Effects of nutrient enrichment on surface microbial community gene expression in the oligotrophic North Pacific Subtropical Gyre.

Marine microbial communities are critical for biogeochemical cycles and the productivity of ocean ecosystems. Primary productivity in the surface ocean is constrained by nutrients which are supplied, in part, by mixing with deeper water. Little is known about the time scales, frequency, or impact of mixing on microbial communities. We combined in situ sampling using the Environmental Sample Processor and a small-scale mixing experiment with lower euphotic zone water to determine how individual populations respond to mixing. Transcriptional responses were measured using the MicroTOOLs (Microbiological Targets for Ocean Observing Laboratories) microarray, which targets all three domains of life and viruses. The experiment showed that mixing substantially affects photosynthetic taxa as expected, but surprisingly also showed that populations respond differently to unfiltered deep water which contains particles (organisms and detritus) compared to filtered deep water that only contains nutrients and viruses, pointing to the impact of biological interactions associated with these events. Comparison between experimental and in situ population transcription patterns indicated that manipulated populations can serve as analogs for natural populations, and that natural populations may be frequently or continuously responding to nutrients from deeper waters. Finally, this study also shows that the microarray approach, which is complementary to metatranscriptomic sequencing, is useful for determining the physiological status of in situ microbial communities.

Vertical distribution of nitrogen-fixing phylotypes in a meromictic, hypersaline lake.

We investigated the diversity of nitrogenase genes in the alkaline, moderately hypersaline Mono Lake, California to determine (1) whether nitrogen-fixing (diazotrophic) populations were similar to those in other aquatic environments and (2) if there was a pattern of distribution of phylotypes that reflected redox conditions, as well as (3) to identify populations that could be important in N dynamics in this nitrogen-limited lake. Mono Lake has been meromictic for almost a decade and has steep gradients in oxygen and reduced compounds that provide a wide range of aerobic and anaerobic habitats. We amplified a fragment of the nitrogenase gene (nifH) from planktonic DNA samples collected at three depths representing oxygenated surface waters, the oxycline, and anoxic, ammonium-rich deep waters. Forty-three percent of the 90 sequences grouped in nifH Cluster I. The majority of clones (57%) grouped in Cluster III, which contains many known anaerobic bacteria. Cluster I and Cluster III sequences were retrieved at every depth indicating little vertical zonation in sequence types related to the prominent gradients in oxygen and ammonia. One group in Cluster I was found most often at every depth and accounted for 29% of all the clones. These sequences formed a subcluster that contained other environmental clones, but no cultivated representatives. No significant nitrogen fixation was detected by the 15N2 method after 48 h of incubation of surface, oxycline, or deep waters, suggesting that pelagic diazotrophs were contributing little to nitrogen fluxes in the lake. The failure to measure any significant nitrogen fixation, despite the detection of diverse and novel nitrogenase genes throughout the water column, raises interesting questions about the ecological controls on diazotrophy in Mono Lake and the distribution of functional genes in the environment.

Nitrogen-fixing phylotypes of Chesapeake Bay and Neuse River estuary sediments.

Sediments often exhibit low rates of nitrogen fixation, despite the presence of elevated concentrations of inorganic nitrogen. The organisms that potentially fix nitrogen in sediments have not previously been identified. Amplification of nifH genes with degenerate primers was used to assess the diversity of diazotrophs in two distinct sediment systems, anoxic muds of Chesapeake Bay and shallow surficial sediments of the Neuse River. Phylogenetic analysis revealed that sequences obtained from mid-Chesapeake Bay, which receive high organic loading and are highly reducing, clustered closely with each other and with known anaerobic microorganisms, suggesting a low abundance of aerobic or facultative diazotrophs in these sediments. Sulfate reduction dominates in the surface, but methanogenesis becomes more important with depth. A thin (<1 cm) oxidized layer is present only in the spring. No archaeal nifH sequences were obtained from Chesapeake Bay. Sequences of nifH amplified from surficial sediments of the Neuse River were distant from Chesapeake Bay sequences and included nif phylotypes related to sequences previously reported from marine mats and the Spartina rhizosphere. Differences in environmental site characteristics appear to select for different types of sediment diazotrophs, which is reflected in the phylogenetic composition of amplified nifH sequences.

Diversity and detection of nitrate assimilation genes in marine bacteria.

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.

Unicellular cyanobacteria fix N2 in the subtropical North Pacific Ocean.

