Roseobacter weihaiensis sp. nov., a cellulose-degrading bacterium isolated from intestinal content of Nipponacmea schrenckii collected from golden beach in Weihai, China.

A Gram-staining-negative, dark pink, rod-shaped, amastigote and cellulose-degrading strain, designated H9T, was isolated from intestinal contents of Nipponacmea schrenckii. The isolate was able to grow at 4-42 °C (optimum, 25 °C), at pH 6.5-9.0 (optimum, pH 7.0), and with 0.0-11.0% (w/v) NaCl (optimum, 3.0-5.0%). Phylogenetic analysis of the 16S rRNA gene sequence suggested that isolate H9T belongs to the genus Roseobacter, neighboring Roseobacter insulae YSTF-M11T, Roseobacter cerasinus AI77T and Roseobacter ponti MM-7 T, and the pairwise sequence showed the highest similarity of 99.1% to Roseobacter insulae YSTF-M11T. The major fatty acid was summed feature 8 (C18:1ω7c and/or C18:1ω6c; 81.08%). The predominant respiratory quinone was Q-10. The polar lipids consisted of phosphatidylcholine, phosphatidylglycerol, an unknown lipid, and a small amount of an unknown phospholipid. The genome of strain H9T was 5,351,685 bp in length, and the DNA G + C content was 59.8%. The average amino acid identity (AAI), average nucleotide identity (ANI), and digital DNA hybridization (dDDH) values between strain H9T and closely related strains were 63.4-76.8%, 74.7-78.8%, and 13.4-19.7%, respectively. On the basis of the phenotypic, chemical taxonomic, and phylogenetic data, it is suggested that strain H9T should represent a novel species in the genus Roseobacter, for which the name Roseobacter weihaiensis sp. nov. is proposed. The type strain is H9T (= KCTC 82507 T = MCCC 1K04354T).

Roseobacter fucihabitans sp. nov., isolated from the brown alga Fucus spiralis.

A Gram-negative, aerobic, pink-pigmented, and bacteriochlorophyll a-containing bacterial strain, designated B14T, was isolated from the macroalga Fucus spiralis sampled from the southern North Sea, Germany. Based on 16S rRNA gene sequences, species of the genera Roseobacter and Sulfitobacter were most closely related to strain B14T with sequence identities ranging from 98.15 % (Roseobacter denitrificans Och 114T) to 99.11 % (Roseobacter litoralis Och 149T), whereas Sulfitobacter mediterraneus CH-B427T exhibited 98.52 % sequence identity. Digital DNA-DNA hybridization and average nucleotide identity values between the genome of the novel strain and that of closely related Roseobacter and Sulfitobacter type strains were <20 % and <77 %, respectively. The novel strain contained ubiquinone-10 as the only respiratory quinone and C18 : 1 ω7c, C16 : 0, C18 : 0, C12 : 1 ω7c, C18 : 2 ω7,13c, and C10 : 0 3-OH as the major cellular fatty acids. The predominant polar lipids of strain B14T were phosphatidylcholine, phosphatidylethanolamine, and phosphatidylglycerol. The genome of strain B14T comprises a chromosome with a size of 4.5 Mbp, one chromid, and four plasmids. The genome contains the complete gene cluster for aerobic anoxygenic photosynthesis required for a photoheterotrophic lifestyle. The results of this study indicate that strain B14T (=DSM 116946T=LMG 33352T) represents a novel species of the genus Roseobacter for which the name Roseobacter fucihabitans sp. nov. is proposed.

Novel phage infecting the Roseobacter CHUG lineage reveals a diverse and globally distributed phage family.

