Evolutionary analysis of a streamlined lineage of surface ocean Roseobacters

The vast majority of surface ocean bacteria are uncultivated. Compared with their cultured relatives, they frequently exhibit a streamlined genome, reduced G+C content and distinct gene repertoire. These genomic traits are relevant to environmental adaptation, and have generally been thought to become fixed in marine bacterial populations through selection. Using single-cell genomics, we sequenced four uncultivated cells affiliated with the ecologically relevant Roseobacter clade and used a composition-heterogeneous Bayesian phylogenomic model to resolve these single-cell genomes into a new clade. This lineage has no representatives in culture, yet accounts for ∼35% of Roseobacters in some surface ocean waters. Analyses of multiple genomic traits, including genome size, G+C content and percentage of noncoding DNA, suggest that these single cells are representative of oceanic Roseobacters but divergent from isolates. Population genetic analyses showed that substitution of physicochemically dissimilar amino acids and replacement of G+C-rich to G+C-poor codons are accelerated in the uncultivated clade, processes that are explained equally well by genetic drift as by the more frequently invoked explanation of natural selection. The relative importance of drift vs selection in this clade, and perhaps in other marine bacterial clades with streamlined G+C-poor genomes, remains unresolved until more evidence is accumulated.     

Ecological genomics of marine Roseobacters

Bacterioplankton of the marine Roseobacter clade have genomes that reflect a dynamic environment and diverse interactions with marine plankton. Comparative genome sequence analysis of three cultured representatives suggests that cellular requirements for nitrogen are largely provided by regenerated ammonium and organic compounds (polyamines, allophanate, and urea), while typical sources of carbon include amino acids, glyoxylate, and aromatic metabolites. An unexpectedly large number of genes are predicted to encode proteins involved in the production, degradation, and efflux of toxins and metabolites. A mechanism likely involved in cell-to-cell DNA or protein transfer was also discovered: vir-related genes encoding a type IV secretion system typical of bacterial pathogens. These suggest a potential for interacting with neighboring cells and impacting the routing of organic matter into the microbial loop. Genes shared among the three roseobacters and also common in nine draft Roseobacter genomes include those for carbon monoxide oxidation, dimethylsulfoniopropionate demethylation, and aromatic compound degradation. Genes shared with other cultured marine bacteria include those for utilizing sodium gradients, transport and metabolism of sulfate, and osmoregulation.     

Genomic insights into the uncultivated marine Zetaproteobacteria at Loihi Seamount

The Zetaproteobacteria are a candidate class of marine iron-oxidizing bacteria that are typically found in high iron environments such as hydrothermal vent sites. As much remains unknown about these organisms due to difficulties in cultivation, single-cell genomics was used to learn more about this elusive group at Loihi Seamount. Comparative genomics of 23 phylogenetically diverse single amplified genomes (SAGs) and two isolates indicate niche specialization among the Zetaproteobacteria may be largely due to oxygen tolerance and nitrogen transformation capabilities. Only Form II ribulose 1,5-bisphosphate carboxylase (RubisCO) genes were found in the SAGs, suggesting that some of the uncultivated Zetaproteobacteria may be adapted to low oxygen and/or high carbon dioxide concentrations. There is also genomic evidence of oxygen-tolerant cytochrome c oxidases and oxidative stress-related genes, indicating that others may be exposed to higher oxygen conditions. The Zetaproteobacteria also have the genomic potential for acquiring nitrogen from numerous sources including ammonium, nitrate, organic compounds, and nitrogen gas. Two types of molybdopterin oxidoreductase genes were found in the SAGs, indicating that those found in the isolates, thought to be involved in iron oxidation, are not consistent among all the Zetaproteobacteria. However, a novel cluster of redox-related genes was found to be conserved in 10 SAGs as well as in the isolates warranting further investigation. These results were used to isolate a novel iron-oxidizing Zetaproteobacteria. Physiological studies and genomic analysis of this isolate were able to support many of the findings from SAG analyses demonstrating the value of these data for designing future enrichment strategies.     

