The key monooxygenase involved in phenanthrene degradation of Ruegeria sp. PrR005.

As a typical polycyclic aromatic hydrocarbon (PAH), phenanthrene is often present in diverse environments, leading to severe environmental contamination. However, bacterial degradation plays a crucial role in remediating phenanthrene contamination and has been widely adopted. The widely distributed marine Roseobacter-clade bacteria are frequently found in phenanthrene-contaminated environments, but their catalyzing ability and related molecular mechanism have been rarely elucidated. Our previous work showed Ruegeria sp. PrR005 isolated from the Pearl River Estuary sediment could degrade phenanthrene and other PAHs. Integrated approaches including multi-omics and biochemical analysis were applied here to explore its catabolism mechanism. The genomic and transcriptomic analysis indicated that six new P450 monooxygenase proteins could be closely associated with phenanthrene degradation. Heterologous expression of P450 monooxygenase candidates revealed that PrR005_00615, PrR005_04282, PrR005_04577 have considerable activity in phenanthrene removal, with PrR005_00615 being the primary contributor. Further, the biochemical and metabolic analysis revealed that PrR005_00615 could catalyze phenanthrene to phenanthrene-9,10-epoxide by introducing an oxygen atom at 9,10-carbon positions, which functioned as a monooxygenase. The present study provides compelling evidences of a novel enzyme responsible for catalyzing the initial step of phenanthrene transformation in PrR005. These findings hold significant importance in unraveling the mechanism behind phenanthrene degradation by Roseobacter-clade bacteria.

Developing a Base Editing System for Marine Roseobacter Clade Bacteria.

The Roseobacter clade bacteria are of great significance in marine ecology and biogeochemical cycles, and they are potential microbial chassis for marine synthetic biology due to their versatile metabolic capabilities. Here, we adapted a CRISPR-Cas-based system, base editing, with the combination of nuclease-deactivated Cas9 and deaminase for Roseobacter clade bacteria. Taking the model roseobacter Roseovarius nubinhibens as an example, we achieved precise and efficient genome editing at single-nucleotide resolution without generating double-strand breaks or requesting donor DNAs. Since R. nubinhibens can metabolize aromatic compounds, we interrogated the key genes in the ß-ketoadipate pathway with our base editing system via the introduction of premature STOP codons. The essentiality of these genes was demonstrated, and for the first time, we determined PcaQ as a transcription activator experimentally. This is the first report of CRISPR-Cas-based genome editing in the entire clade of Roseobacter bacteria. We believe that our work provides a paradigm for interrogating marine ecology and biogeochemistry with direct genotype-and-phenotype linkages and potentially opens a new avenue for the synthetic biology of marine Roseobacter bacteria.

Roseobacters in a Sea of Poly- and Paraphyly: Whole Genome-Based Taxonomy of the Family Rhodobacteraceae and the Proposal for the Split of the "Roseobacter Clade" Into a Novel Family, Roseobacteraceae fam. nov.

The family Rhodobacteraceae consists of alphaproteobacteria that are metabolically, phenotypically, and ecologically diverse. It includes the roseobacter clade, an informal designation, representing one of the most abundant groups of marine bacteria. The rapid pace of discovery of novel roseobacters in the last three decades meant that the best practice for taxonomic classification, a polyphasic approach utilizing phenotypic, genotypic, and phylogenetic characteristics, was not always followed. Early efforts for classification relied heavily on 16S rRNA gene sequence similarity and resulted in numerous taxonomic inconsistencies, with several poly- and paraphyletic genera within this family. Next-generation sequencing technologies have allowed whole-genome sequences to be obtained for most type strains, making a revision of their taxonomy possible. In this study, we performed whole-genome phylogenetic and genotypic analyses combined with a meta-analysis of phenotypic data to review taxonomic classifications of 331 type strains (under 119 genera) within the Rhodobacteraceae family. Representatives of the roseobacter clade not only have different environmental adaptions from other Rhodobacteraceae isolates but were also found to be distinct based on genomic, phylogenetic, and in silico-predicted phenotypic data. As such, we propose to move this group of bacteria into a new family, Roseobacteraceae fam. nov. In total, reclassifications resulted to 327 species and 128 genera, suggesting that misidentification is more problematic at the genus than species level. By resolving taxonomic inconsistencies of type strains within this family, we have established a set of coherent criteria based on whole-genome-based analyses that will help guide future taxonomic efforts and prevent the propagation of errors.