Bacteriophages play an essential role in shaping the diversity and metabolism of bacterial communities. Marine Roseobacter group is an abundant heterotrophic bacterial group that is involved in many major element cycles, especially carbon and sulfur. Members of the Roseobacter CHUG (Clade Hidden and Underappreciated Globally) lineage are globally distributed and are activated in pelagic marine environments. In this study, we isolated and characterized a phage, CRP-810, that infects the CHUG strain FZCC0198. The genome of CRP-810 was dissimilar to those of other known phages. Additionally, 251 uncultured viral genomes (UViGs) closely related to CRP-810 were obtained from the uncultivated marine viral contig databases. Comparative genomic and phylogenetic analyses revealed that CRP-810 and these related UViGs exhibited conserved genome synteny, representing a new phage family with at least eight subgroups. Most of the CRP-810-type phages contain an integrase gene, and CRP-810 can be integrated into the host genome. Further analysis revealed that three CRP-810-type members were prophages found in the genomes of marine SAR11, Poseidonocella, and Sphingomonadaceae. Finally, viromic read-mapping analysis showed that CRP-810-type phages were globally distributed and displayed distinct biogeographic patterns related to temperature and latitude. Many members with a lower G + C content were mainly distributed in the trade station, whereas members with a higher G + C content were mainly distributed in polar and westerlies station, indicating that the niche differentiation of phages was subject to host adaptation. Collectively, these findings identify a novel phage family and expand our understanding of phylogenetic diversity, evolution, and biogeography of marine phages. IMPORTANCE: The Roseobacter CHUG lineage, affiliated with the Pelagic Roseobacter Cluster (PRC), is widely distributed in the global oceans and is active in oligotrophic seawater. However, knowledge of the bacteriophages that infect CHUG members is limited. In this study, a CHUG phage, CRP-810, that infects the CHUG strain FZCC0198, was isolated and shown to have a novel genomic architecture. In addition, 251 uncultured viral genomes closely related to CRP-810 were recovered and included in the analyses. Phylogenomic analyses revealed that the CRP-810-type phages represent a new phage family containing at least eight genus-level subgroups. Members of this family were predicted to infect various marine bacteria. We also demonstrated that the CRP-810-type phages are widely distributed in global oceans and display distinct biogeographic patterns related to latitude. Collectively, this study provides important insights into the genomic organization, diversity, and ecology of a novel phage family that infect ecologically important bacteria in the global ocean.

Genome sequences of the first Autographiviridae phages infecting marine Roseobacter.

The ubiquitous and abundant marine phages play critical roles in shaping the composition and function of bacterial communities, impacting biogeochemical cycling in marine ecosystems. Autographiviridae is among the most abundant and ubiquitous phage families in the ocean. However, studies on the diversity and ecology of Autographiviridae phages in marine environments are restricted to isolates that infect SAR11 bacteria and cyanobacteria. In this study, ten new roseophages that infect marine Roseobacter strains were isolated from coastal waters. These new roseophages have a genome size ranging from 38 917 to 42 634 bp and G+C content of 44.6-50 %. Comparative genomics showed that they are similar to known Autographiviridae phages regarding gene content and architecture, thus representing the first Autographiviridae roseophages. Phylogenomic analysis based on concatenated conserved genes showed that the ten roseophages form three distinct subgroups within the Autographiviridae, and sequence analysis revealed that they belong to eight new genera. Finally, viromic read-mapping showed that these new Autographiviridae phages are widely distributed in global oceans, mostly inhabiting polar and estuarine locations. This study has expanded the current understanding of the genomic diversity, evolution and ecology of Autographiviridae phages and roseophages. We suggest that Autographiviridae phages play important roles in the mortality and community structure of roseobacters, and have broad ecological applications.

Structure Revision of a Widespread Marine Sulfonolipid Class Based on Isolation and Total Synthesis.

