Ligand cross-feeding resolves bacterial vitamin B12 auxotrophies.

Cobalamin (vitamin B12, herein referred to as B12) is an essential cofactor for most marine prokaryotes and eukaryotes1,2. Synthesized by a limited number of prokaryotes, its scarcity affects microbial interactions and community dynamics2-4. Here we show that two bacterial B12 auxotrophs can salvage different B12 building blocks and cooperate to synthesize B12. A Colwellia sp. synthesizes and releases the activated lower ligand α-ribazole, which is used by another B12 auxotroph, a Roseovarius sp., to produce the corrin ring and synthesize B12. Release of B12 by Roseovarius sp. happens only in co-culture with Colwellia sp. and only coincidently with the induction of a prophage encoded in Roseovarius sp. Subsequent growth of Colwellia sp. in these conditions may be due to the provision of B12 by lysed cells of Roseovarius sp. Further evidence is required to support a causative role for prophage induction in the release of B12. These complex microbial interactions of ligand cross-feeding and joint B12 biosynthesis seem to be widespread in marine pelagic ecosystems. In the western and northern tropical Atlantic Ocean, bacteria predicted to be capable of salvaging cobinamide and synthesizing only the activated lower ligand outnumber B12 producers. These findings add new players to our understanding of B12 supply to auxotrophic microorganisms in the ocean and possibly in other ecosystems.

Description of Palleronia rufa sp. nov., a biofilm-forming and AHL-producing Rhodobacteraceae, reclassification of Hwanghaeicola aestuarii as Palleronia aestuarii comb. nov., Maribius pontilimi as Palleronia pontilimi comb. nov., Maribius salinus as Palleronia salina comb. nov., Maribius pelagius as Palleronia pelagia comb. nov. and emended description of the genus Palleronia.

Strain MOLA 401T was isolated from marine waters in the southwest lagoon of New Caledonia and was shown previously to produce an unusual diversity of quorum sensing signaling molecules. This strain was Gram-negative, formed non-motile cocci and colonies were caramel. Optimum growth conditions were 30°C, pH 8 and 3% NaCl (w/v). Based on 16S rRNA gene sequence analysis, this strain was found to be closely related to Pseudomaribius aestuariivivens NBRC 113039T (96.9% of similarity), Maribius pontilimi DSM 104950T (96.4% of similarity) and Palleronia marisminoris LMG 22959T (96.3% of similarity), belonging to the Roseobacter group within the family Rhodobacteraceae. As its closest relatives, strain MOLA 401T is able to form a biofilm on polystyrene, supporting the view of Roseobacter group strains as prolific surface colonizers. An in-depth genomic study allowed us to affiliate strain MOLA 401T as a new species of genus Palleronia and to reaffiliate some of its closest relatives in this genus. Consequently, we describe strain MOLA 401T (DSM 106827T=CIP 111607T=BBCC 401T) for which we propose the name Palleronia rufa sp. nov. We also propose to emend the description of the genus Palleronia and to reclassify Maribius and Hwanghaeicola species as Palleronia species.

Role of RpoN from Labrenzia aggregata LZB033 (Rhodobacteraceae) in Formation of Flagella and Biofilms, Motility, and Environmental Adaptation.

