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.

Occurrence of antibiotic-resistant bacteria in the sea surface microlayer of coastal waters in the southern North Sea.

The emergence of antibiotic-resistant bacteria in coastal waters is a global health problem posing potential risks to the health of humans who depend on coastal resources. Monitoring and increased efforts are needed to maintain the health of marine ecosystems. The sea surface microlayer (SML) is poorly studied for antibiotic resistance of the inhabiting bacteria. Therefore, we examined the antibiotic resistance patterns of 41 bacterial strains isolated from the SML in a harbor in the southern North Sea. The strains are affiliated with 17 genera typically found in the marine environment. Using the disc diffusion assay, we found extensive resistance, particularly to gentamycin, kanamycin, nalidixic acid, penicillin, sulfadimidine, and streptomycin. A broth microdilution assay showed high minimum inhibitory concentrations (MICs) for most isolates for amikacin, aztreonam, ceftazidime, cefepime, minocycline, and tobramycin. Genome analysis of three strains affiliated with the genera Pseudoseohaeicola, Nereida, and Vibrio, all showing a highly resistant phenotype, revealed the presence of 57, 42, and 90 genes, respectively, associated with antibiotic resistance. Over 50 % of these genes are multidrug efflux pumps. Our study shows that the SML in anthropogenic-influenced coastal regions harbors a wide diversity of antibiotic-resistant bacteria equipped with a broad range of multidrug efflux pumps.

Different utilization of alginate and other algal polysaccharides by marine Alteromonas macleodii ecotypes.

The marine bacterium Alteromonas macleodii is a copiotrophic r-strategist, but little is known about its potential to degrade polysaccharides. Here, we studied the degradation of alginate and other algal polysaccharides by A. macleodii strain 83-1 in comparison to other A. macleodii strains. Cell densities of strain 83-1 with alginate as sole carbon source were comparable to those with glucose, but the exponential phase was delayed. The genome of 83-1 was found to harbour an alginolytic system comprising five alginate lyases, whose expression was induced by alginate. The alginolytic system contains additional CAZymes, including two TonB-dependent receptors, and is part of a 24 kb genomic island unique to the A. macleodii 'surface clade' ecotype. In contrast, strains of the 'deep clade' ecotype contain only a single alginate lyase in a separate 7 kb island. This difference was reflected in an eightfold greater efficiency of surface clade strains to grow on alginate. Strain 83-1 furthermore hydrolysed laminarin, pullulan and xylan, and corresponding polysaccharide utilization loci were detected in the genome. Alteromonas macleodii alginate lyases were predominantly detected in Atlantic Ocean metagenomes. The demonstrated hydrolytic capacities are likely of ecological relevance and represent another level of adaptation among A. macleodii ecotypes.

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.

Usage of antibiotics in aquaculture and the impact on coastal waters.

For decades, coastal marine ecosystems have been threatened by a wide range of anthropogenic pollutants. Recently, there has been increasing concern about the accumulation and impacts of antibiotic compounds on marine ecosystems. However, information regarding the accumulation of antibiotics and the impacts they may have on microbial communities in coastal water bodies and on human health is sparse in literature. Antibiotics from aquacultures are constantly discharged into marine environments via rivers. Large rivers transport tons of antibiotics every year into coastal waters, e.g., 12 tons of sulfonamide by the river Mekong. Here, we discuss a potential influence of such imported antibiotics on bacterial communities in coastal waters. Potential accumulation of antibiotics in the uppermost surface layer of aquatic ecosystems, the so-called sea surface microlayer (SML), is of interest. Because of the ability of the SML to accumulate anthropogenic pollutants, it may serve as a pool for antibiotics and correspondingly also for resistant organisms. Also, due to its biofilm-like structure, the SML could serve as a hotspot for horizontal gene transfer, speeding up the spread of antibiotic resistant strains to encompassing marine environments. The emergence of antibiotic resistant bacteria is a global threat and scientists projected that it could pave the way for the next pandemic that could ravage the world in the next decades. For this reason, it is time to focus research on understanding and minimizing the impact of antibiotics on the sustainability of coastal waters and on the health of humans who depend on coastal resources for food and recreational purposes. Also, knowledge about antibiotics in the SML is necessary to understand the effects they are likely to have on bacterial abundance, diversity, and metabolic activities in coastal water bodies.

