In previous works we showed that a V. cholera wild-type strain expresses a number of phosphate (Pi) starvation-induced functions not directly related to Pi metabolism. Those were not expressed by an isogenic mutant in phoB. PhoB is a member of the system Pho/PhoR, which regulates expression of the Pho regulon genes, involved in the acquisition and transport of Pi. However, a crescent number of PhoB-dependent functions, not clearly related to Pi metabolism, have been described in many other bacterial species, suggesting that PhoB/PhoR might have additional roles in those cells.

In order to have a broader picture of the PhoB/PhoR functions in V. cholerae, we initiated a proteomic analysis of a wild-type V. cholerae strain and of an isogenic phoB mutant in cells grown overnight in a rich medium (LB) and in a medium with high levels of Pi (TGHP) at 37ºC. Lysate proteins of cells grown in TGHP were then separated by two dimensional gel electrophoresis (2D) at a pH range 4-7 and SDS-polyacrilamide gradient gel (12-14%). Most proteins on the gels presented pIs between 4,5-6,5 and Mr from 97-20 KDa and many of them were differentially expressed. These proteins were removed from the gels, trypsin digested, analyzed by mass spectrometry (MALDI-TOF) and identified in the vibrio sps databank entries.

Simultaneously, we compared the abilities of both strains to form biofilm, also in LB and TGHP. The biofilm assay was carried out in microtiter plates, for 24h at 22, 30 and 37ºC. After incubation, loose cells were removed, the wells were washed and the biofilm on the wells surfaces were stained with a solution of crystal violet. The coloured biofilm was solubilized in chloroform and biofilm production was estimated by OD ₅₇₀ nm. Both strains formed similar amounts of biofilm in TGHP at 22, 30 and 37ºC. In LB, however, the phoB mutant produced higher quantities of biofilm at the three temperatures, with a maximum at 37ºC. The proteins in the biofilms produced by the wild-type and phoB mutant in LB at 37ºC, as well as, proteins from cells grown in liquid LB and TGHP, will be further analysed by 2D electrophoresis, so that we can extend our understanding of the roles of PhoR/PhoR in V. cholerae.