Salmonella typhimurium prop Gene Encodes a Transport System for

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1 JOURNAL OF BACTERIOLOGY, Dec. 1985, p Vol. 164, No /85/ $2./ Copyright 1985, American Society for Microbiology Salmonella typhimurium prop Gene Encodes a Transport System for the Osmoprotectant Betaine JOHN CAIRNEY,1 IAN R. BOOTH,2 AND CHRISTOPHER F. HIGGINS'* Department ofbiochemistry, University of Dundee, Dundee DDJ 4HN,l and Department of Microbiology, Marischal College, University of Aberdeen, Aberdeen AB9 las,2 Scotland Received 11 June 1985/Accepted 22 August 1985 Betaine (N,N,N-trimethylglycine) can be accumulated to high intracellular concentrations and serves an important osmoprotective function in enteric bacteria. We found that the prop gene of Salmonella typhimurium, originally identified as encoding a minor transport system for proline (permease PP-II), plays an important role in betaine uptake. Mutations in prop reduced the ability of betaine to serve as an osmoprotectant. Transport of betaine into the cells was also severely impaired in these mutants. The kinetics of uptake via PP-II suggest that betaine, rather than proline, is the important physiological substrate for this transport system. Betaine uptake via PP-II was regulated by osmotic pressure at two different levels: transcription of the prop gene was increased by increasing osmolarity, and, in addition, activity of the transport system itself was dependent upon the osmotic pressure of the medium. The specificity of the transport system was also altered by increasing osmolarity which enhanced the affinity for betaine while reducing that for proline. A number of diverse organisms have evolved similar strategies to combat the osmotic stresses they encounter when their environment is altered by increasing concentrations of salts or other substances (9, 13, 14, 25). In particular, many species accumulate high concentrations of compatible solutes in response to osmotic stress which are thought to restore turgor pressure across the cell membrane as well as stabilize enzyme function. It has been known for many years that L-proline can stimulate the growth of enteric bacteria in media of otherwise inhibitory osmotic strength (6). In addition, proline overproduction enhances osmotolerance of Salmonella typhimurium (7). More recently, betaine (N,N,N-trimethylglycine) has been shown to be a more effective osmoprotectant than proline and to accumulate to high concentrations in both halophilic bacteria and in the enteric bacteria Escherichia coli and Klebsiella pneumoniae in response to osmotic stress (11, 12, 18). Exogenous betaine is taken up by an active transport process in E. coli, and the rate of uptake is stimulated by increasing the osmotic pressure of the medium (18). However, the number of betaine transport systems, the genes encoding them, and the mechanisms of osmoregulation are unknown. In this paper we show that a major pathway for betaine uptake requires the function of the prop gene which was originally identified as encoding a minor transport system for proline. Three genetically distinct proline transport systems have been characterized in S. typhimurium. The major profile permease (PP-I) is encoded by the putp gene, located at 22 min on the chromosome adjacent to the puta gene which encodes proline oxidase (4, 19, 2). The puta gene product also serves as a negative effector of putp expression (15). Strains deficient in PP-I exhibit low-level proline transport via a second system, PP-II, which is abolished by mutations in the prop gene (1, 16). prop maps at 92 min on the S. typhimurium chromosome. In addition, a third proline permease, PP-III, encoded by the prou gene, has been reported to function in media of high osmolarity (8). However, we have recently shown that prou encodes a highaffinity transport system with betaine, rather than proline, as * Corresponding author its primary substrate (3). The precise roles of the multiple proline transport systems is unclear. In particular, PP-Il has a very poor affinity for proline (Km = 3,uM) and would not seem to contribute significantly to proline uptake in cells in which PP-I is