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.

Origin and evolution of a novel DnaA-like plasmid replication type in Rhodobacterales.

Large extrachromosomal elements are widespread among Alphaproteobacteria, but it is unclear how up to a dozen low-copy plasmids can stably coexist within the same cell. We systematically analyzed the distribution of different replicons in about 40 completely sequenced genomes of the Roseobacter clade (Rhodobacterales) and surprisingly identified a novel plasmid replicon type. The conserved replication module comprises the characteristic partitioning operon (parAB) and a hitherto unknown replicase. The latter shows a weak homology to the chromosomal replication initiator DnaA and was accordingly named "DnaA-like." Phylogenetic analyses of the adjacent parAB genes document a common ancestry with repA- and repB-type plasmids and moreover indicate the presence of two dnaA-like compatibility groups. This conclusion is supported by conserved palindrome sequences within the replication module that probably represent crucial centromeric anchors for plasmid partitioning. The functionality of dnaA-like replicons was proven by transformation experiments in Phaeobacter gallaeciensis BS107 (DSM 17395). This Roseobacter strain furthermore allows the phenotypical monitoring of plasmid incompatibility, based on a 262-kb dnaA-like replicon required for the brown pigmentation of the bacterium. Uptake of an incompatible construct induces its loss, hence resulting in white colonies. Accordingly, we could substantiate the in silico predictions about stable maintenance of dnaA-like plasmids and thereby functionally validate our approach of plasmid classification based on phylogenetic analyses.

Genetic analysis of the upper phenylacetate catabolic pathway in the production of tropodithietic acid by Phaeobacter gallaeciensis.

Production of the antibiotic tropodithietic acid (TDA) depends on the central phenylacetate catabolic pathway, specifically on the oxygenase PaaABCDE, which catalyzes epoxidation of phenylacetyl-coenzyme A (CoA). Our study was focused on genes of the upper part of this pathway leading to phenylacetyl-CoA as precursor for TDA. Phaeobacter gallaeciensis DSM 17395 encodes two genes with homology to phenylacetyl-CoA ligases (paaK1 and paaK2), which were shown to be essential for phenylacetate catabolism but not for TDA biosynthesis and phenylalanine degradation. Thus, in P. gallaeciensis another enzyme must produce phenylacetyl-CoA from phenylalanine. Using random transposon insertion mutagenesis of a paaK1-paaK2 double mutant we identified a gene (ior1) with similarity to iorA and iorB in archaea, encoding an indolepyruvate:ferredoxin oxidoreductase (IOR). The ior1 mutant was unable to grow on phenylalanine, and production of TDA was significantly reduced compared to the wild-type level (60%). Nuclear magnetic resonance (NMR) spectroscopic investigations using (13)C-labeled phenylalanine isotopomers demonstrated that phenylalanine is transformed into phenylacetyl-CoA by Ior1. Using quantitative real-time PCR, we could show that expression of ior1 depends on the adjacent regulator IorR. Growth on phenylalanine promotes production of TDA, induces expression of ior1 (27-fold) and paaK1 (61-fold), and regulates the production of TDA. Phylogenetic analysis showed that the aerobic type of IOR as found in many roseobacters is common within a number of different phylogenetic groups of aerobic bacteria such as Burkholderia, Cupriavidis, and Rhizobia, where it may also contribute to the degradation of phenylalanine.

Impact of Quorum Sensing and Tropodithietic Acid Production on the Exometabolome of Phaeobacter inhibens.

