Genomes of diverse isolates of the marine cyanobacterium Prochlorococcus

The marine cyanobacterium Prochlorococcus is the numerically dominant photosynthetic organism in the oligotrophic oceans, and a model system in marine microbial ecology. Here we report 27 new whole genome sequences (2 complete and closed; 25 of draft quality) of cultured isolates, representing five major phylogenetic clades of Prochlorococcus. The sequenced strains were isolated from diverse regions of the oceans, facilitating studies of the drivers of microbial diversity—both in the lab and in the field. To improve the utility of these genomes for comparative genomics, we also define pre-computed clusters of orthologous groups of proteins (COGs), indicating how genes are distributed among these and other publicly available Prochlorococcus genomes. These data represent a significant expansion of Prochlorococcus reference genomes that are useful for numerous applications in microbial ecology, evolution and oceanography.Gordon and Betty Moore Foundation (Grant GBMR #495.01)National Science Foundation (U.S.) (Grant OCE-1153588)National Science Foundation (U.S.) (Grant OCE-0425602)National Science Foundation (U.S.) (Grant DBI-0424599)Center for Microbial Oceanography: Research and Educatio

Bridging the gap between omics and earth system science to better understand how environmental change impacts marine microbes

The advent of genomic-, transcriptomic- and proteomic-based approaches has revolutionized our ability to describe marine microbial communities, including biogeography, metabolic potential and diversity, mechanisms of adaptation, and phylogeny and evolutionary history. New interdisciplinary approaches are needed to move from this descriptive level to improved quantitative, process-level understanding of the roles of marine microbes in biogeochemical cycles and of the impact of environmental change on the marine microbial ecosystem. Linking studies at levels from the genome to the organism, to ecological strategies and organism and ecosystem response, requires new modelling approaches. Key to this will be a fundamental shift in modelling scale that represents micro-organisms from the level of their macromolecular components. This will enable contact with omics data sets and allow acclimation and adaptive response at the phenotype level (i.e. traits) to be simulated as a combination of fitness maximization and evolutionary constraints. This way forward will build on ecological approaches that identify key organism traits and systems biology approaches that integrate traditional physiological measurements with new insights from omics. It will rely on developing an improved understanding of ecophysiology to understand quantitatively environmental controls on microbial growth strategies. It will also incorporate results from experimental evolution studies in the representation of adaptation. The resulting ecosystem-level models can then evaluate our level of understanding of controls on ecosystem structure and function, highlight major gaps in understanding and help prioritize areas for future research programs. Ultimately, this grand synthesis should improve predictive capability of the ecosystem response to multiple environmental drivers

Diversity and Distribution of Marine Synechococcus: Multiple Gene Phylogenies for Consensus Classification and Development of qPCR Assays for Sensitive Measurement of Clades in the Ocean

Marine Synechococcus is a globally significant genus of cyanobacteria that is comprised of multiple genetic lineages or clades. These clades are thought to represent ecologically distinct units, or ecotypes. Because multiple clades often co-occur together in the oceans, Synechococcus are ideal microbes to explore how closely related bacterial taxa within the same functional guild of organisms co-exist and partition marine habitats. Here we sequenced multiple gene loci from cultured strains to confirm the congruency of clade classifications between the 16S–23S rDNA internally transcribed spacer (ITS), 16S rDNA, narB, ntcA, and rpoC1 loci commonly used in Synechococcus diversity studies. We designed quantitative PCR (qPCR) assays that target the ITS for 10 Synechococcus clades, including four clades, XV, XVI, CRD1, and CRD2, not covered by previous assays employing other loci. Our new qPCR assays are very sensitive and specific, detecting down to tens of cells per ml. Application of these qPCR assays to field samples from the northwest Atlantic showed clear shifts in Synechococcus community composition across a coastal to open-ocean transect. Consistent with previous studies, clades I and IV dominated cold, coastal Synechococcus communities. Clades II and X were abundant at the two warmer, off-shore stations, and at all stations multiple Synechococcus clades co-occurred. qPCR assays developed here provide valuable tools to further explore the dynamics of microbial community structure and the mechanisms of co-existence

REPK: an analytical web server to select restriction endonucleases for terminal restriction fragment length polymorphism analysis

