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.     

Nitrogenase gene expression in the Chesapeake Bay Estuary

Like many estuaries, the Chesapeake Bay has pronounced gradients in salinity and nutrients. Previous studies have shown that there is a high diversity of nitrogenase (nifH) genes in the estuary, and that there are specific distributions of individual nifH phylotypes. In contrast to previous work that revealed the remarkable diversity of nifH phylotypes in the Chesapeake estuary, in this study of nifH expression we only detected two phylotypes, and both were phylogenetically related to cyanobacterial nifH genes. One of the phylotypes was closely related to a nifH sequence from the filamentous, heterocystous cyanobacterium Anabaena cylindrica, and was found at the head of the estuary. The other phylotype was found in a sample collected near the mouth of the estuary and was closely related to nifH sequences from Group A unicellular cyanobacteria, which has previously been reported in oceanic waters only. These nifH phylotypes had distinct patterns of expression that were restricted to different regions of the Chesapeake Bay. This study provides the first evidence of nifH expression in the Chesapeake Bay, and suggests that diazotrophic unicellular cyanobacteria have a broader distribution and activity than previously recognized.     

Co-occurring Synechococcus ecotypes occupy four major oceanic regimes defined by temperature,macronutrients and iron

Marine picocyanobacteria, comprised of the genera Synechococcus and Prochlorococcus, are the most abundant and widespread primary producers in the ocean. More than 20 genetically distinct clades of marine Synechococcus have been identified, but their physiology and biogeography are not as thoroughly characterized as those of Prochlorococcus. Using clade-specific qPCR primers, we measured the abundance of 10 Synechococcus clades at 92 locations in surface waters of the Atlantic and Pacific Oceans. We found that Synechococcus partition the ocean into four distinct regimes distinguished by temperature, macronutrients and iron availability. Clades I and IV were prevalent in colder, mesotrophic waters; clades II, III and X dominated in the warm, oligotrophic open ocean; clades CRD1 and CRD2 were restricted to sites with low iron availability; and clades XV and XVI were only found in transitional waters at the edges of the other biomes. Overall, clade II was the most ubiquitous clade investigated and was the dominant clade in the largest biome, the oligotrophic open ocean. Co-occurring clades that occupy the same regime belong to distinct evolutionary lineages within Synechococcus, indicating that multiple ecotypes have evolved independently to occupy similar niches and represent examples of parallel evolution. We speculate that parallel evolution of ecotypes may be a common feature of diverse marine microbial communities that contributes to functional redundancy and the potential for resiliency.     

Diazotrophy in Modern Marine Bahamian Stromatolites

N2 fixation (nitrogenase activity), primary production, and diazotrophic community composition of stromatolite mats from Highborne Cay, Exuma, Bahamas, were examined over a 2-year period (1997-1998). The purpose of the study was to characterize the ecophysiology of N2 fixation in modern marine stromatolites. Microbial mats are an integral surface component of these stromatolites and are hypothesized to have a major role in stromatolite formation and growth. The stromatolite mats contained active photosynthetic and diazotrophic assemblages that exhibited temporal separation of nitrogenase activity (NA) and photosynthesis. Maximal NA was detected at night. Seasonal differences in NA and net O2 production were observed. Photosynthetic activity and the availability of reduced organic carbon appear to be the key determinants of NA. Additions of the de novo protein synthesis inhibitor chloramphenicol did not inhibit NA in March 1998, but greatly inhibited NA in August 1998. Partial sequence analysis of the nifH gene indicates that a broad diversity of diazotrophs may be responsible for NA in the stromatolites.     

Nitrogen fixation in denitrified marine waters

Nitrogen fixation is an essential process that biologically transforms atmospheric dinitrogen gas to ammonia, therefore compensating for nitrogen losses occurring via denitrification and anammox. Currently, inputs and losses of nitrogen to the ocean resulting from these processes are thought to be spatially separated: nitrogen fixation takes place primarily in open ocean environments (mainly through diazotrophic cyanobacteria), whereas nitrogen losses occur in oxygen-depleted intermediate waters and sediments (mostly via denitrifying and anammox bacteria). Here we report on rates of nitrogen fixation obtained during two oceanographic cruises in 2005 and 2007 in the eastern tropical South Pacific (ETSP), a region characterized by the presence of coastal upwelling and a major permanent oxygen minimum zone (OMZ). Our results show significant rates of nitrogen fixation in the water column; however, integrated rates from the surface down to 120 m varied by ～30 fold between cruises (7.5±4.6 versus 190±82.3 μmol m(-2) d(-1)). Moreover, rates were measured down to 400 m depth in 2007, indicating that the contribution to the integrated rates of the subsurface oxygen-deficient layer was ～5 times higher (574±294 μmol m(-2) d(-1)) than the oxic euphotic layer (48±68 μmol m(-2) d(-1)). Concurrent molecular measurements detected the dinitrogenase reductase gene nifH in surface and subsurface waters. Phylogenetic analysis of the nifH sequences showed the presence of a diverse diazotrophic community at the time of the highest measured nitrogen fixation rates. Our results thus demonstrate the occurrence of nitrogen fixation in nutrient-rich coastal upwelling systems and, importantly, within the underlying OMZ. They also suggest that nitrogen fixation is a widespread process that can sporadically provide a supplementary source of fixed nitrogen in these regions.     