Microbial interactions shape ecosystem diversity and chemistry through production and exchange of organic compounds, but the impact of regulatory mechanisms on production and release of these exometabolites is largely unknown. We studied the extent and nature of impact of two signaling molecules, tropodithietic acid (TDA) and the quorum sensing molecule acyl homoserine lactone (AHL) on the exometabolome of the model bacterium Phaeobacter inhibens DSM 17395, a member of the ubiquitous marine Roseobacter group. Exometabolomes of the wild type, a TDA and a QS (AHL-regulator) negative mutant were analyzed via Fourier-transform ion cyclotron resonance mass spectrometry (FT-ICR-MS). Based on a total of 996 reproducibly detected molecular masses, exometabolomes of the TDA and QS negative mutant were ∼70% dissimilar to each other, and ∼90 and ∼60% dissimilar, respectively, to that of the wild type. Moreover, at any sampled growth phase, 40-60% of masses detected in any individual exometabolome were unique to that strain, while only 10-12% constituted a shared "core exometabolome." Putative annotation revealed exometabolites of ecological relevance such as vitamins, amino acids, auxins, siderophore components and signaling compounds with different occurrence patterns in the exometabolomes of the three strains. Thus, this study demonstrates that signaling molecules, such as AHL and TDA, extensively impact the composition of bacterial exometabolomes with potential consequences for species interactions in microbial communities.

Tropodithietic acid production in Phaeobacter gallaeciensis is regulated by N-acyl homoserine lactone-mediated quorum sensing.

The production of N-acyl homoserine lactones (AHLs) is widely distributed within the marine Roseobacter clade, and it was proposed that AHL-mediated quorum sensing (QS) is one of the most common cell-to-cell communication mechanisms in roseobacters. The traits regulated by AHL-mediated QS are yet not known for members of the Roseobacter clade, but production of the antibiotic tropodithietic acid (TDA) was supposed to be controlled by AHL-mediated QS in Phaeobacter spp. We describe here for the first time the functional role of luxR and luxI homologous genes of an organism of the Roseobacter clade, i.e., pgaR and pgaI in Phaeobacter gallaeciensis. Our results demonstrate that the AHL synthase gene pgaI is responsible for production of N-3-hydroxydecanoylhomoserine lactone (3OHC(10)-HSL). Insertion mutants of pgaI and pgaR are both deficient in TDA biosynthesis and the formation of a yellow-brown pigment when grown in liquid marine broth medium. This indicates that in P. gallaeciensis the production of both secondary metabolites is controlled by AHL-mediated QS. Quantitative real-time PCR showed that the transcription level of tdaA, which encodes an essential transcriptional regulator for TDA biosynthesis, decreased 28- and 51-fold in pgaI and pgaR genetic backgrounds, respectively. These results suggest that both the response regulator PgaR and the 3OHC(10)-HSL produced by PgaI induce expression of tdaA, which in turn positively regulates expression of the tda genes. Moreover, we confirmed that TDA can also act as autoinducer in P. gallaeciensis, as previously described for Silicibacter sp. strain TM1040, but only in the presence of the response regulator PgaR.

Genomic, metabolic and phenotypic variability shapes ecological differentiation and intraspecies interactions of Alteromonas macleodii.

Ecological differentiation between strains of bacterial species is shaped by genomic and metabolic variability. However, connecting genotypes to ecological niches remains a major challenge. Here, we linked bacterial geno- and phenotypes by contextualizing pangenomic, exometabolomic and physiological evidence in twelve strains of the marine bacterium Alteromonas macleodii, illuminating adaptive strategies of carbon metabolism, microbial interactions, cellular communication and iron acquisition. In A. macleodii strain MIT1002, secretion of amino acids and the unique capacity for phenol degradation may promote associations with Prochlorococcus cyanobacteria. Strain 83-1 and three novel Pacific isolates, featuring clonal genomes despite originating from distant locations, have profound abilities for algal polysaccharide utilization but without detrimental implications for Ecklonia macroalgae. Degradation of toluene and xylene, mediated via a plasmid syntenic to terrestrial Pseudomonas, was unique to strain EZ55. Benzoate degradation by strain EC673 related to a chromosomal gene cluster shared with the plasmid of A. mediterranea EC615, underlining that mobile genetic elements drive adaptations. Furthermore, we revealed strain-specific production of siderophores and homoserine lactones, with implications for nutrient acquisition and cellular communication. Phenotypic variability corresponded to different competitiveness in co-culture and geographic distribution, indicating linkages between intraspecific diversity, microbial interactions and biogeography. The finding of "ecological microdiversity" helps understanding the widespread occurrence of A. macleodii and contributes to the interpretation of bacterial niche specialization, population ecology and biogeochemical roles.