functional (1, 4). We show here that PP-Il plays a major role in betaine uptake and that mutations in prop reduce the ability of betaine to serve as an osmoprotectant. prop-dependent betaine transport is regulated in response to osmotic pressure both at the level of transcription and by an alteration in the activity of the transport protein. The kinetics of betaine uptake via PP-II suggest that betaine, rather than proline, is the most important physiological substrate for this transport system. MATERIALS AND METHODS Bacterial strains and mnedia. All bacterial strains were derivatives of S. typhimurium LT2. Their genotypes and construction are listed in Table 1. Cells were grown with aeration in LB medium (17) at 3'C to avoid induction of Mu dl lysogens. Minimal glucose plates were based on the E mediun of Vogel and Bonner as described by Roth (22). Antibiotics were used in both rich and minimal medium at the following concentrations: tetracycline, 15,ug ml-'; carbenicillin (an analog of ampicillin, resistance to which is referred to in the text as Apr), 5,xg ml-'; kanamycin, 5 jig ml-'. For growth curves and transport assays cells were grown in low-osmolarity medium (LOM) containing 1 mnm glucose and any appropriate osmolytes. LOM is based on KONO medium (4) and contains.4 mm MgSO4, 6,uM (NH4)2SO4 FeSO4, 2 mm (NH4)2HP4, 1 mm bistrispropane, 1,ug of biotin ml-', 1 mm KCI, and 5 mm NaCl. Genetic techniques and strain construction. Transductions were carried out by using a high-transducing derivative of phage P22 int4 as described by Roth (22). When used as donors or recipients for transduction, gale strains were grown in LB medium supplemented with.2% galactose and.2% glucose to ensure efficient synthesis of phage receptors. After transduction of TnS or TnJO insertions from one strain to another, the location of the transposon insertion and the presence ofjust a single insertion in the transductant

2 VOL. 164, 1985 UPTAKE OF THE OSMOPROTECTANT BETAINE 1219 TABLE 1. Bacterial strains Strain' Genotype Construction or source CH223 gaie53 bio CH486 gale53 bio-561 putp21::mu dl propj667::tn5 4 CH5 gale53 bio-561 putp2j4::tns propj673::mu dl 4 CH51 gale53 bio-561 putp214::tns propj674::mu dl 4 CH579 gale53 bio-561 puta81::tnjo Transduction donor TT946; recipient CH223 CH627 gale53 bio-561 AputPA23 Tets derivative of CH579; this study CH638 gale53 bio-561 AputPA23 propi667::tns Transduction donor CH486; recipient CH627 CH784 propj673::mu dl prouj697::tnlo 3 CH992 gale53 bio-561 AputPA23 prouj697::tnjo 3 TT946 puta8i::tnlo J. Roth a All strains are derivatives of S. typhimurium LT2. were verified by marker rescue. All strains were derivatives of CH223 (4) except where stated. Deletion of the putpa genes was achieved by aberrant excision of the puta81::tnjo transposon from strain CH579 by the fusaric acid selection (2). Tetracycline-sensitive derivatives were screened for PutP and PutA phenotypes as described below. One PutP- PutA- derivative, CH627 (A6putPA23), was shown to be a deletion rather than an inversion by its inability to recombine with put point mutations and was used for all further studies. Identification of genotypes and phenotypes. Mutations in the proline transport genes were identified by their resistance to appropriate toxic proline analogs when radially streaked on minimal glucose plates around a filter disk inpregnated with L-azetidine-2-carboxylic acid (AC) or 3,4- dehydro-dl-proline (DHP) (3, 8, 19). putp+ prop+ strains are sensitive to both AC (15,ug) and DHP (6,ug); putp prop+ strains are AC resistant but DHP sensitive; putp prop derivatives are resistant to both analogs. Strains carrying puta mutations were identified by their inability to utilize L-prolyl-L-valine as the sole nitrogen source as described previously (4, 2). Growth curves. Cells were grown overnight in LOMglucose medium and diluted 1:1 into the same medium (containing.8 M NaCl when appropriate), and their growth was monitored by following the A6Eo in a 1-in. (2.5-cm) Spectronic tube with a Spectronic 2 spectrophotometer (Bausch & Lomb, Inc., Rochester, N.Y.). Proline and betaine, when used as osmoprotectants, were added at a concentration of 1 