Cyanobacterial assimilatory nitrate reductase gene diversity in coastal and oligotrophic marine environments

Cyanobacteria are important primary producers in many marine ecosystems and their abundances and growth rates depend on their ability to assimilate various nitrogen sources. To examine the diversity of nitrate-utilizing marine cyanobacteria, we developed PCR primers specific for cyanobacterial assimilatory nitrate reductase (narB) genes. We obtained amplification products from diverse strains of cultivated cyanobacteria and from several marine environments. Phylogenetic trees constructed with the narB gene are congruent with those based on ribosomal RNA genes and RNA polymerase genes. Analysis of sequence library data from coastal and oligotrophic marine environments shows distinct groups of Synechococcus sp. in each environment; some of which are represented by sequences from cultivated organisms and others that are unrelated to known sequences and likely represent novel phylogenetic groups. We observed spatial differences in the distribution of sequences between two sites in Monterey Bay and differences in the vertical distribution of sequence types at the Hawai'i Ocean Time-series Station ALOHA, suggesting that nitrogen assimilation in Synechococcus living in different ecological niches can be followed with the nitrate reductase gene.     

Phylogenetic analyses of ribosomal DNA-containing bacterioplankton genome fragments from a 4000 m vertical profile in the North Pacific Subtropical Gyre

High-throughput identification of rRNA gene-containing clones in large insert metagenomic libraries is difficult, because of the high background of host ribosomal RNA (rRNA) and rRNA genes. To address this challenge, a membrane hybridization method was developed to identify all bacterial small subunit rRNA-containing fosmid clones of microbial community DNA from seven different depths in the North Pacific Subtropical Gyre. Out of 101,376 clones screened, 751 rDNA-containing clones were identified that grouped in ∼60 different clades. Several rare sequences only remotely related to known groups were detected, including a Wolbachia -related sequence containing a putative intron or intervening sequence, as well as seven sequences from Order Myxococcales not previously detected in pelagic habitats. Stratified, depth-specific population structure was evident within both cultured and uncultured lineages. Conversely, some eurybathyal members of the genera Alcanivorax and Rhizobium shared identical small subunit ribosomal DNA sequences that were distributed from surface waters to the 4000 m depth. Comparison with similar analyses in Monterey Bay microbial communities revealed previously recognized, as well as some distinctive, depth-stratified partitioning that distinguished coastal from open ocean bacterioplankton populations. While some bias was evident in fosmid clone recovery in a few particular lineages, the overall phylogenetic group recovery and distributions were consistent with previous studies, as well as with direct shotgun sequence data from the same source DNA.     

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.     

Diversity and expression of cyanobacterial hupS genes in pure cultures and in a nitrogen-limited phototrophic biofilm

A PCR was developed for conserved regions within the cyanobacterial small subunit uptake hydrogenase (hupS) gene family. These primers were used to PCR amplify partial hupS sequences from 15 cyanobacterial strains. HupS clone libraries were constructed from PCR-amplified genomic DNA and reverse-transcribed mRNA extracted from phototrophic biofilms cultivated under nitrate-limiting conditions. Partial hupS gene sequences derived from cyanobacteria, some of which were not previously known to contain hup genes were used for phylogenetic analysis. Phylogenetic trees constructed with partial hupS genes were congruent with those based on 16S rRNA genes, indicating that hupS sequences can be used to identify cyanobacteria expressing hup. Sequences from heterocystous and nonheterocystous cyanobacteria formed two separate clusters. Analysis of clone library data showed a discrepancy between the presence and the activity of cyanobacterial hupS genes in phototrophic biofilms. The results showed that the hupS gene can be used to characterize the diversity of natural populations of diazotrophic cyanobacteria, and to characterize gene expression patterns of individual species and strains.     

CHARACTERIZATION OF DIATOM (BACILLARIOPHYCEAE) NITRATE REDUCTASE GENES AND THEIR DETECTION IN MARINE PHYTOPLANKTON COMMUNITIES1

The complete assimilatory nitrate reductase (NR) gene from the pennate diatom Phaeodactylum triconutum Bohlin was sequenced from cDNA and compared with NR sequences from fungi, green algae, vascular plants, and the recently sequenced genome of the centric diatom Thalassiosira pseudonana Hasle and Heimdal CCMP1335. In all the major eukaryotic nitrate reductase (Euk‐NR) functional domains, diatom NR gene sequences are generally 50%–60% identical to plant and alga sequences at the amino acid level. In the less conserved N‐terminal, hinge 1, and hinge 2 regions, homology to other NR sequences is weak, generally<30%. Two PCR primer sets capable of amplifying Euk‐NR from plants, algae, and diatoms were designed. One primer set was used to amplify a 750‐base pair (bp) NR fragment from the cDNA of five additional diatom strains. The PCR amplicon spans part of the well‐conserved dimer interface region, the more variable hinge 1 region, and part of the conserved cytochrome b heme binding region. The second primer set, targeted to the dimer region, was used to amplify an approximately 400‐bp fragment of the NR gene from DNA samples collected in Monterey Bay, California and in central New Jersey inner continental shelf (LEO‐15 site) waters. Only diatom‐like NR sequences were recovered from Monterey Bay samples, whereas LEO‐15 samples yielded NR sequences from a range of photosynthetic eukaryotes. The prospect of using DNA‐ and RNA‐based methods to target the NR genes of diatoms specifically is a promising approach for future physiological and ecological experiments.     

