixing microorganisms in natural environments have often relied on PCR amplification and sequence analysis of a portion of one of the key enzymes in nitrogen fixation, dinitrogenase reductase, encoded by nifH. Recent work has suggested that DNA macroarrays provide semiquantitative fingerprints of diversity within mixtures of nifH amplicons (G. F. Steward, B. D. Jenkins, B. B. Ward, and J. P. Zehr, Appl. Environ. Microbiol. 70:1455-1465, 2004). Here we report the application of macroarrays for a study in the Chesapeake Bay. Samples from different locations in the bay yielded distinct fingerprints. Analysis of replicates and samples from different locations by cluster analysis showed that replicates clustered together, whereas different samples formed distinct clusters. There was a correspondence between the hybridization pattern observed and that predicted from the distribution of sequence types in a corresponding clone library. Some discrepancies between the methods were observed which are likely a result of the high nifH sequence diversity in the Chesapeake Bay and the limited number of sequences represented on this version of the array. Analyses of sequences in the clone library indicate that the Chesapeake Bay harbors unique, phylogenetically diverse diazotrophs. The macroarray hybridization patterns suggest that there are spatially variable communities of diazotrophs, which have been confirmed by quantitative PCR methods (S. M. Short, B. D. Jenkins, and J. P. Zehr, Appl. Environ. Microbiol., in press). The results show that DNA macroarrays have great potential for mapping the spatial and temporal variability of functional gene diversity in the environment.
doi:10.1128/AEM.70.3.1767-1776.2004
PMCID: PMC368353  PMID: 15006803
19.  Development and Testing of a DNA Macroarray To Assess Nitrogenase (nifH) Gene Diversity 
A DNA macroarray was developed and evaluated for its potential to distinguish variants of the dinitrogenase reductase (nifH) gene. Diverse nifH gene fragments amplified from a clone library were spotted onto nylon membranes. Amplified, biotinylated nifH fragments from individual clones or a natural picoplankton community were hybridized to the array and detected by chemiluminescence. A hybridization test with six individual targets mixed in equal proportions resulted in comparable relative signal intensities for the corresponding probes (standard deviation, 14%). When the targets were mixed in unequal concentrations, there was a predictable, but nonlinear, relationship between target concentration and relative signal intensity. Results implied a detection limit of roughly 13 pg of target ml−1, a half-saturation of signal at 0.26 ng ml−1, and a dynamic range of about 2 orders of magnitude. The threshold for cross-hybridization varied between 78 and 88% sequence identity. Hybridization patterns were reproducible with significant correlations between signal intensities of duplicate probes (r = 0.98, P < 0.0001, n = 88). A mixed nifH target amplified from a natural Chesapeake Bay water sample hybridized strongly to 6 of 88 total probes and weakly to 17 additional probes. The natural community results were well simulated (r = 0.941, P < 0.0001, n = 88) by hybridizing a defined mixture of six individual targets corresponding to the strongly hybridizing probes. Our results indicate that macroarray hybridization can be a highly reproducible, semiquantitative method for assessing the diversity of functional genes represented in mixed pools of PCR products amplified from the environment.
doi:10.1128/AEM.70.3.1455-1465.2004
PMCID: PMC368376  PMID: 15006766
20.  Nitrogen Cycling in the Ocean: New Perspectives on Processes and Paradigms 
doi:10.1128/AEM.68.3.1015-1024.2002
PMCID: PMC123768  PMID: 11872445
21.  Diversity and Detection of Nitrate Assimilation Genes in Marine Bacteria 
Applied and Environmental Microbiology  2001;67(11):5343-5348.
