Bacillus subtilis is known to accumulate large amounts of the compatible solute proline via de novo synthesis as a stress protectant when it faces high-salinity environments. We elucidated the genetic determinants required for the osmoadaptive proline production from the precursor glutamate. This proline biosynthesis route relies on the proJ-encoded ␥-glutamyl kinase, the proA-encoded ␥-glutamyl phosphate reductase, and the proH-encoded ⌬ 1 -pyrroline-5-caboxylate reductase. Disruption of the proHJ operon abolished osmoadaptive proline production and strongly impaired the ability of B. subtilis to cope with high-osmolarity growth conditions. Disruption of the proA gene also abolished osmoadaptive proline biosynthesis but caused, in contrast to the disruption of proHJ, proline auxotrophy. Northern blot analysis demonstrated that the transcription of the proHJ operon is osmotically inducible, whereas that of the proBA operon is not. Reporter gene fusion studies showed that proHJ expression is rapidly induced upon an osmotic upshift. Increased expression is maintained as long as the osmotic stimulus persists and is sensitively linked to the prevalent osmolarity of the growth medium. Primer extension analysis revealed the osmotically controlled proHJ promoter, a promoter that resembles typical SigA-type promoters of B. subtilis. Deletion analysis of the proHJ promoter region identified a 126-bp DNA segment carrying all sequences required in cis for osmoregulated transcription. Our data disclose the presence of ProA-interlinked anabolic and osmoadaptive proline biosynthetic routes in B. subtilis and demonstrate that the synthesis of the compatible solute proline is a central facet of the cellular defense to high-osmolarity surroundings for this soil bacterium.The soil-dwelling bacterium Bacillus subtilis (20) is frequently exposed to osmotic fluctuations in its environment as a consequence of wetting and drying cycles of the upper layers of the soil (8). As a result of these changes in the osmolarity and salinity of the habitat, the B. subtilis cell has to cope with osmotically instigated water fluxes across the cytoplasmic membrane. Consequently, the integrity of the cell is threatened under hypo-osmotic conditions, or its growth is impaired under hyperosmotic conditions (8). No microorganism can actively pump water in or out of the cell to compensate for water fluxes caused by changes in the external osmotic condition. Instead, microorganisms determine the direction and scale of water permeation across the cytoplasmic membrane indirectly by actively controlling the osmotic potential of their cytoplasm (9,66). This is often accomplished under high-osmolarity conditions by first importing large amounts of potassium as an emergency stress reaction (9,41,66). Subsequently, the cell replaces part of the accumulated potassium by a distinct class of highly water-soluble organic compounds, the compatible solutes (11). These osmolytes have been specifically selected in the course of evolution as effective osmo-and cytoprote...

The Gram-positive endospore-forming bacterium Bacillus licheniformis can be found widely in nature and it is exploited in industrial processes for the manufacturing of antibiotics, specialty chemicals, and enzymes. Both in its varied natural habitats and in industrial settings, B. licheniformis cells will be exposed to increases in the external osmolarity, conditions that trigger water efflux, impair turgor, cause the cessation of growth, and negatively affect the productivity of cell factories in biotechnological processes. We have taken here both systems-wide and targeted physiological approaches to unravel the core of the osmostress responses of B. licheniformis. Cells were suddenly subjected to an osmotic upshift of considerable magnitude (with 1 M NaCl), and their transcriptional profile was then recorded in a time-resolved fashion on a genome-wide scale. A bioinformatics cluster analysis was used to group the osmotically up-regulated genes into categories that are functionally associated with the synthesis and import of osmostress-relieving compounds (compatible solutes), the SigB-controlled general stress response, and genes whose functional annotation suggests that salt stress triggers secondary oxidative stress responses in B. licheniformis. The data set focusing on the transcriptional profile of B. licheniformis was enriched by proteomics aimed at identifying those proteins that were accumulated by the cells through increased biosynthesis in response to osmotic stress. Furthermore, these global approaches were augmented by a set of experiments that addressed the synthesis of the compatible solutes proline and glycine betaine and assessed the growth-enhancing effects of various osmoprotectants. Combined, our data provide a blueprint of the cellular adjustment processes of B. licheniformis to both sudden and sustained osmotic stress.