mm. Transport assays. Proline and betaine transport was assayed as described previously (4). Growth of cells at low osmolarity was in LOM-glucose. Growth at high osmolarity was achieved by growing the cells in LOM-glucose to an optical density at 6 nm of.6, adding NaCl to a final concentration of.3 M, and growing the cells for a further 1 h. Cells were grown at either high or low osmolarity to an optical density at 6 nm of.8, harvested by centrifugation, and washed twice in the medium in which they were grown. Cells were finally suspended to about.5 mg of cells ml-' in preincubation buffer (LOM containing 1 mm glucose, 5,ug of chloramphenicol ml-', and, when appropriate, additional NaCI to raise the osmolarity). Cell suspensions were equilibrated at 3 C for 5 min, and transport was initiated by adding [U-'4C]proline (2 mci mmol-1) or [methyl-'4c] betaine (6.6 mci mmol-1). Samples (1,ul) were removed at the indicated time intervals, the cells were collected by passing the samples through a Whatman GFF glass fiber filter and washed with 2.5 ml of preincubation buffer (at the same osmolarity as that used for the transport assay), and the accumulated proline or betaine was determined by scintillation counting. Each datum point was determined at least in duplicate, and each experiment was repeated with at least two independent cell suspensions. Because of the variation in cell volume between cells grown at different osmolarities, the protein content of cell suspensions was measured routinely, and transport rates were expressed as nanomoles per milligram of protein. Protein assays. Protein was assayed by the Bradford procedure, using reagents obtained from Bio-Rad Laboratories (Richmond, Calif.) and bovine serum albumin as a standard. The protein present in cell suspensions was determined after breaking the cells by sonication. P-Galactosidase assays. Assay of,b-galactosidase activity was carried out as described by Miller (17). Cells were permeabilized by the chloroform-sodium dodecyl sulfate procedure. RESULTS Osmotically induced transport via PP-II. Strains of S. typhimurium which were deficient in the major proline permease (PP-I, putp) were resistant to 4,ug of AC ml-1. However, if the osmotic pressure was raised by incorporating.3 M NaCl into minimal agar plates, sensitivity to the analog was restored. We have shown previously that this sensitivity is due to increased uptake of AC through PP-Il. Thus, mutants defective in PP-II (prop) can be selected by their resistance to 4 pug of AC ml-' at high osmolarity (4). This effect is not to be confused with the osmotically induced uptake of AC through PP-III (prolu), which requires very much higher concentrations of the toxic analog (3, 8). The increased uptake of AC through PP-II at high osmotic pressure could be due to an increase in activity of the transport system or, alternatively, to induction of expression of the prop gene. To distinguish between these two possibilities, we measured PP-1I-dependent uptake of proline at different osmolarities. The strain used, CH627, was deleted for putp to exclude proline uptake via PP-I. In this strain all proline transport is dependent upon PP-II; no uptake can be detected when prop is mutated (strain CH638). Thus, prou (PP-Ill) plays no significant role in proline uptake under these conditions. Strain CH627 (prop+) was grown in the presence or absence of.3 M NaCl, and in both cases, proline transport was assayed at low osmolarity. Proline transport was enhanced about threefold by growth of cells at high osmolarity (Fig. 1). However, when proline transport was assayed at high osmolarity (in the presence of.3 M NaCl), uptake was slightly reduced compared with assays performed at low osmolarity; the activity of PP-II was not increased by increased osmolarity. Indeed, when assayed at