Application of a nifH oligonucleotide microarray for profiling diversity of N2-fixing microorganisms in marine microbial mats

Diazotrophic community structure in microbial mats from Guerrero Negro (GN), Baja California, Mexico, was studied using polymerase chain reaction amplification of the nifH gene and a newly developed nifH oligonucleotide microarray. Ninety-six oligonucleotide probes designed for nifH sequences from cultivated isolates and the environment were printed on glass microarrays. Phylogenetic analysis showed that the probes represented all of the main nifH clusters. Specificity was tested by (i) evaluation of cross hybridization using individual targets, and (ii) comparison of the observed hybridization signals and those predicted from the sequences cloned from microbial mats. Signal intensity had a positive relationship with target concentration and the percentage identity between probe and target. Under moderate stringency and high target concentration, specificity of the probes varied from 77% to 100% with the individual targets tested. At the end of a 7-month long nutrient manipulation experiment in GN microbial mats, no expression of nitrogen fixation under nitrogen loading was detected, although a diverse community of diazotrophs was detected. The diversity in diazotrophic population present was higher than in the population expressing the nifH gene, and there were taxa specific differences in response to nutrients. The nifH microarray is a powerful tool for diazotroph community analysis in the marine environment.     

Comparative genomics reveals surprising divergence of two closely related strains of uncultivated UCYN-A cyanobacteria

Marine planktonic cyanobacteria capable of fixing molecular nitrogen (termed ‘diazotrophs'') are key in biogeochemical cycling, and the nitrogen fixed is one of the major external sources of nitrogen to the open ocean. Candidatus Atelocyanobacterium thalassa (UCYN-A) is a diazotrophic cyanobacterium known for its widespread geographic distribution in tropical and subtropical oligotrophic oceans, unusually reduced genome and symbiosis with a single-celled prymnesiophyte alga. Recently a novel strain of this organism was also detected in coastal waters sampled from the Scripps Institute of Oceanography pier. We analyzed the metagenome of this UCYN-A2 population by concentrating cells by flow cytometry. Phylogenomic analysis provided strong bootstrap support for the monophyly of UCYN-A (here called UCYN-A1) and UCYN-A2 within the marine Crocosphaera sp. and Cyanothece sp. clade. UCYN-A2 shares 1159 of the 1200 UCYN-A1 protein-coding genes (96.6%) with high synteny, yet the average amino-acid sequence identity between these orthologs is only 86%. UCYN-A2 lacks the same major pathways and proteins that are absent in UCYN-A1, suggesting that both strains can be grouped at the same functional and ecological level. Our results suggest that UCYN-A1 and UCYN-A2 had a common ancestor and diverged after genome reduction. These two variants may reflect adaptation of the host to different niches, which could be coastal and open ocean habitats.     

Effects of ecological engineered oxygenation on the bacterial community structure in an anoxic fjord in western Sweden

Oxygen-depleted bodies of water are becoming increasingly common in marine ecosystems. Solutions to reverse this trend are needed and under development, for example, by the Baltic deep-water OXygenation (BOX) project. In the framework of this project, the Swedish Byfjord was chosen for a pilot study, investigating the effects of an engineered oxygenation on long-term anoxic bottom waters. The strong stratification of the water column of the Byfjord was broken up by pumping surface water into the deeper layers, triggering several inflows of oxygen-rich water and increasing oxygen levels in the lower water column and the benthic zone up to 110 μmol l⁻¹.We used molecular ecologic methods to study changes in bacterial community structure in response to the oxygenation in the Byfjord. Water column samples from before, during and after the oxygenation as well as from two nearby control fjords were analyzed. Our results showed a strong shift in bacterial community composition when the bottom water in the Byfjord became oxic. Initially dominant indicator species for oxygen minimum zones such as members of the SUP05 clade declined in abundance during the oxygenation event and nearly vanished after the oxygenation was accomplished. In contrast, aerobic species like SAR11 that initially were restricted to surface waters could later be detected deep into the water column. Overall, the bacterial community in the formerly anoxic bottom waters changed to a community structure similar to those found in oxic waters, showing that an engineered oxygenation of a large body of anoxic marine water is possible and emulates that of a natural oxygenation event.     