Lipidomic Analysis of Roseobacters of the Pelagic RCA Cluster and Their Response to Phosphorus Limitation.

The marine roseobacter-clade affiliated cluster (RCA) represents one of the most abundant groups of bacterioplankton in the global oceans, particularly in temperate and sub-polar regions. They play a key role in the biogeochemical cycling of various elements and are important players in oceanic climate-active trace gas metabolism. In contrast to copiotrophic roseobacter counterparts such as Ruegeria pomeroyi DSS-3 and Phaeobacter sp. MED193, RCA bacteria are truly pelagic and have smaller genomes. We have previously shown that RCA bacteria do not appear to encode the PlcP-mediated lipid remodeling pathway, whereby marine heterotrophic bacteria remodel their membrane lipid composition in response to phosphorus (P) stress by substituting membrane glycerophospholipids with alternative glycolipids or betaine lipids. In this study, we report lipidomic analysis of six RCA isolates. In addition to the commonly found glycerophospholipids such as phosphatidylglycerol (PG) and phosphatidylethanolamine (PE), RCA bacteria synthesize a relatively uncommon phospholipid, acylphosphatidylglycerol, which is not found in copiotrophic roseobacters. Instead, like the abundant SAR11 clade, RCA bacteria upregulate ornithine lipid biosynthesis in response to P stress, suggesting a key role of this aminolipid in the adaptation of marine heterotrophs to oceanic nutrient limitation.

Characteristics and Evolutionary Analysis of Photosynthetic Gene Clusters on Extrachromosomal Replicons: from Streamlined Plasmids to Chromids.

Aerobic anoxygenic photoheterotrophic bacteria (AAPB) represent a bacteriochlorophyll a-containing functional group. Substantial evidence indicates that highly conserved photosynthetic gene clusters (PGCs) of AAPB can be transferred between species, genera, and even phyla. Furthermore, analysis of recently discovered PGCs carried by extrachromosomal replicons (exPGCs) suggests that extrachromosomal replicons (ECRs) play an important role in the transfer of PGCs. In this study, 13 Roseobacter clade genomes from seven genera that harbored exPGCs were used to analyze the characteristics and evolution of PGCs. The identification of plasmid-like and chromid-like ECRs among PGC-containing ECRs revealed two different functions: the spread of PGCs among strains and the maintenance of PGCs within genomes. Phylogenetic analyses indicated two independent origins of exPGCs, corresponding to PufC-containing and PufX-containing puf operons. Furthermore, the two different types of operons were observed within different strains of the same Tateyamaria and Jannaschia genera. The PufC-containing and PufX-containing operons were also differentially carried by chromosomes and ECRs in the strains, respectively, which provided clear evidence for ECR-mediated PGC transfer. Multiple recombination events of exPGCs were also observed, wherein the majority of exPGCs were inserted by replication modules at the same genomic positions. However, the exPGCs of the Jannaschia strains comprised superoperons without evidence of insertion and therefore likely represent an initial evolutionary stage where the PGC was translocated from chromosomes to ECRs without further combinations. Finally, a scenario of PGC gain and loss is proposed that specifically focuses on ECR-mediated exPGC transfer to explain the evolution and patchy distribution of AAPB within the Roseobacter clade.IMPORTANCE The evolution of photosynthesis was a significant event during the diversification of biological life. Aerobic anoxygenic photoheterotrophic bacteria (AAPB) share physiological characteristics with chemoheterotrophs and represent an important group associated with bacteriochlorophyll-dependent phototrophy in the environment. Here, characterization and evolutionary analyses were conducted for 13 bacterial strains that contained photosynthetic gene clusters (PGCs) carried by extrachromosomal replicons (ECRs) to shed light on the evolution of chlorophototrophy in bacteria. This report advances our understanding of the importance of ECRs in the transfer of PGCs within marine photoheterotrophic bacteria.

