Meinhard Simon

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.

Biomass and production of heterotrophic bacterioplankton in the oceanic subarctic Pacific

Abstract As part of the Subarctic Pacific Ecosystem Research (SUPER) program, we measured the abundance and biomass production of heterotrophic bacterioplankton in the subarctic Pacific and compared these parameters with those of phytoplankton during four cruises in 1987 and 1988. Bacterial biomass was about equal to phytoplankton biomass during all cruises. Based on rates of bacterial biomass production and assuming a growth efficiency of 50%, we estimate that heterotrophic bacteria consumed 10% (June 1987) to 24% (August 1988) of primary production in the euphotic zone. These percentages are low compared with other aquatic ecosystems, apparently due to low bacterial growth rates ( −1 ) iin the subarctic Pacific. In contrast, phytoplankton growth rates were much higher (0.1–8.8 day −1 ). Bacterial growth rates were limited by the supply of dissolved organic matter and temperature. Even with these low growth rates, however, bacterial biomass and rates of biomass production increased by 2–5-fold in May and August 1988, changes that were not obviously related to corresponding changes in phytoplankton biomass nor primary production. Heterotrophic bacterioplankton constitutes a large reservoir of carbon and nitrogen that needs to be considered in modelling ecosystem dynamics of the subarctic Pacific.

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.

α- and β-Proteobacteria Control the Consumption and Release of Amino Acids on Lake Snow Aggregates

ABSTRACT We analyzed the composition of aggregate (lake snow)-associated bacterial communities in Lake Constance from 1994 until 1996 between a depth of 25 m and the sediment surface at 110 m by fluorescent in situ hybridization with rRNA-targeted oligonucleotide probes of various specificity. In addition, we experimentally examined the turnover of dissolved amino acids and carbohydrates together with the microbial colonization of aggregates formed in rolling tanks in the lab. Generally, between 40 and more than 80% of the microbes enumerated by DAPI staining (4′,6′-diamidino-2-phenylindole) were detected asBacteria by the probe EUB338. At a depth of 25 m, 10.5% ± 7.9% and 14.2% ± 10.2% of the DAPI cell counts were detected by probes specific for α- and β-Proteobacteria. These proportions increased to 12.0% ± 3.3% and 54.0% ± 5.9% at a depth of 50 m but decreased again at the sediment surface at 110 m to 2.7% ± 1.4% and 41.1% ± 8.4%, indicating a clear dominance of β-Proteobacteria at depths of 50 and 110 m, where aggregates have an age of 3 to 5 and 8 to 11 days, respectively. From 50 m to the sediment surface, cells detected by aCytophaga/Flavobacteria-specific probe (CF319a) comprised increasing proportions up to 18% of the DAPI cell counts. γ-Proteobacteria always comprised minor proportions of the aggregate-associated bacterial community. Using only two probes highly specific for clusters of bacteria closely related toSphingomonas species and Brevundimonas diminuta, we identified between 16 and 60% of the α-Proteobacteria. In addition, with three probes highly specific for close relatives of the β-Proteobacteria Duganella zoogloeoides (formerly Zoogloea ramigera),Acidovorax facilis, and Hydrogenophaga palleroni, bacteria common in activated sludge, 42 to 70% of the β-Proteobacteria were identified. In the early phase (<20 h) of 11 of the 15 experimental incubations of aggregates, dissolved amino acids were consumed by the aggregate-associated bacteria from the surrounding water. This stage was followed by a period of 1 to 3 days during which dissolved amino acids were released into the surrounding water, paralleled by an increasing dominance of β-Proteobacteria. Hence, our results show that lake snow aggregates are inhabited by a community dominated by a limited number of α- and β-Proteobacteria, which undergo a distinct succession. They successively decompose the amino acids bound in the aggregates and release substantial amounts into the surrounding water during aging and sinking.

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 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.

Inefficient microbial production of refractory dissolved organic matter in the ocean

Dissolved organic matter (DOM) in the oceans constitutes a major carbon pool involved in global biogeochemical cycles. More than 96% of the marine DOM resists microbial degradation for thousands of years. The composition of this refractory DOM (RDOM) exhibits a molecular signature ubiquitously detected in the deep oceans. Surprisingly efficient microbial transformation of labile into stable forms of DOM has been shown previously, implying that microorganisms apparently produce far more RDOM than needed to sustain the global pool. Here we show, by assessing the microbial formation and transformation of DOM in unprecedented molecular detail for 3 years, that most of the microbial DOM is different from RDOM in the ocean. Only <0.4% of the net community production is channelled into a form of DOM that is undistinguishable from oceanic RDOM. Our study provides a molecular background for global models on the production, turnover and accumulation of marine DOM.

Phylogenomics of Rhodobacteraceae reveals evolutionary adaptation to marine and non-marine habitats

Marine Rhodobacteraceae (Alphaproteobacteria) are key players of biogeochemical cycling, comprise up to 30% of bacterial communities in pelagic environments and are often mutualists of eukaryotes. As ‘Roseobacter clade’, these ‘roseobacters’ are assumed to be monophyletic, but non-marine Rhodobacteraceae have not yet been included in phylogenomic analyses. Therefore, we analysed 106 genome sequences, particularly emphasizing gene sampling and its effect on phylogenetic stability, and investigated relationships between marine versus non-marine habitat, evolutionary origin and genomic adaptations. Our analyses, providing no unequivocal evidence for the monophyly of roseobacters, indicate several shifts between marine and non-marine habitats that occurred independently and were accompanied by characteristic changes in genomic content of orthologs, enzymes and metabolic pathways. Non-marine Rhodobacteraceae gained high-affinity transporters to cope with much lower sulphate concentrations and lost genes related to the reduced sodium chloride and organohalogen concentrations in their habitats. Marine Rhodobacteraceae gained genes required for fucoidan desulphonation and synthesis of the plant hormone indole 3-acetic acid and the compatible solutes ectoin and carnitin. However, neither plasmid composition, even though typical for the family, nor the degree of oligotrophy shows a systematic difference between marine and non-marine Rhodobacteraceae. We suggest the operational term ‘Roseobacter group’ for the marine Rhodobacteraceae strains.