Multi-omics analysis reveals the molecular response to heat stress in a "red tide" dinoflagellate.

BACKGROUND: "Red tides" are harmful algal blooms caused by dinoflagellate microalgae that accumulate toxins lethal to other organisms, including humans via consumption of contaminated seafood. These algal blooms are driven by a combination of environmental factors including nutrient enrichment, particularly in warm waters, and are increasingly frequent. The molecular, regulatory, and evolutionary mechanisms that underlie the heat stress response in these harmful bloom-forming algal species remain little understood, due in part to the limited genomic resources from dinoflagellates, complicated by the large sizes of genomes, exhibiting features atypical of eukaryotes. RESULTS: We present the de novo assembled genome (~ 4.75 Gbp with 85,849 protein-coding genes), transcriptome, proteome, and metabolome from Prorocentrum cordatum, a globally abundant, bloom-forming dinoflagellate. Using axenic algal cultures, we study the molecular mechanisms that underpin the algal response to heat stress, which is relevant to current ocean warming trends. We present the first evidence of a complementary interplay between RNA editing and exon usage that regulates the expression and functional diversity of biomolecules, reflected by reduction in photosynthesis, central metabolism, and protein synthesis. These results reveal genomic signatures and post-transcriptional regulation for the first time in a pelagic dinoflagellate. CONCLUSIONS: Our multi-omics analyses uncover the molecular response to heat stress in an important bloom-forming algal species, which is driven by complex gene structures in a large, high-G+C genome, combined with multi-level transcriptional regulation. The dynamics and interplay of molecular regulatory mechanisms may explain in part how dinoflagellates diversified to become some of the most ecologically successful organisms on Earth.

Thiorhodovibrio frisius and Trv. litoralis spp. nov., Two Novel Members from a Clade of Fastidious Purple Sulfur Bacteria That Exhibit Unique Red-Shifted Light-Harvesting Capabilities.

In the pursuit of cultivating anaerobic anoxygenic phototrophs with unusual absorbance spectra, a purple sulfur bacterium was isolated from the shoreline of Baltrum, a North Sea island of Germany. It was designated strain 970, due to a predominant light harvesting complex (LH) absorption maximum at 963-966 nm, which represents the furthest infrared-shift documented for such complexes containing bacteriochlorophyll a. A polyphasic approach to bacterial systematics was performed, comparing genomic, biochemical, and physiological properties. Strain 970 is related to Thiorhodovibrio winogradskyi DSM 6702T by 26.5, 81.9, and 98.0% similarity via dDDH, ANI, and 16S rRNA gene comparisons, respectively. The photosynthetic properties of strain 970 were unlike other Thiorhodovibrio spp., which contained typical LH absorbing characteristics of 800-870 nm, as well as a newly discovered absorption band at 908 nm. Strain 970 also had a different photosynthetic operon composition. Upon genomic comparisons with the original Thiorhodovibrio strains DSM 6702T and strain 06511, the latter was found to be divergent, with 25.3, 79.1, and 97.5% similarity via dDDH, ANI, and 16S rRNA gene homology to Trv. winogradskyi, respectively. Strain 06511 (=DSM 116345T) is thereby described as Thiorhodovibrio litoralis sp. nov., and the unique strain 970 (=DSM 111777T) as Thiorhodovibrio frisius sp. nov.

Volatiles of the Apicomplexan Alga Chromera velia and Associated Bacteria.

Volatiles released by the apicomplexan alga Chromera velia CCAP1602/1 and their associated bacteria have been investigated. A metagenome analysis allowed the identification of the most abundant heterotrophic bacteria of the phycosphere, but the isolation of additional strains showed that metagenomics underestimated the complexity of the algal microbiome, However, a culture-independent approach revealed the presence of a planctomycete that likely represents a novel bacterial family. We analysed algal and bacterial volatiles by open-system-stripping analysis (OSSA) on Tenax TA desorption tubes, followed by thermodesorption, cryofocusing and GC-MS-analysis. The analyses of the alga and the abundant bacterial strains Sphingopyxis litoris A01A-101, Algihabitans albus A01A-324, "Coraliitalea coralii" A01A-333 and Litoreibacter sp. A01A-347 revealed sulfur- and nitrogen-containing compounds, ketones, alcohols, aldehydes, aromatic compounds, amides and one lactone, as well as the typical algal products, apocarotenoids. The compounds were identified by gas chromatographic retention indices, comparison of mass spectra and syntheses of reference compounds. A major algal metabolite was 3,4,4-trimethylcyclopent-2-en-1-one, an apocarotenoid indicating the presence of carotenoids related to capsanthin, not reported from algae so far. A low overlap in volatiles bouquets between C. velia and the bacteria was found, and the xenic algal culture almost exclusively released algal components.