Fixed nitrogen (N) often limits the growth of organisms in terrestrial and aquatic biomes, and N availability has been important in controlling the CO2 balance of modern and ancient oceans. The fixation of atmospheric dinitrogen gas (N2) to ammonia is catalysed by nitrogenase and provides a fixed N for N-limited environments. The filamentous cyanobacterium Trichodesmium has been assumed to be the predominant oceanic N2-fixing microorganism since the discovery of N2 fixation in Trichodesmium in 1961 (ref. 6). Attention has recently focused on oceanic N2 fixation because nitrogen availability is generally limiting in many oceans, and attempts to constrain the global atmosphere-ocean fluxes of CO2 are based on basin-scale N balances. Biogeochemical studies and models have suggested that total N2-fixation rates may be substantially greater than previously believed but cannot be reconciled with observed Trichodesmium abundances. It is curious that there are so few known N2-fixing microorganisms in oligotrophic oceans when it is clearly ecologically advantageous. Here we show that there are unicellular cyanobacteria in the open ocean that are expressing nitrogenase, and are abundant enough to potentially have a significant role in N dynamics.

Distribution of Nitrogen-Fixing Microorganisms along the Neuse River Estuary, North Carolina.

Nitrogen fixation genes (nifH) were amplified and sequenced from DNA extracted from surface water samples collected from six stations along the length of the Neuse River Estuary, North Carolina, in order to determine the distribution of nitrogen-fixing organisms in the transition from fresh- to saltwater. Nitrogenase genes were detected in all samples by a nested polymerase chain reaction method, and the amplification products from the upriver, midriver, and downriver stations were cloned, sequenced, and used for phylogenetic analysis. The composition of nifH clone libraries from upriver, midriver, and downriver stations (each composed of 14 randomly selected clones) were very diverse (samples from upriver and midriver stations were composed of 14 unique sequences, downriver station composed of 7 unique sequences) and differed among the stations. Some phylotypes were found at more than one station, but were usually found in the upriver and midriver stations or in the midriver and downriver stations, indicating that the phylotypes were probably transported along the river. Cyanobacterial nifH were not found at the most upriver site, but were a large fraction of sequences (50%) recovered from the downriver station, where nitrate concentration was an order of magnitude lower and salinity was higher. In contrast, g proteobacteria nifH sequences were much more common at the midriver and upriver sites (58% and 64%, respectively), compared to the downriver site (14%). Results indicate that substantially different nitrogen-fixing assemblages are present along the river, reflecting differential watershed hydrological inputs, sedimentation, and environmental selection pressures, along the salinity gradient.

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-microm 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 alpha- and gamma-subdivisions of the division Proteobacteria (alpha- and gamma-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, alpha-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.

New perspectives on nitrogen-fixing microorganisms in tropical and subtropical oceans.

New molecular and microscopic evidence indicates that the open ocean harbors a diverse range of novel free-living and symbiotic nitrogen-fixing microorganisms. Although the extent to which these microorganisms fix nitrogen is currently unclear, ongoing research indicates that they might make a substantial contribution to the open ocean nitrogen budget.

Expression of photosynthesis genes in relation to nitrogen fixation in the diazotrophic filamentous nonheterocystous cyanobacterium Trichodesmium sp. IMS 101.

The daily cycle of nitrogenase expression in the marine filamentous nonheterocystous cyanobacterium Trichodesmium spp. is controlled by a circadian rhythm. We evaluated the rhythm of two key photosynthesis genes, psbA of photosystem II and psaA of photosystem I, in Trichodesmium sp. IMS 101 using the 3 criteria for an endogenous rhythm. The transcript abundance of psbA and psaA transcripts oscillated with a period of ca. 24 h under a 12 h light/12 h dark regime. At 24 degrees C and 28 degrees C the cyclic pattern of transcript abundance was maintained for at least 58 h under constant light conditions, whereas the periods were about 24 h at 24 degrees C, and 26-30 h at the higher temperature. The cycles of psbA and psaA gene expression were entrained using light-dark cues. Transcription of nifHDK was initiated prior to the light period, followed by psbA and finally psaA. There was a 90 degrees (6 h) phase difference between the net accumulation of nifHDK and psbA transcripts, as well as between that of psbA and psaA transcripts. Results of inhibitor experiments indicated that psbA and psaA transcription was regulated differently by initiation and degradation during the light period. Short-term changes of light conditions resulted in significant effects on psbA transcription and nitrogenase activity, but had less of an effect on psaA and nifHDK transcription.

