Osmostress response in Bacillus subtilis: characterization of a proline uptake system (OpuE) regulated by high osmolarity and the alternative transcription factor sigma B

Abstract

SummaryExogenously provided proline has been shown to serve as an osmoprotectant in Bacillus subtilis. Uptake of proline is under osmotic control and functions independently of the known transport systems for the osmoprotectant glycine betaine. We cloned the structural gene (opuE ) for this 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 longer 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 mutant revealed the presence of a second proline transport activity in B. subtilis. DNA sequence analysis of the opuE region showed that the OpuE transporter (492 residues) consists of a single integral membrane protein. 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 þ symport. The highest similarity was detected to the PutP proline permeases, which are used in Escherichia coli, Salmonella typhimurium and Staphylococcus aureus for the acquisition of proline as a carbon and nitrogen source, but not for osmoprotective purposes. An elevation of the osmolarity of the growth medium by either ionic or nonionic osmolytes resulted in a strong increase in the OpuE-mediated proline uptake. This osmoregulated proline transport activity was entirely dependent on de novo protein synthesis, suggesting a transcriptional control mechanism. Primer 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 the general stress transcription factor sigma B.

Osmoregulation of the opuE proline transport gene from Bacillus subtilis: contributions of the sigma A‐ and sigma B‐dependent stress‐responsive promoters

“…The ¹10 region of opuE P-1 (5Ј TAGGTT) was replaced by 5Ј CGACGA, a sequence completely different from the canonical ¹10 regions of A -dependent promoters. The ¹35 sequence of the opuE P-2 (5Ј GTTTCA) was altered to 5Ј GGTTGG, thereby changing a highly conserved T at the second position in ¹35 regions of B -dependent promoters to a G von Blohn et al, 1997). These alterations were introduced into the 330 bp opuE promoter fragment, which contains all cis regulatory elements necessary for opuE expression (Fig.…”

T-box-mediated control of the anabolic proline biosynthetic genes of Bacillus subtilis

“…Proline also serves as an important osmostress protectant (Bremer, 2002). For this task, proline can either be taken up by B. subtilis from the environment via the osmotically inducible OpuE transporter (Spiegelhalter & Bremer, 1998;von Blohn et al, 1997) or be produced in very large quantities from the precursor glutamate (Belitsky et al, 2001;Whatmore et al, 1990). Proline belongs to a selected group of organic osmolytes, the compatible solutes, which are amassed by many micro-organisms to adapt to high-osmolarity growth conditions (Kempf & Bremer, 1998).…”

Role of anionic phospholipids in the adaptation of Bacillus subtilis to high salinity

“…Osmoprotectants like glycine betaine and proline increase the cytoplasmic volume and the free water content of the cells at high salinity; therefore their accumulation allows cell proliferation under adverse environmental conditions (Record et al, 1998). In the case of B. subtilis, a family of transporters (OpuA-E) is involved in the transport of these compounds (Kappes et al, 1996;von Blohn et al, 1997).…”

“…Until recently studies concerning the adaptation of cells against osmotic stress have been essentially oriented to genetic and physiological regulation aspects (Csonka & Hanson, 1991;von Blohn et al, 1997;Glassker et al, 1998;Kappes et al, 1996;Price et al, 2001). Recently, a transcriptome analysis of the adaptation of B. subtilis cells to high salinity showed that at least 125 genes are induced, thus revealing the complexity of this response (Steil et al, 2003).…”