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.     

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.     

Molecular genetic variation within and among isolates of QPX (Thraustochytridae), a parasite of the hard clam Mercenaria mercenaria

The thraustochytrid known as QPX (Quahog Parasite Unknown) has sporadically caused disease in the hard clam Mercenaria mercenaria along the east coast of North America since the 1960s. We hypothesized that genetically distinct QPX strains might be responsible for outbreaks of QPX disease in different areas and tested this hypothesis by comparing several QPX isolates recovered from the recent outbreak in Raritan Bay, New York with QPX strains isolated from 2 outbreaks in Massachusetts, USA. There was no variation in small subunit rDNA (SSU rDNA), 5.8S rDNA, or 4 mitochondrial gene sequences. In contrast, both of the ribosomal ribonucleic acid (rRNA) operon intergenic spacers, internal transcribed spacers 1 and 2 (ITS1 and ITS2), revealed substantial sequence variation. However, strain-specific sequences were not detected because the ITS sequence variation within QPX isolates was comparable to the variation between isolates. ITS1 sequences recovered from an infected clam by amplification with a QPX ITS2-specific primer were identical to those recovered from the QPX isolates.     

Quantitative distribution of presumptive archaeal and bacterial nitrifiers in Monterey Bay and the North Pacific Subtropical Gyre

The recent isolation of the ammonia-oxidizing crenarchaeon Nitrosopumilus maritimus has expanded the known phylogenetic distribution of nitrifying phenotypes beyond the domain Bacteria. To further characterize nitrification in the marine environment and explore the potential crenarchaeal contribution to this process, we quantified putative nitrifying genes and phylotypes in picoplankton genomic libraries and environmental DNA samples from coastal and open ocean habitats. Betaproteobacteria ammonia monooxygenase subunit A (amoA) gene copy numbers were low or undetectable, in stark contrast to crenarchaeal amoA-like genes that were broadly distributed and reached up to 6 x 10(4) copies ml(-1). Unexpectedly, in the North Pacific Subtropical Gyre, a deeply branching crenarchaeal group related to a hot spring clade (pSL12) was at times abundant below the euphotic zone. Quantitative data suggested that the pSL12 relatives also contain archaeal amoA-like genes. In both coastal and open ocean habitats, close relatives of known nitrite-oxidizing Nitrospina species were well represented in genomic DNA libraries and quantitative PCR profiles. Planktonic Nitrospina depth distributions correlated with those of Crenarchaea. Overall, the data suggest that amoA-containing Crenarchaea are more phylogenetically diverse than previously reported. Additionally, distributional patterns of planktonic Crenarchaea and Nitrospina species suggest potential metabolic interactions between these groups in the ocean's water column.     

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.     

Abundant presence of the gamma-like Proteobacterial pufM gene in oxic seawater

Known anoxygenic photosynthetic bacteria (APB) affiliated to Gammaproteobacteria usually use anaerobic metabolism and are restricted to oxygen-free habitats. Here, we report abundant (average of 34.5%) presence of diverse APB related to gamma-like Proteobacteria in oxic oceanic surface water as indicated by the pufM gene, that encodes the M subunit of the light reaction centre complex. Thus, our sequences were most likely derived from aerobic anoxygenic phototrophs (AAnP). Two genetically distinct genotypes were revealed: one was from the oligotrophic North Pacific Ocean Gyre and the other, was from the trophic East China Sea and Bering Sea. The discovery of abundant presence of novel gamma-like Proteobacterial pufM gene in the oxic seawater extends the functional ecotypes of AAnP.     

