Kendra A. Turk-Kubo

Genetic diversity of the unicellular nitrogen-fixing cyanobacteria UCYN-A and its prymnesiophyte host.

Symbiotic interactions between nitrogen-fixing prokaryotes and photosynthetic eukaryotes are an integral part of biological nitrogen fixation at a global scale. One of these partnerships involves the cyanobacterium UCYN-A, which has been found in partnership with an uncultivated unicellular prymnesiophyte alga in open-ocean and coastal environments. Phylogenetic analysis of the UCYN-A nitrogenase gene (nifH) showed that the UCYN-A lineage is represented by three distinct clades, referred to herein as UCYN-A1, UCYN-A2 and UCYN-A3, which appear to have overlapping and distinct geographic distributions. The relevance of UCYN-As genetic diversity to its symbiosis and ecology was explored through combining flow cytometric cell sorting and molecular techniques to determine the host identity, nifH expression patterns and host cell size of one newly discovered clade, UCYN-A2, at a coastal site. UCYN-A2 nifH expression peaked during daylight hours, which is consistent with expression patterns of the UCYN-A1 clade in the open ocean. However, the cell size of the UCYN-A2 host was significantly larger than UCYN-A1 and host, suggesting adaptation to different environmental conditions. Like the UCYN-A1 host, the UCYN-A2 host was closely related to the genus Braarudosphaera; however, the UCYN-A1 and UCYN-A2 host rRNA sequences clustered into two distinct clades suggesting co-evolution of symbiont and host.

Aphotic N2 Fixation in the Eastern Tropical South Pacific Ocean

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.

The paradox of marine heterotrophic nitrogen fixation: abundances of heterotrophic diazotrophs do not account for nitrogen fixation rates in the Eastern Tropical South Pacific.

Results of recent modelling efforts imply denitrification-influenced waters, such as those in the Eastern Tropical South Pacific (ETSP), may support high rates of biological nitrogen fixation (BNF), yet little is known about the N2 -fixing microbial community in this region. Our characterization of the ETSP diazotrophic community along a gradient from upwelling-influenced to oligotrophic waters did not detect cyanobacterial diazotrophs commonly found in other open ocean regions. Most of the nifH genes amplified by polymerase chain reaction (PCR) from DNA and RNA samples clustered with γ-proteobacterial nifH sequences, although a novel Trichodesmium phylotype was also recovered. Three quantitative PCR assays were developed to target γ-proteobacterial phylotypes, but all were found to be present at low abundances. An analysis of the expected BNF rates based on abundances and plausible cell-specific N2 fixation rates indicates that these γ-proteobacteria are unlikely to be responsible for previously reported BNF rates from corresponding samples. Therefore, the organisms responsible for the measured BNF rates remain poorly understood. Furthermore, there is little direct evidence, at this time, to support the hypothesis that heterotrophic N2 fixation contributes significantly to oceanic BNF rates based on our analysis of heterotrophic cell-specific N2 fixation rates required to explain BNF rates reported in previously published studies.

Contrasted geographical distribution of N2 fixation rates and nifH phylotypes in the Coral and Solomon Seas (southwestern Pacific) during austral winter conditions

Biological dinitrogen (N2) fixation and the distribution of diazotrophic phylotypes were investigated during two cruises in the Coral Sea and the Solomon Sea (southwestern Pacific) during austral winter conditions. N2 fixation rates were measurable at every station, but integrated (0–150 m) rates were an order of magnitude higher in the Solomon Sea (30 to 5449 µmol N m−2 d−1) compared to those measured in the Coral Sea (2 to 109 µmol N m−2 d−1). Rates measured in the Solomon Sea were in the upper range (100–1000 µmol N m−2 d−1) or higher than rates compiled in the global MARine Ecosystem biomass DATa database, indicating that this region has some of the highest N2 fixation rates reported in the global ocean. While unicellular diazotrophic cyanobacteria from group A (UCYN-A1 and UCYN-A2) and the proteobacteria γ-24774A11 dominated in the Coral Sea and were correlated with N2 fixation rates (p < 0.05), Trichodesmium and UCYN-B dominated in the Solomon Sea and were correlated (p < 0.05) with N2 fixation rates. UCYN-A were totally absent in the Solomon Sea. The biogeographical distribution of diazotrophs is discussed within the context of patterns in measured environmental parameters.

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.

ARBitrator: a software pipeline for on-demand retrieval of auto-curated nifH sequences from GenBank

MOTIVATION Studies of the biochemical functions and activities of uncultivated microorganisms in the environment require analysis of DNA sequences for phylogenetic characterization and for the development of sequence-based assays for the detection of microorganisms. The numbers of sequences for genes that are indicators of environmentally important functions such as nitrogen (N2) fixation have been rapidly growing over the past few decades. Obtaining these sequences from the National Center for Biotechnology Informations GenBank database is problematic because of annotation errors, nomenclature variation and paralogues; moreover, GenBanks structure and tools are not conducive to searching solely by function. For some genes, such as the nifH gene commonly used to assess community potential for N2 fixation, manual collection and curation are becoming intractable because of the large number of sequences in GenBank and the large number of highly similar paralogues. If analysis is to keep pace with sequence discovery, an automated retrieval and curation system is necessary. RESULTS ARBitrator uses a two-step process composed of a broad collection of potential homologues followed by screening with a best hit strategy to conserved domains. 34 420 nifH sequences were identified in GenBank as of November 20, 2012. The false-positive rate is ∼0.033%. ARBitrator rapidly updates a public nifH sequence database, and we show that it can be adapted for other genes. AVAILABILITY AND IMPLEMENTATION Java source and executable code are freely available to non-commercial users at http://pmc.ucsc.edu/∼wwwzehr/research/database/. CONTACT zehrj@ucsc.edu SUPPLEMENTARY INFORMATION SUPPLEMENTARY INFORMATION is available at Bioinformatics online.

