Quantitative RT-PCR was used to evaluate the expression of genes involved in the production of EPS I and EPS II in biofilms of Rm1021 and its mucR mutant. Expression of expE2
or exoY gene in biofilms grown in RDM medium (0.015 M sucrose, 12.5 mM phosphate), and RDM supplemented with 0.3 M sucrose or 25 mM phosphate was analyzed as described in M&M. The expE2 gene encodes a glycosyltransferase involved in the synthesis of EPS II. The exoY gene www.selleckchem.com/products/PD-98059.html encodes a galactosyltransferase responsible for incorporation of the first galactose into the intermediate lipid in EPS I biosynthesis. Introduction of a mutation in the MucR regulator in Rm1021 led to increased transcription of the expE2 gene, relative to the wild type. Transcription of expE2 in biofilms KPT-330 nmr formed by the mucR mutant was enhanced by addition of 0.3 M sucrose to culture medium, but was reduced by a high phosphate concentration (Fig. 5). This is consistent with the findings that increased phosphate availability in planktonic bacteria blocks EPS II synthesis (Zhan et al., 1991; Mendrygal & González, 2000), and thereby reduces expression of the genes responsible for EPS II production. Because expE2 is actively transcribed in biofilms of Rm1021 mucR (a strain that produces HMW EPS II) grown in RDM medium, and biofilm formation in Rm1021 mucR
is similar to that in the wild type (present study, and Rinaudi & González, 2009), the above finding confirms that the HMW fraction of EPS II produced by the mucR mutant is not involved in biofilm formation. exoY expression in biofilms of the mucR mutant was less than that in Rm1021 (Fig. 5). This result is consistent with previous observations that MucR promotes EPS I synthesis in planktonic bacteria (Bertram-Drogatz et al., 1998). On the other hand, exoY expression was not activated by 25 mM phosphate (Fig. 5), suggesting that higher concentrations of phosphate are needed for induction of EPS I production. Mendrygal & González (2000) reported that S. meliloti achieves the maximal production of EPS I at phosphate concentrations higher than those used in the present study. In conclusion, HAS1 our findings suggest that in vitro polyvinylchloride attachment
by Rm1021 does not depend on exopolysaccharide synthesis under our experimental conditions. In contrast, Fujishige et al. (2006) found that succinoglycan (EPS I) is involved in biofilm development. This apparent discrepancy may be explained by the fact that sucrose concentration in RDM medium for S. meliloti growth was 2% in the Fujishige study, but only 0.5% in the present study. High levels of sucrose in culture medium have been reported to cause increased exopolysaccharide synthesis in other microorganisms (van Geel-Schutten et al., 1998; Lee et al., 2003; Gross & Rudolph, 2008), probably as a result of facilitated carbon uptake. Under our assay conditions, mucR gene expression and regulation of exopolysaccharide biosynthesis do not appear to be crucial for biofilm formation in S.