The Sixth Element: a 102-kb RepABC Plasmid of Xenologous Origin Modulates Chromosomal Gene Expression in Dinoroseobacter shibae.

The model organism Dinoroseobacter shibae and many other marine Rhodobacterales (Roseobacteraceae, Alphaproteobacteria) are characterized by a multipartite genome organization. Here, we show that the original isolate (Dshi-6) contained six extrachromosomal replicons (ECRs), whereas the strain deposited at the DSMZ (Dshi-5) lacked a 102-kb plasmid. To determine the role of the sixth plasmid, we investigated the genomic and physiological differences between the two strains. Therefore, both genomes were (re)sequenced, and gene expression, growth, and substrate utilization were examined. For comparison, we included additional plasmid-cured strains in the analysis. In the Dshi-6 population, the conjugative 102-kb RepABC-9 plasmid was present in only about 50% of the cells, irrespective of its experimentally validated stability. In the presence of the sixth plasmid, copy number changes of other ECRs, in particular, a decrease of the 86-kb plasmid, were observed. The most conspicuous finding was the strong influence of plasmids on chromosomal gene expression, especially the repression of the CtrA regulon and the activation of the denitrification gene cluster. Expression is inversely controlled by either the presence of the 102-kb plasmid or the absence of the 86-kb plasmid. We identified regulatory genes on both plasmids, i.e., a sigma 70 factor and a quorum sensing synthase, that might be responsible for these major changes. The tremendous effects that were probably even underestimated challenge the current understanding of the relevance of volatile plasmids not only for the original host but also for new recipients after conjugation. IMPORTANCE Plasmids are small DNA molecules that replicate independently of the bacterial chromosome. The common view of the role of plasmids is dominated by the accumulation of resistance genes, which is responsible for the antibiotic crisis in health care and livestock breeding. Beyond rapid adaptations to a changing environment, no general relevance for the host cell's regulome was attributed to these volatile ECRs. The current study shows for the model organism D. shibae that its chromosomal gene expression is strongly influenced by two plasmids. We provide evidence that the gain or loss of plasmids not only results in minor alterations of the genetic repertoire but also can have tremendous effects on bacterial physiology. The central role of some plasmids in the regulatory network of the host could also explain their persistence despite fitness costs, which has been described as the "plasmid paradox."

Beyond the ABCs-Discovery of Three New Plasmid Types in Rhodobacterales (RepQ, RepY, RepW).

Copiotrophic marine bacteria of the Roseobacter group (Rhodobacterales, Alphaproteobacteria) are characterized by a multipartite genome organization. We sequenced the genomes of Sulfitobacter indolifex DSM 14862T and four related plasmid-rich isolates in order to investigate the composition, distribution, and evolution of their extrachromosomal replicons (ECRs). A combination of long-read PacBio and short-read Illumina sequencing was required to establish complete closed genomes that comprised up to twelve ECRs. The ECRs were differentiated in stably evolving chromids and genuine plasmids. Among the chromids, a diagnostic RepABC-8 replicon was detected in four Sulfitobacter species that likely reflects an evolutionary innovation that originated in their common ancestor. Classification of the ECRs showed that the most abundant plasmid system is RepABC, followed by RepA, DnaA-like, and RepB. However, the strains also contained three novel plasmid types that were designated RepQ, RepY, and RepW. We confirmed the functionality of their replicases, investigated the genetic inventory of the mostly cryptic plasmids, and retraced their evolutionary origin. Remarkably, the RepY plasmid of S. pontiacus DSM 110277 is the first high copy-number plasmid discovered in Rhodobacterales.

