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...

Positive and negative regulatory elements involved in the synthesis of colanic acid, the capsular polysaccharide ofEscherichia coli K-12, have been identified previously. RcsB, a positive regulator for transcription of the structural genes of colanic acid synthesis (cps), is a protein of about 26 kilodaltons which probably acts as a multimer. rcsC, which maps close to rcsB at 48 min on the E. coli chromosome, exerts a negative effect on expression of the structural genes and codes for a protein of about 100 kilodaltons. The two genes appear to be transcribed in opposite directions, with the C-terminal ends of the genes being less than 0.3 kilobases apart. Multicopy expression of rcsB is lethal in rcsC mutants which carry cps-lac fusions, probably owing to accumulation of intermediates in the capsule synthesis pathway in these cells. Examination of double mutants and cells carrying multicopy rcsB+ plasmids reveal an rcsA-independent pathway for capsule synthesis. We hypothesize that RcsC may act as an environmental sensor, transmitting information to the RcsB positive regulator.The capsular polysaccharide colanic acid is synthesized by a variety of enteric bacteria. Although a function for colanic acid has not been clearly demonstrated, similar capsules in other gram-negative organisms serve to protect cells from dehydration, from bacteriophage infection, and from cellular immunity systems (3, 10, 17). In plant pathogens such as Erwinia stewartii, the capsule acts as an important virulence factor (4, 9). Capsular polysaccharides have also been implicated in the plant-bacterium interactions of Rhizobium species (21).We have been investigating the regulation of colanic acid synthesis in Escherichia coli K-12 by monitoring the expression of lac operon fusions to genes necessary for colanic acid synthesis (cpsA to cpsF) (13,39,41). Using these fusions, we have identified four loci which have major effects on cps-lac expression. Two negative regulators, lon and rcsC, and two positive regulators, rcsA and rcsB, have been identified (13,41).Lon is a major ATP-dependent protease in E. coli (6,8,11). Among the pleiotropic effects of lon are filamentation after DNA-damaging treatments such as UV irradiation and methyl methanesulfonate treatment, failure to degrade abnormal and some normal proteins, and the mucoidy associated with overproduction of colanic acid (12,15,16,25). We have hypothesized that the stabilization of regulatory proteins in lon mutants is responsible for the phenotypes of lon cells (14). SulA, an inhibitor of cell septation induced after DNA damage, seems to be responsible for the filamentation phenotype; it has a half-life of 1. The other two regulators of capsule synthesis, rcsB and rcsC, map near to each other at 48 min and have opposite effects on the synthesis of capsular polysaccharide. Mutations in rcsB, like those in rcsA, reduce synthesis in lon cells, whereas rcsC mutations increase expression in lon+ hosts. Preliminary complementation studies with these mutations, carried out with a cosmid v...

The complete Bacillus subtilis genome contains four genes (proG, proH, proI, and comER) with the potential to encode ⌬ 1 -pyrroline-5-carboxylate reductase, a proline biosynthetic enzyme. Simultaneous defects in three of these genes (proG, proH, and proI) were required to confer proline auxotrophy, indicating that the products of these genes are mostly interchangeable with respect to the last step in proline biosynthesis.The pathway of proline synthesis from glutamate, the most common mechanism of proline biosynthesis, comprises three enzymatic steps (Fig. 1). The corresponding genes of Escherichia coli, proB, proA, and proC, encode ␥-glutamyl kinase, ␥-glutamyl phosphate reductase, and ⌬ 1 -pyrroline-5-carboxylate (P5C) reductase, respectively (21). The proBA-dependent pathway of proline synthesis was shown to function also in Bacillus subtilis; mutations within the proBA locus cause auxotrophy for proline (8,29). While B. subtilis has a single proAlike gene, a second proB-like gene, proJ of the proHJ locus (B. R. Belitsky and A. L. Sonenshein, GenBank accession number AF006720) has been found. In a manner unique to this bacterium, either ProB-like enzyme can provide enough ␥-glutamyl kinase activity to support growth in the absence of exogenous proline (unpublished results). Apparently, previously described mutations to auxotrophy in the proBA locus either affect proA or are proB alleles that are polar on proA expression. No proC mutant of B. subtilis has been described, and four genes have the potential to encode ProC-like proteins with P5C reductase activity: proH (also called orf257 and proC), comER (also called comED), proI (also called yqjO), and ykeA (here renamed proG) (1,14,20). The four genes are located at 172.3°, 225.5°, 211.2°, and 116.1°on the chromosomal map (http://genolist.pasteur.fr/SubtiList [26]) and code for proteins of 271, 273, 278, and 272 amino acids, respectively (the originally reported coding region of proH [1,20] was extended by resequencing the proH 3Ј end [GenBank accession number AF006720]). ProH and ProI are 42% identical to each other and up to 35% identical to many other P5C reductases from bacteria, archaea, and eukaryotes. ProG and ComER have more limited similarity to other P5C reductases and to each other. The functions of the four B. subtilis genes are not known. In this work we sought to identify the gene(s) responsible for the last step of proline biosynthesis.Construction and properties of a proG (ykeA) null mutant. To create pBB1081, the 1.56-kb PvuII-EcoRI fragment from pCM103 (23) containing most of the proG gene and the 5Ј end of the dppA gene was cloned between the PstI (blunt-ended) and EcoRI sites of pJPM1, a derivative of pBS (Stratagene) containing a chloramphenicol resistance marker (27). Methods for plasmid isolation, agarose gel electrophoresis, use of restriction and DNA modification enzymes, DNA ligation, PCR, Southern hybridization with digoxigenin-labeled DNA probes, and electroporation of E. coli JM107 or DH5␣ cells were as described by Sambrook et al....

Bacillus subtilis can attain cellular protection against the detrimental effects of high osmolarity through osmotically induced de novo synthesis and uptake of the compatible solute L-proline. We have now found that B. subtilis can also exploit exogenously provided proline-containing peptides of various lengths and compositions as osmoprotectants. Osmoprotection by these types of peptides is generally dependent on their import via the peptide transport systems (

Bacillus subtilis possesses interlinked routes for the synthesis of proline. The ProJ-ProA-ProH route is responsible for the production of proline as an osmoprotectant, and the ProB-ProA-ProI route provides proline for protein synthesis. We show here that the transcription of the anabolic proBA and proI genes is controlled in response to proline limitation via a T-box-mediated termination/antitermination regulatory mechanism, a tRNA-responsive riboswitch. Primer extension analysis revealed mRNA leader transcripts of 270 and 269 nt for the proBA and proI genes, respectively, both of which are synthesized from SigA-type promoters. These leader transcripts are predicted to fold into two mutually exclusive secondary mRNA structures, forming either a terminator or an antiterminator configuration. Northern blot analysis allowed the detection of both the leader and the full-length proBA and proI transcripts. Assessment of the level of the proBA transcripts revealed that the amount of the full-length mRNA species strongly increased in proline-starved cultures. Genetic studies with a proB-treA operon fusion reporter strain demonstrated that proBA transcription is sensitively tied to proline availability and is derepressed as soon as cellular starvation for proline sets in. Both the proBA and the proI leader sequences contain a CCU proline-specific specifier codon prone to interact with the corresponding uncharged proline-specific tRNA. By replacing the CCU proline specifier codon in the proBA T-box leader with UUC, a codon recognized by a Phe-specific tRNA, we were able to synthetically re-engineer the proline-specific control of proBA transcription to a control that was responsive to starvation for phenylalanine.