The cosmopolitan marine Roseobacter clade is of global biogeochemical importance. Members of this clade produce sulfur-containing amino lipids (SALs) involved in biofilm formation and marine surface colonization processes. Despite their physiological relevance and abundance, SALs have only been explored through genomic mining approaches and lipidomic studies based on mass spectrometry, which left the relative and absolute structures of SALs unresolved, hindering progress in biochemical and functional investigations. Herein, we report the structural revision of a new group of SALs, which we named cysteinolides, using a combination of analytical techniques, isolation and degradation experiments and total synthetic efforts. Contrary to the previously proposed homotaurine-based structures, cysteinolides are composed of an N,O-acylated cysteinolic acid-containing head group carrying various different (α-hydroxy)carboxylic acids. We also performed the first validated targeted-network based analysis, which allowed us to map the distribution and structural diversity of cysteinolides across bacterial lineages. Beyond offering structural insight, our research provides SAL standards and validated analytical data. This information holds significance for forthcoming investigations into bacterial sulfonolipid metabolism and biogeochemical nutrient cycling within marine environments.

Metagenome-assembled genomes reveal greatly expanded taxonomic and functional diversification of the abundant marine Roseobacter RCA cluster.

BACKGROUND: The RCA (Roseobacter clade affiliated) cluster belongs to the family Roseobacteracea and represents a major Roseobacter lineage in temperate to polar oceans. Despite its prevalence and abundance, only a few genomes and one described species, Planktomarina temperata, exist. To gain more insights into our limited understanding of this cluster and its taxonomic and functional diversity and biogeography, we screened metagenomic datasets from the global oceans and reconstructed metagenome-assembled genomes (MAG) affiliated to this cluster. RESULTS: The total of 82 MAGs, plus five genomes of isolates, reveal an unexpected diversity and novel insights into the genomic features, the functional diversity, and greatly refined biogeographic patterns of the RCA cluster. This cluster is subdivided into three genera: Planktomarina, Pseudoplanktomarina, and the most deeply branching Candidatus Paraplanktomarina. Six of the eight Planktomarina species have larger genome sizes (2.44-3.12 Mbp) and higher G + C contents (46.36-53.70%) than the four Pseudoplanktomarina species (2.26-2.72 Mbp, 42.22-43.72 G + C%). Cand. Paraplanktomarina is represented only by one species with a genome size of 2.40 Mbp and a G + C content of 45.85%. Three novel species of the genera Planktomarina and Pseudoplanktomarina are validly described according to the SeqCode nomenclature for prokaryotic genomes. Aerobic anoxygenic photosynthesis (AAP) is encoded in three Planktomarina species. Unexpectedly, proteorhodopsin (PR) is encoded in the other Planktomarina and all Pseudoplanktomarina species, suggesting that this light-driven proton pump is the most important mode of acquiring complementary energy of the RCA cluster. The Pseudoplanktomarina species exhibit differences in functional traits compared to Planktomarina species and adaptations to more resource-limited conditions. An assessment of the global biogeography of the different species greatly expands the range of occurrence and shows that the different species exhibit distinct biogeographic patterns. They partially reflect the genomic features of the species. CONCLUSIONS: Our detailed MAG-based analyses shed new light on the diversification, environmental adaptation, and global biogeography of a major lineage of pelagic bacteria. The taxonomic delineation and validation by the SeqCode nomenclature of prominent genera and species of the RCA cluster may be a promising way for a refined taxonomic identification of major prokaryotic lineages and sublineages in marine and other prokaryotic communities assessed by metagenomics approaches. Video Abstract.

DMSOP-cleaving enzymes are diverse and widely distributed in marine microorganisms.

Dimethylsulfoxonium propionate (DMSOP) is a recently identified and abundant marine organosulfur compound with roles in oxidative stress protection, global carbon and sulfur cycling and, as shown here, potentially in osmotolerance. Microbial DMSOP cleavage yields dimethyl sulfoxide, a ubiquitous marine metabolite, and acrylate, but the enzymes responsible, and their environmental importance, were unknown. Here we report DMSOP cleavage mechanisms in diverse heterotrophic bacteria, fungi and phototrophic algae not previously known to have this activity, and highlight the unappreciated importance of this process in marine sediment environments. These diverse organisms, including Roseobacter, SAR11 bacteria and Emiliania huxleyi, utilized their dimethylsulfoniopropionate lyase 'Ddd' or 'Alma' enzymes to cleave DMSOP via similar catalytic mechanisms to those for dimethylsulfoniopropionate. Given the annual teragram predictions for DMSOP production and its prevalence in marine sediments, our results highlight that DMSOP cleavage is likely a globally significant process influencing carbon and sulfur fluxes and ecological interactions.

