MarFERReT, an open-source, version-controlled reference library of marine microbial eukaryote functional genes.

Metatranscriptomics generates large volumes of sequence data about transcribed genes in natural environments. Taxonomic annotation of these datasets depends on availability of curated reference sequences. For marine microbial eukaryotes, current reference libraries are limited by gaps in sequenced organism diversity and barriers to updating libraries with new sequence data, resulting in taxonomic annotation of about half of eukaryotic environmental transcripts. Here, we introduce Marine Functional EukaRyotic Reference Taxa (MarFERReT), a marine microbial eukaryotic sequence library designed for use with taxonomic annotation of eukaryotic metatranscriptomes. We gathered 902 publicly accessible marine eukaryote genomes and transcriptomes and assessed their sequence quality and cross-contamination issues, selecting 800 validated entries for inclusion in MarFERReT. Version 1.1 of MarFERReT contains reference sequences from 800 marine eukaryotic genomes and transcriptomes, covering 453 species- and strain-level taxa, totaling nearly 28 million protein sequences with associated NCBI and PR2 Taxonomy identifiers and Pfam functional annotations. The MarFERReT project repository hosts containerized build scripts, documentation on installation and use case examples, and information on new versions of MarFERReT.

Comparative Genomic Analysis of Vibrio diabolicus and Six Taxonomic Synonyms: A First Look at the Distribution and Diversity of the Expanded Species.

Vibrio is a diverse genus of Gammaproteobacteria autochthonous to marine environments worldwide. Vibrio diabolicus and V. antiquarius were originally isolated from deep-sea hydrothermal fields in the East Pacific Rise. These species are closely related to members of the Harveyi clade (e.g., V. alginolyticus and V. parahaemolyticus) that are commonly isolated from coastal systems. This study reports the discovery and draft genome sequence of a novel isolate (Vibrio sp. 939) cultured from Pacific oysters (Crassostrea gigas). Questions surrounding the identity of Vibrio sp. 939 motivated a genome-scale taxonomic analysis of the Harveyi clade. A 49-genome phylogeny based on 1,109 conserved coding sequences and a comparison of average nucleotide identity (ANI) values revealed a clear case of synonymy between Vibrio sp. 939, V. diabolicus Art-Gut C1 and CNCM I-1629, V. antiquarius EX25 and four V. alginolyticus strains (E0666, FF273, TS13, and V2). This discovery expands the V. diabolicus species and makes available six additional genomes for comparative genomic analyses. The distribution of the expanded species is thought to be global given the range of isolation sources (horse mackerel, seawater, sediment, dentex, oyster, artemia and polycheate) and origins (China, India, Greece, United States, East Pacific Rise, and Chile). A subsequent comparative genomic analysis of this new eight-genome subclade revealed a high degree of individual genome plasticity and a large repertoire of genes related to virulence and defense. These findings represent a significant revision to the understanding of V. diabolicus and V. antiquarius as both have long been regarded as distinct species. This first look at the expanded V. diabolicus subclade suggests that the distribution and diversity of this species mirrors that of other Harveyi clade species, which are notable for their ubiquity and diversity.

Ocean acidification conditions increase resilience of marine diatoms.

The fate of diatoms in future acidified oceans could have dramatic implications on marine ecosystems, because they account for ~40% of marine primary production. Here, we quantify resilience of Thalassiosira pseudonana in mid-20th century (300 ppm CO2) and future (1000 ppm CO2) conditions that cause ocean acidification, using a stress test that probes its ability to recover from incrementally higher amount of low-dose ultraviolet A (UVA) and B (UVB) radiation and re-initiate growth in day-night cycles, limited by nitrogen. While all cultures eventually collapse, those growing at 300 ppm CO2 succumb sooner. The underlying mechanism for collapse appears to be a system failure resulting from "loss of relational resilience," that is, inability to adopt physiological states matched to N-availability and phase of the diurnal cycle. Importantly, under elevated CO2 conditions diatoms sustain relational resilience over a longer timeframe, demonstrating increased resilience to future acidified ocean conditions. This stress test framework can be extended to evaluate and predict how various climate change associated stressors may impact microbial community resilience.