Uncultured Gammaproteobacteria and Desulfobacteraceae Account for Major Acetate Assimilation in a Coastal Marine Sediment.

Acetate is a key intermediate in anaerobic mineralization of organic matter in marine sediments. Its turnover is central to carbon cycling, however, the relative contribution of different microbial populations to acetate assimilation in marine sediments is unknown. To quantify acetate assimilation by in situ abundant bacterial populations, we incubated coastal marine sediments with 14C-labeled acetate and flow-sorted cells that had been labeled and identified by fluorescence in situ hybridization. Subsequently, scintillography determined the amount of 14C-acetate assimilated by distinct populations. This approach fostered a high-throughput quantification of acetate assimilation by phylogenetically identified populations. Acetate uptake was highest in the oxic-suboxic surface layer for all sorted bacterial populations, including deltaproteobacterial sulfate-reducing bacteria (SRB), which accounted for up to 32% of total bacterial acetate assimilation. We show that the family Desulfobulbaceae also assimilates acetate in marine sediments, while the more abundant Desulfobacteraceae dominated acetate assimilation despite lower uptake rates. Unexpectedly, members of Gammaproteobacteria accounted for the highest relative acetate assimilation in all sediment layers with up to 31-62% of total bacterial acetate uptake. We also show that acetate is used to build up storage compounds such as polyalkanoates. Together, our findings demonstrate that not only the usual suspects SRB but a diverse bacterial community may substantially contribute to acetate assimilation in marine sediments. This study highlights the importance of quantitative approaches to reveal the roles of distinct microbial populations in acetate turnover.

Genomic, proteomic and bioinformatic analysis of two temperate phages in Roseobacter clade bacteria isolated from the deep-sea water.

BACKGROUND: Marine phages are spectacularly diverse in nature. Dozens of roseophages infecting members of Roseobacter clade bacteria were isolated and characterized, exhibiting a very high degree of genetic diversity. In the present study, the induction of two temperate bacteriophages, namely, vB_ThpS-P1 and vB_PeaS-P1, was performed in Roseobacter clade bacteria isolated from the deep-sea water, Thiobacimonas profunda JLT2016 and Pelagibaca abyssi JLT2014, respectively. Two novel phages in morphological, genomic and proteomic features were presented, and their phylogeny and evolutionary relationships were explored by bioinformatic analysis. RESULTS: Electron microscopy showed that the morphology of the two phages were similar to that of siphoviruses. Genome sequencing indicated that the two phages were similar in size, organization, and content, thereby suggesting that these shared a common ancestor. Despite the presence of Mu-like phage head genes, the phages are more closely related to Rhodobacter phage RC1 than Mu phages in terms of gene content and sequence similarity. Based on comparative genomic and phylogenetic analysis, we propose a Mu-like head phage group to allow for the inclusion of Mu-like phages and two newly phages. The sequences of the Mu-like head phage group were widespread, occurring in each investigated metagenomes. Furthermore, the horizontal exchange of genetic material within the Mu-like head phage group might have involved a gene that was associated with phage phenotypic characteristics. CONCLUSIONS: This study is the first report on the complete genome sequences of temperate phages that infect deep-sea roseobacters, belonging to the Mu-like head phage group. The Mu-like head phage group might represent a small but ubiquitous fraction of marine viral diversity.

Boosted Membrane Potential as Bioenergetic Response to Anoxia in Dinoroseobacter shibae.

