The pellicles were prevented from formation in the presence

of 100 μg/ml proteinase K (Figure 2A). Consistently, 100 μg/ml of the proteinase K was able to degrade the developed pellicles in 24 h, resulting in the semi-transparent membrane-like complexes (Figure 2A). In the control experiment, proteinase K at concentrations up to 300 μg/ml did not show a noticeable inhibitory influence on growth of S. oneidensis under agitated conditions. On the contrary, DNase I (up to 1000 U/ml) was not effective to inhibit pellicle formation or to degrade of the developed pellicles (data not shown), suggesting that DNA plays a negligible role in the process. Since proteinase K unspecifically removes polypeptides in the extracellular space and in the outer-membrane exposed to environments, the results could not conclude whether specific extracellular proteins are required for the process. Figure 2 EPS analysis. (A) Effects of proteinase K on pellicle RO4929097 formation and developed pellicles. Upper-panel, pellicle formation of the WT in static LB, in which the proteinase K was added at inoculation to 100 mg/ml (final concentration). Lower panel, developed pellicles of the WT (48 h after inoculation) were treated with 100 mg/ml (final concentration). (B) TLC analysis of monosaccharide in pellicles and supernatants. P and S represent JQ1 pellicle and supernatant, respectively. Man, gal, and glu

represent mannose, galactose, and glucose, respectively. Supernatants of the aggA mutant culture were included in the analysis. Attempts were made to solve the major polysaccharide components of S. oneidensis

pellicles by the thin layer chromatography (TLC) analysis. Culture supernatants and pellicles were collected independently after 36 h of growth and pellicles were then treated with 100 μg/ml proteinase K to removed cells. Polysaccharides were extracted and subjected to TLC analysis as described in Methods. A preliminary experiment was performed with six monosaccharides as standards, including ribose, mannose, glucose, galactose, rhamnose, and N-acetyl-glucosamine. The monosaccharides visualized on the TLC plates were close to mannose, glucose, and galactose (data not shown). To further confirm the observation, the experiment was conducted again with these three PRKACG monosaccharide standards only. As shown in Figure 2B the major monosaccharides identified were most likely to be mannose in both supernatants and pellicles. To validate this result, the aggA mutant, a pellicle-less strain was included in the analysis and the same result was obtained. These data suggest that the mannose-rich polysaccharides identified in pellicles are not pellicle specific. Certain metal cations are required for pellicle formation in S. oneidensis On the basis that metal cations are of general importance in biofilm formation, we examined the effects of certain metal cations on pellicle formation of S. oneidensis.