Fatal affairs - conjugational transfer of a dinoflagellate-killing plasmid between marine Rhodobacterales.

The roseobacter group of marine bacteria is characterized by a mosaic distribution of ecologically important phenotypes. These are often encoded on mobile extrachromosomal replicons. So far, conjugation had only been experimentally proven between the two model organisms Phaeobacter inhibens and Dinoroseobacter shibae. Here, we show that two large natural RepABC-type plasmids from D. shibae can be transferred into representatives of all known major Rhodobacterales lineages. Complete genome sequencing of the newly established Phaeobacter inhibens transconjugants confirmed their genomic integrity. The conjugated plasmids were stably maintained as single copy number replicons in the genuine as well as the new host. Co-cultivation of Phaeobacter inhibens and the transconjugants with the dinoflagellate Prorocentrum minimum demonstrated that Phaeobacter inhibens is a probiotic strain that improves the yield and stability of the dinoflagellate culture. The transconjugant carrying the 191 kb plasmid, but not the 126 kb sister plasmid, killed the dinoflagellate in co-culture.

Diversity and substrate-specificity of green algae and other micro-eukaryotes colonizing amphibian clutches in Germany, revealed by DNA metabarcoding.

Amphibian clutches are colonized by diverse but poorly studied communities of micro-organisms. One of the most noted ones is the unicellular green alga, Oophila amblystomatis, but the occurrence and role of other micro-organisms in the capsular chamber surrounding amphibian clutches have remained largely unstudied. Here, we undertook a multi-marker DNA metabarcoding study to characterize the community of algae and other micro-eukaryotes associated with agile frog (Rana dalmatina) clutches. Samplings were performed at three small ponds in Germany, from four substrates: water, sediment, tree leaves from the bottom of the pond, and R. dalmatina clutches. Sampling substrate strongly determined the community compositions of algae and other micro-eukaryotes. Therefore, as expected, the frog clutch-associated communities formed clearly distinct clusters. Clutch-associated communities in our study were structured by a plethora of not only green algae, but also diatoms and other ochrophytes. The most abundant operational taxonomic units (OTUs) in clutch samples were taxa from Chlamydomonas, Oophila, but also from Nitzschia and other ochrophytes. Sequences of Oophila "Clade B" were found exclusively in clutches. Based on additional phylogenetic analyses of 18S rDNA and of a matrix of 18 nuclear genes derived from transcriptomes, we confirmed in our samples the existence of two distinct clades of green algae assigned to Oophila in past studies. We hypothesize that "Clade B" algae correspond to the true Oophila, whereas "Clade A" algae are a series of Chlorococcum species that, along with other green algae, ochrophytes and protists, colonize amphibian clutches opportunistically and are often cultured from clutch samples due to their robust growth performance. The clutch-associated communities were subject to filtering by sampling location, suggesting that the taxa colonizing amphibian clutches can drastically differ depending on environmental conditions.

Filling the Gaps in the Cyanobacterial Tree of Life-Metagenome Analysis of Stigonema ocellatum DSM 106950, Chlorogloea purpurea SAG 13.99 and Gomphosphaeria aponina DSM 107014.

Cyanobacteria represent one of the most important and diverse lineages of prokaryotes with an unparalleled morphological diversity ranging from unicellular cocci and characteristic colony-formers to multicellular filamentous strains with different cell types. Sequencing of more than 1200 available reference genomes was mainly driven by their ecological relevance (Prochlorococcus, Synechococcus), toxicity (Microcystis) and the availability of axenic strains. In the current study three slowly growing non-axenic cyanobacteria with a distant phylogenetic positioning were selected for metagenome sequencing in order to (i) investigate their genomes and to (ii) uncover the diversity of associated heterotrophs. High-throughput Illumina sequencing, metagenomic assembly and binning allowed us to establish nearly complete high-quality draft genomes of all three cyanobacteria and to determine their phylogenetic position. The cyanosphere of the limnic isolates comprises up to 40 heterotrophic bacteria that likely coexisted for several decades, and it is dominated by Alphaproteobacteria and Bacteriodetes. The diagnostic marker protein RpoB ensured in combination with our novel taxonomic assessment via BLASTN-dependent text-mining a reliable classification of the metagenome assembled genomes (MAGs). The detection of one new family and more than a dozen genera of uncultivated heterotrophic bacteria illustrates that non-axenic cyanobacteria are treasure troves of hidden microbial diversity.

