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.     

Diazotrophic diversity and distribution in the tropical and subtropical Atlantic Ocean

To understand the structure of marine diazotrophic communities in the tropical and subtropical Atlantic Ocean, the molecular diversity of the nifH gene was studied by nested PCR amplification using degenerate primers, followed by cloning and sequencing. Sequences of nifH genes were amplified from environmental DNA samples collected during three cruises (November-December 2000, March 2002, and October-November 2002) covering an area between 0 to 28.3 degrees N and 56.6 to 18.5 degrees W. A total of 170 unique sequences were recovered from 18 stations and 23 depths. Samples from the November-December 2000 cruise contained both unicellular and filamentous cyanobacterial nifH phylotypes, as well as gamma-proteobacterial and cluster III sequences, so far only reported in the Pacific Ocean. In contrast, samples from the March 2002 cruise contained only phylotypes related to the uncultured group A unicellular cyanobacteria. The October-November 2002 cruise contained both filamentous and unicellular cyanobacterial and gamma-proteobacterial sequences. Several sequences were identical at the nucleotide level to previously described environmental sequences from the Pacific Ocean, including group A sequences. The data suggest a community shift from filamentous cyanobacteria in surface waters to unicellular cyanobacteria and/or heterotrophic bacteria in deeper waters. With one exception, filamentous cyanobacterial nifH sequences were present within temperatures ranging between 26.5 and 30 degrees C and where nitrate was undetectable. In contrast, nonfilamentous nifH sequences were found throughout a broader temperature range, 15 to 30 degrees C, more often in waters with temperature of <26 degrees C, and were sometimes recovered from waters with detectable nitrate concentrations.     

Variability in benthic diazotrophy and cyanobacterial diversity in a tropical intertidal lagoon

Benthic nitrogen fixation has been estimated to contribute 15 Tg N year(-1) to the marine nitrogen budget. With benthic marine nitrogen fixation being largely overlooked in more recent surveys, a refocus on benthic diazotrophy was considered important. Variations in nitrogenase activity (acetylene reduction-gas chromatography) in a tropical lagoon in the western Indian Ocean (Zanzibar, Tanzania) were monitored over a 3-year period (2003-2005) and related to cyanobacterial and diazotrophic microbial diversity using a polyphasic approach. Different nitrogenase activity patterns were discerned, with the predominant pattern being high daytime activities combined with low nighttime activities. Analyses of the morphological and 16S rRNA gene diversity among cyanobacteria revealed filamentous nonheterocystous (Oscillatoriales) and unicellular (Chroococcales) representatives to be predominant. Analyses of the nifH gene diversity showed that the major phylotypes belonged to noncyanobacterial prokaryotes. However, as shown by cyanobacterial selective nifH-denaturing gradient gel electrophoresis analysis, cyanobacterial nifH gene sequences were present at all sites. Several nifH and 16S rRNA gene phylotypes were related to uncultured cyanobacteria or bacteria of geographically distant habitats, stressing the widespread occurrence of still poorly characterized microorganisms in tropical benthic marine communities.     

Diversity of diazotrophic unicellular cyanobacteria in the tropical North Atlantic Ocean

We present data on the genetic diversity and phylogenetic affinities of N2-fixing unicellular cyanobacteria in the plankton of the tropical North Atlantic Ocean. Our dinitrogenase gene (nifH) sequences grouped together with a group of cyanobacteria from the subtropical North Pacific; another subtropical North Pacific group was only distantly related. Most of the 16S ribosomal DNA sequences from our tropical North Atlantic samples were closely allied with sequences from a symbiont of the diatom Climacodium frauenfeldianum. These findings suggest a complex pattern of evolutionary and ecological divergence among unicellular cyanobacteria within and between ocean basins.     

