Thorsten Brinkhoff

A newly discovered Roseobacter cluster in temperate and polar oceans.

Bacterioplankton phylotypes of α-Proteobacteria have been detected in various marine regions, but systematic biogeographical studies of their global distribution are missing. α-Proteobacteria comprise one of the largest fractions of heterotrophic marine bacteria and include two clades, SAR11 and Roseobacter, which account for 26 and 16% of 16S ribosomal RNA gene clones retrieved from marine bacterioplankton. The SAR11 clade attracted much interest because related 16S rRNA gene clones were among the first groups of marine bacteria to be identified by cultivation-independent approaches and appear to dominate subtropical surface bacterioplankton communities. Here we report on the global distribution of a newly discovered cluster affiliated to the Roseobacter clade, comprising only as-yet-uncultured phylotypes. Bacteria of this cluster occur from temperate to polar regions with highest abundance in the Southern Ocean, but not in tropical and subtropical regions. Between the south Atlantic subtropical front and Antarctica, we detected two distinct phylotypes, one north and one south of the polar front, indicating that two adjacent but different oceanic provinces allow the persistence of distinct but closely related phylotypes. These results suggest that the global distribution of major marine bacterioplankton components is related to oceanic water masses and controlled by their environmental and biogeochemical properties.

Antibiotic Production by a Roseobacter Clade-Affiliated Species from the German Wadden Sea and Its Antagonistic Effects on Indigenous Isolates

ABSTRACT A strain affiliated with the Roseobacter clade and producing a new antibiotic named tropodithietic acid (L. Liang, Ph.D. thesis, University of Göttingen, Göttingen, Germany, 2003) was isolated from the German Wadden Sea. The compound showed strong inhibiting properties with respect to marine bacteria of various taxa and marine algae. Antibiotic production was found to occur during the complete growth phase. Strain mutants without antagonistic properties appeared several times spontaneously.

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

Dinoroseobacter shibae DFL12T, 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 DFL12T is able to synthesize the vitamins B1 and B12 for which its host is auxotrophic. Two pathways for the de novo synthesis of vitamin B12 are present, one requiring oxygen and the other an oxygen-independent pathway. The de novo synthesis of vitamin B12 was confirmed to be functional, and D. shibae DFL12T was shown to provide the growth-limiting vitamins B1 and B12 to its dinoflagellate host. The Roseobacter clade has been considered to comprise obligate aerobic bacteria. However, D. shibae DFL12T 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 DFL12T 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 DFL12T shows the most complex viral defense system of all Rhodobacterales sequenced to date.

Antagonistic activity of bacteria isolated from organic aggregates of the German Wadden Sea

Marine aggregates are densely colonized by bacteria, and inter-specific interactions such as inhibition are important for colonization by aggregate-associated bacteria and thus affect the turnover of organic matter in the sea. In order to study antagonistic activities we carried out inhibition tests with 51 isolates obtained exclusively from aggregates of the German Wadden Sea. 16S rRNA gene sequences of all isolates revealed that 35% of the isolates affiliated with the Flavobacteria/Sphingobacteria group, 24% and 16% with alpha- and gamma-Proteobacteria, respectively, 16% with the Bacillus/Clostridium group, and 10% with Actinobacteria. The relatively high percentage of Gram-positive bacteria may be related to specific features of the Wadden Sea environment. After 11 days of incubation using Burkholder agar diffusion assays the percentage of inhibitory isolates was 54.1% and this decreased to 20.7% after 20 days of incubation but it did not decline for members of the Bacillus/Clostridium group. Inhibitory activity was expressed in strain-specific patterns even though some isolates were closely related according to their 16S rRNA gene sequences. Antagonistic activity was lowest for Flavobacteria/Sphingobacteria (35%) and highest for Actinobacteria (80%). We further examined whether growth of isolates was affected when they were placed on lawns of certain other isolates. In parallel with lowest percentage of inhibitory isolates, highest growth occurred on lawns of the Flavobacteria/Sphingobacteria group whereas it was lowest on lawns of Actinobacteria and the Bacillus/Clostridium group. The high inhibitory activity of both groups of Gram-positive bacteria fits well with data from chemical screening using matrix-assisted laser desorption ionization time of flight mass spectrometry. Hence, inhibitory activity greatly influences inter-specific interactions and may impact microbial degradation and remineralization of particulate organic matter in aquatic environments.

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.

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.

Bacteria of the Roseobacter clade show potential for secondary metabolite production

Members of the Roseobacter clade are abundant and widespread in marine habitats and have very diverse metabolisms. Production of acylated homoserine lactones (AHL) and secondary metabolites, e.g., antibiotics has been described sporadically. This prompted us to screen 22 strains of this group for production of signaling molecules, antagonistic activity against bacteria of different phylogenetic groups, and the presence of genes encoding for nonribosomal peptide synthetases (NRPS) and polyketide synthases (PKS), representing enzymes involved in the synthesis of various pharmaceutically important natural products. The screening approach for NRPS and PKS genes was based on polymerase chain reaction (PCR) with degenerate primers specific for conserved sequence motifs. Additionally, sequences from whole genome sequencing projects of organisms of the Roseobacter clade were considered. Obtained PCR products were cloned, sequenced, and compared with genes of known function. With the PCR approach genes showing similarity to known NRPS and PKS genes were found in seven and five strains, respectively, and three PKS and NRPS sequences from genome sequencing projects were obtained. Three strains exhibited antagonistic activity and also showed production of AHL. Overall production of AHL was found in 10 isolates. Phylogenetic analysis of the 16S rRNA gene sequences of the tested organisms showed that several of the AHL-positive strains clustered together. Three strains were positive for three or four categories tested, and were found to be closely related within the genus Phaeobacter. The presence of a highly similar hybrid PKS/NRPS gene locus of unknown function in sequenced genomes of the Roseobacter clade plus the significant similarity of gene fragments from the strains studied to these genes argues for the functional requirement of the encoded hybrid PKS/NRPS complex. Our screening results therefore suggest that the Roseobacter clade is indeed employing PKS/NRPS biochemistry and should thus be further studied as a potential and largely untapped source of secondary metabolites.

