All microorganisms possess a positive turgor, and maintenance of this outward-directed pressure is essential since it is generally considered as the driving force for cell expansion. Exposure of microorganisms to high-osmolality environments triggers rapid fluxes of cell water along the osmotic gradient out of the cell, thus causing a reduction in turgor and dehydration of the cytoplasm. To counteract the outflow of water, microorganisms increase their intracellular solute pool by amassing large amounts of organic osmolytes, the so-called compatible solutes. These osmoprotectants are highly congruous with the physiology of the cell and comprise a limited number of substances including the disaccharide trehalose, the amino acid proline, and the trimethylammonium compound glycine betaine. The intracellular amassing of compatible solutes as an adaptive strategy to high-osmolality environments is evolutionarily well-conserved in Bacteria, Archaea, and Eukarya. Furthermore, the nature of the osmolytes that are accumulated during water stress is maintained across the kingdoms, reflecting fundamental constraints on the kind of solutes that are compatible with macromolecular and cellular functions. Generally, compatible solutes can be amassed by microorganisms through uptake and synthesis. Here we summarise the molecular mechanisms of compatible solute accumulation in Escherichia coli and Bacillus subtilis, model organisms for the gram-negative and gram-positive branches of bacteria.

The accumulation of the osmoprotectant glycine betaine from exogenous sources provides a high degree of osmotic tolerance to Bacillus subtilis. We have identified, through functional complementation of an Escherichia coli mutant defective in glycine betaine uptake, a new glycine betaine transport system from B. subtilis. The DNA sequence of a 2,310-bp segment of the cloned region revealed a single gene (opuD) whose product (OpuD) was essential for glycine betaine uptake and osmoprotection in E. coli. The opuD gene encodes a hydrophobic 56.13-kDa protein (512 amino acid residues). OpuD shows a significant degree of sequence identity to the choline transporter BetT and the carnitine transporter CaiT from E. coli and a BetT-like protein from Haemophilus influenzae. These membrane proteins form a family of transporters involved in the uptake of trimethylammonium compounds. The OpuD-mediated glycine betaine transport activity in B. subtilis is controlled by the environmental osmolarity. High osmolarity stimulates de novo synthesis of OpuD and activates preexisting OpuD proteins to achieve maximal glycine betaine uptake activity. An opuD mutant was constructed by marker replacement, and the OpuD-mediated glycine betaine uptake activity was compared with that of the previously identified multicomponent OpuA and OpuC (ProU) glycine betaine uptake systems. In addition, a set of mutants was constructed, each of which synthesized only one of the three glycine betaine uptake systems. These mutants were used to determine the kinetic parameters for glycine betaine transport through OpuA, OpuC, and OpuD. Each of these uptake systems shows high substrate affinity, with K m values in the low micromolar range, which should allow B. subtilis to efficiently acquire the osmoprotectant from the environment. The systems differed in their contribution to the overall glycine betaine accumulation and osmoprotection. A triple opuA, opuC, and opuD mutant strain was isolated, and it showed no glycine betaine uptake activity, demonstrating that three transport systems for this osmoprotectant operate in B. subtilis.

SummaryBiosynthesis of the compatible solute glycine betaine in Bacillus subtilis confers a considerable degree of osmotic tolerance and proceeds via a two-step oxidation process of choline, with glycine betaine aldehyde as the intermediate. We have exploited the sensitivity of B. subtilis strains defective in glycine betaine production against glycine betaine aldehyde to select for mutants resistant to this toxic intermediate. These strains were also defective in choline uptake, and genetic analysis proved that two mutations affecting different genetic loci (opuB and opuC ) were required for these phenotypes. Molecular analysis allowed us to demonstrate that the opuB and opuC operons each encode a binding protein-dependent ABC transport system that consists of four components. The presumed binding proteins of both ABC transporters were shown to be lipoproteins. Kinetic analysis of [ 14 C]-choline uptake via OpuB (K m 1 mM; V max 21 nmol min À1 mg À1 protein) and OpuC (K m 38 mM;V max 75 nmol min À1 mg À1 protein) revealed that each of these ABC transporters exhibits high af®nity and substantial transport capacity. Western blotting experiments with a polyclonal antiserum cross-reacting with the presumed substrate-binding proteins from both the OpuB and OpuC transporter suggested that the expression of the opuB and opuC operons is regulated in response to increasing osmolality of the growth medium. Primer extension analysis con®rmed the osmotic control of opuB and allowed the identi®cation of the promoter of this operon. The opuB and opuC operons are located close to each other on the B. subtilis chromosome, and their high sequence identity strongly suggests that these systems have evolved from a duplication event of a primordial gene cluster. Despite the close relatedness of OpuB and OpuC, these systems exhibit a striking difference in substrate speci®city for osmoprotectants that would not have been predicted readily for such closely related ABC transporters.