Epilithic cyanobacterial communities of a marine tropical beach rock (Heron Island, Great Barrier Reef): diversity and diazotrophy

The diversity and nitrogenase activity of epilithic marine microbes in a Holocene beach rock (Heron Island, Great Barrier Reef, Australia) with a proposed biological calcification "microbialite" origin were examined. Partial 16S rRNA gene sequences from the dominant mat (a coherent and layered pink-pigmented community spread over the beach rock) and biofilms (nonstratified, differently pigmented microbial communities of small shallow depressions) were retrieved using denaturing gradient gel electrophoresis (DGGE), and a clone library was retrieved from the dominant mat. The 16S rRNA gene sequences and morphological analyses revealed heterogeneity in the cyanobacterial distribution patterns. The nonheterocystous filamentous genus Blennothrix sp., phylogenetically related to Lyngbya, dominated the mat together with unidentified nonheterocystous filaments of members of the Pseudanabaenaceae and the unicellular genus Chroococcidiopsis. The dominance and three-dimensional intertwined distribution of these organisms were confirmed by nonintrusive scanning microscopy. In contrast, the less pronounced biofilms were dominated by the heterocystous cyanobacterial genus Calothrix, two unicellular Entophysalis morphotypes, Lyngbya spp., and members of the Pseudanabaenaceae family. Cytophaga-Flavobacterium-Bacteroides and Alphaproteobacteria phylotypes were also retrieved from the beach rock. The microbial diversity of the dominant mat was accompanied by high nocturnal nitrogenase activities (as determined by in situ acetylene reduction assays). A new DGGE nifH gene optimization approach for cyanobacterial nitrogen fixers showed that the sequences retrieved from the dominant mat were related to nonheterocystous uncultured cyanobacterial phylotypes, only distantly related to sequences of nitrogen-fixing cultured cyanobacteria. These data stress the occurrence and importance of nonheterocystous epilithic cyanobacteria, and it is hypothesized that such epilithic cyanobacteria are the principal nitrogen fixers of the Heron Island beach rock.     

Composition and diversity of nifH genes of nitrogen-fixing cyanobacteria associated with boreal forest feather mosses

Recent studies have revealed that nitrogen fixation by cyanobacteria living in association with feather mosses is a major input of nitrogen to boreal forests. We characterized the community composition and diversity of cyanobacterial nifH phylotypes associated with each of two feather moss species (Pleurozium schreberi and Hylocomium splendens) on each of 30 lake islands varying in ecosystem properties in northern Sweden. Nitrogen fixation was measured using acetylene reduction, and nifH sequences were amplified using general and cyanobacterial selective primers, separated and analyzed using density gradient gel electrophoresis (DGGE) or cloning, and further sequenced for phylogenetic analyses. Analyses of DGGE fingerprinting patterns revealed two host-specific clusters (one for each moss species), and sequence analysis showed five clusters of nifH phylotypes originating from heterocystous cyanobacteria. For H. splendens only, N(2) fixation was related to both nifH composition and diversity among islands. We demonstrated that the cyanobacterial communities associated with feather mosses show a high degree of host specificity. However, phylotype composition and diversity, and nitrogen fixation, did not differ among groups of islands that varied greatly in their availability of resources. These results suggest that moss species identity, but not extrinsic environmental conditions, serves as the primary determinant of nitrogen-fixing cyanobacterial communities that inhabit mosses.     

Spatial-temporal variability in diazotroph assemblages in Chesapeake Bay using an oligonucleotide nifH microarray

The distribution of nitrogen-fixing microorganisms in the Chesapeake Bay was investigated using fingerprints from a nifH microarray comprised of 706 60-mer oligonucleotide nifH probes representing cultivated organisms and environmental clones from different nifH clusters. Diverse nifH targets, amplified from samples using degenerate nifH primers, were detected in water column and sediment samples collected in April and October, 2001-2002. Total nifH richness and diversity (Simpson's and Shannon indices) were highest at the most riverine, oligohaline North Bay station. In most samples, the highest diversity was in nifH Cluster 3, which includes many anaerobes, while Cluster 1 (alpha-, beta- gamma- Proteobacteria, Cyanobacteria) targets had the greatest microarray signal intensities. In a multidimensional scaling analysis, deep water communities from April and October were similar within each of the sampling sites, while the surface communities had more variability. Diazotroph communities in the water column in the North Bay were distinct from the Mid- and South Bay communities, and there was a gradual change in sediment diazotroph assemblages from the North to the South Bay. Diazotrophic assemblages from the majority of the water column samples from the Mid- and South Bay clustered with the sediment assemblage in Mid-Bay. Dissolved inorganic nitrogen, salinity, dissolved organic carbon and dissolved organic phosphorus had a significant relationship with the diazotrophic bacterioplankton community. Higher diversity in the freshwater end of the system may reflect variability in disturbance rates and environmental conditions such as forms and concentrations of organic matter, nutrients and oxygen.     

