cholerae biofilms. Because we showed that phosphate limitation enhanced the expression of HapR, we considered the possibility of PhoB negatively affecting biofilm formation by increasing the expression of HapR. To test this possibility, we allowed the wild
type and ΔphoB mutant to form static biofilms and determined the expression of HapR in the planktonic and adherent subpopulations. As shown in Fig. 2d, no differences were found between the wild BAY 80-6946 purchase type and mutant strain, which leads to the conclusion that expression of PhoB does not negatively affect biofilm formation by enhancing the expression of HapR. Given that HapR and PhoB appear to negatively affect biofilm formation in different ways, we used laser confocal microscopy to examine the three-dimensional architecture of wild-type, ΔphoB and ΔhapR biofilms under phosphate limitation. As shown in Fig. 3, under these conditions the wild-type strain adhered poorly, while the ΔphoB mutant formed a more uniform monolayer. As expected, the ΔhapR mutant displayed an enhanced biofilm-forming phenotype. These findings suggest that PhoB might negatively affect biofilm formation by interfering with early events that mediate adherence and monolayer formation. It has been suggested that surface attachment can trigger the expression of additional genes and regulators required for
exopolysaccharide matrix biosynthesis and development of a mature biofilm (Watnick & Kolter, 1999). Thus, we decided to examine the effect of PhoB on the expression of known regulators of exopolysaccharide biosynthesis and www.selleckchem.com/products/apo866-fk866.html biofilm formation. Given the complex regulatory circuitry controlling biofilm formation and exopolysaccharide biosynthesis, we used qRT-PCR to compare the effects of PhoB and HapR on regulators of exopolysaccharide gene expression and biofilm formation. In agreement with our previous results (Liang
et al., 2007b), deletion of hapR enhanced the expression of vpsA, vpsL and the positive regulator vpsT, but had little effect on the expression of vpsR and cytR (Fig. 4). In concurrence Sodium butyrate with results presented in Fig. 2d, deletion of phoB did not affect the expression of hapR. Deletion of phoB also enhanced the expression of vpsA and vpsL (Fig. 4). However, contrary to the deletion of hapR, elimination of phoB had little effect on the expression of vpsT and cytR, but significantly enhanced vpsR (Fig. 4). These results further support the conclusion that HapR and PhoB independently diminish biofilm formation through distinct pathways that converge to diminish the expression of vpsA and vpsL. In addition to the formation of biofilm communities that provide protection to many environmental stressors at a population or social level, the general stress response contributes to environmental stress survival by armoring individual cells with the biochemical activities required to provide protection against many environmental stressors.