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 alpha and gamma 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.

Circadian rhythm of nitrogenase gene expression in the diazotrophic filamentous nonheterocystous cyanobacterium Trichodesmium sp. strain IMS 101.

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 degrees 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.

Nearly identical 16S rRNA sequences recovered from lakes in North America and Europe indicate the existence of clades of globally distributed freshwater bacteria.

We compared bacterial 16S ribosomal RNA gene sequences recovered from Lake Loosdrecht, the Netherlands, to reported sequences from lakes in Alaska and New York State. In each of the three lake systems, which differ in pH and trophic state, some sequence types were found without related sequences (sequence identity < 90%) in the data sets from the other two systems. Two sequences in the Actinomycetes and Verrucomicrobia radiations were more closely related to sequences from the New York lakes data set than to any other sequence in the global databases. However, the most striking similarities were found in the subdivisions alpha and beta of the Proteobacteria. In these subdivisions three different clusters of highly related bacteria were identified (97-100% sequence identity) that were represented in all three lake regions. The clusters contained no members other than freshwater bacteria. One cluster falls within a monophyletic aquatic supergroup that apparently diverged early in evolution into an exclusive freshwater cluster and an exclusive marine cluster, the so-called SAR11 cluster. The detection of these three bacterial clades in lakes distinguished by geographic distance as well as physical and chemical diversity suggests that these organisms are dispersed globally and that they possess unique functional capabilities enabling successful competition in a wide range of freshwater environments.

New Nitrogen-Fixing Microorganisms Detected in Oligotrophic Oceans by Amplification of Nitrogenase (nifH) Genes.

[This corrects the article on p. 3444 in vol. 64, PMID: 9726895.].

Structural analysis of the Trichodesmium nitrogenase iron protein: implications for aerobic nitrogen fixation activity.

Trichodesmium spp. are marine filamentous nitrogen-fixing cyanobacteria which play an important role in the nitrogen budget of the open ocean. Trichodesmium is unique in that it is nonheterocystous and fixes nitrogen during the day, while evolving oxygen through photosynthesis, even though nitrogenase is sensitive to oxygen inactivation. The sequence of the gene encoding the Fe protein component of nitrogenase from the recently cultivated isolate Trichodesmium sp. IMS 101 was used to construct a 3-dimensional model of the Fe protein, by comparison to the X-ray crystallographic structure of the Fe protein of the gamma-proteobacterium Azotobacter vinelandii. The primary differences in amino acid sequences of the Fe protein from diverse organisms do not impact the critical structural features of the Fe protein. It can be concluded that aerobic nitrogen fixation in Trichodesmium spp. is not facilitated by unique structural features of Trichodesmium Fe protein.

Bacterial diversity in Adirondack mountain lakes as revealed by 16S rRNA gene sequences.

Bacterial communities of seven lakes in the Adirondack Mountains of New York State were characterized by amplification and sequencing of 16S ribosomal DNA. Analysis of over 100 partial sequences revealed a diverse collection of lineages, largely of the class Proteobacteria (19% alpha subdivision, 31% beta subdivision, and 9% gamma subdivision), the phylum Cytophaga-Flavobacteria-Bacteroides (15%), and the order Actinomycetales (18%). Additionally, a number of the sequences were similar to those of the order Verrucomicrobiales. However, few of the sequence types are closely related to those of characterized species. The relative contributions of the groups of sequences differed among the lakes, suggesting that bacterial population structure varies and that it may be possible to relate aquatic bacterial community structure to water chemistry.

Temporal Variability in Nitrogenase Gene Expression in Natural Populations of the Marine Cyanobacterium Trichodesmium thiebautii.

We report a distinct diel periodicity in the abundance of nifH (dinitrogenase reductase) mRNA in natural populations of the nonheterocystous marine cyanobacterium Trichodesmium thiebautii. Our observations show that in addition to translational and posttranslational controls, Trichodesmium nitrogenase expression is also regulated at the transcriptional and/or posttranscriptional level.

Diversity of heterotrophic nitrogen fixation genes in a marine cyanobacterial mat.