Interaction and signalling between a cosmopolitan phytoplankton and associated bacteria.

Interactions between primary producers and bacteria impact the physiology of both partners, alter the chemistry of their environment, and shape ecosystem diversity. In marine ecosystems, these interactions are difficult to study partly because the major photosynthetic organisms are microscopic, unicellular phytoplankton. Coastal phytoplankton communities are dominated by diatoms, which generate approximately 40% of marine primary production and form the base of many marine food webs. Diatoms co-occur with specific bacterial taxa, but the mechanisms of potential interactions are mostly unknown. Here we tease apart a bacterial consortium associated with a globally distributed diatom and find that a Sulfitobacter species promotes diatom cell division via secretion of the hormone indole-3-acetic acid, synthesized by the bacterium using both diatom-secreted and endogenous tryptophan. Indole-3-acetic acid and tryptophan serve as signalling molecules that are part of a complex exchange of nutrients, including diatom-excreted organosulfur molecules and bacterial-excreted ammonia. The potential prevalence of this mode of signalling in the oceans is corroborated by metabolite and metatranscriptome analyses that show widespread indole-3-acetic acid production by Sulfitobacter-related bacteria, particularly in coastal environments. Our study expands on the emerging recognition that marine microbial communities are part of tightly connected networks by providing evidence that these interactions are mediated through production and exchange of infochemicals.

Detection of glycolate oxidase gene glcD diversity among cultured and environmental marine bacteria.

Of eight laboratory cultures of marine gamma- and alpha-Proteobacteria tested, growth on glycolate as a sole carbon source was detected for only three species: Pseudomonas stutzeri, Oceanimonas doudoroffii and Roseobacter sp. isolate Y3F. Degenerate polymerase chain reaction (PCR) primers were designed to amplify glcD, which encodes the D-subunit of the enzyme glycolate oxidase; glcD could be amplified only from those cultures that grew on glycolate. The PCR primers were used to explore glcD diversity in four field samples collected from different ocean environments: an Atlantic Gulf Stream Ring, sampled above and below the thermocline and two Pacific coastal sites, Parks Bay and San Juan Channel, WA. Environmental glcD sequences belonged to six major bacterial phylogenetic groups, with most sequences forming novel clades with no close relatives. Different patterns of glcD diversity were observed within and between the two nutrient regimes. Comparison of glcD and 16S rDNA diversity and analyses of available bacterial genomes and a metgenomic library from the Sargasso Sea show that glycolate-utilizing potential exists in only a subset of bacteria. Glycolate is produced in marine environments mainly by phytoplankton. Examination of glcD diversity will aid in understanding the influence of phytoplankton on bacterial community structure.

Genetic diversity of attached bacteria in the hindgut of the deposit-feeding shrimp Neotrypaea (formerly Callianassa) californiensis (decapoda: thalassinidae).

Microbial colonization of marine invertebrate guts is widespread, but in general the roles that these bacteria play in the nutrition of their hosts are unknown. To examine the diversity and potential nutritional roles of hindgut microbiota in a deposit feeder, PCR-amplified 16S rRNA genes were cloned from the bacterial community attached to the hindguts of the thalassinid shrimp Neotrypaea californiensis exposed to different feeding treatments. Partial 16S rDNA sequences were analyzed for 30 clones for three shrimp per treatment for a total of 270 clones. No effects of host starvation or high-protein diets were apparent on hindgut bacterial community composition. Diversity analyses indicated high variability between bacterial communities in individual shrimp hindguts, but partial 16S rDNA sequences revealed remarkable species-level similarity (>98%) within clusters of sequences from the different shrimp hindguts, and many sequences from different shrimp hindguts were identical. Sequences belonged to three main groups of bacteria: Cytophaga-Flavobacteria-Bacteroides (CFB), proteobacteria, and gram-positives. Of the 270 sequences, 40% belonged to the alpha-proteobacteria, > or = 5% each to the gamma- and epsilon -proteobacteria, and > or =20% each to the gram-positive and CFB groups. All except one sequence are novel with < or = 95% sequence similarity to known genes. Despite weak similarity to known taxa,about 75% of the sequences were most closely related to known symbiotic and sedimentary bacteria. The bacteria in shrimp hindguts represent new species that have not yet been en-countered in other environments, and gut environments may be a rich source of the difficult-to-culture and novel components of marine bacterial diversity.