Genome sequence of the marine bacterium Vibrio campbellii DS40M4, isolated from open ocean water

Vibrio sp. strain DS40M4 is a marine bacterium that was isolated from open ocean water. In this work, using genomic taxonomy, we were able to classify this bacterium as V. campbellii. Our genomic analysis revealed that V. campbellii DS40M4 harbors genes related to iron transport, virulence, and environmental fitness, such as those encoding anguibactin and vanchrobactin biosynthesis proteins, type II, III, IV, and VI secretion systems, and proteorhodopsin.     

Phylogenetic analysis of ribosomal RNA operons from uncultivated coastal marine bacterioplankton

Analyses of small subunit ribosomal RNA genes (SSU rDNAs) have significantly influenced our understanding of the composition of aquatic microbial assemblages. Unfortunately, SSU rDNA sequences often do not have sufficient resolving power to differentiate closely related species. To address this general problem for uncultivated bacterioplankton taxa, we analysed and compared sequences of polymerase chain reaction (PCR)-generated and bacterial artificial chromosome (BAC)-derived clones that contained most of the SSU rDNAs, the internal transcribed spacer (ITS) and the large subunit ribosomal RNA gene (LSU rDNA). The phylogenetic representation in the rRNA operon PCR library was similar to that reported previously in coastal bacterioplankton SSU rDNA libraries. We observed good concordance between the phylogenetic relationships among coastal bacterioplankton inferred from SSU or LSU rDNA sequences. ITS sequences confirmed the close intragroup relationships among members of the SAR11, SAR116 and SAR86 clades that were predicted by SSU and LSU rDNA sequence analyses. We also found strong support for homologous recombination between the ITS regions of operons from the SAR11 clade.     

Bacterial abundance in relation to surface area and organic content of marine sediments

Based on direct measurements on surface sediments collected from an intertidal salt marsh, a positive relationship was demonstrated between bacterial abundance and specific surface area of sediment. While this relationship has been postulated previously, this is the first direct confirmation that it holds over a wide range of sediment types. A laboratory experiment was conducted to determine the effects of specific surface area, distribution of surface area, and organic loading on bacterial colonization. Model sediments included angular silica particles, kaolin, and spherical glass beads, used singly or in mixtures. Organic loading resulted in substantial enhancement of bacterial colonization. Distribution of surface area controlled by textural, shape, and sorting, had a complex effect, with glass bead sediments generally supporting better colonization than silica particles or kaolin. The effect of specific surface area was noted only in restricted comparisons of similarly shaped glass beads.     

Unveiling new microbial eukaryotes in the surface ocean

A decade after molecular techniques were used to discover novel bacteria and archaea in the oceans, the same approach has revealed a wealth of new marine eukaryotic microbes. The approach has been particularly successful with the smallest eukaryotes, where morphological and culture approaches frequently fail. Analysis of samples from the surface ocean, the most accessible and supposedly well-known oceanic region, reveals novel eukaryotic diversity at all different levels: from the highest taxonomic rank to the lowest microdiverse clusters. Moreover, marine eukaryotic assemblages show a large diversity with members belonging to many different lineages. The implication of this large and novel eukaryotic diversity for biodiversity surveys and ecosystem functioning opens new avenues for future research.     

Genomic potential for nitrogen assimilation in uncultivated members of Prochlorococcus from an anoxic marine zone