A novel class of sulfur-containing aminolipids widespread in marine roseobacters.

Marine roseobacter group bacteria are numerically abundant and ecologically important players in ocean ecosystems. These bacteria are capable of modifying their membrane lipid composition in response to environmental change. Remarkably, a variety of lipids are produced in these bacteria, including phosphorus-containing glycerophospholipids and several amino acid-containing aminolipids such as ornithine lipids and glutamine lipids. Here, we present the identification and characterization of a novel sulfur-containing aminolipid (SAL) in roseobacters. Using high resolution accurate mass spectrometry, a SAL was found in the lipid extract of Ruegeria pomeroyi DSS-3 and Phaeobacter inhibens DSM 17395. Using comparative genomics, transposon mutagenesis and targeted gene knockout, we identified a gene encoding a putative lyso-lipid acyltransferase, designated salA, which is essential for the biosynthesis of this SAL. Multiple sequence analysis and structural modeling suggest that SalA is a novel member of the lysophosphatidic acid acyltransferase (LPAAT) family, the prototype of which is the PlsC acyltransferase responsible for the biosynthesis of the phospholipid phosphatidic acid. SAL appears to play a key role in biofilm formation in roseobacters. salA is widely distributed in Tara Oceans metagenomes and actively expressed in Tara Oceans metatranscriptomes. Our results raise the importance of sulfur-containing membrane aminolipids in marine bacteria.

Identification of volatiles from six marine Celeribacter strains.

The volatiles emitted from six marine Rhodobacteraceae species of the genus Celeribacter were investigated by GC-MS. Besides several known compounds including dimethyl trisulfide and S-methyl methanethiosulfonate, the sulfur-containing compounds ethyl (E)-3-(methylsulfanyl)acrylate and 2-(methyldisulfanyl)benzothiazole were identified and their structures were verified by synthesis. Feeding experiments with [methyl-2H3]methionine, [methyl-13C]methionine and [34S]-3-(dimethylsulfonio)propanoate (DMSP) resulted in the high incorporation into dimethyl trisulfide and S-methyl methanethiosulfonate, and revealed the origin of the methylsulfanyl group of 2-(methyldisulfanyl)benzothiazole from methionine or DMSP, while the biosynthetic origin of the benzothiazol-2-ylsulfanyl portion could not be traced. The heterocyclic moiety of this compound is likely of anthropogenic origin, because 2-mercaptobenzothiazole is used in the sulfur vulcanization of rubber. Also in none of the feeding experiments incorporation into ethyl (E)-3-(methylsulfanyl)acrylate could be observed, questioning its bacterial origin. Our results demonstrate that the Celeribacter strains are capable of methionine and DMSP degradation to widespread sulfur volatiles, but the analysis of trace compounds in natural samples must be taken with care.

Dinoroseobacter shibae Outer Membrane Vesicles Are Enriched for the Chromosome Dimer Resolution Site dif.