Genome sequence of Epibacterium ulvae strain DSM 24752T, an indigoidine-producing, macroalga-associated member of the marine Roseobacter group.

Strain U95T (= DSM 24752T = LMG 26464T) is the type strain of Epibacterium ulvae, which is the type species of the genus Epibacterium. This genus belongs to the marine Roseobacter group. E. ulvae Strain U95T was isolated from the macroalga Ulva australis, is Gram-negative, rod-shaped and motile. Here we describe the permanent draft genome sequence and annotation of E. ulvae U95T with a focus on secondary metabolite production and interaction with its host. The genome contains 4,092,893 bp, 3977 protein-coding genes and 60 RNA genes. The genome encodes a gene cluster for synthesis of the blue-pigmented secondary metabolite indigoidine and contains several genes for adhesion mechanisms, putative bacteriocin, siderophores, a type VI secretion system, and enzymes that confer oxidative stress resistance. Combined, these features may aid in the successful colonization and persistence of E. ulvae on host surfaces and in competition with the surrounding microbial consortium.

Phaeobacter gallaeciensis genomes from globally opposite locations reveal high similarity of adaptation to surface life.

Phaeobacter gallaeciensis, a member of the abundant marine Roseobacter clade, is known to be an effective colonizer of biotic and abiotic marine surfaces. Production of the antibiotic tropodithietic acid (TDA) makes P. gallaeciensis a strong antagonist of many bacteria, including fish and mollusc pathogens. In addition to TDA, several other secondary metabolites are produced, allowing the mutualistic bacterium to also act as an opportunistic pathogen. Here we provide the manually annotated genome sequences of the P. gallaeciensis strains DSM 17395 and 2.10, isolated at the Atlantic coast of north western Spain and near Sydney, Australia, respectively. Despite their isolation sites from the two different hemispheres, the genome comparison demonstrated a surprisingly high level of synteny (only 3% nucleotide dissimilarity and 88% and 93% shared genes). Minor differences in the genomes result from horizontal gene transfer and phage infection. Comparison of the P. gallaeciensis genomes with those of other roseobacters revealed unique genomic traits, including the production of iron-scavenging siderophores. Experiments supported the predicted capacity of both strains to grow on various algal osmolytes. Transposon mutagenesis was used to expand the current knowledge on the TDA biosynthesis pathway in strain DSM 17395. This first comparative genomic analysis of finished genomes of two closely related strains belonging to one species of the Roseobacter clade revealed features that provide competitive advantages and facilitate surface attachment and interaction with eukaryotic hosts.

The complete genome sequence of the algal symbiont Dinoroseobacter shibae: a hitchhiker's guide to life in the sea.

Dinoroseobacter shibae DFL12(T), a member of the globally important marine Roseobacter clade, comprises symbionts of cosmopolitan marine microalgae, including toxic dinoflagellates. Its annotated 4 417 868 bp genome sequence revealed a possible advantage of this symbiosis for the algal host. D. shibae DFL12(T) is able to synthesize the vitamins B(1) and B(12) for which its host is auxotrophic. Two pathways for the de novo synthesis of vitamin B(12) are present, one requiring oxygen and the other an oxygen-independent pathway. The de novo synthesis of vitamin B(12) was confirmed to be functional, and D. shibae DFL12(T) was shown to provide the growth-limiting vitamins B(1) and B(12) to its dinoflagellate host. The Roseobacter clade has been considered to comprise obligate aerobic bacteria. However, D. shibae DFL12(T) is able to grow anaerobically using the alternative electron acceptors nitrate and dimethylsulfoxide; it has the arginine deiminase survival fermentation pathway and a complex oxygen-dependent Fnr (fumarate and nitrate reduction) regulon. Many of these traits are shared with other members of the Roseobacter clade. D. shibae DFL12(T) has five plasmids, showing examples for vertical recruitment of chromosomal genes (thiC) and horizontal gene transfer (cox genes, gene cluster of 47 kb) possibly by conjugation (vir gene cluster). The long-range (80%) synteny between two sister plasmids provides insights into the emergence of novel plasmids. D. shibae DFL12(T) shows the most complex viral defense system of all Rhodobacterales sequenced to date.