A microarray for assessing transcription from pelagic marine microbial taxa

Metagenomic approaches have revealed unprecedented genetic diversity within microbial communities across vast expanses of the world’s oceans. Linking this genetic diversity with key metabolic and cellular activities of microbial assemblages is a fundamental challenge. Here we report on a collaborative effort to design MicroTOOLs (Microbiological Targets for Ocean Observing Laboratories), a high-density oligonucleotide microarray that targets functional genes of diverse taxa in pelagic and coastal marine microbial communities. MicroTOOLs integrates nucleotide sequence information from disparate data types: genomes, PCR-amplicons, metagenomes, and metatranscriptomes. It targets 19 400 unique sequences over 145 different genes that are relevant to stress responses and microbial metabolism across the three domains of life and viruses. MicroTOOLs was used in a proof-of-concept experiment that compared the functional responses of microbial communities following Fe and P enrichments of surface water samples from the North Pacific Subtropical Gyre. We detected transcription of 68% of the gene targets across major taxonomic groups, and the pattern of transcription indicated relief from Fe limitation and transition to N limitation in some taxa. Prochlorococcus (eHLI), Synechococcus (sub-cluster 5.3) and Alphaproteobacteria SAR11 clade (HIMB59) showed the strongest responses to the Fe enrichment. In addition, members of uncharacterized lineages also responded. The MicroTOOLs microarray provides a robust tool for comprehensive characterization of major functional groups of microbes in the open ocean, and the design can be easily amended for specific environments and research questions.

Coordinated regulation of growth, activity and transcription in natural populations of the unicellular nitrogen-fixing cyanobacterium Crocosphaera

The temporal dynamics of phytoplankton growth and activity have large impacts on fluxes of matter and energy, yet obtaining in situ metabolic measurements of sufficient resolution for even dominant microorganisms remains a considerable challenge. We performed Lagrangian diel sampling with synoptic measurements of population abundances, dinitrogen (N2) fixation, mortality, productivity, export and transcription in a bloom of Crocosphaera over eight days in the North Pacific Subtropical Gyre (NPSG). Quantitative transcriptomic analyses revealed clear diel oscillations in transcript abundances for 34% of Crocosphaera genes identified, reflecting a systematic progression of gene expression in diverse metabolic pathways. Significant time-lagged correspondence was evident between nifH transcript abundance and maximal N2 fixation, as well as sepF transcript abundance and cell division, demonstrating the utility of transcriptomics to predict the occurrence and timing of physiological and biogeochemical processes in natural populations. Indirect estimates of carbon fixation by Crocosphaera were equivalent to 11% of net community production, suggesting that under bloom conditions this diazotroph has a considerable impact on the wider carbon cycle. Our cross-scale synthesis of molecular, population and community-wide data underscores the tightly coordinated in situ metabolism of the keystone N2-fixing cyanobacterium Crocosphaera, as well as the broader ecosystem-wide implications of its activities.

Unusual marine unicellular symbiosis with the nitrogen-fixing cyanobacterium UCYN-A

Nitrogen fixation — the reduction of dinitrogen (N2) gas to biologically available nitrogen (N) — is an important source of N for terrestrial and aquatic ecosystems. In terrestrial environments, N2-fixing symbioses involve multicellular plants, but in the marine environment these symbioses occur with unicellular planktonic algae. An unusual symbiosis between an uncultivated unicellular cyanobacterium (UCYN-A) and a haptophyte picoplankton alga was recently discovered in oligotrophic oceans. UCYN-A has a highly reduced genome, and exchanges fixed N for fixed carbon with its host. This symbiosis bears some resemblance to symbioses found in freshwater ecosystems. UCYN-A shares many core genes with the ‘spheroid bodies’ of Epithemia turgida and the endosymbionts of the amoeba Paulinella chromatophora. UCYN-A is widely distributed, and has diversified into a number of sublineages that could be ecotypes. Many questions remain regarding the physical and genetic mechanisms of the association, but UCYN-A is an intriguing model for contemplating the evolution of N2-fixing organelles.

Non-cyanobacterial nifH phylotypes in the North Pacific Subtropical Gyre detected by flow-cytometry cell sorting

In contrast to cyanobacteria, the significance of bacteria and archaea in oceanic N2 fixation remains unknown, apart from the knowledge that their nitrogenase (nifH) genes are diverse, present in all oceans and at least occasionally expressed. Non-cyanobacterial nifH sequences often occur as contamination from reagents and other sources, complicating the detection and interpretation of environmental phylotypes. We amplified and sequenced partial nifH gene fragments directly from cell populations sorted by fluorescence activated cell sorting from water collected in the North Pacific Subtropical Gyre (NPSG). Sequences recovered (195 total) included presumed heterotrophic or photoheterotrophic non-cyanobacterial nifH phylotypes previously unreported in the NPSG. A nifH sequence previously found in the South Pacific Gyre (HM210397) was exclusively recovered from sorted picoeukaryote populations, and was detected in water column samples using quantitative PCR (qPCR), with 60% of samples detected in the > 10 μm size fraction in addition to the 0.2-10 μm size fraction. A novel cluster 3-like nifH sequence was also recovered from discrete cell sorts and detected by qPCR in environmental samples. This approach enables the detection of rare nifH phylotypes, identifies possible associations with larger cells or particles and offers a possible solution for distinguishing reagent contaminants from real microbial community components.