Phylogenetic diversity of nitrogen-fixing bacteria and the nifH gene from mangrove rhizosphere soil

Nine types of nitrogen-fixing bacterial strains were isolated from 3 rhizosphere soil samples taken from mangrove plants in the Dongzhaigang National Mangrove Nature Reserve of China. Most isolates belonged to Gammaproteobacteria Pseudomonas, showing that these environments constituted favorable niches for such abundant nitrogen-fixing bacteria. New members of the diazotrophs were also found. Using a soil DNA extraction and PCR-cloning-sequencing approach, 135 clones were analyzed by restriction fragment length polymorphism (RFLP) analysis, and 27 unique nifH sequence phylotypes were identified, most of which were closely related to sequences from uncultured bacteria. The diversity of nitrogen-fixing bacteria was assessed by constructing nifH phylogenetic trees from sequences of all isolates and clones in this work, together with related nifH sequences from other mangrove ecosystems in GenBank. The nifH diversity varied among soil samples, with distinct biogeochemical properties within a mangrove ecosystem. When comparing different mangrove ecosystems, the nifH gene sequences from a specific site tended to cluster as individual groups. The results provided interesting data and novel information on our understanding of diazotroph community diversity in the mangrove ecosystems.     

Symbiotic nitrogen fixation in the New Zealand dampwood termite (

This study investigates symbiotic microorganisms in the New Zealand dampwood termite Stolotermes ruficeps using culture-independent techniques to describe the diversity of nitrogen-fixing organisms within this termite. Phylogenetic analysis of a portion of the nifH gene (encoding dinitrogenase reductase) revealed 19 phylotypes (>98% sequence identity) with 77?86% similarity to published nucleotide sequences from uncultured microorganisms described from termite guts. The majority of sequences obtained in this study were most closely related to sequences obtained from basal families Kalotermitidae, Termopsidae and the closely related wood-feeding cockroach species Cryptocercus. This adds to the growing amount of evidence suggesting that the composition of nifH sequences is characteristic of a termite family. This study also identifies wood-dwelling termites as a potentially important source of nitrogen input into temperate forests, something previously neglected and warranting further investigation.     

Diazotrophic Diversity and Distribution in the Tropical and Subtropical Atlantic Ocean

To understand the structure of marine diazotrophic communities in the tropical and subtropical Atlantic Ocean, the molecular diversity of the nifH gene was studied by nested PCR amplification using degenerate primers, followed by cloning and sequencing. Sequences of nifH genes were amplified from environmental DNA samples collected during three cruises (November-December 2000, March 2002, and October-November 2002) covering an area between 0 to 28.3°N and 56.6 to 18.5°W. A total of 170 unique sequences were recovered from 18 stations and 23 depths. Samples from the November-December 2000 cruise contained both unicellular and filamentous cyanobacterial nifH phylotypes, as well as γ-proteobacterial and cluster III sequences, so far only reported in the Pacific Ocean. In contrast, samples from the March 2002 cruise contained only phylotypes related to the uncultured group A unicellular cyanobacteria. The October-November 2002 cruise contained both filamentous and unicellular cyanobacterial and γ-proteobacterial sequences. Several sequences were identical at the nucleotide level to previously described environmental sequences from the Pacific Ocean, including group A sequences. The data suggest a community shift from filamentous cyanobacteria in surface waters to unicellular cyanobacteria and/or heterotrophic bacteria in deeper waters. With one exception, filamentous cyanobacterial nifH sequences were present within temperatures ranging between 26.5 and 30°C and where nitrate was undetectable. In contrast, nonfilamentous nifH sequences were found throughout a broader temperature range, 15 to 30°C, more often in waters with temperature of <26°C, and were sometimes recovered from waters with detectable nitrate concentrations.     

Archaeal Communities in a Tropical Estuarine Ecosystem: Guanabara Bay,Brazil

Guanabara Bay is an eutrophic estuarine system located in a humid tropical region surrounded by the second largest metropolitan area of Brazil. This study explores the contrasting environmental chemistry and microbiological parameters that influence the archaeaplankton diversity in a pollution gradient in Guanabara Bay ecosystem. The environments sampled ranged from completely anoxic waters in a polluted inner channel to the adjacent, relatively pristine, coastal Atlantic Ocean. Partial archaeal 16S rDNA sequences in water samples were retrieved by polymerase chain reaction (PCR) and analyzed using denaturing gradient gel electrophoresis (DGGE), cloning, and sequencing. Sequences were subjected to phylogenetic and diversity analyses. Community structure of the free-living archaeal assemblages was different from that of the particle-attached archaea according to DGGE. Gene libraries revealed that phylotype identification was consistent with environmental setting. Archaeal phylotypes found in polluted anoxic waters and in more pristine waters were closely related to organisms that have previously been found in these environments. However, inner bay archaea were related to organisms found in oil, industrial wastes, and sewage, implying that water pollution controls archaea communities in this system. The detection of a substantial number of uncultured phylotypes suggests that Guanabara Bay harbors a pool of novel archaeaplankton taxa.     

