Phosphate starvation-inducible proteins of Bacillus subtilis: Proteomics and transcriptional analysis

The phosphate starvation response in Bacillus subtilis was analyzed using t wo-dimensional (2D) polyacrylamide gel electrophoresis of cell extracts and supernatants from phosphate-starved cells. Most of the phosphate starvatio n-induced proteins are under the control of sigma(B), the activity of which is increased by energy depletion. In order to define the proteins belongin g to the Pho regulon, which is regulated by the two-component regulatory pr oteins PhoP and PhoR, the 2D protein pattern of the wild type was compared with those of a sigB mutant and a phoR mutant. By matrix-assisted laser des orption ionization-time of flight mass spectrometry, two alkaline phosphata ses (APases) (PhoA and PhoB), an APase-alkaline phosphodiesterase (PhoD), a glycerophosphoryl diester phosphodiesterase (GlpQ), and the lipoprotein Yd hF were identified as very strongly induced PhoPR-dependent proteins secret ed into the extracellular medium. In the cytoplasmic fraction, PstB1, PstB2 , and TuaD were identified as already known PhoPR-dependent proteins, in ad dition to PhoB, PhoD, and the previously described PstS. Transcriptional st udies of glpQ and ydhF confirmed the strong PhoPR dependence. Northern hybr idization and primer extension experiments showed that glpQ is transcribed monocistronically from a sigma(A) promoter which is overlapped by four puta tive TT(A/T)ACA-like PhoP binding sites. Furthermore, ydhF might be cotrans cribed with phoB initiating from the phoB promoter. Only a small group of p roteins remained phosphate starvation inducible in both phoR and sigB mutan t and did not form a unique regulation group. Among these, YfhM and YjbC we re controlled by sigma(B)-dependent and unknown PhoPR-independent mechanism s. Furthermore, YtxH and YvyD seemed to be induced after phosphate starvati on in the wild type in a sigma(B)-dependent manner and in the sigB mutant p robably via sigma(H). YxiE was induced by phosphate starvation independentl y of sigma(B) and PhoPR.