Rapid evolution of a sexual reproduction gene in centric diatoms of the genus Thalassiosira.

Sexual reproduction is commonly assumed to occur in the vast majority of diatoms due to the intimate association of this process with cell size control. Surprisingly, however, little is known about the impact of sexual events on diatom population dynamics. The Sig1 gene is strongly upregulated during sexual reproduction in the centric diatom Thalassiosira weissflogii and has been hypothesized to encode a protein involved in gamete recognition. In the present study, degenerate PCR primers were designed and used to amplify a portion of Sig1 from three closely related species in the cosmopolitan genus Thalassiosira, Thalassiosira oceanica, Thalassiosira guillardii, and Thalassiosira pseudonana. Identification of Sig1 in these three additional species facilitated development of this gene as a molecular marker for diatom sexual events. Examination of the new sequences indicated that multiple copies of Sig1 are probably present in the genome. Moreover, compared to the housekeeping gene beta-tubulin, the Sig1 genes of isolates of T. weissflogii collected from different regions of the Atlantic and Pacific oceans displayed high levels of divergence. The Sig1 genes of the four closely related Thalassiosira species also displayed high levels of sequence divergence compared to the levels observed with a second gene, Fcp, probably explaining why Sig1 could not be amplified from more distantly related species. The high levels of sequence divergence both within and between species suggest that Sig1 is rapidly evolving in a manner reminiscent of the manner observed in other genes that encode gamete recognition proteins. A simple model is presented for Sig1 evolution and the implications of such a rapidly evolving sexual reproduction gene for diatom speciation and population dynamics.

Identification of a new gene family expressed during the onset of sexual reproduction in the centric diatom Thalassiosira weissflogii.

An intriguing feature of the diatom life cycle is that sexual reproduction and the generation of genetic diversity are coupled to the control of cell size. A PCR-based cDNA subtraction technique was used to identify genes that are expressed as small cells of the centric diatom Thalassiosira weissflogii initiate gametogenesis. Ten genes that are up-regulated during the early stages of sexual reproduction have been identified thus far. Three of the sexually induced genes, Sig1, Sig2, and Sig3, were sequenced to completion and are members of a novel gene family. The three polypeptides encoded by these genes possess different molecular masses and charges but display many features in common: they share five highly conserved domains; they each contain three or more cysteine-rich epithelial growth factor (EGF)-like repeats; and they each display homology to the EGF-like region of the vertebrate extracellular matrix glycoprotein tenascin X. Interestingly, the five conserved domains appear in the same order in each polypeptide but are separated by variable numbers of nonconserved amino acids. SIG1 and SIG2 display putative regulatory domains within the nonconserved regions. A calcium-binding, EF-hand motif is found in SIG1, and an ATP/GTP binding motif is present in SIG2. The striking similarity between the SIG polypeptides and extracellular matrix components commonly involved in cell-cell interactions suggests that the SIG polypeptides may play a role in sperm-egg recognition. The SIG polypeptides are thus important molecular targets for determining when and where sexual reproduction occurs in the field.

Phylogenetic analysis of particle-attached and free-living bacterial communities in the Columbia river, its estuary, and the adjacent coastal ocean.