Labrenzia aggregata LZB033 (Rhodobacteraceae), which produces dimethylsulfoniopropionate (DMSP) and reduces nitrate to nitrogen, was isolated from seawater of the East China Sea. Its genome encodes a large number of transcriptional regulators which may be important for its adaptation to diverse marine environments. The alternative σ54 factor (RpoN) is a central regulator of many bacteria, regulating the transcription of multiple genes and controlling important cellular functions. However, the exact role of RpoN in Labrenzia spp. is unknown. In this study, an in-frame rpoN deletion mutant was constructed in LZB033, and the function of RpoN was determined. To systematically identify RpoN-controlled genes, we performed a detailed analysis of gene expression differences between the wild-type strain and the ΔrpoN mutant using RNA sequencing. The expression of 175 genes was shown to be controlled by RpoN. Subsequent phenotypic assays showed that the ΔrpoN mutant was attenuated in flagellar biosynthesis and swimming motility, utilized up to 13 carbon substrates differently, lacked the ability to assimilate malic acid, and displayed markedly decreased biofilm formation. In addition, stress response assays showed that the ΔrpoN mutant was impaired in the ability to survive under different challenge conditions, including osmotic stress, oxidative stress, temperature changes, and acid stress. Moreover, both the DMSP synthesis and catabolism rates of LZB033 decreased after rpoN was knocked out. Our work provides essential insight into the regulatory function of RpoN, revealing that RpoN is a key determinant for LZB033 flagellar formation, motility, biofilm formation, and environmental fitness, as well as DMSP production and degradation.IMPORTANCE This study established an in-frame gene deletion method in the alphaproteobacterium Labrenzia aggregata LZB033 and generated an rpoN gene mutant. A comparison of the transcriptomes and phenotypic characteristics between the mutant and wild-type strains confirmed the role of RpoN in L. aggregata LZB033 flagellar formation, motility, biofilm formation, and carbon usage. Most importantly, RpoN is a key factor for survival under different environmental challenge conditions. Furthermore, the ability to synthesize and metabolize dimethylsulfoniopropionate (DMSP) was related to RpoN. These features revealed RpoN to be an important regulator of stress resistance and survival for L. aggregata LZB033 in marine environments.

Phage infection of an environmentally relevant marine bacterium alters host metabolism and lysate composition.

Viruses contribute to the mortality of marine microbes, consequentially altering biological species composition and system biogeochemistry. Although it is well established that host cells provide metabolic resources for virus replication, the extent to which infection reshapes host metabolism at a global level and the effect of this alteration on the cellular material released following viral lysis is less understood. To address this knowledge gap, the growth dynamics, metabolism and extracellular lysate of roseophage-infected Sulfitobacter sp. 2047 was studied using a variety of techniques, including liquid chromatography-tandem mass spectrometry (LC-MS/MS)-based metabolomics. Quantitative estimates of the total amount of carbon and nitrogen sequestered into particulate biomass indicate that phage infection redirects ∼75% of nutrients into virions. Intracellular concentrations for 82 metabolites were measured at seven time points over the infection cycle. By the end of this period, 71% of the detected metabolites were significantly elevated in infected populations, and stable isotope-based flux measurements showed that these cells had elevated metabolic activity. In contrast to simple hypothetical models that assume that extracellular compounds increase because of lysis, a profile of metabolites from infected cultures showed that >70% of the 56 quantified compounds had decreased concentrations in the lysate relative to uninfected controls, suggesting that these small, labile nutrients were being utilized by surviving cells. These results indicate that virus-infected cells are physiologically distinct from their uninfected counterparts, which has implications for microbial community ecology and biogeochemistry.

The CckA-ChpT-CtrA phosphorelay system is regulated by quorum sensing and controls flagellar motility in the marine sponge symbiont Ruegeria sp. KLH11.

Bacteria respond to their environment via signal transduction pathways, often two-component type systems that function through phosphotransfer to control expression of specific genes. Phosphorelays are derived from two-component systems but are comprised of additional components. The essential cckA-chpT-ctrA phosphorelay in Caulobacter crescentus has been well studied and is important in orchestrating the cell cycle, polar development and flagellar biogenesis. Although cckA, chpT and ctrA homologues are widespread among the Alphaproteobacteria, relatively few is known about their function in the large and ecologically significant Roseobacter clade of the Rhodobacterales. In this study the cckA-chpT-ctrA system of the marine sponge symbiont Ruegeria sp. KLH11 was investigated. Our results reveal that the cckA, chpT and ctrA genes positively control flagellar biosynthesis. In contrast to C. crescentus, the cckA, chpT and ctrA genes in Ruegeria sp. KLH11 are non-essential and do not affect bacterial growth. Gene fusion and transcript analyses provide evidence for ctrA autoregulation and the control of motility-related genes. In KLH11, flagellar motility is controlled by the SsaRI system and acylhomoserine lactone (AHL) quorum sensing. SsaR and long chain AHLs are required for cckA, chpT and ctrA gene expression, providing a regulatory link between flagellar locomotion and population density in KLH11.