Exogenously provided glycine betaine functions as an efficient osmoprotectant for Bacillus subtilis in high-osmolarity environments. This gram-positive soil organism is not able to increase the intracellular level of glycine betaine through de novo synthesis in defined medium (A. M. Whatmore, J. A. Chudek, and R. H. Reed, J. Gen. Microbiol. 136:2527Microbiol. 136: -2535Microbiol. 136: , 1990 Exogenously provided choline significantly increased the growth rate ofB. subtUis in high-osmolarity media and permitted its proliferation under conditions that are otherwise strongly inhibitory for its growth. Choline and glycine betaine were not used as sole sources of carbon or nitrogen, consistent with their functional role in the process of adaptation of B. subtilis to high-osmolarity stress.

Exogenously provided glycine betaine can efficiently protect Bacillus subtilis from the detrimental effects of high osmolarity environments. Through functional complementation of an Escherichia coli mutant deficient in glycine betaine uptake with a gene library from B. subtilis, we have identified a multicomponent glycine betaine transport system, OpuA. Uptake of radiolabeled glycine betaine in B. subtilis was found to be osmotically stimulated and was strongly decreased in a mutant strain lacking the OpuA transport system. DNA sequence analysis revealed that the components of the OpuA system are encoded by anoperon (opuA) comprising three structural genes: opuAA, opuAB, and opuAC. The products of these genes exhibit features characteristic for binding protein-dependent transport systems and in particular show homology to the glycine betaine uptake system ProU from E. coli. Expression of the opuA operon is under osmotic control. The transcriptional initiation sites of opuA were mapped by high resolution primer extension analysis, and two opuA mRNAs were detected that differed by 38 base pairs at their 5' ends. Synthesis of the shorter transcript was strongly increased in cells grown at high osmolarity, whereas the amount of the longer transcript did not vary in response to medium osmolarity. Physical and genetic mapping experiments allowed the positioning the opuA operon at 25 degrees on the genetic map of B. subtilis.

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.

Synthesis of the osmoprotectant glycine betaine from the exogenously provided precursor choline or glycine betaine aldehyde confers considerable osmotic stress tolerance to Bacillus subtilis in high-osmolarity media. Using an Escherichia coli mutant (betBA) defective in the glycine betaine synthesis enzymes, we cloned by functional complementation the genes that are required for the synthesis of the osmoprotectant glycine betaine in B. subtilis. The DNA sequence of a 4.1-kb segment from the cloned chromosomal B. subtilis DNA was established, and two genes (gbsA and gbsB) whose products were essential for glycine betaine biosynthesis and osmoprotection were identified. The gbsA and gbsB genes are transcribed in the same direction, are separated by a short intergenic region, and are likely to form an operon. The deduced gbsA gene product exhibits strong sequence identity with members of a superfamily of specialized and nonspecialized aldehyde dehydrogenases. This superfamily comprises glycine betaine aldehyde dehydrogenases from bacteria and plants with known involvement in the cellular adaptation to high-osmolarity stress and drought. The deduced gbsB gene product shows significant similarity to the family of type III alcohol dehydrogenases. B. subtilis mutants with defects in the chromosomal gbsAB genes were constructed by marker replacement, and the growth properties of these mutant strains in high-osmolarity medium were analyzed. Deletion of the gbsAB genes destroyed the cholineglycine betaine synthesis pathway and abolished the ability of B. subtilis to deal effectively with high-osmolarity stress in choline-or glycine betaine aldehyde-containing medium. Uptake of radiolabelled choline was unaltered in the gbsAB mutant strain. The continued intracellular accumulation of choline or glycine betaine aldehyde in a strain lacking the glycine betaine-biosynthetic enzymes strongly interfered with the growth of B. subtilis, even in medium of moderate osmolarity. A single transcription initiation site for gbsAB was detected by high-resolution primer extension analysis. gbsAB transcription was initiated from a promoter with close homology to A -dependent promoters and was stimulated by the presence of choline in the growth medium.

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