Outer membrane vesicles (OMVs) are universally produced by prokaryotes and play important roles in symbiotic and pathogenic interactions. They often contain DNA, but a mechanism for its incorporation is lacking. Here, we show that Dinoroseobacter shibae, a dinoflagellate symbiont, constitutively secretes OMVs containing DNA. Time-lapse microscopy captured instances of multiple OMV production at the septum during cell division. DNA from the vesicle lumen was up to 22-fold enriched for the region around the terminus of replication (ter). The peak of coverage was located at dif, a conserved 28-bp palindromic sequence required for binding of the site-specific tyrosine recombinases XerC/XerD. These enzymes are activated at the last stage of cell division immediately prior to septum formation when they are bound by the divisome protein FtsK. We suggest that overreplicated regions around the terminus have been repaired by the FtsK-dif-XerC/XerD molecular machinery. The vesicle proteome was clearly dominated by outer membrane and periplasmic proteins. Some of the most abundant vesicle membrane proteins were predicted to be required for direct interaction with peptidoglycan during cell division (LysM, Tol-Pal, Spol, lytic murein transglycosylase). OMVs were 15-fold enriched for the saturated fatty acid 16:00. We hypothesize that constitutive OMV secretion in D. shibae is coupled to cell division. The footprint of the FtsK-dif-XerC/XerD molecular machinery suggests a novel potentially highly conserved route for incorporation of DNA into OMVs. Clearing the division site from small DNA fragments might be an important function of vesicles produced during exponential growth under optimal conditions.IMPORTANCE Gram-negative bacteria continually form vesicles from their outer membrane (outer membrane vesicles [OMVs]) during normal growth. OMVs frequently contain DNA, and it is unclear how DNA can be shuffled from the cytoplasm to the OMVs. We studied OMV cargo in Dinoroseobacter shibae, a symbiont of dinoflagellates, using microscopy and a multi-omics approach. We found that vesicles formed during undisturbed exponential growth contain DNA which is enriched for genes around the replication terminus, specifically, the binding site for an enzyme complex that is activated at the last stage of cell division. We suggest that the enriched genes are the result of overreplication which is repaired by their excision and excretion via membrane vesicles to clear the divisome from waste DNA.

RepC_soli: a novel promiscuous plasmid type of Rhodobacteraceae mediates horizontal transfer of antibiotic resistances in the ocean.

Alphaproteobacteria are typically characterized by a multipartite genome organization with a chromosome, stable chromids and accessory plasmids. Extrachromosomal elements determine the lifestyle of roseobacters and their horizontal transfer was previously correlated with rapid adaptations to novel ecological niches. We characterized the distribution and biology of a novel Rhodobacteraceae-specific plasmid type that was designated RepC_soli according to its diagnostic solitary replicase. This low copy number replicon exhibits an exceptional stability, which is likely ensured by non-canonical separate parA and parB partitioning genes. RepC_soli plasmids occur frequently in the surface-associated marine genus Phaeobacter and comparative genome analyses revealed the emergence of four compatibility groups. The universal presence of conserved type IV secretion systems in RepC_soli plasmids is indicative of their recurrent mobilization, a prediction that was experimentally validated by conjugation of the 57 kb Phaeobacter inhibens P72 plasmid (pP72_e) over genus borders. RepC_soli plasmids harbour a diverse collection of beneficial genes including transporters for heavy metal detoxification, prokaryotic defence systems and a conspicuous abundance of antibiotic resistance genes. The pP72_e-encoded efflux pump FloR conferred an about 50-fold increase of resistance against chloramphenicol. Its specific occurrence in Phaeobacter likely reflects a genetic footprint of (former) antimicrobial use in marine aquaculture.

Phylogenomics reveals the basis of adaptation of Pseudorhizobium species to extreme environments and supports a taxonomic revision of the genus.

The family Rhizobiaceae includes many genera of soil bacteria, often isolated for their association with plants. Herein, we investigate the genomic diversity of a group of Rhizobium species and unclassified strains isolated from atypical environments, including seawater, rock matrix or polluted soil. Based on whole-genome similarity and core genome phylogeny, we show that this group corresponds to the genus Pseudorhizobium. We thus reclassify Rhizobium halotolerans, R. marinum, R. flavum and R. endolithicum as P. halotolerans sp. nov., P. marinum comb. nov., P. flavum comb. nov. and P. endolithicum comb. nov., respectively, and show that P. pelagicum is a synonym of P. marinum. We also delineate a new chemolithoautotroph species, P. banfieldiae sp. nov., whose type strain is NT-26T (=DSM 106348T=CFBP 8663T). This genome-based classification was supported by a chemotaxonomic comparison, with increasing taxonomic resolution provided by fatty acid, protein and metabolic profiles. In addition, we used a phylogenetic approach to infer scenarios of duplication, horizontal transfer and loss for all genes in the Pseudorhizobium pangenome. We thus identify the key functions associated with the diversification of each species and higher clades, shedding light on the mechanisms of adaptation to their respective ecological niches. Respiratory proteins acquired at the origin of Pseudorhizobium were combined with clade-specific genes to enable different strategies for detoxification and nutrition in harsh, nutrient-poor environments.