3 122 CAIRNEY ET AL. J. BACTERIOL. E to 6 )"I CL g cn 3[ 1 ingly, growth was slightly but consistently enhanced by the introduction of a mutation in prop (doubling time = 35 min). This was also found to be true for a variety of other strains carrying various TnS, TnJO, or Mu dl insertions in prop (data not shown). It is not clear why prop mutations should enhance growth at high osmolarity as they do not / affect growth rates in medium of low osmolarity. However, the most probable explanation is that when PP-TI is absent, proline exodus from cells is reduced; consequently, the internal pools of this osmoprotectant are increased and growth is enhanced. / a Although growth of the wild-type strain in the absence of /, A an osmoprotectant was poor, the addition of 1 mm proline to the growth medium considerably enhanced growth, decreas- ing the doubling time to 24 min (Fig. 2B). Deletion ofputpa (CH627) had no effect on osmoprotection by proline, although this is perhaps not surprising since transport through e s Ca ez ;>PP-I is inhibited by high Na+ concentrations (4, 1). How ever, introduction of a prop mutation (strain CH638) considerably impaired the ability of proline to protect cells TIME (mins) against high osmotic pressure. Little or no growth stimulaf osmotic pressure on proline uptake by PP-Il. tion by proline was seen in prop mutants, indicating an FIG. 1. Effect o Cells of CH627 (AplutPA prop+ [A, O, A]) or CH638 (A&putPA prop important role for PP-1I in osmoprotection. [, ]) were grown in LOM-glucose in the presence (open symbols) At a concentration of 1 mm, betaine was a considerably or absence (closed rsymbols) of.3 M NaCI. Proline uptake at 1,uM better osmoprotectant than proline (Fig. 2C). The doubling was assayed at low osmolarity as described in the text except in the time of LT2 decreased from 42 mm in the absence of one case (A) in whiich the assay was performed at high osmolarity. osmoprotectant to 14 min when betaine was added. Again, elimination of putp had no effect on osmoprotection, while very high salt corncentration (.8 M NaCI), proline transport inactivation of prop dramatically reduced the ability of via PP-TI was strc)ngly inhibited (data not shown). Thus, it is betaine to protect against high osmotic pressure. Appropritransport via PP-IT is increased when cells ate controls (data not shown) showed that mutations in prop clear that proline are grown at highi osmolarity and that this increase is due to do not reduce the growth rate of cells at low osmotic induction of syntlhesis of PP-IT, rather than to an increase in pressure. Thus, either prop is required for osmoprotection specific activity c)f the transport system. per se, or, alternatively, PP-II serves as a transport system Osmotic enhan4 cement of prop expression is transcriptional. for both proline and betaine. To determine whether the increase in PP-IT synthesis at high Betaine protection against inhibition by toxic proline anais at the transcriptional level, we made use logs. The toxic proline analog DHP enters the cell via PP-TI osmotic pressure of Mu dl-mediat ed lac fusions (5) to the prop gene. These (8). If betaine also enters the cell through PP-II it might be fusions place the lacz gene under control of the prop expected to compete for uptake and reduce the toxic effects promoter. The construction of Mu dl(apr lac) fusions to of DHP. Figure 3 shows the growth of strain CH627 prop has been dlescribed previously (14). P-Galactosidase (AputPA) in LOM-glucose. After 9 min of growth, DHP or synthesis by tw4 o strains carrying independently isolated betaine or both were added as indicated. The addition of fusions of lacz to prop (CH5, CH51) was increased about betaine alone had no effect on the growth rate. However, three- to fourfolcd by growth in high-osmotic-pressure me- when DHP (1,ug ml-'; approximately.8 mm) was added dium (Table 2). 1Fhis effect was seen whether the osmolyte growth was strongly inhibited. This inhibition was relieved used was NaCI, KCl, choline chloride, or sucrose (data not by the addition of betaine, suggesting that betaine and DHP shown). Thus, iinduction of prop transcription correlates compete for the same uptake system. well with the in( crease in proline transport through PP-II prop encodes a betaine transport system. Betaine is known observed at higih osmotic pressure. Neither proline nor to be taken up into E. coli by an active transport process betaine induced IvroP expression. Indeed, betaine caused a (18). To determine the potential role of prop in betaine