A PCR approach was used to construct a database of nasA genes (called narB genes in cyanobacteria) and to detect the genetic potential for heterotrophic bacterial nitrate utilization in marine environments. A nasA-specific PCR primer set that could be used to selectively amplify the nasA gene from heterotrophic bacteria was designed. Using seawater DNA extracts obtained from microbial communities in the South Atlantic Bight, the Barents Sea, and the North Pacific Gyre, we PCR amplified and sequenced nasA genes. Our results indicate that several groups of heterotrophic bacterial nasA genes are common and widely distributed in oceanic environments.
doi:10.1128/AEM.67.11.5343-5348.2001
PMCID: PMC93313  PMID: 11679368
22.  Expression of nifH Genes in Natural Microbial Assemblages in Lake George, New York, Detected by Reverse Transcriptase PCR 
A modified nested reverse transcriptase PCR (RT-PCR) method was used to detect the expression of nitrogenase genes in meso-oligotrophic Lake George, New York. Net (>20-μm pore size) plankton samples collected from two sites (Dome Island and Hague Marina) were extracted for total RNA and genomic DNA to determine the identity of diazotrophic organisms that were present and those that were actively expressing nitrogenase genes. Phylogenetic analysis of individual sequences cloned from PCR amplifications showed that there were phylogenetically diverse groups of bacteria that possessed a nifH gene, including representatives of unicellular and filamentous cyanobacteria, the α- and γ-subdivisions of the division Proteobacteria (α- and γ-proteobacteria), and a previously undefined group of bacteria. The phylotypes cloned from RT-PCR amplifications, which were actively expressing nifH transcripts, clustered with the unicellular and filamentous cyanobacteria, α-proteobacteria, and the novel bacterial cluster. No bacterial sequences were found which clustered with sequences from cluster II (alternative nitrogenases), III (nitrogenases in strict anaerobes), or IV (nifH-like sequences). These results indicate that there were several distinct groups of nitrogen-fixing microorganisms in the net plankton from both sampling sites and that most of the groups had representative phylotypes that were actively expressing nitrogenase genes.
PMCID: PMC92123  PMID: 10877818
24.  New Nitrogen-Fixing Microorganisms Detected in Oligotrophic Oceans by Amplification of Nitrogenase (nifH) Genes 
Oligotrophic oceanic waters of the central ocean gyres typically have extremely low dissolved fixed inorganic nitrogen concentrations, but few nitrogen-fixing microorganisms from the oceanic environment have been cultivated. Nitrogenase gene (nifH) sequences amplified directly from oceanic waters showed that the open ocean contains more diverse diazotrophic microbial populations and more diverse habitats for nitrogen fixers than previously observed by classical microbiological techniques. Nitrogenase genes derived from unicellular and filamentous cyanobacteria, as well as from the α and γ subdivisions of the class Proteobacteria, were found in both the Atlantic and Pacific oceans. nifH sequences that cluster phylogenetically with sequences from sulfate reducers or clostridia were found associated with planktonic crustaceans. Nitrogenase sequence types obtained from invertebrates represented phylotypes distinct from the phylotypes detected in the picoplankton size fraction. The results indicate that there are in the oceanic environment several distinct potentially nitrogen-fixing microbial assemblages that include representatives of diverse phylotypes.
PMCID: PMC106745  PMID: 9726895
25.  Circadian Rhythm of Nitrogenase Gene Expression in the Diazotrophic Filamentous Nonheterocystous Cyanobacterium Trichodesmium sp. Strain IMS 101 
Journal of Bacteriology  1998;180(14):3598-3605.
Recent studies suggested that the daily cycle of nitrogen fixation activity in the marine filamentous nonheterocystous cyanobacterium Trichodesmium sp. is controlled by a circadian rhythm. In this study, we evaluated the rhythm of nitrogen fixation in Trichodesmium sp. strain IMS 101 by using the three criteria for an endogenous rhythm. Nitrogenase transcript abundance oscillated with a period of approximately 24 h, and the cycle was maintained even under constant light conditions. The cyclic pattern of transcript abundance was maintained when the culture was grown at 24 and 28.5°C, although the period was slightly longer (26 h) at the higher temperature. The cycle of gene expression could be entrained with light-dark cues. Results of inhibitor experiments indicated that transcript abundance was regulated primarily by transcription initiation, rather than by degradation. The circadian rhythm, the first conclusively demonstrated endogenous rhythm in a filamentous cyanobacterium, was also reflected in nitrogenase MoFe protein abundance and patterns of Fe protein posttranslational modification-demodification.
PMCID: PMC107328  PMID: 9658003

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