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.

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.

It was found that low concentrations of the naturally occurring and structurally related betaines L-carnitine, crotonobetaine and y-butyrobetaine conferred a high degree of osmotic tolerance to Bacillus subtilis. Kinetic analysis of ~-[N-rnethyl-~~C]carnitine uptake in cells grown in minimal medium revealed the presence of a high-aff inity transport system with a K, value of 5 pM and a maximum rate of transport (V,,) of 41 nmol min'l (mg protein)''. A rise in medium osmolarity moderately increased the maximum velocity [V,, 71 nmol min'l (mg protein) 'l] of this transport system, but had little effect on its affinity. Growth and transport studies with a set of strains that carried defined mutations in the previously identified glycine betaine transport systems OpuA, OpuC and OpuD allowed the identification of the ATP-binding cassette (ABC) transport system OpuC as the only uptake route for L-carnitine in B. subtilis. Competition experiments with crotonobetaine and ybutyrobetaine revealed that the OpuC system also exhibited a high affinity for these trimethylammonium compounds with Ki values of 6 4 pM. Tracer experiments with radiolabelled L-carnitine and 13C-NMR tracings of cell extracts demonstrated that these betaines are accumulated by B. subtilis in an unmodified form. In contrast, the /?-substituted acylcarnitine esters acetylcarnitine and octanoylcarnitine both functioned as osmoprotectants for B. subtilis but were found to be accumulated as carnitine by the cells. None of these trimethylammonium compounds were used as sole carbon or nitrogen sources. The results thus characterize L-carnitine, crotonobetaine and y-butyrobetaine as effective compatible solutes for B. subtilis and establish a crucial role of the ABC transport system OpuC for the supply of B. subtilis with a variety of osmoprotectants.Keywords : compatible solutes, osmoprotection, ABC transporters, carnitine, acylcarnitines INTRODUCTIONA decrease in the water content of soil imposes a considerable stress on soil-living micro-organisms : water exits from the cells, resulting in decreased turgor and cessation of growth (Miller & Wood, 1996). Bacillus subtilis actively modulates the osmolarity of its cytoplasm under these adverse circumstances through uptake of K+ (Whatmore & Reed, 1990) (Kappes et al., 1996). A glycine betaine transporter closely related to OpuD has recently also been described in the soil bacterium Corynebacterium glutamicum (Peter et al., 1996). The Growth conditions, media and chemicals. The bacterial strains were grown in Spizizen's minimal medium (SMM) with 0 5 % (w/v) glucose as the carbon source and supplemented with L-tryptophan (20 pg ml-') , L-phenylalanine (18 pg ml-l) and a solution of trace elements (Harwood & Archibald, 1990). The osmotic strength of SMM was increased by the addition of NaCl from stock solutions. The osmolarity of growth media was determined with a vapour pressure osmometer (model 5500 ; Wescor) and the osmolarity of SMM, SMM with 0-4M NaCl and SMM with 1.2 M NaCl was 340 mosmol kg-l, 1100 mosm...