reduction in prop) expression in medium of high osmolarity. This could be a result of the intracellular accumulation of betaine causing. a reduction in turgor pressure differences which may deterrmine the osmoinduction of gene expression Regulation prop expression (3). Expression c _-C~~~~._7 TABLE_ 2. Reuato of Dro t pror, as juugec ty p-gatactosiuase proese fusions, was also unaffected by the 13-Galactosidase (U) -r duction from th Medium additives introduction of 1putP, puta, or prou mutations into the CH5 CH51 fusion strains (datta not shown). None Major role of prop in osmoprotection. Because of the NaCI (.3 M) increase in prop expression at high osmotic pressure, it Betaine (1 mm) seemed possible that this transport system plays a role in NaCl (.3 M) + betaine (1 mm) osmoprotection. We therefore examined the role of PP-II in Proline (1 mm) osmoprotection. Figure 2A shows the growth of cells in NaCl (.3 M) + proline (1 mm) LOM-glucose c( )ntaining.8 M NaCl but lacking any a Cells were grown in LOM with additives as indicated. Strains CH5 and osmoprotectant. As expected, growth in this medium was CH51 contain independently isolated prop::mu dl(apr lac) fusions. Both very poor (doub] ling time = 42 min). However, interest- strains are also putp.

4 VOL. 164, 1985 UPTAKE OF THE OSMOPROTECTANT BETAINE 1221 A. B. C TIME (hours) FIG. 2. Osmoprotection by proline and betaine. Cells were grown in LOM-glucose containing.8 M NaCl, and growth was monitored by determining the optical density of the culture at 6 nm. Cells were grown in the absence of any osmoprotectants (A); with 1 mm proline (B); with 1 mm betaine (C). The strains used were LT2 (putpa+ prop+ []), CH627 (AputPA prop+ [A]), and CH638 (AputPA prop [A]). uptake, transport was measured directly (Fig. 4). Because pressure. However, when transport was assayed in highprop expression is enhanced at high osmo)tic pressure, cells osmolarity medium, betaine transport was detected. Since were grown and assayed for betaine tranisport at both low significant betaine uptake could only be detected when (LOM) and high (LOM plus.3 M NaC1) osmolarity. No assayed at high osmolarity, all subsequent assays were betaine uptake by strains CH627 or CH638,could be detected carried out under these conditions. when transport was assayed in medium c)f low osmolarity, To determine whether putp and prop play a role in betaine whether or not the cells were grown at low or at high osmotic transport, uptake was measured in strains carrying mutations in these genes (Fig. 4). Cells of LT2 and CH627 (AputPA) were grown in medium of high osmolarity, and betaine uptake was determined at 3,uM concentration. Clearly, putp plays no role in betaine uptake as betaine uptake was identical in these two strains. Because expression of prop was enhanced by growth at high osmotic pressure we wished to determine if betaine transport was similarly enhanced. Thus, CH627 and CH638 cells were grown at both low and high osmotic pressure, and in both cases betaine uptake was determined at high osmotic pressure. In cells grown at low osmotic pressure all betaine uptake was dependent upon prop; no uptake could be detected in CH638 in which prop is mutated. However, in cells grown at high osmolarity an additional, propindependent betaine uptake system was induced. The rate of betaine uptake through PP-IT for cells grown at low osmolarity can be calculated to be 2.2 nmol min-' mg of protein-'. The rate of prop-dependent betaine uptake in cells grown at high osmolarity can be calculated from the difference in uptake between the parent (CH627) and the prop mutant (CH638), and is about 9 nmol min-' mg of protein-1. Thus, growth at high osmotic pressure increases betaine uptake through PP-TI by about fourfold, in good agreement with the increases in proline transport through PP-II and transcrip tion of prop observed under the same conditions. TIME (hours) It should be noted that these experiments were carried out FIG. 3. Betaine protects against inhibition by DHP. Cells of with 3,uM betaine. At higher betaine concentrations uptake strain CH627 (AputPA) were grown in LOM-gluicose in the presence could be detected even when assayed at low osmotic pres- (open symbols) or absence (closed symbols) of ] L Lg of DHP ml-' sure (data not shown). This is compatible with data pre- density (OD) at sented above which show that, even in LOM, 1 mm betaine and growth was followed by measuring the opttical 6 nm. After 1.5 h betaine was added to the cultures at the can compete with DHP uptake via PP-IT. In addition, while following concentrations: 1 mm (I, *), 5 p LM (, ), and no mutations in prop reduce betaine uptake considerably, it is addition (A, A). clear that there is an additional prop-independent betaine

