OSMOSTRESS RESPONSE IN BACILLUS-SUBTILIS - CHARACTERIZATION OF A PROLINE UPTAKE SYSTEM (OPUE) REGULATED BY HIGH OSMOLARITY AND THE ALTERNATIVE TRANSCRIPTION FACTOR SIGMA-B

Exogenously provided proline has been shown to serve as an osmoprotect ant in Bacillus subtilis. Uptake of proline is under osmotic control a nd functions independently of the known transport systems for the osmo protectant glycine betaine. We cloned the structural gene (opuE) for t his proline transport system and constructed a chromosomal opuE mutant by marker replacement. The resulting B. subtilis strain was entirely deficient in osmoregulated proline transport activity and was no longe r protected by exogenously provided proline, attesting to the central importance of OpuE for proline uptake in high-osmolarity environments. The transport characteristics and growth properties of the opuE mutan t revealed the presence of a second proline transport activity in B. s ubtilis. DNA sequence analysis of the opuE region showed that the OpuE transporter (492 residues) consists of a single integral membrane pro tein. Database searches indicated that OpuE is a member of the sodium/ solute symporter family, comprising proteins from both prokaryotes and eukaryotes that obligatorily couple substrate uptake to Na+ symport. The highest similarity was detected to the PutP proline permeases, whi ch are used in Escherichia coli, Salmonella typhimurium and Staphyloco ccus aureus for the acquisition of proline as a carbon and nitrogen so urce, but not for osmoprotective purposes. An elevation of the osmolar ity of the growth medium by either ionic or non-ionic osmolytes result ed in a strong increase in the OpuE-mediated proline uptake. This osmo regulated proline transport activity was entirely dependent on de novo protein synthesis, suggesting a transcriptional control mechanism. Pr imer extension analysis revealed the presence of two osmoregulated and tightly spaced opuE promoters. The activity of one of these promoters was dependent on sigma A and the second promoter was controlled by th e general stress transcription factor sigma B.