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.

Proteome analysis of Bacillus subtilis cells grown at low and high salinity revealed the induction of 16 protein spots and the repression of 2 protein spots, respectively. Most of these protein spots were identified by mass spectrometry. Four of the 16 high-salinity-induced proteins corresponded to DhbA, DhbB, DhbC, and DhbE, enzymes that are involved in the synthesis of 2,3-dihydroxybenzoate (DHB) and its modification and esterification to the iron siderophore bacillibactin. These proteins are encoded by the dhbACEBF operon, which is negatively controlled by the central iron regulatory protein Fur and is derepressed upon iron limitation. We found that iron limitation and high salinity derepressed dhb expression to a similar extent and that both led to the accumulation of comparable amounts of DHB in the culture supernatant. DHB production increased linearly with the degree of salinity of the growth medium but could still be reduced by an excess of iron. Such an excess of iron also partially reversed the growth defect exhibited by salt-stressed B. subtilis cultures. Taken together, these findings strongly suggest that B. subtilis cells grown at high salinity experience iron limitation. In support of this notion, we found that the expression of several genes and operons encoding putative iron uptake systems was increased upon salt stress. The unexpected finding that high-salinity stress has an iron limitation component might be of special ecophysiological importance for the growth of B. subtilis in natural settings, in which bioavailable iron is usually scarce.The soil bacterium Bacillus subtilis often experiences fluctuations in the osmolality of its habitat due to the drying and flooding of the upper layers of the soil (7,40). A rise in the external salinity and osmolality triggers the outflow of water from the cell, resulting in a reduction in turgor and dehydration of the cytoplasm. To cope with these unfavorable osmotic conditions, B. subtilis initiates a two-step adaptation response (7, 32). Initially, K ϩ is rapidly taken up (56) and subsequently replaced in part by proline (55), a member of the so-called compatible solutes (19). These osmolytes can be accumulated to high levels through either de novo synthesis or uptake of preformed osmoprotectants from the environment without interfering with central cellular functions. For B. subtilis, proline serves as the primary endogenously synthesized compatible solute (55), and large quantities are produced via a dedicated osmostress-responsive synthesis pathway (J. Brill and E. Bremer, unpublished results). In addition, B. subtilis can efficiently scavenge a wide variety of compatible solutes from environmental sources by means of five osmoregulated transport systems (OpuA to OpuE) (29-31, 41, 54) and can acquire choline for the production of the osmoprotectant glycine betaine (4, 30). The accumulation of compatible solutes offsets the detrimental effects of high osmolality on cell physiology and permits growth over a wide range of osmotic conditions (3,36).Under...

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