You are what you talk: quorum sensing induces individual morphologies and cell division modes in Dinoroseobacter shibae.

Dinoroseobacter shibae, a member of the Roseobacter clade abundant in marine environments, is characterized by a pronounced pleomorphism. Cell shapes range from variable-sized ovoid rods to long filaments with a high copy number of chromosomes. Time-lapse microscopy shows cells dividing either by binary fission or by budding from the cell poles. Here we demonstrate that this morphological heterogeneity is induced by quorum sensing (QS). D. shibae utilizes three acylated homoserine lactone (AHL) synthases (luxI1-3) to produce AHLs with unsaturated C18 side chains. A ΔluxI1-knockout strain completely lacking AHL biosynthesis was uniform in morphology and divided by binary fission only. Transcriptome analysis revealed that expression of genes responsible for control of cell division was reduced in this strain, providing the link between QS and the observed phenotype. In addition, flagellar biosynthesis and type IV secretion system (T4SS) were downregulated. The wild-type phenotype and gene expression could be restored through addition of synthetic C18-AHLs. Their effectiveness was dependent on the number of double bonds in the acyl side chain and the regulated trait. The wild-type expression level of T4SS genes was fully restored even by an AHL with a saturated C18 side chain that has not been detected in D. shibae. QS induces phenotypic individualization of D. shibae cells rather than coordinating the population. This strategy might be beneficial in unpredictably changing environments, for example, during algal blooms when resource competition and grazing exert fluctuating selective pressures. A specific response towards non-native AHLs might provide D. shibae with the capacity for complex interspecies communication.

Patch-clamp characterization of the MscS-like mechanosensitive channel from Silicibacter pomeroyi.

Based on sequence similarity, the sp7 gene product, MscSP, of the sulfur-compound-decomposing Gram-negative marine bacterium Silicibacter pomeroyi belongs to the family of MscS-type mechanosensitive channels. To investigate MscSP channel properties, we measured its response to membrane tension using the patch-clamp technique on either a heterologous expression system using giant spheroplasts of MJF465 Escherichia coli strain (devoid of mechanosensitive channels MscL, MscS, and MscK), or on purified MscSP protein reconstituted in azolectin liposomes. These experiments showed typical pressure-dependent gating properties of a stretch-activated channel with a current/voltage plot indicating a rectifying behavior and weak preference for anions similar to the MscS channel of E. coli. However, the MscSP channel exhibited functional differences with respect to conductance and desensitization behavior, with the most striking difference between the two channels being the lack of inactivation in MscSP compared with MscS. This seems to result from the fact that although MscSP has a Gly in an equivalent position to MscS (G113), a position that is critical for inactivation, MscSP has a Glu residue instead of an Asn in a position that was recently shown to allosterically influence MscS inactivation, N117. To our knowledge, this study describes the first electrophysiological characterization of an MscS-like channel from a marine bacterium belonging to sulfur-degrading α-proteobacteria.

Light enhances survival of Dinoroseobacter shibae during long-term starvation.