Unveiling of novel radiations within Trichodesmium cluster by hetR gene sequence analysis

The filamentous nonheterocystous cyanobacterial genus Katagnymene is a common diazotrophic component of tropical and subtropical oceans. To assess the phylogenetic affiliation of this taxon, two partial 16S rRNA gene sequences and 25 partial hetR gene sequences originating from the genera Katagnymene and Trichodesmium collected from open, surface waters of the Atlantic, Indian, and Pacific oceans were compared. Single trichomes or colonies were identified morphologically by using light microscopy and then used directly as templates in hetR PCR analyses. In addition, three cultured strains, identified as Katagnymene pelagica, Katagnymene spiralis, and Trichodesmium sp., were examined. The data show that the genus Katagnymene is in the Trichodesmium cluster and that K. pelagica Lemmermann and K. spiralis Lemmermann are most likely one species, despite their different morphologies. Phylogenetic analyses also unveiled four distinct clusters in the Trichodesmium cluster, including one novel cluster. Our findings emphasize the conclusion that known morphological traits used to differentiate marine nonheterocystous cyanobacteria at the genus and species levels correlate poorly with genetic data, and a revision is therefore suggested.     

Assessing the phylogeny of Frankia-actinorhizal plant nitrogen-fixing root nodule symbioses with Frankia 16S rRNA and glutamine synthetase gene sequences

Actinomycetes from the genus Frankia induce nitrogen-fixing root nodules on actinorhizal plants in the "core rosid" clade of eudicots. Reported here are nine partial Frankia 16S rRNA gene sequences including the first from host plants of the rosaceous genera Cercocarpus and Chamaebatia, 24 partial glutamine synthetase (GSI; glnA) sequences from Frankia in nodules of 17 of the 23 actinorhizal genera, and the partial glnA sequence of Acidothermus cellulolyticus. Phylogenetic analyses of combined Frankia 16S rDNA and glnA sequences indicate that infective strains belong to three major clades (I-III) and that Clade I strains consisting of unisolated symbionts from the Coriariaceae, Datiscaceae, Rosaceae, and Ceanothus of the Rhamnaceae are basal to the other clades. Clock-like mutation rates in glnA sequence alignments indicate that all three major Frankia clades diverged early during the emergence of eudicots in the Cretaceous period, and suggest that present-day symbioses are the result of an ancestral symbiosis that emerged before the divergence of extant actinorhizal plants.     

A novel cohabitation between two diazotrophic cyanobacteria in the oligotrophic ocean

The cyanobacterial genus Trichodesmium is biogeochemically significant because of its dual role in nitrogen and carbon fixation in the oligotrophic ocean. Trichodesmium species form colonies that can be easily enriched from the water column and used for shipboard rate measurements to estimate their contribution to oceanic carbon and nitrogen budgets. During a July 2010 cruise near the Hawaiian Islands in the oligotrophic North Pacific Subtropical Gyre, a specific morphology of Trichodesmium puff-form colonies were examined under epifluorescent microscopy and found to harbor a colonial endobiont, morphologically identified as the heterocystous diazotrophic cyanobacterium Calothrix. Using unialgal enrichments obtained from this cruise, we show that these Calothrix-like heterocystous cyanobionts (hetDA for ‘Trichodesmium-associated heterocystous diazotroph'') fix nitrogen on a diurnal cycle (maximally in the middle of the light cycle with a detectable minimum in the dark). Gene sequencing of nifH from the enrichments revealed that this genus was likely not quantified using currently described quantitative PCR (qPCR) primers. Guided by the sequence from the isolate, new hetDA-specific primers were designed and subsequent qPCR of environmental samples detected this diazotroph from surface water to a depth of 150 m, reaching densities up to ∼9 × 10³ l⁻¹. Based on phylogenetic relatedness of nifH and 16S rRNA gene sequences, it is predicted that the distribution of this cyanobiont is not limited to subtropical North Pacific but likely reaches to the South Pacific and Atlantic Oceans. Therefore, this previously unrecognized cohabitation, if it reaches beyond the oligotrophic North Pacific, could potentially influence Trichodesmium-derived nitrogen fixation budgets in the world ocean.     

Evolution of gene orders in genomes of cyanobacteria

Genomes of 23 strains of cyanobacteria were comparatively analyzed using quantitative methods of estimation of gene order similarity. It has been found that reconstructions of phylogenesis of cyanobacteria based on the comparison of the orders of genes in chromosomes and nucleotide sequences appear to be similar. This confirms the applicability of quantitative measures of similarity of gene orders for phylogenetic reconstructions. In the evolution of marine unicellular planktonic cyanobacteria, genome rearrangements are fixed with a low rate (about 3% of gene order changes per 1% of 16S rRNA changes), whereas in other groups of cyanobacteria the gene order can change several times more rapidly. The gene orders in genomes of cyanobacteria and chloroplasts preserve a considerable degree of similarity. The closest relatives of chloroplasts among the analyzed cyanobacteria are likely to be strains from hot springs belonging to the genus Synechococcus. Comparative analysis of gene orders and nucleotide sequences strongly suggests that Synechococcus strains from different environments (sea, fresh waters, hot springs) are not related and belong to evolutionally distant lines.