Dinoroseobacter shibae DFL 12T is a metabolically versatile member of the world-wide abundant Roseobacter clade. As an epibiont of dinoflagellates D. shibae is subjected to rigorous changes in oxygen availability. It has been shown that it loses up to 90% of its intracellular ATP when exposed to anoxic conditions. Yet, D. shibae regenerates its ATP level quickly when oxygen becomes available again. In the present study we focused on the bioenergetic aspects of the quick recovery and hypothesized that the proton-motive force decreases during anoxia and gets restored upon re-aeration. Therefore, we analyzed ΔpH and the membrane potential (ΔΨ) during the oxic-anoxic transitions. To visualize changes of ΔΨ we used fluorescence microscopy and the carbocyanine dyes DiOC2 (3; 3,3'-Diethyloxacarbocyanine Iodide) and JC-10. In control experiments the ΔΨ-decreasing effects of the chemiosmotic inhibitors CCCP (carbonyl cyanide m-chlorophenyl hydrazone), TCS (3,3',4',5-tetrachlorosalicylanilide) and gramicidin were tested on D. shibae and Gram-negative and -positive control bacteria (Escherichia coli and Micrococcus luteus). We found that ΔpH is not affected by short-term anoxia and does not contribute to the quick ATP regeneration in D. shibae. By contrast, ΔΨ was increased during anoxia, which was astonishing since none of the control organisms behaved that way. Our study shows physiological and bioenergetical aspects comparing to previous studies on transcriptomic responses to the transition from aerobic to nitrate respiration in D. shibae. For the lifestyle as an epibiont of a dinoflagellate, the ability to stand phases of temporary oxygen depletion is beneficial. With a boosted ΔΨ, the cells are able to give their ATP regeneration a flying start, once oxygen is available again.

Dimethylsulfoniopropionate Promotes Process Outgrowth in Neural Cells and Exerts Protective Effects against Tropodithietic Acid.

The marine environment harbors a plethora of bioactive substances, including drug candidates of potential value in the field of neuroscience. The present study was undertaken to investigate the effects of dimethylsulfoniopropionate (DMSP), produced by several algae, corals and higher plants, on cells of the mammalian nervous system, i.e., neuronal N2a and OLN-93 cells as model system for nerve cells and glia, respectively. Additionally, the protective capabilities of DMSP were assessed in cells treated with tropodithietic acid (TDA), a marine metabolite produced by several Roseobacter clade bacteria. Both cell lines, N2a and OLN-93, have previously been shown to be a sensitive target for the action of TDA, and cytotoxic effects of TDA have been connected to the induction of oxidative stress. Our data shows that DMSP promotes process outgrowth and microtubule reorganization and bundling, accompanied by an increase in alpha-tubulin acetylation. Furthermore, DMSP was able to prevent the cytotoxic effects exerted by TDA, including the breakdown of the mitochondrial membrane potential, upregulation of heat shock protein Hsp32 and activation of the extracellular signal-regulated kinases 1/2 (ERK1/2). Our study points to the conclusion that DMSP provides an antioxidant defense, not only in algae but also in mammalian neural cells.

Draft genome sequence of Thalassobius mediterraneus CECT 5383(T), a poly-beta-hydroxybutyrate producer.

Thalassobius mediterraneus is the type species of the genus Thalassobius and a member of the Roseobacter clade, an abundant representative of marine bacteria. T. mediterraneus XSM19(T) (= CECT 5383(T)) was isolated from the Western Mediterranean coast near Valencia (Spain) in 1989. We present here the draft genome sequence and annotation of this strain (ENA/DDBJ/NCBI accession number CYSF00000000), which is comprised of 3,431,658 bp distributed in 19 contigs and encodes 10 rRNA genes, 51 tRNA genes and 3276 protein coding genes. Relevant findings are commented, including the complete set of genes required for poly-beta-hydroxybutyrate (PHB) synthesis and genes related to degradation of aromatic compounds.

Bacterial influence on alkenones in live microalgae.