bThe data presented here reveal a new facet of the physiological adjustment processes through which Bacillus subtilis can derive osmostress protection. We found that the import of proteogenic (Glu, Gln, Asp, Asn, and Arg) and of nonproteogenic (Orn and Cit) amino acids and their metabolic conversion into proline enhances growth under otherwise osmotically unfavorable conditions. Osmoprotection by amino acids depends on the functioning of the ProJ-ProA-ProH enzymes, but different entry points into this biosynthetic route are used by different amino acids to finally yield the compatible solute proline. Glu, Gln, Asp, and Asn are used to replenish the cellular pool of glutamate, the precursor for proline production, whereas Arg, Orn, and Cit are converted into ␥-glutamic semialdehyde/⌬ 1 -pyrroline-5-carboxylate, an intermediate in proline biosynthesis. The import of Glu, Gln, Asp, Asn, Arg, Orn, and Cit did not lead to a further increase in the size of the proline pool that is already present in osmotically stressed cells. Hence, our data suggest that osmoprotection of B. subtilis by this group of amino acids rests on the savings in biosynthetic building blocks and energy that would otherwise have to be devoted either to the synthesis of the proline precursor glutamate or of proline itself. Since glutamate is the direct biosynthetic precursor for proline, we studied its uptake and found that GltT, an Na ؉ -coupled symporter, is the main uptake system for both glutamate and aspartate in B. subtilis. Collectively, our data show how effectively B. subtilis can exploit environmental resources to derive osmotic-stress protection through physiological means. Bacillus subtilis is a resident of the upper layers of the soil and of the rhizosphere, and it can also efficiently colonize root surfaces (1-3). The blueprint of its genome (4) bears the hallmarks of a bacterium that can exploit many plant-produced compounds for its growth. Accordingly, a considerable portion of the coding capacity of the B. subtilis chromosome (5) is devoted to highaffinity import systems (6) that allow the scavenging of a wide spectrum of nutrients. Reoccurring and persisting high osmolarity in the soil ecosystem (7) is a situation in which B. subtilis can take advantage of the import of plant-produced compounds (8-10) for its physiological adjustment to these unfavorable environmental conditions (7, 11).As in many bacterial species (11-13), cellular adaptation of B. subtilis to both sudden and sustained increases in the external osmolarity involves a two-stage process (7,14). It initially encompasses the uptake of large quantities of potassium as an emergency stress reaction to curb water efflux (14, 15) and, subsequently, the replacement of part of this ion by organic osmolytes, such as proline (Pro) and glycine betaine (GB), to decrease the ionic strength of the cytoplasm and to optimize its solvent properties (14,(16)(17)(18)(19). These organic osmolytes, commonly referred to as compatible solutes, are highly compliant with cellular physiolog...

The ProJ and ProH enzymes of Bacillus subtilis catalyse together with ProA (ProJ-ProA-ProH), osmostress-adaptive synthesis of the compatible solute proline. The proA-encoded gamma-glutamyl phosphate reductase is also used for anabolic proline synthesis (ProB-ProA-ProI). Transcription of the proHJ operon is osmotically inducible whereas that of the proBA operon is not. Targeted and quantitative proteome analysis revealed that the amount of ProA is not limiting for the interconnected anabolic and osmostress-responsive proline production routes. A key player for enhanced osmostress-adaptive proline production is the osmotically regulated proHJ promoter. We used site-directed mutagenesis to study the salient features of this stress-responsive promoter. Two important features were identified: (i) deviations of the proHJ promoter from the consensus sequence of SigA-type promoters serve to keep transcription low under non-inducing growth conditions, while still allowing a finely tuned induction of transcriptional activity when the external osmolarity is increased and (ii) a suboptimal spacer length for SigA-type promoters of either 16-bp (the natural proHJ promoter), or 18-bp (a synthetic promoter variant) is strictly required to allow regulation of promoter activity in proportion to the external salinity. Collectively, our data suggest that changes in the local DNA structure at the proHJ promoter are important determinants for osmostress-inducibility of transcription.

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