Distinct ecological niches of marine symbiotic N2‐fixing cyanobacterium Candidatus Atelocyanobacterium thalassa sublineages

A recently described symbiosis between the metabolically streamlined nitrogen‐fixing cyanobacterium UCYN‐A and a single‐celled eukaryote prymnesiophyte alga is widely distributed throughout tropical and subtropical marine waters, and is thought to contribute significantly to nitrogen fixation in these regions. Several UCYN‐A sublineages have been defined based on UCYN‐A nitrogenase (nifH) sequences. Due to the low abundances of UCYN‐A in the global oceans, currently existing molecular techniques are limited for detecting and quantifying these organisms. A targeted approach is needed to adequately characterize the diversity of this important marine cyanobacterium, and to advance understanding of its ecological importance. We present findings on the distribution of UCYN‐A sublineages based on high throughput sequencing of UCYN‐A nifH PCR amplicons from 78 samples distributed throughout many major oceanic provinces. These UCYN‐A nifH fragments were used to define oligotypes, alternative taxonomic units defined by nucleotide positions with high variability. The data set was dominated by a single oligotype associated with the UCYN‐A1 sublineage, consistent with previous observations of relatively high abundances in tropical and subtropical regions. However, this analysis also revealed for the first time the widespread distribution of the UCYN‐A3 sublineage in oligotrophic waters. Furthermore, distinct assemblages of UCYN‐A oligotypes were found in oligotrophic and coastally influenced waters. This unique data set provides a framework for determining the environmental controls on UCYN‐A distributions and the ecological importance of the different sublineages.     

Biogeography and phylogeny of the NOR5/OM60 clade of Gammaproteobacteria

The phylogeny, abundance, and biogeography of the NOR5/OM60 clade was investigated. This clade includes “Congregibacter litoralis” strain KT71, the first cultured representative of marine aerobic anoxygenic phototrophic Gammaproteobacteria. More than 500 16S rRNA sequences affiliated with this clade were retrieved from public databases. By comparative sequence analysis, 13 subclades could be identified, some of which are currently restricted to discrete habitat types. Almost all sequences in the largest subclade NOR5-1 and related subclade NOR5-4 originated from marine surface water samples. Overall, most of the NOR5/OM60 sequences were retrieved from marine coastal settings, whereas there were fewer from open-ocean surface waters, deep-sea sediment, freshwater, saline lakes and soil.     

Phylogenetic diversity of nitrogen fixation genes in the symbiotic microbial community in the gut of diverse termites.

Nitrogen fixation by the microorganisms in the gut of termites is one of the crucial aspects of symbiosis, since termites usually thrive on a nitrogen-poor diet. The phylogenetic diversity of the nitrogen-fixing organisms within the symbiotic community in the guts of various termite species was investigated without culturing the resident microorganisms. A portion of the dinitrogenase reductase gene (nifH) was directly amplified from DNA extracted from the mixed population in the termite gut. Analysis of deduced amino acid sequences of the products of the clonally isolated nifH genes revealed the presence of diverse nifH sequences in most of the individual termite species, and their constituents were considerably different among termite species. A majority of the nifH sequences from six lower termites, which showed significant levels of nitrogen fixation activity, could be assigned to either the anaerobic nif group (consisting of clostridia and sulfur reducers) or the alternative nif methanogen group among the nifH phylogenetic groups. In the case of three higher termites, which showed only low levels of nitrogen fixation activity, a large number of the sequences were assigned to the most divergent nif group, probably functioning in some process other than nitrogen fixation and being derived from methanogenic archaea. The nifH groups detected were similar within each termite family but different among the termite families, suggesting an evolutionary trend reflecting the diazotrophic habitats in the symbiotic community. Within these phylogenetic groups, the sequences from the termites formed lineages distinct from those previously recognized in studies using classical microbiological techniques, and several sequence clusters unique to termites were found. The results indicate the presence of diverse potentially nitrogen-fixing microbial assemblages in the guts of termites, and the majority of them are as yet uncharacterized.     