The microalga Emiliania huxleyi produces alkenone lipids that are important proxies for estimating past sea surface temperatures. Field calibrations of this proxy are robust but highly variable results are obtained in culture. Here, we present results suggesting that algal-bacterial interactions may be responsible for some of this variability. Co-cultures of E. huxleyi and the bacterium Phaeobacter inhibens resulted in a 2.5-fold decrease in algal alkenone-containing lipid bodies. In addition levels of unsaturated alkenones increase in co-cultures. These changes result in an increase in the reconstructed growth temperature of up to 2°C relative to axenic algal cultures.

Expansion of Cultured Bacterial Diversity by Large-Scale Dilution-to-Extinction Culturing from a Single Seawater Sample.

High-throughput cultivation (HTC) based on a dilution-to-extinction method has been applied broadly to the cultivation of marine bacterial groups, which has often led to the repeated isolation of abundant lineages such as SAR11 and oligotrophic marine gammaproteobacteria (OMG). In this study, to expand the phylogenetic diversity of HTC isolates, we performed a large-scale HTC with a single surface seawater sample collected from the East Sea, the Western Pacific Ocean. Phylogenetic analyses of the 16S rRNA genes from 847 putative pure cultures demonstrated that some isolates were affiliated with not-yet-cultured clades, including the OPB35 and Puniceicoccaceae marine group of Verrucomicrobia and PS1 of Alphaproteobacteria. In addition, numerous strains were obtained from abundant clades, such as SAR11, marine Roseobacter clade, OMG (e.g., SAR92 and OM60), OM43, and SAR116, thereby increasing the size of available culture resources for representative marine bacterial groups. Comparison between the composition of HTC isolates and the bacterial community structure of the seawater sample used for HTC showed that diverse marine bacterial groups exhibited various growth capabilities under our HTC conditions. The growth response of many bacterial groups, however, was clearly different from that observed with conventional plating methods, as exemplified by numerous isolates of the SAR11 clade and Verrucomicrobia. This study showed that a large number of novel bacterial strains could be obtained by an extensive HTC from even a small number of samples.

Cytotoxic Effects of Tropodithietic Acid on Mammalian Clonal Cell Lines of Neuronal and Glial Origin.

The marine metabolite tropodithietic acid (TDA), produced by several Roseobacter clade bacteria, is known for its broad antimicrobial activity. TDA is of interest not only as a probiotic in aquaculture, but also because it might be of use as an antibacterial agent in non-marine or non-aquatic environments, and thus the potentially cytotoxic influences on eukaryotic cells need to be evaluated. The present study was undertaken to investigate its effects on cells of the mammalian nervous system, i.e., neuronal N2a cells and OLN-93 cells as model systems for nerve cells and glia. The data show that in both cell lines TDA exerted morphological changes and cytotoxic effects at a concentration of 0.3-0.5 µg/mL (1.4-2.4 µM). Furthermore, TDA caused a breakdown of the mitochondrial membrane potential, the activation of extracellular signal-regulated kinases ERK1/2, and the induction of the small heat shock protein HSP32/HO-1, which is considered as a sensor of oxidative stress. The cytotoxic effects were accompanied by an increase in intracellular Ca(2+)-levels, the disturbance of the microtubule network, and the reorganization of the microfilament system. Hence, mammalian cells are a sensitive target for the action of TDA and react by the activation of a stress response resulting in cell death.

Comparative genomics of Roseobacter clade bacteria isolated from the accessory nidamental gland of Euprymna scolopes.