Distribution of Roseobacter RCA and SAR11 lineages in the North Sea and characteristics of an abundant RCA isolate

The Roseobacter group and SAR11 clade constitute high proportions of the marine bacterioplankton, but only scarce information exists on the abundance of distinct populations of either lineage. Therefore, we quantified the abundance of the largest cluster of the Roseobacter group, the RCA (Roseobacter clade affiliated) cluster together with the SAR11 clade by quantitative PCR in the southern and eastern North Sea. The RCA cluster constituted up to 15 and 21% of total bacterial 16S ribosomal RNA (rRNA) genes in September 2005 and May 2006, respectively. At a few stations, the RCA cluster exceeded the SAR11 clade, whereas at most stations, SAR11 constituted higher fractions with maxima of 37%. In most samples, only one RCA ribotype was detected. RCA abundance was positively correlated with phaeopigments, chlorophyll, dissolved and particulate organic carbon (POC), turnover rates of dissolved free amino acids (DFAAs), temperature, and negatively correlated with salinity. The SAR11 clade was only correlated with POC (negatively, May) and with DFAA turnover rates (positively, September). An abundant RCA strain, ‘Candidatus Planktomarina temperata’, was isolated from the southern North Sea. This strain has an identical 16S rRNA gene sequence to the dominant RCA ribotype. Detection of the pufM gene, coding for a subunit of the reaction center of bacteriochlorophyll a, indicates the potential of the isolate for aerobic anoxygenic photosynthesis. Our study shows that a distinct population of the RCA cluster constitutes an abundant bacterioplankton group in a neritic sea of the temperate zone and indicates that this population has an important role during decaying phytoplankton blooms.

Distribution of Roseobacter RCA and SAR11 lineages and distinct bacterial communities from the subtropics to the Southern Ocean

We assessed the composition of the bacterioplankton in the Atlantic sector of the Southern Ocean in austral fall and winter and in New Zealand coastal waters in summer. The various water masses between the subtropics/Agulhas-Benguela boundary region and the Antarctic coastal current exhibited distinct bacterioplankton communities with the highest richness in the polar frontal region, as shown by denaturing gradient gel electrophoresis of 16S rRNA gene fragments. The SAR11 clade and the Roseobacter clade-affiliated (RCA) cluster were quantified by real-time quantitative PCR. SAR11 was detected in all samples analysed from subtropical waters to the coastal current and to depths of > 1000 m. In fall and winter, this clade constituted < 3% to 48% and 4-28% of total bacterial 16S rRNA genes respectively, with highest fractions in subtropical to polar frontal regions. The RCA cluster was only present in New Zealand coastal surface waters not exceeding 17 degrees C, in the Agulhas-Benguela boundary region (visited only during the winter cruise), in subantarctic waters and in the Southern Ocean. In fall, this cluster constituted up to 36% of total bacterial 16S rRNA genes with highest fractions in the Antarctic coastal current and outnumbered the SAR11 clade at most stations in the polar frontal region and further south. In winter, the RCA cluster constituted lower proportions than the SAR11 clade and did not exceed 8% of total bacterial 16S rRNA genes. In fall, the RCA cluster exhibited significant positive correlations with latitude and ammonium concentrations and negative correlations with concentrations of nitrate, phosphate, and for near-surface samples also with chlorophyll a, biomass production of heterotrophic prokaryotes and glucose turnover rates. The findings show that the various water masses between the subtropics and the Antarctic coastal current harbour distinct bacterioplankton communities. They further indicate that the RCA cluster, despite the narrow sequence similarity of > 98% of its 16S rRNA gene, is an abundant component of the heterotrophic bacterioplankton in the Southern Ocean, in particular in its coldest regions.

Growth phase-dependent global protein and metabolite profiles of Phaeobacter gallaeciensis strain DSM 17395, a member of the marine Roseobacter-clade.

The marine heterotrophic roseobacter Phaeobacter gallaeciensis DSM 17395 was grown with glucose in defined mineral medium. Relative abundance changes of global protein (2‐D DIGE) and metabolite (GC‐MS) profiles were determined across five different time points of growth. In total, 215 proteins were identified and 147 metabolites detected (101 structurally identified), among which 60 proteins and 87 metabolites displayed changed abundances upon entry into stationary growth phase. Glucose breakdown to pyruvate apparently proceeds via the Entner–Doudoroff (ED) pathway, since phosphofructokinase of the Embden–Meyerhof–Parnas pathway is missing and the key metabolite of the ED‐pathway, 2‐keto‐3‐desoxygluconate, was detected. The absence of pfk in other genome‐sequenced roseobacters suggests that the use of the ED pathway is an important physiological property among these heterotrophic marine bacteria. Upon entry into stationary growth phase (due to glucose starvation), sulfur assimilation (including cysteine biosynthesis) and parts of cell envelope synthesis (e.g. the lipid precursor 1‐monooleoylglycerol) were down‐regulated and cadaverine formation up‐regulated. In contrast, central carbon catabolism remained essentially unchanged, pointing to a metabolic “stand‐by” modus as an ecophysiological adaptation strategy. Stationary phase response of P. gallaeciensis differs markedly from that of standard organisms such as Escherichia coli, as evident e.g. by the absence of an rpoS gene.