Aerobic anoxygenic phototrophs (AAPs) as being photoheterotrophs require organic substrates for growth and use light as a supplementary energy source under oxic conditions. We hypothesized that AAPs benefit from light particularly under carbon and electron donor limitation. The effect of light was determined in long-term starvation experiments with Dinoroseobacter shibae DFL 12(T) in both complex marine broth and defined minimal medium with succinate as the sole carbon source. The cells were starved over six months under three conditions: continuous darkness (DD), continuous light (LL), and light/dark cycle (LD, 12 h/12 h, 12 µmol photons m(-2) s(-1)). LD starvation at low light intensity resulted in 10-fold higher total cell and viable counts, and higher bacteriochlorophyll a and polyhydroxyalkanoate contents. This coincided with better physiological fitness as determined by respiration rates, proton translocation and ATP concentrations. In contrast, LD starvation at high light intensity (>22 µmol photons m(-2) s(-1), LD conditions) resulted in decreasing cell survival rates but increasing carotenoid concentrations, indicating a photo-protective response. Cells grown in complex medium survived longer starvation (more than 20 weeks) than those grown in minimal medium. Our experiments show that D. shibae benefits from the light and dark cycle, particularly during starvation.

Marivita roseacus sp. nov., of the family Rhodobacteraceae, isolated from a temperate estuary and an emended description of the genus Marivita.

A gram-negative, non-motile, pigmented, rod-shaped and strictly aerobic bacterium (CB1052(T)) was isolated from a temperate estuary. On the basis of 16S rRNA gene sequence similarity, strain CB1052(T) belongs to the α-3 subclass of the Proteobacteria, within the family Rhodobacteraceae, having the highest similarity to members of the genus Marivita (97.8%) of the Roseobacter lineage. Pylogenetic analysis showed CB1052(T) to be a distinct sister clade to M. litorea and M. cryptomonadis and DNA-DNA relatedness was quite low amongst the strains (< 35%). Strain CB1052(T) cells are non-motile and display a needle-like filamentous form, where individual cells can become quite elongated (up to 15 µm). Similar to M. litorea and M. cryptomonadis, CB1052(T) harbors aerobic anoxygenic photosynthesis genes. However, in contrast to other described Marivita species, strain CB1052(T) actively produces bacteriochlorophyll a. Further physiological features, including antibiotic sensitivities, differentiate strain CB1052(T) from the other members of the genus. Therefore, strain CB1052(T) is considered to represent a novel species of the genus Marivita, for which the name Marivita roseacus sp. nov. is proposed, with the type strain CB1052(T) (=DSM 23118(T) =ATCC BAA 1914(T)).

Isolation and physiological characterization of two novel, piezophilic, thermophilic chemolithoautotrophs from a deep-sea hydrothermal vent chimney.

Two novel, thermophilic piezophiles, capable of chemolithoautotrophic growth, are successfully cultivated and isolated from a black smoker chimney at the TAG field (Mid Atlantic Ridge: MAR) by using a piezophilic cultivation technique. Both strains (strains 106 and 108) represent dominant cultivated populations of the microbial communities in the chimney surface habitat. Strain 106 represents typically thin, long spiral cells under the piezophilic growth condition but short bent cells under the non-piezophilic condition. It is a strictly chemolithoautotrophic gammaproteobacterium using reduced sulfur compounds as the electron donors, and nitrate and O(2) as the electron acceptors. Based on the 16S rRNA gene sequence, strain 106 would represent a novel genus of the previously uncultivated group (Symbiont Group I; a potentially novel family) within the Gammaproteobacteria, and 'Thioprofundum lithotrophica' gen. nov., sp. nov. is proposed. Strain 108 is a short, oval rod at any of the growth pressures. It is a facultative chemoautotroph, capable of both chemolithoautotrophic growth with H(2) and S oxidations and organotrophic growth with complex organics or organic acids using nitrate and O(2) as the electron acceptors. The chemolithoautotrophic growth is strictly piezophilic and under the organotrophic growth condition, it grows at conventional pressures (0.1 MPa). Strain 108 is phylogenetically distinctive from any of the previously described genera of the family Rhodobacteraceae within the Alphaproteobacteria, and 'Piezobacter thermophilus' gen. nov., sp. nov. is proposed. The piezophilic cultivation technique can be a powerful tool to isolate and characterize the previously uncultivated phylotypes in the deep-sea hydrothermal vent environments.