Global Response of Phaeobacter inhibens DSM 17395 to Deletion of Its 262-kb Chromid Encoding Antibiotic Synthesis.

The marine alphaproteobacterium Phaeobacter inhibens DSM 17395, a member of the Roseobacter group, was recently shown to markedly enhance growth upon deletion of its 262-kb chromid encoding biosynthesis of tropodithietic acid (TDA). To scrutinize the metabolic/regulatory adaptations that underlie enhanced growth of the Δ262 mutant, its transcriptome and proteome compared to the wild type were investigated in process-controlled bioreactors with Casamino Acids as growth substrate. Genome resequencing revealed only few additional genetic changes (a heterogenic insertion, prophage activation, and several point mutations) between wild type and Δ262 mutant, albeit with no conceivable effect on the studied growth physiology. The abundances of the vast majority of transcripts and proteins involved in the catabolic network for complete substrate oxidation to CO2 were found to be unchanged, suggesting that the enhanced amino acid utilization of the Δ262 mutant did not require elevated synthesis of most enzymes of the catabolic network. Similarly, constituents of genetic information processing and cellular processes remained mostly unchanged. In contrast, 426 genes displayed differential expression, of which 410 were localized on the 3.2-Mb chromosome, 5 on the 65-kb chromid, and 11 on the 78-kb chromid. Notably, the branched-chain amino transferase IlvE acting on rapidly utilized Val, Ile, and Leu was upregulated. Moreover, the transportome was reconfigured, as evidenced from increased abundances of transcripts and proteins of several uptake systems for amino acids and inorganic nutrients (e.g., phosphate). Some components of the respiratory chain were also upregulated, which correlates with the higher respiration rates of the Δ262 mutant. Furthermore, chromosomally encoded transcripts and proteins that are peripherally related to TDA biosynthesis (e.g., the serine acyl transferase CysE) were strongly downregulated in the Δ262 mutant. Taken together, these observations reflect adaptations to enhanced growth as well as the functional interconnectivity of the replicons of P. inhibens DSM 17395.

Author Correction: The planctomycete Stieleria maiorica Mal15T employs stieleriacines to alter the species composition in marine biofilms.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Author Correction: The planctomycete Stieleria maiorica Mal15T employs stieleriacines to alter the species composition in marine biofilms.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

The planctomycete Stieleria maiorica Mal15T employs stieleriacines to alter the species composition in marine biofilms.

Bacterial strains of the phylum Planctomycetes occur ubiquitously, but are often found on surfaces of aquatic phototrophs, e.g. alga. Despite slower growth, planctomycetes are not outcompeted by faster-growing bacteria in biofilms on such surfaces; however, strategies allowing them to compensate for slower growth have not yet been investigated. Here, we identified stieleriacines, a class of N-acylated tyrosines produced by the novel planctomycete Stieleria maiorica Mal15T, and analysed their effects on growth of the producing strain and bacterial species likely co-occurring with strain Mal15T. Stieleriacines reduced the lag phase of Mal15T and either stimulated or inhibited biofilm formation of two bacterial competitors, indicating that Mal15T employs stieleriacines to specifically alter microbial biofilm composition. The genetic organisation of the putative stieleriacine biosynthetic cluster in strain Mal15T points towards a functional link of stieleriacine biosynthesis to exopolysaccharide-associated protein sorting and biofilm formation.

A marine plasmid hitchhiking vast phylogenetic and geographic distances.