The Columbia River estuary is a dynamic system in which estuarine turbidity maxima trap and extend the residence time of particles and particle-attached bacteria over those of the water and free-living bacteria. Particle-attached bacteria dominate bacterial activity in the estuary and are an important part of the estuarine food web. PCR-amplified 16S rRNA genes from particle-attached and free-living bacteria in the Columbia River, its estuary, and the adjacent coastal ocean were cloned, and 239 partial sequences were determined. A wide diversity was observed at the species level within at least six different bacterial phyla, including most subphyla of the class Proteobacteria. In the estuary, most particle-attached bacterial clones (75%) were related to members of the genus Cytophaga or of the alpha, gamma, or delta subclass of the class Proteobacteria. These same clones, however, were rare in or absent from either the particle-attached or the free-living bacterial communities of the river and the coastal ocean. In contrast, about half (48%) of the free-living estuarine bacterial clones were similar to clones from the river or the coastal ocean. These free-living bacteria were related to groups of cosmopolitan freshwater bacteria (beta-proteobacteria, gram-positive bacteria, and Verrucomicrobium spp.) and groups of marine organisms (gram-positive bacteria and alpha-proteobacteria [SAR11 and Rhodobacter spp.]). These results suggest that rapidly growing particle-attached bacteria develop into a uniquely adapted estuarine community and that free-living estuarine bacteria are similar to members of the river and the coastal ocean microbial communities. The high degree of diversity in the estuary is the result of the mixing of bacterial communities from the river, estuary, and coastal ocean.

A mating type-linked mutation that disrupts the uniparental inheritance of chloroplast DNA also disrupts cell-size control in Chlamydomonas.

An intriguing feature of early zygote development in Chlamydomonas reinhardtii is the active elimination of chloroplast DNA from the mating-type minus parent due presumably to the action of a zygote-specific nuclease. Meiotic progeny thus inherit chloroplast DNA almost exclusively from the mating-type plus parent. The plus-linked nuclear mutation mat3 prevents this selective destruction of minus chloroplast DNA and generates progeny that display a biparental inheritance pattern. Here we show that the mat3 mutation creates additional phenotypes not previously described: the cells are much smaller than wild type and they possess substantially reduced amounts of both mitochondrial and chloroplast DNA. We propose that the primary defect of the mat3 mutation is a disruption of cell-size control and that the inhibition of the uniparental transmission of chloroplast genomes is a secondary consequence of the reduced amount of chloroplast DNA in the mat3 parent.

A mating type-linked gene cluster expressed in Chlamydomonas zygotes participates in the uniparental inheritance of the chloroplast genome.

A characteristic feature of early zygote development in Chlamydomonas is the selective degradation of chloroplast DNA from the mating type minus parent. The zygote-specific gene cluster ezy-1 is linked to the mating type locus and is transcribed almost immediately upon zygote formation. We show here that the acidic Ezy-1 polypeptide is rapidly transported to both the plus and minus chloroplasts, where it interacts with each chloroplast nucleoid. Expression of ezy-1 is selectively inhibited when plus, but not minus, gametes are briefly ultraviolet irradiated just prior to mating, a treatment known to disrupt the uniparental inheritance of chloroplast traits. We propose that the Ezy-1 polypeptide participates in the destruction of the minus chloroplast DNA in zygotes and thus the uniparental inheritance of chloroplast traits. The ezy-1 gene represents a valuable molecular probe for dissecting mechanisms underlying organelle inheritance.

Effect of light on the cell cycle of a marine synechococcus strain.

Light-dependent regulation of cell cycle progression in the marine cyanobacterium Synechococcus strain WH-8101 was demonstrated through the use of flow cytometry. Our results show that, similar to eucaryotic cells, marine Synechococcus spp. display two gaps in DNA synthesis, at the beginning and at the end of the cell cycle. Progression through each of these gaps requires light, and their durations lengthen under light limitation.