Cyanobacteria of the genus Prochlorococcus are the most abundant photosynthetic marine organisms and key factors in the global carbon cycle. The understanding of their distribution and ecological importance in oligotrophic tropical and subtropical waters, and their differentiation into distinct ecotypes, is based on genetic and physiological information from several isolates. Currently, all available Prochlorococcus genomes show their incapacity for nitrate utilization. However, environmental sequence data suggest that some uncultivated lineages may have acquired this capacity. Here we report that uncultivated low-light-adapted Prochlorococcus from the nutrient-rich, low-light, anoxic marine zone (AMZ) of the eastern tropical South Pacific have the genetic potential for nitrate uptake and assimilation. All genes involved in this trait were found syntenic with those present in marine Synechococcus. Genomic and phylogenetic analyses also suggest that these genes have not been aquired recently, but perhaps were retained from a common ancestor, highlighting the basal characteristics of the AMZ lineages within Prochlorococcus.Cyanobacteria of the genus Prochlorococcus are the most abundant photosynthetic microorganisms inhabiting the oceans, key factors in the carbon cycle and a model organism in environmental microbiology (Partensky and Garczarek, 2010). They can be broadly classified into high-light and low-light (LL)-adapted ecotypes (Rocap et al., 2002). These ecotypes exhibit distinct distributions both vertically in the water column and geographically across oligotrophic tropical and subtropical waters (Bouman et al., 2006; Johnson et al., 2006; Zwirglmaier et al., 2008).In past years, the genomes of over a dozen isolates of Prochlorocococus have been fully sequenced (for example, Kettler et al., 2007) and over a hundred single-cell-amplified partial genomes have been described (Malmstrom et al., 2013; Kashtan et al., 2014). All of them have revealed that they cannot use nitrate as a nitrogen source. However, new uncultivated lineages of Prochlorocococus have been identified in the environment using culture-independent techniques based on the sequencing of the 16S rRNA gene and related genomic regions (Lavin et al., 2010; West et al., 2011; Mühling, 2012; Malmstrom et al., 2013). On the other hand, nitrate assimilation rates were reported for uncultivated deep populations of Prochlorococcus in the Western Atlantic Ocean (Casey et al., 2007). In adition, genes necessary for nitrate assimilation associated to Prochlorococcus were identified in the global ocean sampling metagenomic database (Martiny et al., 2009) and in metagenomes of flow-cytometry-sorted Prochlorococcus populations (Batmalle et al., 2014).Important uncultivated Prochlorococcus lineages include those thriving in anoxic marine zones (AMZs), where oxygen concentrations fall below the detection limit of modern sensors, light is scarce, but inorganic nutrients are plentiful (Goericke et al., 2000; Ulloa et al., 2012). Phylogenetic analysis using the 16S–23S rRNA internal transcribed spacer region revealed that the AMZ-associated Prochlorococcus assemblages are mainly composed of two novel LL ecotypes (termed LL-V and LL-VI), which correspond to basal groups linking Prochlorococcus with marine Synechococcus (Lavin et al., 2010), the other dominant marine picocyanobacterium. However, no genomic or physiological information exists for these AMZ lineages.Here we report results from a metagenomic analysis carried out on environmental genomic sequences retrieved from a sample collected at 60 m depth within the AMZ of the eastern tropical South Pacific (Supplementary Figure S1), where dissolved oxygen was undetectable and inorganic nutrients were abundant (Supplementary Figure S2a; Thamdrup et al., 2012). The microbial community was enriched in Prochlorococcus, shown to comprise ~10% of cell abundance, versus ~0.7% of Synechococcus, assessed by flow cytometry (Supplementary Figure S2b). Blast analysis of the taxonomic affiliation of sequences matching the rpoC region 1, a taxonomic marker for cyanobacteria based on a single-copy gene (Palenik, 1994), showed an rpoC gene relative abundance of 86% for Prochlorococcus and 14% for Synechococcus (Supplementary Table S1), supporting the flow cytometry results. Moreover, of the 15% protein-coding sequences assigned to cyanobacteria, 10% binned with Prochlorococcus and 5% with Synechococcus (Supplementary Figure S3). Of those assigned to Prochlorococcus, 90% were related to the LL ecotypes MIT9313 and MIT9303, the closest reported relatives to the