The diversity of nitrogenase genes in a marine cyanobacterial mat was investigated through amplification of a fragment of nifH, which encodes the Fe protein of the nitrogenase complex. The amplified nifH products were characterized by DNA sequencing and were compared with the sequences of nitrogenase genes from cultivated organisms. Phylogenetic analysis showed that similar organisms clustered together, with the exception that anaerobic bacteria clustered together, even though they represented firmicutes, (delta)-proteobacteria, and (gamma)-proteobacteria. Mat nifH sequences were most closely related to those of the anaerobes, with a few being most closely related to the cluster of (gamma)-proteobacteria containing Klebsiella and Azotobacter species. No cyanobacterial nifH sequences were found from the mat collected in November when Microcoleus chthonoplastes was the dominant cyanobacterium, but sequences closely related to the cyanobacterium Lyngbya lagerheimeii were found during summer, when a Lyngbya strain was dominant. The results indicate that there is a high diversity of heterotrophic nitrogen-fixing organisms in marine cyanobacterial mats.

Detection and characterization of cyanobacterial nifH genes.

The DNA sequence of a 359-bp fragment of nifH was determined for the heterocystous strains Anabaena sp. strain CA (ATCC 33047), Nostoc muscorum UTEX 1933, a Nostoc sp., Gloeothece sp. strain ATCC 27152, Lyngbya lagerheimii UTEX 1930, and Plectonema boryanum IU 594. Results confirmed that the DNA sequence of the 359-bp segment is sufficiently variable to distinguish cyanobacterial nifH genes from other eubacterial and arachaeobacterial nifH genes, as well as to distinguish heterocystous from nonheterocystous nifH genes. Nonheterocystous cyanobacterial nifH sequences were greater than 70 and 82% identical on the DNA and amino acid levels, respectively, whereas corresponding values for heterocystous cyanobacterial nifH sequences were 84 and 91%. The amplified nifH fragments can be used as DNA probes to differentiate between species, although there was substantial cross-reactivity between the nifH amplification products of some strains. However, an oligonucleotide designed from a sequence conserved within the heterocystous cyanobacteria hybridized primarily with the amplification product from heterocystous strains. The use of oligonucleotides designed from amplified nifH sequences shows great promise for characterizing assemblages of diazotrophs.

Modification of the Fe Protein of Nitrogenase in Natural Populations of Trichodesmium thiebautii.

The Fe protein of nitrogenase in the marine nonheterocystous cyanobacterium Trichodesmium thiebautii is interconverted between two forms, which is reminiscent of the ADP-ribosylation described in the purple bacterium Rhodospirillum rubrum. In natural populations of T. thiebautii during the day, when nitrogenase activity (NA) is present and while photosynthetic rates are high, a low-molecular-mass form of the Fe protein is present. In the late afternoon, the low-molecular-mass form is partially converted to a higher-molecular-mass form (approximately equal distribution of high- and low-molecular-mass forms of the Fe protein subunits), concurrent with cessation of NA. Some of the higher-molecular-mass form persists through the night until the very early morning, when the lower-molecular-mass form appears. New synthesis of both the Fe and MoFe proteins of nitrogenase appears to occur at this time. The higher-molecular-mass form of the Fe protein is also produced rapidly in response to artificially elevated external O(2) levels (40%) during the day. T. thiebautii is capable of recovery of NA in less than 1 h following exposure to 40% O(2), which is correlated with the return of the Fe protein to the lower-molecular-mass form. Recovery from exposure to O(2) is not dependent upon protein synthesis. The modification of the Fe protein is clearly involved in regulation of NA during the diel cycle of NA in T. thiebautii but may also be involved in protecting the Fe protein during transient O(2) concentration increases.

Arrangement of nitrogenase structural genes in an aerobic filamentous nonheterocystous cyanobacterium.

Members of the marine filamentous, nonheterocystous cyanobacterial genus Trichodesmium not only are capable of fixing nitrogen aerobically in the light but when grown under a light-dark cycle will fix nitrogen only during the light phase. In this study, we constructed a restriction map of the structural nitrogen fixation genes (nifHDK) in Trichodesmium sp. strain NIBB 1067. We found that the organization of the nif genes in Trichodesmium sp. strain NIBB 1067 is contiguous, as found in other nonheterocystous cyanobacteria and in heterocysts. Furthermore, the nif gene arrangement was identical when the cultures were grown with combined nitrogen or under nitrogen-fixing conditions. Therefore, no gene rearrangements occur, such as those that occur during the development of heterocysts in heterocystous species.