Phylogenetic classification of heterotrophic bacteria associated with filamentous marine cyanobacteria in culture.

Fifty-one heterotrophic bacterial strains were isolated from the marine cyanobacterial cultures of heterocystous Nodularia harveyana strain Bo53 and non-heterocystous Oscillatoria brevis strain Bo10. Fluorescence in situ hybridisation and fingerprinting methods were used for a preliminary taxonomical classification of 44 of the 51 isolates. The strains obtained from Bo53 were mostly Alphaproteobacteria (10/24), followed by Bacteroidetes (7/24), and Gammaproteobacteria (3/24). The affiliation of the isolates originating from Bo10 was dominated by Alphaproteobacteria (8/20) and Bacteroidetes (7/20), followed by Gammaproteobacteria (3/20). The 16S rRNA genes of four selected isolates were sequenced. A red-coloured bacterium from Bo53 grouped with the alphaproteobacterial genus Porphyrobacter, while the other three strains, obtained from Bo10, belonged to the alphaproteobacterial genera Roseobacter (pink) and Rhodobacter (colourless), and to the genus Muricauda (yellow) of Bacteroidetes. The findings indicated that the aerobic anoxygenic phototroph Porphyrobacter and its relatives only occurred in Bo10 culture, whereas members of the Roseobacter clade and the Bacteroidetes bacterium Muricauda sp. seemed to be more ubiquitous.

Sulfitobacter marinus sp. nov., isolated from seawater of the East Sea in Korea.

A Gram-negative, non-motile, rod- or oval-shaped Sulfitobacter-like bacterial strain, SW-265T, was isolated from seawater at Hwajinpo, Korea, and was subjected to a polyphasic taxonomic study. Strain SW-265T grew optimally at pH 7.0-8.0 and 30 degrees C in the presence of 2 % (w/v) NaCl. It contained Q-10 as the predominant ubiquinone and C(18 : 1)omega7c as the major fatty acid. The DNA G+C content was 57.8 mol%. A phylogenetic analysis based on 16S rRNA gene sequences showed that strain SW-265T fell within the cluster comprising Sulfitobacter species. The levels of 16S rRNA gene sequence similarity between strain SW-265T and the type strains of Sulfitobacter species ranged from 97.1 to 98.7 %. DNA-DNA relatedness data and differential phenotypic properties, together with the phylogenetic distinctiveness, demonstrated that strain SW-265T differs from recognized Sulfitobacter species. On the basis of the phenotypic, phylogenetic and genetic data, strain SW-265T represents a novel species of the genus Sulfitobacter, for which the name Sulfitobacter marinus sp. nov. is proposed. The type strain is SW-265T (=KCTC 12738T=JCM 13602T).

Identification of "Haematobacter," a new genus of aerobic Gram-negative rods isolated from clinical specimens, and reclassification of Rhodobacter massiliensis as "Haematobacter massiliensis comb. nov.".

Twelve strains of gram-negative, nonfermenting rods recovered mainly from septicemic patients were studied using conventional and molecular methods. The phenotypic profiles of these strains most closely resembled Psychrobacter phenylpyruvicus. They produced catalase, oxidase, urease, and H(2)S (lead acetate paper) but did not produce indole, reduce nitrate or nitrite, or hydrolyze gelatin or esculin. No acid production was observed in a King's oxidation-fermentation base containing d-glucose, d-xylose, d-mannitol, sucrose, lactose, or maltose. All strains were nonmotile and nonpigmented. Most strains produced green discoloration on blood agar. All strains grew at 25 degrees C and 35 degrees C and most grew on MacConkey agar. They shared a common cellular fatty acid (CFA) profile characterized by large amounts (56% to 90%) of 18:1omega7c and the presence of 3-OH-10:0, 16:1omega7c, 16:0, and 19:0cycomega8c that overall was most similar to that of Rhodobacter species but was quite distinct from that of P. phenylpyruvicus. The MICs for most beta-lactams, fluoroquinolones, aminoglycosides, and carbapenems were low. MICs for aztreonam and piperacillin were higher, with MICs for some strains of > 64 mg/liter and > 128 mg/liter, respectively. Polyphasic analysis of these strains, including morphological, biochemical, CFA composition, DNA-DNA hybridization, 16S rRNA gene sequencing, and percent guanine-plus-cytosine (G+C) content analysis, demonstrated that these strains and Rhodobacter massiliensis represent a new genus, "Haematobacter" (proposed name), with the species H. missouriensis (type strain H1892(T) = CCUG 52307(T) = CIP 109176(T)) and H. massiliensis comb. nov. (type strain Framboise(T) = CCUG 47968(T) = CIP 107725(T)) and an unnamed genomospecies.