AMZ lineages with genomes fully sequenced (Lavin et al., 2010). General statistics of this AMZ metagenome are shown in Supplementary Tables S2 and S3.Analysis of de novo-assembled contigs revealed the presence of several large contigs that binned with Prochlorocococus. In particular, a single contig was found to encode genes related to urea and nitrate uptake and assimilation (contig 51148, GenBank accession number {"type":"entrez-nucleotide","attrs":{"text":"KM282015","term_id":"729772065","term_text":"KM282015"}}KM282015; 10 300 bp; Figure 1), in synteny with those in Synechococcus WH8102. The genes in the urease gene cluster (ureABCD) presented high identity to those described for Prochlorococcus MIT9313 and MIT9303 (Rocap et al., 2003; Supplementary Figure S4). Notably, the nitrate/nitrite transporter napA and assimilatory nitrate reductase narB were also found within the same contig (Figure 1a), as well as the genes moeA and mobA (Supplementary Figure S5) involved in the biosynthesis of the Mo-cofactor and necessary for the narB function (Flores et al., 2005). None of these genes have been found in any of the genomes of Prochlorococcus sequenced and described so far. However, homologues that presumably come from uncultivated relatives of Prochlorococcus have been found in the global ocean sampling database (Martiny et al., 2009) and in metagenomes of uncultured, sorted Prochlorococcus populations (Batmalle et al., 2014).Open in a separate windowFigure 1Genomic characteristics of the nitrogen assimilation operon found in contig 51148. (a) Schematic representation of syntenies among contig 51148, Prochlorococcus MIT9313 and MIT9303 genomes, and Synechococcus WH7803 and WH8102 genomes centered on nitrate and urea assimilation genes. Identities (%) among sequences are shown in gray. (b) GC content. (c) Contig coverage. (d) Proximity matrix (Euclidean distance) of the difference in codon usage pattern for the genomes of Prochlorococcus (Pro) and Synechococcus (Syn), and of contig 51148. The shortest distance (dark blue) indicates the highest proximity. (e) Spearman rank-order correlation between tetranucleotide frequency of contig 51148 and those of genomes of marine Prochlorococcus (Pro) and marine Synechococcus (Syn). The highest correlation is shown in dark green.The GC content of contig 51148 was ~51.1% (Figure 1b) and similar to that of LL Prochlorococcus and some marine Synechococcus (Kettler et al., 2007). Likewise, the narB gene had a GC content of 52%, which is less than the ~60% of those in the marine Synechococcus strains WH8102 and WH7803 (to which it presented the highest nucleotide identity), but significantly higher than the ~40% GC of the global ocean sampling high-light Prochlorococcus narB (Supplementary Figure S6). Analysis of codon usage patterns (Yu et al., 2012) and tetranucleotide frequencies (see Supplementary Material and Methods) showed that the cyanobacterial portion of the metagenome and contig 51148 exhibit the highest similarity with LL Prochlorococcus MIT9303 (Figures 1d and e). Additionally, nucleotide identities and phylogenetic analysis confirmed that the urease genes of contig 51148 were associated more closely with Prochlorococcus than Synechococcus (Supplementary Table S4 and Supplementary Figure S4).The homogeneous GC content of contig 51148, the differences in codon usage bias with Synechococcus and phylogenetic analyses of AMZ narB and napA (Figures 2a and b) all suggest that the genetic potential for nitrate uptake and assimilation was not obtained recently by horizontal gene transfer, but instead potentially were retained from a common ancestor with Synechococcus. Mapping the presence/absence of the different nitrate utilization genes onto the cyanobacteria 16S rRNA phylogenetic tree is consistent with this hypothesis (Supplementary Figure S7).Open in a separate windowFigure 2Phylogenetic trees for nitrate assimilation and uptake genes. Maximum-likelihood phylogenetic trees of (a) narB- and (b) napA-predicted amino acid sequences found in contig 51148. Evolutionary history was inferred using neighbour joining (NJ), maximum parsimony (MP) and maximum likelihood (ML). Bootstrap support values for 100 replications are shown at the nodes (NJ/MP/ML).In summary, our results indicate that AMZ Prochlorococcus lineages have the genetic potential for urea and nitrate assimilation, likely an adaptation to the unique nutrient-rich environment where they thrive. Additional genomic characteristics that could explain their high abundance in the oxygen-deficient and very-LL waters of AMZs remain to be assessed.     