The accessory nidamental gland (ANG) of the female Hawaiian bobtail squid, Euprymna scolopes, houses a consortium of bacteria including members of the Flavobacteriales, Rhizobiales, and Verrucomicrobia but is dominated by members of the Roseobacter clade (Rhodobacterales) within the Alphaproteobacteria. These bacteria are deposited into the jelly coat of the squid's eggs, however, the function of the ANG and its bacterial symbionts has yet to be elucidated. In order to gain insight into this consortium and its potential role in host reproduction, we cultured 12 Rhodobacterales isolates from ANGs of sexually mature female squid and sequenced their genomes with Illumina sequencing technology. For taxonomic analyses, the ribosomal proteins of 79 genomes representing both roseobacters and non-roseobacters along with a separate MLSA analysis of 33 housekeeping genes from Roseobacter organisms placed all 12 isolates from the ANG within two groups of a single Roseobacter clade. Average nucelotide identity analysis suggests the ANG isolates represent three genera (Leisingera, Ruegeria, and Tateyamaria) comprised of seven putative species groups. All but one of the isolates contains a predicted Type VI secretion system, which has been shown to be important in secreting signaling and/or effector molecules in host-microbe associations and in bacteria-bacteria interactions. All sequenced genomes also show potential for secondary metabolite production, and are predicted to be involved with the production of acyl homoserine lactones (AHLs) and/or siderophores. An AHL bioassay confirmed AHL production in three tested isolates and from whole ANG homogenates. The dominant symbiont, Leisingera sp. ANG1, showed greater viability in iron-limiting conditions compared to other roseobacters, possibly due to higher levels of siderophore production. Future comparisons will try to elucidate novel metabolic pathways of the ANG symbionts to understand their putative role in host development.

A glutathione peroxidase (GpoA) plays a role in the pathogenicity of Nautella italica strain R11 towards the red alga Delisea pulchra.

Oxidative bursts are a common mechanism used by higher organisms to defend themselves against bacterial attacks, but some pathogenic bacteria have evolved resistance mechanisms to counteract this. The role of oxidative stress resistance as a virulence trait in macroalgal disease is however unknown. Here, we demonstrate that the gene gpoA, encoding for a glutathione peroxidase, is important for the oxidative stress response of the macroalgal pathogen Nautella italica R11. We also show that a lack of gpoA prevents N. italica R11 from inducing a bleaching disease in the red alga Delisea pulchra. These results show that a defense against oxidative stress is likely to be an important feature enabling pathogenic bacteria to infect macroalgae.

Genome sequence and emended description of Leisingera nanhaiensis strain DSM 24252(T) isolated from marine sediment.

Leisingera nanhaiensis DSM 24252(T) is a Gram-negative, motile, rod-shaped marine Alphaproteobacterium, isolated from sandy marine sediments. Here we present the non-contiguous genome sequence and annotation together with a summary of the organism's phenotypic features. The 4,948,550 bp long genome with its 4,832 protein-coding and 64 RNA genes consists of one chromosome and six extrachromosomal elements with lengths of 236 kb, 92 kb, 61 kb, 58 kb, 56 kb, and 35 kb, respectively. The analysis of the genome showed that DSM 24252(T) possesses all genes necessary for dissimilatory nitrite reduction, and the strain was shown to be facultatively anaerobic, a deviation from the original description that calls for an emendation of the species. Also present in the genome are genes coding for a putative prophage, for gene-transfer agents and for the utilization of methylated amines. Phylogenetic analysis and intergenomic distances indicate that L. nanhaiensis might not belong to the genus Leisingera.

Genome sequence of the exopolysaccharide-producing Salipiger mucosus type strain (DSM 16094(T)), a moderately halophilic member of the Roseobacter clade.

Salipiger mucosus Martínez-Cànovas et al. 2004 is the type species of the genus Salipiger, a moderately halophilic and exopolysaccharide-producing representative of the Roseobacter lineage within the alphaproteobacterial family Rhodobacteraceae. Members of this family were shown to be the most abundant bacteria especially in coastal and polar waters, but were also found in microbial mats and sediments. Here we describe the features of the S. mucosus strain DSM 16094(T) together with its genome sequence and annotation. The 5,689,389-bp genome sequence consists of one chromosome and several extrachromosomal elements. It contains 5,650 protein-coding genes and 95 RNA genes. The genome of S. mucosus DSM 16094(T) was sequenced as part of the activities of the Transregional Collaborative Research Center 51 (TRR51) funded by the German Research Foundation (DFG).

