To determine the relationship between PhoB and the general stress response in V. cholerae, we compared the ability of isogenic wild type, ΔphoB and ΔrpoS mutants to withstand environmental stresses. In Fig. 5 we show that, while strain SZS007 (wild type) and its isogenic ΔrpoS were similarly sensitive to 0.5 mM hydrogen peroxide, the ΔphoB was significantly more sensitive to this level of oxidative stress. The ΔrpoS mutant was more sensitive

than the wild type to higher hydrogen peroxide concentrations (data not shown). This result indicates that expression of PhoB provides protection to oxidative stress by a mechanism different from RpoS. As expected from previous studies (Yildiz & Schoolnik, BMS907351 1998; Jahid et al., 2006), the ΔrpoS mutant was more sensitive than the wild type to 1.2 M NaCl, while the ΔphoB mutant was more resistant (Fig. 5). The ΔphoB mutant was also more resistant than the wild-type strain and the ΔrpoS mutant to pH 4.5. Finally, the ΔrpoS mutant was more sensitive Caspase inhibitor to carbon starvation

compared with wild type and ΔphoB that behaved similarly with regard to this stress. Taken together, the above data are consistent with PhoB modulating environmental stress in a manner independent of the general stress response regulator RpoS. The finding that V. cholerae builds large poly-P stores and our previous observation that a mutant impaired in poly-P biosynthesis was more sensitive to environmental stressors in low-phosphate medium (Ogawa et al., 2000; Jahid et al., 2006) points toward phosphate starvation as a critical environmental stress that could impact the survival of V. cholerae in its aquatic Mannose-binding protein-associated serine protease habitat. Unfortunately, very little is known about how phosphate starvation affects V. cholerae behavior and life style. Extracellular orthophosphate is the major source of

high-energy phosphate for biosynthesis and many signal transduction pathways such as quorum sensing. Therefore, it was not surprising that phosphate limitation enhanced HapR expression, which is repressed when high-energy phosphate is transferred to LuxO. Phosphate starvation is known to activate PhoB and induce the transcription of the PhoB regulon (Lamarche et al., 2008). The fact that HapR represses biofilm formation (Waters et al., 2008) and PhoB has been shown to negatively affect biofilm formation in other Gram-negative bacteria (Monds et al., 2001, 2007) prompted us to examine the relationship between HapR, PhoB and biofilm formation. To this end, we constructed a ΔphoB mutant of strain SZS007. We did not observe any phenotype for this mutant in LB or high-phosphate medium (i.e. growth rate, motility, extracellular protease production). However, the mutant did exhibit reduced growth and alkaline phosphatase expression in low-phosphate medium containing the chromogenic substrate 5-bromo-4-chloro-3-indolyl phosphate (data not shown).