Genome sequence of the unclassified marine Gammaproteobacterium BDW918

The unclassified marine gammaproteobacterium BDW918, which utilizes volatile fatty acids but not most common carbohydrates and amino acids, was isolated from Dokdo seawater in South Korea. Here we present a draft genome of the strain BDW918, which encodes many putative genes related to fatty acid metabolism and aromatic hydrocarbon degradation.     

The response of marine picoplankton to ocean acidification

Since industrialization global CO(2) emissions have increased, and as a consequence oceanic pH is predicted to drop by 0.3-0.4 units before the end of the century - a process coined 'ocean acidification'. Consequently, there is significant interest in how pH changes will affect the ocean's biota and integral processes. We investigated marine picoplankton (0.2-2?μm diameter) community response to predicted end of century CO(2) concentrations, via a 'high-CO(2) ' (～?750?ppm) large-volume (11?000?l) contained seawater mesocosm approach. We found little evidence of changes occurring in bacterial abundance or community composition due to elevated CO(2) under both phytoplankton pre-bloom/bloom and post-bloom conditions. In contrast, significant differences were observed between treatments for a number of key picoeukaryote community members. These data suggested a key outcome of ocean acidification is a more rapid exploitation of elevated CO(2) levels by photosynthetic picoeukaryotes. Thus, our study indicates the need for a more thorough understanding of picoeukaryote-mediated carbon flow within ocean acidification experiments, both in relation to picoplankton carbon sources, sinks and transfer to higher trophic levels.     

Distribution patterns and phylogeny of marine stramenopiles in the north pacific ocean

Marine stramenopiles (MASTs) are a diverse suite of eukaryotic microbes found in marine environments. Several MAST lineages are thought to contain heterotrophic nanoflagellates. However, MASTs remain uncultured and data on distributions and trophic modes are limited. We investigated MASTs in provinces on the west and east sides of the North Pacific Subtropical Gyre, specifically the East China Sea (ECS) and the California Current system (CALC). For each province, DNA was sampled from three zones: coastal, mesotrophic transitional, and more oligotrophic euphotic waters. Along with diatoms, chrysophytes, and other stramenopiles, sequences were recovered from nine MAST lineages in the six ECS and four CALC 18S rRNA gene clone libraries. All but one of these libraries were from surface samples. MAST clusters 1, 3, 7, 8, and 11 were identified in both provinces, with MAST cluster 3 (MAST-3) being found the most frequently. Additionally, MAST-2 was detected in the ECS and MAST-4, -9, and -12 were detected in the CALC. Phylogenetic analysis indicated that some subclades within these lineages differ along latitudinal gradients. MAST-1A, -1B, and -1C and MAST-4 size and abundance estimates obtained using fluorescence in situ hybridization on 79 spring and summer ECS samples showed a negative correlation between size of MAST-1B and MAST-4 cells and temperature. MAST-1A was rarely detected, but MAST-1B and -1C and MAST-4 were abundant in summer and MAST-1C and MAST-4 were more so at the coast, with maximum abundances of 543 and 1,896 cells ml(-1), respectively. MAST-4 and Synechococcus abundances were correlated, and experimental work showed that MAST-4 ingests Synechococcus. Together with previous studies, this study helps refine hypotheses on distribution and trophic modes of MAST lineages.     

Evidence of biogeography in surface ocean bacterioplankton assemblages

Regardless of the importance of bacterial assemblages as essential components of ecosystems, little is known about how their populations are structured. We analyzed the composition and turnover rates, based on 16S rDNA sequences, of surface water oceanic bacterial assemblages of the fraction between 0.1 and 0.8 μm along a latitudinal gradient (45°6′42′′N in the North Atlantic to 15°8′37′′S in the South Pacific) including geographic distance, temperature, chlorophyll a and salinity. Here we show that oceanic bacterial assemblages between 0.1 and 0.8 μm, can be structured by a variety of environmental interactions that include separation by distance and chlorophyll a concentration in temperate North Atlantic coastal samples and temperature in tropical Atlantic and Pacific coastal and open ocean samples. Bacterial phyla composition diverged between temperate and tropical regions. This study suggests that some bacterial assemblages could be structured both by environmental and spatial factors, while others by environmental factors alone.     