Thioclava pacifica gen. nov., sp. nov., a novel facultatively autotrophic, marine, sulfur-oxidizing bacterium from a near-shore sulfidic hydrothermal area.

Strain TL 2(T) was isolated on mineral medium with thiosulfate from a near-shore sulfidic hydrothermal area in Matupi Harbour on the island of New Britain, Papua New Guinea. The cells varied from long filaments with swollen ends, often aggregated, to short rods, depending on the growth conditions. The bacterium was obligately aerobic and grew autotrophically with thiosulfate as energy source or heterotrophically with organic acids and sugars. In thiosulfate-limited continuous culture, mu(max) and Y(max) for autotrophic growth were 0.1 h(-1) and 3 g protein mol(-1), respectively. From the various reduced sulfur compounds tested, only thiosulfate and sulfide supported active respiration. Inorganic carbon was assimilated via the Calvin cycle. Presence of the 'green'-type of form I RubisCO gene was detected. Growth was possible from 15 to 47 degrees C with an optimum at 35 degrees C, pH 6.5-8.5 with an optimum at pH 8.0, and between 10 and 90 g NaCl l(-1) with an optimum at 35 g l(-1). Phylogenetic analysis based on 16S rRNA and cbbL gene sequences demonstrated that strain TL 2(T) forms a separate lineage within the alpha-3 subdivision of the Proteobacteria, distantly related to the genera Rhodovulum and Rhodobacter. On the basis of these results, a novel genus and species, Thioclava pacifica gen. nov., sp. nov., is proposed to accommodate strain TL 2(T) (= DSM 10166(T) = UNIQEM 229(T)).

Dinoroseobacter shibae gen. nov., sp. nov., a new aerobic phototrophic bacterium isolated from dinoflagellates.

A novel group of aerobic anoxygenic phototrophic bacteria was isolated from marine dinoflagellates, and two strains were characterized in detail. Cells were Gram-negative cocci or ovoid rods and were motile by means of a single, polarly inserted flagellum. They were obligate aerobes requiring 1-7 % salinity. The optimal pH range for growth was 6.5-9.0 and the temperature optimum was 33 degrees C. The bacteria contained bacteriochlorophyll a and spheroidenone as the only carotenoid. The in vivo absorption spectrum displayed two maxima in the infrared region at 804 and 868 nm. The distinct 804 nm band indicates the presence of light-harvesting system 2. Various organic carbon sources were assimilated, including many carboxylic acids, glucose and glycerol, but not butyrate, ethanol or methanol. Dissimilatory nitrate reduction was found for both strains. The physiological characteristics of the new strains resembled those of Roseobacter denitrificans, but there were differences in the lipid composition. Based on 16S rRNA gene sequence analysis the new strains are relatively distant from other recognized species, with the closest relatives Jannaschia helgolandensis, Ruegeria atlantica and Rhodobacter veldkampii showing 94.1-93.4 % similarity. Similarity to Roseobacter denitrificans was only 92.2 %, in line with numerous other species of the Roseobacter group. Therefore, it is proposed to classify the strains in a new genus and species within the Roseobacter clade, Dinoroseobacter shibae gen. nov., sp. nov. The type strain is DFL 12(T) (=DSM 16493(T)=NCIMB 14021(T)).

