Osmotic Control of opuA Expression in Bacillus subtilis and Its Modulation in Response to Intracellular Glycine Betaine and Proline Pools

Abstract

bGlycine betaine is an effective osmoprotectant for Bacillus subtilis. Its import into osmotically stressed cells led to the buildup of large pools, whose size was sensitively determined by the degree of the osmotic stress imposed. The amassing of glycine betaine caused repression of the formation of an osmostress-adaptive pool of proline, the only osmoprotectant that B. subtilis can synthesize de novo. The ABC transporter OpuA is the main glycine betaine uptake system of B. subtilis. Expression of opuA was upregulated in response to both sudden and sustained increases in the external osmolarity. Nonionic osmolytes exerted a stronger inducing effect on transcription than ionic osmolytes, and this was reflected in the development of corresponding OpuA-mediated glycine betaine pools. Primer extension analysis and site-directed mutagenesis pinpointed the osmotically controlled opuA promoter. Deviations from the consensus sequence of SigA-type promoters serve to keep the transcriptional activity of the opuA promoter low in the absence of osmotic stress. opuA expression was downregulated in a finely tuned manner in response to increases in the intracellular glycine betaine pool, regardless of whether this osmoprotectant was imported or was newly synthesized from choline. Such an effect was also exerted by carnitine, an effective osmoprotectant for B. subtilis that is not a substrate for the OpuA transporter. opuA expression was upregulated in a B. subtilis mutant that was unable to synthesize proline in response to osmotic stress. Collectively, our data suggest that the intracellular solute pool is a key determinant for the osmotic control of opuA expression.

Dimethylglycine Provides Salt and Temperature Stress Protection to Bacillus subtilis

“…As a result of this two-tier level of regulation, microbial cells are provided with an increased capacity to import glycine betaine under both acute and sustained osmotic stress conditions. The accumulation of compatible solutes is sensitively tied to the severity of the environmentally imposed osmotic stress (20,21) and thereby allows the cell to relieve the harmful effects of high external salinity and osmolarity on turgor, physiology, and growth (14).…”

“…However, it served as an only modestly effective osmotic stress protectant. We identified the osmotically controlled ABC transporter OpuA (21,43) as the main entry route of DMG into the B. subtilis cell under osmotic and temperature stress conditions. Sarcosine, on the other hand, had no noticeable stress-relieving potential for either osmotically or thermally challenged cells.…”

The γ-Aminobutyrate Permease GabP Serves as the Third Proline Transporter of Bacillus subtilis

“…The intracellular proline content of B. subtilis cells was determined by a colorimetric assay that detects L-proline as a colored proline-ninhydrin complex that can be quantified by measuring the absorption of the solution at 480 nm (45). For these assays, the B. subtilis cells were grown in SMM with 0.4 M NaCl; harvesting and processing of the cells, the details of the assay conditions, and the calculation of the intracellular volume have been described previously (15,21,53). Consistent with previous measurements (21), the proline pool of strain JH642 was found to be 95 Ϯ 12 mM in cells that had been grown in SMM containing 0.4 M NaCl; the concentrations of proline pools detected in the 15 tested DHP-resistant mutant strains ranged from 79 Ϯ 4 mM to 98 Ϯ 5 mM.…”

“…The anabolic proline synthesis route (ProB-ProA-ProI [ProG]) is interconnected with the osmostress-relieving production route for proline (ProJ-ProA-ProH) via the ␥-glutamyl phosphate reductase (ProA) (14). The latter pathway is osmotically controlled and allows B. subtilis to produce very large amounts of the compatible solute proline (20) to fend off the detrimental effects of high salinity on cellular hydration, turgor, and physiology (13)(14)(15)21). Proline-mediated osmoprotection can also be achieved through uptake; the osmotically inducible OpuE transporter of B. subtilis serves this function (15,16).…”

Liberate and Grab It, Ingest and Digest It: the GbdR Regulon of the Pathogen Pseudomonas aeruginosa

“…Transporters for glycine betaine allow microorganisms to make effective use of precious environmental resources (10), and their high affinity and osmotic control (5,9,11,12) permit the amassing of their substrate(s) against steep concentration gradients and to pool sizes that are correlated with the severity of the imposed osmotic stress (13). Many microorganisms can also synthesize glycine betaine.…”