Assessing marine ecosystem acoustic diversity across ocean basins

Concurrent with the elevation of the concern over the state of sound in the ocean, advances in terrestrial acoustic monitoring techniques have produced concepts and tools that may be applicable to the underwater world. Several index values that convey information related to acoustic diversity with a single numeric measurement made from acoustic recordings have been proposed for rapidly assessing community biodiversity. Here we apply the acoustic biodiversity index method to low frequency recordings made from three different ocean basins to assess its appropriateness for characterizing species richness in the marine environment. Initial results indicated that raw acoustic entropy (H) values did not correspond to biological patterns identified from individual signal detections and classification. Noise from seismic airgun activity masked the weaker biological signals and confounded the entropy calculation. A simple background removal technique that subtracted an average complex spectrum characteristic of seismic exploration signals from the average spectra of each analysis period that contained seismic signals was applied to compensate for salient seismic airgun signals present in all locations. The noise compensated (H_N) entropy index was more reflective of biological patterns and holds promise for the use of rapid acoustic biodiversity in the marine environment as an indicator of habitat biodiversity and health.     

Genome sequence of the ubiquitous hydrocarbon-degrading marine bacterium Alcanivorax borkumensis

Alcanivorax borkumensis is a cosmopolitan marine bacterium that uses oil hydrocarbons as its exclusive source of carbon and energy. Although barely detectable in unpolluted environments, A. borkumensis becomes the dominant microbe in oil-polluted waters. A. borkumensis SK2 has a streamlined genome with a paucity of mobile genetic elements and energy generation-related genes, but with a plethora of genes accounting for its wide hydrocarbon substrate range and efficient oil-degradation capabilities. The genome further specifies systems for scavenging of nutrients, particularly organic and inorganic nitrogen and oligo-elements, biofilm formation at the oil-water interface, biosurfactant production and niche-specific stress responses. The unique combination of these features provides A. borkumensis SK2 with a competitive edge in oil-polluted environments. This genome sequence provides the basis for the future design of strategies to mitigate the ecological damage caused by oil spills.     

Genome organisation of the marine Roseobacter clade member Marinovum algicola

The Roseobacter clade, belonging to the family Rhodobacteraceae of the class Alphaproteobacteria, is one of the major bacterial groups in marine environments. A remarkable wealth of diverse large plasmids has been detected in members of this lineage. Here, we analysed the genome structure and extrachromosomal DNA content of four strains of the roseobacter species Marinovum algicola by pulsed-field gel electrophoresis. They were originally isolated from toxic dinoflagellates and possess multireplicon genomes with sizes between 5.20 and 5.35 Mb. In addition to the single circular chromosomes (3.60–3.74 Mb), whose organisation seem to be conserved, 9 to 12 extrachromosomal replicons have been detected for each strain. This number is unprecedented for roseobacters and proposes a sophisticated regulation of replication and partitioning to ensure stable maintenance. The plasmid lengths range from 7 to 477 kb and our analyses document a circular conformation for all but one of them, which might represent a linear plasmid-like prophage. In striking contrast to other roseobacters, up to one-third of the genomic information (1.75 Mb) is plasmid borne in Marinovum algicola. The plasmid patterns of some strains are conspicuously different, indicating that recombination and conjugative gene transfer are dominant mechanisms for microevolution within the Roseobacter clade.     

Genome sequence of the halotolerant marine bacterium Myxococcus fulvus HW-1

Myxococcus fulvus HW-1 (ATCC BAA-855) is a halotolerant marine myxobacterium. This strain exhibits complex social behaviors in the presence of low concentrations of seawater but adopts an asocial living pattern under oceanic conditions. The whole genome of M. fulvus HW-1 will enable us to further investigate the details of its evolution.