Terminal restriction fragment length polymorphism (T-RFLP) analysis is a widespread technique for rapidly fingerprinting microbial communities. Users of T-RFLP frequently overlook the resolving power of well-chosen restriction endonucleases and often fail to report how they chose their enzymes. REPK (Restriction Endonuclease Picker) assists in the rational choice of restriction endonucleases for T-RFLP by finding sets of four restriction endonucleases that together uniquely differentiate user-designated sequence groups. With REPK, users can provide their own sequences (of any gene, not just 16S rRNA), specify the taxonomic rank of interest and choose from a number of filtering options to further narrow down the enzyme selection. Bug tracking is provided, and the source code is open and accessible under the GNU Public License v.2, at http://code.google.com/p/repk. The web server is available without access restrictions at http://rocaplab.ocean.washington.edu/tools/repk

Multiple scales of diversification within natural populations of archaea in hydrothermal chimney biofilms

Author Posting. © The Author(s), 2009. This is the author's version of the work. It is posted here by permission of Blackwell and Society for Applied Microbiology for personal use, not for redistribution. The definitive version was published in Environmental Microbiology Reports 2 (2010): 236-242, doi:10.1111/j.1758-2229.2009.00097.x.Corroborative data collected from 16S rRNA clone libraries, intergenic transcribed spacer (ITS) region clone libraries, and 16S rRNA hypervariable region tag pyrosequencing demonstrate microdiversity within single-species archaeal biofilms of the Lost City Hydrothermal Field. Both 16S rRNA clone libraries and pyrosequencing of the V6 hypervariable region show that Lost City Methanosarcinales (LCMS) biofilms are dominated by a single sequence, but the pyrosequencing dataset also reveals the presence of an additional 1654 rare sequences. Clone libraries constructed with DNA spanning the V6 hypervariable region and ITS show that multiple ITS sequences are associated with the same dominant V6 sequence. Furthermore, ITS variability differed among three chimney samples, and the sample with the highest ITS diversity also contained the highest V6 diversity as measured by clone libraries as well as tag pyrosequencing. These results indicate that the extensive microdiversity detected in V6 tag sequences is an underestimate of genetic diversity within the archaeal biofilms.This research was supported by the W.M. Keck Foundation to MLS, the NASA Astrobiology Institute through the Carnegie Institution for Science to JAB and through the MBL to MLS

Time-series analyses of Monterey Bay coastal microbial picoplankton using a ‘genome proxy’ microarray

To investigate the temporal, spatial and phylogenetic resolution of marine microbial community structure and variability, we designed and expanded a genome proxy array (an oligonucleotide microarray targeting marine microbial genome fragments and genomes), evaluated it against metagenomic sequencing, and applied it to time-series samples from the Monterey Bay. The expanded array targeted 268 microbial genotypes across much of the known diversity of cultured and uncultured marine microbes. The target abundances measured by the array were highly correlated to pyrosequence-based abundances (linear regression R2 = 0.85–0.91, P < 0.0001). Fifty-seven samples from ∼4 years in Monterey Bay were examined with the array, spanning the photic zone (0 m), the base of the surface mixed layer (30 m) and the subphotic zone (200 m). A significant portion of the expanded genome proxy array's targets showed signal (95 out of 268 targets present in ≥ 1 sample). The multi-year community survey showed the consistent presence of a core group of common and abundant targeted taxa at each depth in Monterey Bay, higher variability among shallow than deep samples, and episodic occurrences of more transient marine genotypes. The abundance of the most dominant genotypes peaked after strong episodic upwelling events. The genome-proxy array's ability to track populations of closely related genotypes indicated population shifts within several abundant target taxa, with specific populations in some cases clustering by depth or oceanographic season. Although 51 cultivated organisms were targeted (representing 19% of the array) the majority of targets detected and of total target signal (85% and ∼92% respectively) were from uncultivated genotypes, often those derived from Monterey Bay. The array provided a relatively cost-effective approach (∼$15 per array) for surveying the natural history of uncultivated lineages.Gordon and Betty Moore FoundationNational Science Foundation (U.S.) (Science and Technology Center Award EF0424599)National Science Foundation (U.S.) (Microbial Observatory Award MCB-0348001)United States. Dept. of Energy. Office of Scienc

Production of cobalt binding ligands in a Synechococcus feature at the Costa Rica upwelling dome

