Fresh antibiotic stock solutions (10 mg/ml) were made for every experiment. Test tubes were inoculated this website to an OD578 of 0.05 with over night cultures of the Roseobacter strains in MB at 30°C. The results represent the mean of

three independent experiments performed in duplicate. Amp, ampicillin; Carb, carbenicillin; Cm, chloramphenicol; Gm, gentamicin; Kan, kanamycin; Spec, spectinomycin; Strep, streptomycin; Tc, tetracycline All tested species showed different susceptibilities to the antibiotics (Table 2). As expected, the seven D. shibae strains followed the same trend, with slight variations. They were all resistant to the β-lactam antibiotics ampicillin and carbenicillin. The level of tolerance to ampicillin was up to 500 μg/ml. The Phaeobacter strains, R. denitrificans and R. litoralis also showed resistance to ampicillin, whereas, in contrast to D.

shibae, they were sensitive to carbenicillin. Initially, we hypothesised, that the unexpected high ampicillin tolerance might occur due to instability of this antibiotic. It has been ACP-196 in vitro reported that ampicillin lost 28% of activity after 24 h at room temperature [30]. However, control experiments with the E. coli strain DH5α revealed complete activity of ampicillin even after incubation for five days at 30°C (data not shown). Analysing the annotated genomes of the strains by BLAST search and functional predictions (for details see Methods section), we identified genes encoding for β-lactamases, indicating that they are widespread over the Roseobacter clade. They were also found in R. denitrificans,

R. litoralis, P. gallaeciensis, O. indolifex and D. shibae. For the latter strain, three β-lactamases encoding genes were identified [using ROSY; [12]]. Thus, the inactivation of the antibiotics via degradation by β-lactamases seems to be an intrinsic resistance mechanism. Susceptibility not of the Roseobacter strains differed towards the four tested aminoglycosides. R. denitrificans showed no susceptibility to all tested aminoglycosides. In contrast R. litoralis and P. gallaeciensis were sensitive to this group of antibiotics. Growth of P. inhibens was inhibited by high concentrations of kanamycin, but the bacterium reacted very sensitive to spectinomycin and gentamicin. The D. shibae strains were resistant to kanamycin, but relatively sensitive to the three other aminoglycosides. O. indolifex was susceptible to all aminoglycosides. The resistance to the aminoglycoside gentamicin was already reported by Shiba [1991] as one of the characteristic properties of R. denitrificans. The corresponding genome exhibits a gene encoding for a putative aminoglycoside phosphotransferase, a type of enzyme inactivating aminoglycosides via modification [using IMG; [35], and ROSY; [12]].