Loktanella hongkongensis sp. nov., a novel member of the alpha-Proteobacteria originating from marine biofilms in Hong Kong waters.

A Gram-negative, non-motile, non-spore-forming, short rod-shaped bacterium (UST950701-009P(T)) was isolated from a marine biofilm in Hong Kong waters. Colonies are pink in colour, convex with a smooth surface and entire edge. Brown diffusible pigment is produced. Whitish colonies, with otherwise identical morphology, emerge from every culture upon ageing. The white colonies can be maintained as separate cultures (UST950701-009W) without turning pink. UST950701-009P(T) and UST950701-009W have identical 16S rRNA gene sequences and similar G+C (65.9-66.2 mol%) and fatty acid (86.22-88.52 % 18 : 1omega7c) contents. Phylogenetic analysis of the 16S rRNA gene sequence places UST950701-009P(T) within the Rhodobacter group of the alpha-subclass of the Proteobacteria. The nearest neighbours belong to the genus Loktanella, with similarity values ranging from 94.5 to 95.5 %. Data on G+C and fatty acid contents support the affiliation to the genus Loktanella. UST950701-009P(T) and -009W are heterotrophic, strictly aerobic and require NaCl for growth (2.0-14.0 %). Both grow in pH 5.0-10.0 and at 8-44 degrees C. Both are positive in oxidase, catalase and beta-galactosidase tests, but they differ in the pattern of carbohydrate oxidation and assimilation. Molecular evidence together with phenotypic characteristics shows that UST950701-009P(T) constitutes a novel species within the genus Loktanella. The name Loktanella hongkongensis sp. nov. is proposed; the type strain is UST950701-009P(T) (=NRRL B-41039(T)=JCM 12479(T)) and a morphovar is UST950701-009W (=NRRL B-41040=JCM 12480).

Pseudovibrio denitrificans gen. nov., sp. nov., a marine, facultatively anaerobic, fermentative bacterium capable of denitrification.

Two denitrifying strains of heterotrophic, facultatively anaerobic, marine bacteria, designated DN34(T) and DN33, were isolated from sea-water samples collected in Nanwan Bay, Kenting National Park, Taiwan. They were Gram-negative. Cells in late exponential to early stationary phase of growth were predominantly straight or curved rods, but Y- or V-shaped forms were also observed. They were motile by means of one to several lateral or subpolar flagella. Both strains required NaCl for growth and exhibited optimal growth at about 30 degrees C, pH 8 and 3 % NaCl. They were capable of anaerobic growth by carrying out denitrifying metabolism using nitrate, nitrite or nitrous oxide as terminal electron acceptors or, alternatively, by fermenting glucose, mannose, sucrose or trehalose as substrates. Anaerobic fermentative growth on glucose resulted in formation of various organic acids, including formate, lactate, acetate, pyruvate and fumarate. The major cellular fatty acids were 2-OH-14 : 0, 3-OH-14 : 0 and 16 : 0. DN34(T) and DN33 had DNA G+C contents of 51.7 and 51.6 mol%, respectively. Physiological characterization, together with phylogenetic analysis based on 16S rRNA gene sequence analysis, revealed that the two denitrifying strains could be accommodated in a novel genus, for which the name Pseudovibrio gen. nov. is proposed. Pseudovibrio denitrificans sp. nov. is the type species, with DN34(T) (=BCRC 17323(T)=JCM 12308(T)) as the type strain.

Oceanicola granulosus gen. nov., sp. nov. and Oceanicola batsensis sp. nov., poly-beta-hydroxybutyrate-producing marine bacteria in the order 'Rhodobacterales'.