Environmental forcing of nitrogen fixation in the eastern tropical and sub-tropical North Atlantic Ocean

During the winter of 2006 we measured nifH gene abundances, dinitrogen (N(2)) fixation rates and carbon fixation rates in the eastern tropical and sub-tropical North Atlantic Ocean. The dominant diazotrophic phylotypes were filamentous cyanobacteria, which may include Trichodesmium and Katagnymene, with up to 10(6) L(-1)nifH gene copies, unicellular group A cyanobacteria with up to 10(5) L(-1)nifH gene copies and gamma A proteobacteria with up to 10(4) L(-1)nifH gene copies. N(2) fixation rates were low and ranged between 0.032-1.28 nmol N L(-1) d(-1) with a mean of 0.30 ± 0.29 nmol N L(-1) d(-1) (1σ, n = 65). CO(2)-fixation rates, representing primary production, appeared to be nitrogen limited as suggested by low dissolved inorganic nitrogen to phosphate ratios (DIN:DIP) of about 2 ± 3.2 in surface waters. Nevertheless, N(2) fixation rates contributed only 0.55 ± 0.87% (range 0.03-5.24%) of the N required for primary production. Boosted regression trees analysis (BRT) showed that the distribution of the gamma A proteobacteria and filamentous cyanobacteria nifH genes was mainly predicted by the distribution of Prochlorococcus, Synechococcus, picoeukaryotes and heterotrophic bacteria. In addition, BRT indicated that multiple a-biotic environmental variables including nutrients DIN, dissolved organic nitrogen (DON) and DIP, trace metals like dissolved aluminum (DAl), as a proxy of dust inputs, dissolved iron (DFe) and Fe-binding ligands as well as oxygen and temperature influenced N(2) fixation rates and the distribution of the dominant diazotrophic phylotypes. Our results suggest that lower predicted oxygen concentrations and higher temperatures due to climate warming may increase N(2) fixation rates. However, the balance between a decreased supply of DIP and DFe from deep waters as a result of more pronounced stratification and an enhanced supply of these nutrients with a predicted increase in deposition of Saharan dust may ultimately determine the consequences of climate warming for N(2) fixation in the North Atlantic.     

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.     

Metagenomic Characterization of Chesapeake Bay Virioplankton

Viruses are ubiquitous and abundant throughout the biosphere. In marine systems, virus-mediated processes can have significant impacts on microbial diversity and on global biogeocehmical cycling. However, viral genetic diversity remains poorly characterized. To address this shortcoming, a metagenomic library was constructed from Chesapeake Bay virioplankton. The resulting sequences constitute the largest collection of long-read double-stranded DNA (dsDNA) viral metagenome data reported to date. BLAST homology comparisons showed that Chesapeake Bay virioplankton contained a high proportion of unknown (homologous only to environmental sequences) and novel (no significant homolog) sequences. This analysis suggests that dsDNA viruses are likely one of the largest reservoirs of unknown genetic diversity in the biosphere. The taxonomic origin of BLAST homologs to viral library sequences agreed well with reported abundances of cooccurring bacterial subphyla within the estuary and indicated that cyanophages were abundant. However, the low proportion of Siphophage homologs contradicts a previous assertion that this family comprises most bacteriophage diversity. Identification and analyses of cyanobacterial homologs of the psbA gene illustrated the value of metagenomic studies of virioplankton. The phylogeny of inferred PsbA protein sequences suggested that Chesapeake Bay cyanophage strains are endemic in that environment. The ratio of psbA homologous sequences to total cyanophage sequences in the metagenome indicated that the psbA gene may be nearly universal in Chesapeake Bay cyanophage genomes. Furthermore, the low frequency of psbD homologs in the library supports the prediction that Chesapeake Bay cyanophage populations are dominated by Podoviridae.