A heterotrophic, antibiotic-producing bacterium, strain T5T, was isolated from the German Wadden Sea, located in the southern region of the North Sea. Sequence analysis of the 16S rRNA gene of this strain demonstrated close affiliation with Roseobacter gallaeciensis BS107T (99 % similarity), but the results of genotypic (DNA–DNA hybridization and DNA G+C content) and phenotypic characterization revealed that strain T5T represents a novel species. The novel organism is strictly aerobic, Gram-negative, rod-shaped, motile and forms brown-pigmented colonies. Strain T5T produces the antibiotic tropodithietic acid throughout the exponential phase which inhibits the growth of bacteria from different taxa, as well as marine algae. Strain T5T requires sodium ions and utilizes a wide range of substrates, including oligosaccharides, sugar alcohols, organic acids and amino acids. The DNA G+C content is 55.7 mol%. Comparative 16S rRNA gene sequence analysis revealed that strains T5T and Roseobacter gallaeciensis BS107T group with Leisingera methylohalidivorans as their closest described relative within the Roseobacter clade (97.9 and 97.6 % sequence similarity, respectively) and with Ruegeria algicola (96.6 and 96.5 % similarity, respectively) of the Alphaproteobacteria. Comparison of strains T5T and Roseobacter gallaeciensis BS107T with Roseobacter denitrificans and Roseobacter litoralis showed striking differences in 16S rRNA gene sequence similarities, chemical composition, pigmentation, presence of bacteriochlorophyll a and antibiotic production. On the basis of these results, it is proposed that Roseobacter gallaeciensis is reclassified as the type species of a new genus, Phaeobacter, as Phaeobacter gallaeciensis comb. nov. (type strain BS107T=CIP 105210T=ATCC 700781T=NBRC 16654T=DSM 17395T). Strain T5T (=LMG 22475T=DSM 16374T) is proposed as the type strain of a novel species of this genus, Phaeobacter inhibens sp. nov. At the same time, emended descriptions are provided of the genera Roseobacter, Ruegeria and Leisingera, as well as reclassifying Ruegeria algicola as the type species of a new genus, Marinovum, with the name Marinovum algicola comb. nov.

The North Sea Streptomyces strain GWS-BW-H5 was investigated by analyzing headspace extracts of agar-plate cultures (HE) or liquid cultures (LCE), obtained with a closed-loop stripping apparatus (CLSA), by GC/MS (Table 1). The volatile profile of the HE is dominated by the known volatiles (-)-geosmin (4) and 2-methyisoborneol (1). Small amounts of sesquiterpenes occur, which are present in a more-diverse structural variety and in higher quantities in the LCE. The different structures can be rationalized by few cationic intermediates along their biosynthetic pathway. The most-prominent difference between the two culture methods were the presence of the Me-branched gamma- and delta-lactones 31-38, not previously reported from nature, in the LCE. Major components were 10-methyldodecan-5-olide (34), 10-methyldodec-2-en-4-olide (36), and 10-methyldodec-3-en-4-olide (38). The structures of all new lactones were verified by synthesis. Furthermore, more volatiles in higher amounts were produced by the liquid culture than by to the agar plate culture. Since 36 showed inhibitory growth effects against strain GWS-BW-H5, growth inhibition against twelve other strains isolated from the same habitat was tested. Antagonistic activity against four of the strains was observed, with a slightly higher threshold level than found for penicillin G, which was used in control experiments (Table 2).

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.

We studied the composition of free-living and particle-associated bacterial communities during the course of the phytoplankton succession in spring and early summer in the Wadden Sea, a tidal flat ecosystem in the southern North Sea. We applied the denaturing gradient gel electrophoresis (DGGE) approach based on PCR-amplified 16S rRNA gene fragments and, in addition to Bacteriaspecific primers, used primer sets targeting Alphaproteobacteria, the Roseobacter-clade and the Bacteroidetes phylum. Even though the application of the primer sets targeting Bacteria and Alphaproteobacteria detected some changes, they were most pronounced with those targeting the Roseobacter-clade and Bacteroidetes. The changes were supported by a correspondence analysis, which showed a statistically significant correlation of the DGGE banding patterns of the Roseobacter-specific PCR with the composition of the phytoplankton (p = 0.03). This indicates that changes in the phytoplankton composition in this habitat are not reflected by changes in the most abundant or most readily amplifiable phylotypes. The findings rather suggest that few, specialized, heterotrophic bacteria are most responsive to the organic matter supplied by distinct phytoplankton communities and that the main part of organic matter in the Wadden Sea is utilized by generalists. Sequence analyses of excised bands revealed a high diversity for the Bacteria-and Bacteroidetes-targeted approaches. The bacterial community detected by the primer set targeting Alphaproteobacteria, however, was mainly composed of bacteria affiliated to the Roseobacter-clade.KEY WORDS: Free-living bacteria · Particle-attached bacteria · Bacteroidetes · Roseobacter · Phytoplankton · Denaturing gradient gel electrophoresis · Wadden Sea Resale or republication not permitted without written consent of the publisherAquat Microb Ecol 48: [47][48][49][50][51][52][53][54][55][56][57][58][59][60] 2007 tions (LeBaron et al. 1999, Riemann et al. 2000, Schäfer et al. 2001, Pinhassi et al. 2004, Abell & Bowman 2005. The specific organic matter profile of various algae appears also to be an important selection factor for distinct bacterial communities and populations evolving in the phycosphere of algae (Grossart 1999, Grossart et al. 2005. In fact, Alphaproteobacteria, in particular the Roseobacter-clade, and the Sphingobacteria/Flavobacteria group appear to be most responsive to inputs of phytoplankton-born DOC and organic sulfur compounds (Riemann et al. 2000, Fandino et al. 2001, Schäfer et al. 2001, Moran et al. 2003, Pinhassi et al. 2004, Grossart et al. 2005.It is also well established that the community composition of particle-associated (PA) bacteria differs from that of free-living (FL) bacteria. Several studies have shown that Sphingobacteria and Flavobacteria preferentially colonize particles whereas Alpha-and Gammaproteobacteria mainly dwell in free-living marine bacterial communities (Fandino et al. 2001, Simon et al. 2002, Grossart et al. 2005. Our knowledge on the development and su...