Culture Conditions of Roseobacter Strain 27-4 Affect Its Attachment and Biofilm Formation as Quantified by Real-Time PCR

Abstract

The fish probiotic bacterium Roseobacter strain 27-4 grows only as rosettes and produces its antibacterial compound under static growth conditions. It forms three-dimensional biofilms when precultured under static conditions. We quantified attachment of Roseobacter strain 27-4 using a direct real-time PCR method and demonstrated that the bacteria attached more efficiently to surfaces during static growth than under aerated conditions. Bacteria belonging to the marine Roseobacter clade may produce antibacterial compounds (4, 5, 6) and have been suggested for use as a probiotic treatment in aquaculture (12,19,23,24). Roseobacter strains were isolated from a turbot rearing facility, and very similar DNA types were repeatedly isolated from tank walls over a 1-year period (13). One of these strains, Roseobacter strain 27-4, inhibits fish pathogenic bacteria by a sulfur-containing antibacterial compound (5, 12) and improves the survival of turbot larvae infected with Vibrio anguillarum (19). The antibacterial compound is produced only under static growth conditions, where the bacterium forms a thick biofilm at the air-liquid interface. The biofilm produced by Roseobacter strain 27-4 appears to be correlated to a multicellular growth pattern in which 9 to 10 cells grow in a rosette shape. The rosette is unstable, and shaking will break up the structure; during growth under aerated conditions, the organism grows as single cells and no inhibitory activity is detected (5).It is not known if the probiotic Roseobacter strain 27-4 retains its antibacterial properties when it attaches to inert surfaces; however, marine bacteria such as Roseobacter gallaeciensis and Pseudoalteromonas tunicata can prevent other bacteria from forming a biofilm (20). We hypothesize that interaction between Roseobacter and fish pathogenic bacteria may take place on surfaces in biofilms and that it potentially can exert its effect as a probiotic bacterium by colonizing the turbot larva rearing facilities. Here we describe the development of a realtime PCR method that allows quantification of Roseobacter strain 27-4 on solid surfaces. We also describe how culture conditions may influence biofilm formation of Roseobacter strain 27-4.Effect of preculture growth conditions on pigmentation. In this study, as in previous studies (5), Roseobacter strain 27-4 grown in marine broth (MB) (Difco 279110) under static conditions grew as rosettes consisting of 9 to 10 cells, produced a brown pigment, and formed a biofilm at the air-liquid interface. However, if the statically grown preculture was vigorously shaken before inoculation or if an aerated preculture was used, almost no pigment and biofilm formation were produced (data not shown). Furthermore, when an aerated preculture was used, almost no rosette-forming cell clusters were observed even after several days of static growth.Roseobacter strain 27-4 biofilm formation on plastic surfaces. Statically grown overnight cultures of Roseobacter strain 27-4 grown in MB were diluted to an optical density at 600 nm...

Tropodithietic Acid Production in Phaeobacter gallaeciensis Is Regulated by N-Acyl Homoserine Lactone-Mediated Quorum Sensing

“…This was supported by detection of antimicrobial activity only at high cell densities in the AHL producing Phaeobacter strain 27-4 (6,7). In contrast, Silicibacter sp.…”

Global occurrence and heterogeneity of the Roseobacter-clade species Ruegeria mobilis

“…), none of the R. mobilis strains produced pigment and none of the sterile-filtered supernatants were inhibitory to V. anguillarum. TDA, which is believed to be the prime agent of Roseobacter antibacterial activity Bruhn et al, 2006), was detected in all R. mobilis by liquid chromatography-tandem mass spectrometry and correlates with the presence of the central genes required for the biosynthesis of TDA, tdaBCDE, in all strains (Supplementary Table S8). In F1926, tdaBCDE are located on a plasmid, the 147 kb plasmid, as has been demonstrated for other TDA producers (Thole et al, 2012).…”

Expression of Tropodithietic Acid Biosynthesis Is Controlled by a Novel Autoinducer

“…Unexpectedly, we also found that TDA itself is required for the maximal expression of tda genes and that non-TM1040 Roseobacter clade and Pseudovibrio species producing TDA can cross-feed and elicit an increase in tda gene expression from TM1040. The latter suggests that TDA, which has antibiotic activity against many nonroseobacter marine bacteria (7,9,10,18), also functions as a chemical signal or quorum-sensing autoinducer molecule among several marine Rhodobacterales species. The use of TDA as an autoinducer in the marine bacteria has profound implications for their survival and adaptation, for the symbioses many of them engage in, and for marine ecosystems.…”