Three Gram-negative, chemoheterotrophic, non-motile, rod-shaped bacterial strains that accumulate poly-beta-hydroxybutyrate granules were isolated from the Bermuda Atlantic Time-series Study site by high-throughput culturing methods and characterized by polyphasic approaches. DNA-DNA hybridization, DNA G+C content and phylogenetic analyses based on 16S rRNA gene sequences divided the three isolates into two distinct genospecies that were clearly differentiated by fatty acid profiles, carbon source utilization patterns, antibiotic susceptibility and biochemical characteristics. The strains utilized a wide range of substrates, including pentoses, hexoses, oligosaccharides, sugar alcohols, organic acids and amino acids. DNA G+C contents were 71.5, 70.9 and 67.3 mol% for strains HTCC2516T, HTCC2523 and HTCC2597T, respectively. The most dominant fatty acid was 18 : 1omega7c in strains HTCC2516T and HTCC2523, and cyclo 19 : 0 in strain HTCC2597T. The type strains HTCC2516T and HTCC2597T were clearly differentiated by the presence or absence of 12 : 0, 12 : 1omega11c, 14 : 0, 15 : 0 and methyl 18 : 1. Phylogenetic analyses indicated that the strains formed a distinct monophyletic lineage within the Roseobacter clade in the order 'Rhodobacterales' of the Alphaproteobacteria, and which did not associate with any of the described genera. Genotypic and phenotypic differences of the isolates from the previously described genera support the description of Oceanicola granulosus gen. nov., sp. nov. with the type strain HTCC2516T (=ATCC BAA-861T=DSM 15982T=KCTC 12143T) and of Oceanicola batsensis sp. nov. with the type strain HTCC2597T (=ATCC BAA-863T=DSM 15984T=KCTC 12145T).

Oceanibulbus indolifex gen. nov., sp. nov., a North Sea alphaproteobacterium that produces bioactive metabolites.

A water sample from the North Sea was used to isolate the abundant heterotrophic bacteria that are able to grow on complex marine media. Isolation was by serial dilution and spread plating. Phylogenetic analysis of nearly complete 16S rRNA gene sequences revealed that one of the strains, HEL-45T, had 97.4% sequence similarity to Sulfitobacter mediterraneus and 96.5 % sequence similarity to Staleya guttiformis. Strain HEL-45T is a Gram-negative, non-motile rod and obligate aerobe and requires sodium and 1-7% sea salts for growth. It contains storage granules and does not produce bacteriochlorophyll. Optimal growth temperatures are 25-30 degrees C. The DNA base composition (G+C content) is 60.1 mol%. Strain HEL-45T has Q10 as the dominant respiratory quinone. The major polar lipids are phosphatidyl glycerol, diphosphatidyl glycerol, phosphatidyl choline, phosphatidyl ethanolamine and an aminolipid. The fatty acids comprise 18 : 1omega7c, 18 : 0, 16 : 1omega7c, 16 : 0, 3-OH 10 : 0, 3-OH 12 : 1 (or 3-oxo 12 : 0) and traces of an 18 : 2 fatty acid. Among the hydroxylated fatty acids only 3-OH 12 : 1 (or 3-oxo 12 : 0) appears to be amide linked, whereas 3-OH 10 : 0 appears to be ester linked. The minor fatty acid components (between 1 and 7%) allow three subgroups to be distinguished in the Sulfitobacter/Staleya clade, placing HEL-45T into a separate lineage characterized by the presence of 3-OH 12 : 1 (or 3-oxo 12 : 0) and both ester- and amide-linked 16 : 1omega7c phospholipids. HEL-45T produces indole and derivatives thereof, several cyclic dipeptides and thryptanthrin. Phylogenetic analysis of 16S rRNA gene sequences and chemotaxonomic data support the description of a new genus and species, to include Oceanibulbus indolifex gen. nov., sp. nov., with the type strain HEL-45T (=DSM 14862T=NCIMB 13983T).