KATAGNYMENE: CHARACTERIZATION OF A NOVEL MARINE DIAZOTROPH

The marine planktonic cyanobacterial genus Katagnymene was described by Lemmermann in 1900 and is found in oligotrophic tropical and subtropical oceans. The genus comprises two species, K. pelagica Lemmermann and K. spiralis Lemmermann, and both were observed in most stations sampled during a cruise in the southwest Pacific Ocean in 1998. Katagnymene is nonheterocystous and characterized by single ensheathed trichomes that do not form colonies. Acetylene reduction-GC demonstrated that natural populations of Katagnymene fixed nitrogen and that nitrogenase activity occurred exclusively in the light during a 12:12 light:dark cycle. Whole cell immunolocalization revealed that nitrogenase (the Fe protein) appeared in 7% of the total number of cells and that these were arranged in zones composed of consecutively arranged cells. At least one zone of nitrogenase-containing cells per trichome was found. Nitrogenase was present throughout both the day and night, as shown by Western blotting, and in the same percentage of cells. Ultrastructural immunolocalization on sectioned trichomes also confirmed the presence and localization of nitrogenase in Katagnymene. Cultures of Katagnymene are able to grow on nitrogen-free media and fix nitrogen only in the light. Finally, cloning and sequencing of nifH verified the diazotrophic nature of the genus Katagnymene and demonstrated a close relationship to members of the marine genus Trichodesmium.     

Evolutionary placement of Xanthomonadales based on conserved protein signature sequences

Xanthomonadales comprises one of the largest phytopathogenic bacterial groups, and is currently classified within the gamma-proteobacteria. However, the phylogenetic placement of this group is not clearly resolved, and the results of different studies contradict one another. In this work, the evolutionary position of Xanthomonadales was determined by analyzing the presence of shared insertions and deletions (INDELs) in highly conserved proteins. Several distinctive insertions found in most of the members of the gamma-proteobacteria are absent in Xanthomonadales and groups such as Legionelalles, Chromatiales, Methylococcales, Thiotrichales and Cardiobacteriales. These INDELs were most likely introduced after the branching of Xanthomonadales from most of the gamma-proteobacteria and provide evidence for the phylogenetic placement of the early gamma-proteobacteria. Moreover, other proteins contain insertions exclusive to the Xanthomonadales order, confirming that this is a monophyletic group and provide important specific genetic markers. Thus, the data presented clearly support the Xanthomonadales group as an independent subdivision, and constitute one of the deepest branching lineage within the gamma-proteobacteria clade.     

Abundant proteorhodopsin genes in the North Atlantic Ocean

Proteorhodopsin (PR) is a light-driven proton pump that has been found in a variety of marine bacteria, including Pelagibacter ubique , a member of the ubiquitous SAR11 clade. The goals of this study were to explore the diversity of PR genes and to estimate their abundance in the North Atlantic Ocean using quantitative polymerase chain reaction (QPCR). We found that PR genes in the western portion of the Sargasso Sea could be grouped into 27 clusters, but five clades had the most sequences. Sets of specific QPCR primers were designed to examine the abundance of PR genes in the following four of the five clades: SAR11 ( P. ubique and other SAR11 Alphaproteobacteria ), BACRED17H8 ( Alphaproteobacteria ), HOT2C01 ( Alphaproteobacteria ) and an uncultured subgroup of the Flavobacteria . Two groups (SAR11 and HOT2C01) dominated PR gene abundance in oligotrophic waters, but were significantly less abundant in nutrient- and chlorophyll-rich waters. The other two groups (BACRED17H8 and Flavobacteria subgroup NASB) were less abundant in all waters. Together, these four PR gene types were found in 50% of all bacteria in the Sargasso Sea. We found a significant negative correlation between total PR gene abundance and nutrients and chlorophyll but no significant correlation with light intensity for three of the four PR types in the depth profiles north of the Sargasso Sea. Our data suggest that PR is common in the North Atlantic Ocean, especially in SAR11 bacteria and another marine alphaproteobacterial group (HOT2C01), and that these PR-bearing bacteria are most abundant in oligotrophic waters.     

Effect of N-fertilization, plant genotype and environmental conditions on nifH gene pools in roots of rice

Terminal restriction fragment length polymorphism (T-RFLP) analysis of PCR-amplified nitrogenase gene (nifH) fragments is a rapid technique for profiling of diazotrophic microbial communities without the necessity of cultures for study. Here, we examined the impact of N-fertilization, plant genotype and environmental conditions on diazotrophic microbial populations in association with roots of rice (Oryza species) by T-RFLP community profiling and found marked effects on the composition of the microbial community. We found a rapid change of the diazotrophic population structure within 15 days after application of nitrogen fertilizer and a strong effect of environmental conditions and plant genotype. Control experiments revealed that phylogenetically distantly related nifH genes were proportionately amplified, and that signal strength reflected the relative abundance of nifH genes in the sample within a 10-fold range of template concentrations. These results clearly demonstrated that our T-RFLP method was suitable to reflect compositional differences in the diazotrophic community in a semiquantitative manner and that the diazotrophic rhizosphere communities of rice are not static but presumably rather highly dynamic.