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.

Anaerobic thiosulfate oxidation by the Roseobacter group is prevalent in marine biofilms.

Thiosulfate oxidation by microbes has a major impact on global sulfur cycling. Here, we provide evidence that bacteria within various Roseobacter lineages are important for thiosulfate oxidation in marine biofilms. We isolate and sequence the genomes of 54 biofilm-associated Roseobacter strains, finding conserved sox gene clusters for thiosulfate oxidation and plasmids, pointing to a niche-specific lifestyle. Analysis of global ocean metagenomic data suggests that Roseobacter strains are abundant in biofilms and mats on various substrates, including stones, artificial surfaces, plant roots, and hydrothermal vent chimneys. Metatranscriptomic analysis indicates that the majority of active sox genes in biofilms belong to Roseobacter strains. Furthermore, we show that Roseobacter strains can grow and oxidize thiosulfate to sulfate under both aerobic and anaerobic conditions. Transcriptomic and membrane proteomic analyses of biofilms formed by a representative strain indicate that thiosulfate induces sox gene expression and alterations in cell membrane protein composition, and promotes biofilm formation and anaerobic respiration. We propose that bacteria of the Roseobacter group are major thiosulfate-oxidizers in marine biofilms, where anaerobic thiosulfate metabolism is preferred.

Roseobacter insulae sp. nov. and Loktanella gaetbuli sp. nov., isolated from tidal flats in the Yellow Sea in Korea.

Two bacterial strains (designated as YSTF-M11T and TSTF-M6T) were isolated from tidal flat sediments of the Yellow Sea, Republic of Korea, and taxonomically characterized. A neighbour-joining phylogenetic tree based on 16S rRNA gene sequences showed that strain YSTF-M11T clusters with the type strains of Roseobacter species and strain TSTF-M6T clusters with the type strains of Loktanella salsilacus, Loktanella fryxellensis and Loktanella atrilutea. Strains YSTF-M11T and TSTF-M6T exhibited 16S rRNA gene sequence similarity values of 97.5-98.9 % and 94.1-97.2 % to the type strains of four Roseobacter species and to the type strains of four Loktanella species, respectively. An UBCG tree based on genomic sequences and a tree based on AAI showed that strains YSTF-M11T and TSTF-M6T form a cluster with the type strains of Roseobacter species and with the type strains of L. salsilacus, L. fryxellensis and L. atrilutea, respectively. The ANI and dDDH values between genomic sequences of strain YSTF-M11T and the type strains of four Roseobacter species and between those of strain TSTF-M6T and the type strains of the three Loktanella species were in ranges of 74.0-75.9 and 18.2-19.7 % and 74.7-75.5 and 18.8-19.3 %, respectively. The DNA G+C contents of strains YSTF-M11T and TSTF-M6T were 60.3 and 61.9 % based on their genomic sequences. Both strains contained Q-10 as the predominant ubiquinone and C18 : 1 ω7c as the major fatty acid. Strains YSTF-M11T and TSTF-M6T were separated from recognized Roseobacter species and L. salsilacus, L. fryxellensis and L. atrilutea, respectively, by their phenotypic properties together with the phylogenetic and genetic distinctiveness. Based on data presented in this study, strains YSTF-M11T (=KACC 21642T =NBRC 115155T) and TSTF-M6T (=KACC 21643T =NBRC 115154T) are considered to represent novel species of the genera Roseobacter and Loktanella, respectively, for which the names Roseobacter insulae sp. nov. and Loktanella gaetbuli sp. nov. are proposed.