Author Posting. © American Society of Limnology and Oceanography, 2005. This is the author's version of the work. It is posted here by permission of American Society of Limnology and Oceanography for personal use, not for redistribution. The definitive version was published in Limnology and Oceanography 50 (2005): 279-290.The Costa Rica upwelling dome (CRD; ~8.67ºN and 90.6ºW) was characterized chemically for cobalt and nickel abundances and speciation, and biologically using cyanobacterial abundances and phylogeny. Total dissolved cobalt was 93 pmol L-1at 90 m depth and decreased in surface waters to 45 pmol L-1 at 10 m. Cobalt was 40% labile at 90 m, but was completely complexed by strong ligands at 10 m. A surface transect out of the dome showed decreasing total dissolved cobalt from 57 pmol L-1 to 12 pmol L-1. Detection window studies showed that natural cobalt ligand complexes have conditional stability constants greater than 1016.8, and that competition with nickel did not release cobalt bound to organic complexes, consistent with natural cobalt ligands being Co(III)-complexes. Synechococcus cell densities at the CRD are among the highest reported in nature, varying between 1.2 x 106 to 3.7 x 106 cells ml-1. Phylogenetic analysis using the 16S-23S rDNA internally transcribed spacer showed the majority of clones were related to Synechococcus strain MIT S9220, while the remaining subset form a novel group within the marine Synechococcus lineage. In a bottle incubation experiment chlorophyll increased with cobalt and iron additions relative to each element alone and the unamended control treatment. Cobalt speciation analysis of incubation experiments revealed large quantities of strong cobalt ligand complexes in the cobalt addition treatments (401 pmol L-1), whereas cobalt added to a 0.2 mm filtered control remained predominantly labile (387 pmol L-1), demonstrating that the Synechococcus-dominated community is a source of strong cobalt ligands.This research was funded by NSF OCE-9618729, OCE-0327225, and OCE-0220826

Complete arsenic-based respiratory cycle in the marine microbial communities of pelagic oxygen-deficient zones.

Author Posting. © The Author(s), 2019. This is the author's version of the work. It is posted here by permission of National Academy of Sciences for personal use, not for redistribution. The definitive version was published in Proceedings of the National Academy of Sciences of the United States of America 116(20), (2019):9925-9930, doi:10.1073/pnas.1818349116.Microbial capacity to metabolize arsenic is ancient, arising in response to its pervasive presence in the environment, which was largely in the form of As(III) in the early anoxic ocean. Many biological arsenic transformations are aimed at mitigating toxicity; however, some microorganisms can respire compounds of this redox-sensitive element to reap energetic gains. In several modern anoxic marine systems concentrations of As(V) are higher relative to As(III) than what would be expected from the thermodynamic equilibrium, but the mechanism for this discrepancy has remained unknown. Here we present evidence of a complete respiratory arsenic cycle, consisting of dissimilatory As(V) reduction and chemoautotrophic As(III) oxidation, in the pelagic ocean. We identified the presence of genes encoding both subunits of the respiratory arsenite oxidase AioA and the dissimilatory arsenate reductase ArrA in the Eastern Tropical North Pacific (ETNP) oxygen-deficient zone (ODZ). The presence of the dissimilatory arsenate reductase gene arrA was enriched on large particles (>30 um), similar to the forward bacterial dsrA gene of sulfate-reducing bacteria, which is involved in the cryptic cycling of sulfur in ODZs. Arsenic respiratory genes were expressed in metatranscriptomic libraries from the ETNP and the Eastern Tropical South Pacific (ETSP) ODZ, indicating arsenotrophy is a metabolic pathway actively utilized in anoxic marine water columns. Together these results suggest arsenic-based metabolisms support organic matter production and impact nitrogen biogeochemical cycling in modern oceans. In early anoxic oceans, especially during periods of high marine arsenic concentrations, they may have played a much larger role.We thank John Baross and Rika Anderson for helpful discussions and feedback on this project. We also thank the chief scientists of the research cruise, Al Devol and Bess Ward, as well as the captain and crew of the R/V Thomas G. Thompson. This work was supported through a NASA Earth and Space Sciences Graduate Research Fellowship to J.K.S. and National Science Foundation Grant OCE-1138368 (to G.R.).2019-10-2