Horizontal gene transfer (HGT) plays an important role in bacterial evolution and serves as a driving force for bacterial diversity and versatility. HGT events often involve mobile genetic elements like plasmids, which can promote their own dissemination by associating with adaptive traits in the gene pool of the so-called mobilome. Novel traits that evolve through HGT can therefore lead to the exploitation of new ecological niches, prompting an adaptive radiation of bacterial species. In this study, we present phylogenetic, biogeographic, and functional analyses of a previously unrecognized RepL-type plasmid found in diverse members of the marine Roseobacter group across the globe. Noteworthy, 100% identical plasmids were detected in phylogenetically and geographically distant bacteria, revealing a so-far overlooked, but environmentally highly relevant vector for HGT. The genomic and functional characterization of this plasmid showed a completely conserved backbone dedicated to replication, stability, and mobilization as well as an interchangeable gene cassette with highly diverse, but recurring motifs. The majority of the latter appear to be involved in mechanisms coping with toxins and/or pollutants in the marine environment. Furthermore, we provide experimental evidence that the plasmid has the potential to be transmitted across bacterial orders, thereby increasing our understanding of evolution and microbial niche adaptation in the environment.

Cobaviruses - a new globally distributed phage group infecting Rhodobacteraceae in marine ecosystems.

Bacteriophages are widely considered to influence bacterial communities, however most phages are still unknown or not studied well enough to understand their ecological roles. We have isolated two phages infecting Lentibacter sp. SH36, affiliated with the marine Roseobacter group, and retrieved similar phage genomes from publicly available metagenomics databases. Phylogenetic analysis placed the new phages within the Cobavirus group, in the here newly proposed genus Siovirus and subfamily Riovirinae of the Podoviridae. Gene composition and presence of direct terminal repeats in cultivated cobaviruses point toward a genome replication and packaging strategy similar to the T7 phage. Investigation of the genomes suggests that viral lysis of the cell proceeds via the canonical holin-endolysin pathway. Cobaviral hosts include members of the genera Lentibacter, Sulfitobacter and Celeribacter of the Roseobacter group within the family Rhodobacteraceae (Alphaproteobacteria). Screening more than 5,000 marine metagenomes, we found cobaviruses worldwide from temperate to tropical waters, in the euphotic zone, mainly in bays and estuaries, but also in the open ocean. The presence of cobaviruses in protist metagenomes as well as the phylogenetic neighborhood of cobaviruses in glutaredoxin and ribonucleotide reductase trees suggest that cobaviruses could infect bacteria associated with phototrophic or grazing protists. With this study, we expand the understanding of the phylogeny, classification, genomic organization, biogeography and ecology of this phage group infecting marine Rhodobacteraceae.

Day and Night: Metabolic Profiles and Evolutionary Relationships of Six Axenic Non-Marine Cyanobacteria.

Cyanobacteria are dominant primary producers of various ecosystems and they colonize marine as well as freshwater and terrestrial habitats. On the basis of their oxygenic photosynthesis they are known to synthesize a high number of secondary metabolites, which makes them promising for biotechnological applications. State-of-the-art sequencing and analytical techniques and the availability of several axenic strains offer new opportunities for the understanding of the hidden metabolic potential of cyanobacteria beyond those of single model organisms. Here, we report comprehensive genomic and metabolic analyses of five non-marine cyanobacteria, that is, Nostoc sp. DSM 107007, Anabaena variabilis DSM 107003, Calothrix desertica DSM 106972, Chroococcidiopsis cubana DSM 107010, Chlorogloeopsis sp. PCC 6912, and the reference strain Synechocystis sp. PCC 6803. Five strains that are prevalently belonging to the order Nostocales represent the phylogenetic depth of clade B1, a morphologically highly diverse sister lineage of clade B2 that includes strain PCC 6803. Genome sequencing, light and scanning electron microscopy revealed the characteristics and axenicity of the analyzed strains. Phylogenetic comparisons showed the limits of the 16S rRNA gene for the classification of cyanobacteria, but documented the applicability of a multilocus sequence alignment analysis based on 43 conserved protein markers. The analysis of metabolites of the core carbon metabolism showed parts of highly conserved metabolic pathways as well as lineage specific pathways such as the glyoxylate shunt, which was acquired by cyanobacteria at least twice via horizontal gene transfer. Major metabolic changes were observed when we compared alterations between day and night samples. Furthermore, our results showed metabolic potential of cyanobacteria beyond Synechocystis sp. PCC 6803 as model organism and may encourage the cyanobacterial community to broaden their research to related organisms with higher metabolic activity in the desired pathways.