5 1222 CAIRNEY ET AL a 6 C.. ' E 5 E C a, m 4. a, c co // o r~i.-r TIME (mins) FIG. 4. Betaine uptake by S. typhimurium. Cells of strain CH627 (closed symbols: AputPA prop+) or CH638 (open symbols; AputPA prop) were grown at either low (LOM-glucose) or high (LOMglucose plus.3 M NaCI) osmolarity as described in the text, and betaine transport at 3,uM was assayed in the presence or absence of.3 M NaCl as follows: cells were grown and assayed at high osmolarity (, ); cells were grown at high osmolarity and assayed at low osmolarity (A, A); cells were grown at low osmolarity and assayed at high osmolarity (U, O). Transport was also assayed at high osmolarity in wild-type cells (LT2; putpa+ prop+) grown in medium of high osmolarity (x). transport system which is only active in cells grown at high osmolarity. We have shown in an accompanying paper that this residual betaine uptake is dependent upon the function of the prou gene (3). It is therefore clear that PP-II, encoded by prop, functions as a betaine transport system. The osmolarity of the medium influences uptake via PP-Il in two ways. First, transcription ofprop is induced approximately threefold by growth at high osmolarity, and second, even when induced, the transport system only functions in medium of high osmolarity. Kinetics of betaine transport through PP-II. Strain CH992 (putp prou prop+) is deficient in pro U, and in this strain all betaine transport at 3,uM has been shown to be via PP-II (3). CH992 was grown in high-osmotic-pressure medium (LOM-glucose plus.3 M NaCI), and betaine transport was assayed in the same medium at a variety of different concentrations (Fig. 5). Eadie-Hofstee and Lineweaver-Burk plots of these data give kinetic parameters of Km = 44,uM and Vmax = 37 nmol min-1 mg of protein-'. DISCUSSION It has been known for many years that betaine plays an osmoprotective role in plants, and more recently a similar role has been demonstrated in the enteric bacteria (12, 13). E. coli has been shown to accumulate betaine by an osmotic pressure-dependent active transport process, although the genes involved and the mechanism of this osmotic stimulation are obscure (18). In this paper we demonstrate that the prop gene, originally identified as encoding a minor proline permease, PP-II, plays a major role in betaine uptake. Several lines of evidence show that betaine uptake is mediated by PP-II. First, the osmoprotective effect of betaine toward cells grown at high osmotic pressure was reduced in strains defective in prop. Second, the uptake of toxic proline analogs through prop was inhibited by excess betaine. Finally, the uptake of betaine itself was considerably reduced in strains mutated for prop. The kinetics of betaine uptake through PP-II suggest that betaine, rather than proline, is the primary substrate for this transport system. The prop gene was originally identified as encoding a minor proline permease in both S. typhimurium and E. coli (1, 16, 24). The Km for proline uptake via PP-Il is exceptionally high (3,M), especially when compared with proline uptake through the major proline permease (PP-I; Km = 2,uM). Thus, the physiological role of this second system has been obscure. However, we show here that the affinity of PP-II for betaine is about 1-fold greater than its affinity for proline, suggesting that betaine is the physiological substrate for this transport system. Although it is perhaps surprising that the two amino acids proline and betaine should be substrates for the same transport system, both are N- substituted amino acids. In contrast to PP-II, the major proline permease (PP-I, putp) shows no detectable affinity for betaine and appears to play no role in betaine uptake or osmoprotection. We determined the following kinetic parameters for betaine transport via PP-II: Vmax = 37 nmol min-' mg of protein-' and Km = 44,uM. In addition to PP-II, there is a second betaine transport component in wild-type cells. This is mediated by a high-affinity uptake system dependent upon the osmotically induced prou gene (3). Thus, S. typhimurium has two genetically distinct betaine transport systems. It seems likely that similar betaine transport systems function in E. coli. First, the kinetics of betaine uptake by wild-type E. coli (Vmax = 42 nmol min-' mg of protein-'; Km = 35,uM [18]) are similar to those we obtained for uptake via PP-II in S. typhimurium. The slight differences between the two sets of data can readily be accounted for by the 25 4 a, ' -15.c cn.c 5 / ~~~~~~~ J. BACTERIOL. I/ Betaine conc (,um) FIG. 5. Kinetics of betaine uptake through PP-II. Cells of strain CH992 (AputPA prop+ prou) were grown in LOM-glucose containing.3 M NaCl, and betaine transport was assayed in medium of the same high osmolarity. Eadie-Hoffstee and Lineweaver-Burk transformations of these data give the following kinetic parameters: Vmax = 37 nmol min-1 mg of protein-'; Km = 44,LM.

6 VOL. 164, 1985 additional prou-dependent component of transport present in wild-type cells (3). Proline has been shown to compete with betaine for uptake by E. coli (18). In addition, a gene corresponding to prop and encoding a minor proline permease has been identified in E. coli, although the chromosomal location of this gene is still in some doubt (24). Regulation of PP-II function is intriguing. We found that transport of both betaine and proline via PP-II was stimulated about threefold by growth at high osmolarity. High osmotic pressure also increased transcription of prop to a similar extent. Thus, the increase in PP-Il function in cells grown at high osmolarity seems to be wholly due to increased transcription of the prop gene. Anderson et al. (1) reported that prop expression is also enhanced threefold by amino acid starvation; whether these two regulatory effects are related remains speculative. However, it is interesting to note that osmotic shock causes a rapid increase in the cellular ppgpp pool (1). In addition to transcriptional regulation, the activity of the PP-I1 permease itself is affected by the osmotic pressure of the medium. Thus, even when fully induced, PP-II is only able to transport betaine to a significant extent in medium of high osmolarity. In contrast, the function of many other transport systems is also inhibited at high osmolarity (23; unpublished data). This strongly suggests that increased osmotic stress results in a conformational change in the permease protein, either nonspecifically by virtue of cell and membrane shrinkage or via a specific regulatory circuit. Particularly interesting is the observation that while increased osmolarity increases betaine uptake via PP-II it decreases proline uptake. Thus, not only is transport activity altered, but there is a change in substrate specificity. Potassium transport via the trk system in E. coli, which can also serve an osmoprotective function, has been shown to exhibit similar regulatory properties (21). The mechanisms by which betaine protects against osmotic pressure are unclear. It can be accumulated in large amounts in response to stress (12, 18) and may therefore provide an osmotic balance across the membrane. In addition, there is also considerable evidence that betaine can specifically protect enzymes from adverse concentrations of salt (25) and can also reverse the inhibition of many carbohydrate transport systems by NaCl (23). We show here that uptake of betaine into the cell is important for its osmoprotective function. A further characterization of the mechanism and regulation of the betaine uptake systems is therefore of considerable importance in understanding the mechanisms and evolution of osmotolerance. ACKNOWLEDGMENTS We are grateful to the Medical Research Council for financial support. 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