Phylogeny-driven target selection for large-scale genome-sequencing (and other) projects.

Despite the steadily decreasing costs of genome sequencing, prioritizing organisms for sequencing remains important in large-scale projects. Phylogeny-based selection is of interest to identify those organisms whose genomes can be expected to differ most from those that have already been sequenced. Here, we describe a method that infers a phylogenetic scoring independent of which set of organisms has previously been targeted, which is computationally simple and easy to apply in practice. The scoring itself, as well as pre- and post-processing of the data, is illustrated using two real-world examples in which the method has already been applied for selecting targets for genome sequencing. These projects are the JGI CSP Genomic Encyclopedia of Bacteria and Archaea phase I, targeting 1,000 type strains, and, on a smaller-scale, the phylogenomics of the Roseobacter clade. Potential artifacts of the method are discussed and compared to a selection approach based on the taxonomic classification.

Isotopically labeled sulfur compounds and synthetic selenium and tellurium analogues to study sulfur metabolism in marine bacteria.

Members of the marine Roseobacter clade can degrade dimethylsulfoniopropionate (DMSP) via competing pathways releasing either methanethiol (MeSH) or dimethyl sulfide (DMS). Deuterium-labeled [(2)H6]DMSP and the synthetic DMSP analogue dimethyltelluriopropionate (DMTeP) were used in feeding experiments with the Roseobacter clade members Phaeobacter gallaeciensis DSM 17395 and Ruegeria pomeroyi DSS-3, and their volatile metabolites were analyzed by closed-loop stripping and solid-phase microextraction coupled to GC-MS. Feeding experiments with [(2)H6]DMSP resulted in the incorporation of a deuterium label into MeSH and DMS. Knockout of relevant genes from the known DMSP demethylation pathway to MeSH showed in both species a residual production of [(2)H3]MeSH, suggesting that a second demethylation pathway is active. The role of DMSP degradation pathways for MeSH and DMS formation was further investigated by using the synthetic analogue DMTeP as a probe in feeding experiments with the wild-type strain and knockout mutants. Feeding of DMTeP to the R. pomeroyi knockout mutant resulted in a diminished, but not abolished production of demethylation pathway products. These results further corroborated the proposed second demethylation activity in R. pomeroyi. Isotopically labeled [(2)H3]methionine and (34)SO4 (2-), synthesized from elemental (34)S8, were tested to identify alternative sulfur sources besides DMSP for the MeSH production in P. gallaeciensis. Methionine proved to be a viable sulfur source for the MeSH volatiles, whereas incorporation of labeling from sulfate was not observed. Moreover, the utilization of selenite and selenate salts by marine alphaproteobacteria for the production of methylated selenium volatiles was explored and resulted in the production of numerous methaneselenol-derived volatiles via reduction and methylation. The pathway of selenate/selenite reduction, however, proved to be strictly separated from sulfate reduction.

Genome sequence of the phage-gene rich marine Phaeobacter arcticus type strain DSM 23566(T.).

Phaeobacter arcticus Zhang et al. 2008 belongs to the marine Roseobacter clade whose members are phylogenetically and physiologically diverse. In contrast to the type species of this genus, Phaeobacter gallaeciensis, which is well characterized, relatively little is known about the characteristics of P. arcticus. Here, we describe the features of this organism including the annotated high-quality draft genome sequence and highlight some particular traits. The 5,049,232 bp long genome with its 4,828 protein-coding and 81 RNA genes consists of one chromosome and five extrachromosomal elements. Prophage sequences identified via PHAST constitute nearly 5% of the bacterial chromosome and included a potential Mu-like phage as well as a gene-transfer agent (GTA). In addition, the genome of strain DSM 23566(T) encodes all of the genes necessary for assimilatory nitrate reduction. Phylogenetic analysis and intergenomic distances indicate that the classification of the species might need to be reconsidered.