INTRODUCTION

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Water flows across biological membranes in response to osmotic pressure (osmolality) gradients. Turgor pressure develops if cell walls resist osmotically induced water influx. Osmoregulatory mechanisms adjust cytoplasmic osmolality by modulating the synthesis, catabolism, uptake, or efflux of appropriate solutes in response to osmolality changes. Compatible solutes are organic solutes, accumulated by bacteria exposed to hypertonic environments, which do not impair cellular functions. Physiologists reason that in the absence of osmoregulatory mechanisms, cytoplasmic osmolality would follow environmental osmolality, causing unacceptable fluctuations in cytoplasmic composition, cell volume, and/or turgor pressure (3, 4, 42).

ProP is an osmoregulatory transporter which mediates the active accumulation of diverse compatible solutes, including proline, glycine betaine (N-trimethyl glycine), stachydrine (N-dimethyl proline) (13, 21), pipecolic acid (8), ectoine (1,4,5,6-tetrahydro-2-methyl-4-pyrimidine carboxylic acid) (16), and taurine (23). Gene proP (located at 93 centisomes) is expressed from ⁷⁰- and ^S-dependent promoters. Transcription of proP is modulated by medium osmolality, carbon source, and culture growth phase (24, 43-45). ProP is activated by an osmotic upshift in whole bacteria (11), cytoplasmic membrane vesicles (26), and proteoliposomes (30). An H⁺-compatible solute symporter and member of the major facilitator superfamily, the 500-amino-acid ProP protein differs from sequence homologues not implicated in osmoregulation by possessing a 46-amino-acid carboxyl-terminal extension that is capable of forming a homodimeric -helical coiled coil of limited stability in vitro (5, 38a).

Mutations pro-219 and pro-220::Tn5 were selected as increasing the resistance of Escherichia coli K-12 derivative RM2 [(putPA)101] to toxic proline analogue 3,4-dehydroproline. Mutation proQ220::Tn5 defined a new gene located, by transduction, at 40.4 min on the chromosomal linkage map (27). Whereas no ProP activity could be detected when proQ220::Tn5 bacteria were cultivated in a medium of low osmolality, a partial restoration of ProP activity (41%) was observed when they were cultivated in a hypertonic medium (0.3 M NaCl [27]). The mutation did not alter the transcription of a chromosomal proP::lacZ operon fusion (9) in response to increased medium osmolality, however (27). This report shows that mutation proQ220::Tn5 impairs the osmotic activation of ProP by acting at a posttranslational level, demonstrates the expression of ProQ by wild-type bacteria, and reveals the predicted sequence of protein ProQ.

	MATERIALS AND METHODS

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Bacterial strains, plasmids, molecular biological techniques, and growth conditions. The strains and plasmids used for this study are listed in Table 1. Construction of a prc deletion strain was carried out through a P1-mediated transduction of E. coli RM2 from strain KS1000, yielding strain WG703. Transductants were selected on Luria-Bertani (LB) agar containing kanamycin at a concentration of 50 µg/ml.

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Preparation, solubilization, and analysis of cells and subcellular fractions. To analyze ProQ expression in cells on a small scale, a 1-ml overnight culture was centrifuged in a Microfuge for 1 min. Cells were resuspended and boiled in 50 µl of sample buffer (15.625 mM Tris-HCl [pH 6.8], 2% [vol/vol] glycerol, 0.5% [wt/vol] sodium dodecyl sulfate [SDS], 0.05% [wt/vol] bromphenol blue, 1.25% [vol/vol] mercaptoethanol) following a modification of the method of Sambrook et al. (31). ProP expression was analyzed as described above, except that the boiling step was replaced by a 30-min incubation at 37°C. Cells were sheared by repeated passage through a 26-gauge syringe and centrifuged for 5 min in a Microfuge. The supernatant was analyzed by SDS-polyacrylamide gel electrophoresis (PAGE). For larger-scale preparations, cells cultured in LB medium were harvested by centrifugation (Sorvall GS3 rotor; 5,000 rpm for 20 min at 4°C), washed twice with saline (0.85% [wt/vol] NaCl), and resuspended in a 1/300 volume of potassium phosphate buffer (0.1 M; pH 7.1). Washed cells were passed three times through a French pressure cell at a pressure of 1.6 × 10⁸ Pa. The lysate was centrifuged at a low speed (Sorvall SS34 rotor; 10,000 rpm for 20 min at 4°C) to remove cellular debris and inclusion bodies. Soluble and particulate fractions were obtained by ultracentrifugation of the resulting supernatant (Beckman 45 Ti rotor; 36,000 rpm [145,000 × g] for 2 h at 4°C). All fractions were stored at 70°C after resuspension of the pellets in the same buffer. Appropriately diluted samples of these fractions were dissolved in sample buffer as described above. SDS-PAGE analysis of ProQ was performed with gels comprised of 12% (wt/vol) acrylamide and 2.6% bis-acrylamide according to the method of Laemmli (19) with a MiniProtean II cell (Bio-Rad, Mississauga, Ontario, Canada). SDS-PAGE of proteins to resolve ProP was performed with 4 to 15% polyacrylamide gradient Tris-HCl gels (Bio-Rad).

Affinity purification of anti-ProP antibodies. Anti-ProP antibodies were raised against the partially purified ProP protein, the antibodies were adsorbed with an extract of a proP mutant E. coli strain, and ProP(His)₆ (the ProP protein with six additional, carboxyl-terminal histidine residues) was purified by nickel chelate affinity chromatography as described by Racher et al. (30). Purified ProP(His)₆ (2.5 mg) was coupled to CNBr-activated Sepharose 4B (Pharmacia Biotech) according to the manufacturer's instructions. To bind the anti-ProP antibodies to the active resin, 0.5 ml of adsorbed serum was incubated with the resulting resin for 18 h at 4°C on a rotating platform. Bound antibody was eluted from the column by washing it with 0.1 M glycine-HCl buffer, pH 2.5. The eluate was immediately neutralized with 1 M Na₂CO₃. Fractions containing the affinity-purified antibodies were pooled and stored at 40°C.

Anti-ProQ antibody preparation. The protein overexpressed by E. coli SG13009(pJK1) was purified as follows. The fraction recovered by the first low-speed centrifugation of a French press lysate (described above) was washed twice as described by Neugebauer (29). The insoluble residue was dissolved by boiling it for 15 min in Tris-HCl buffer (pH 8.0)-5% (wt/vol) SDS-40 mM dithiothreitol and resolved by SDS-PAGE as described above. The gel was stained with 0.3 M CuCl₂, the gel slice containing the overexpressed protein was excised, and the staining was reversed by washing it with 0.25 M EDTA and 0.25 M Tris, pH 9.0 (15). A Bio-Rad electroeluter (model 422) was used to recover the protein from the gel slice by electroelution for 4 h at a constant current of 10 mA in a solution of 25 mM Tris, 192 mM glycine, and 0.1% (wt/vol) SDS. Five milliliters of preimmune serum was taken from each of two New Zealand White female rabbits, before each rabbit was injected intramuscularly with the protein purified from strain SG13009(pJK1). Further immunization and serum collection were conducted as described previously (15), and the antibodies were purified as follows. Cells from a 1-liter overnight culture of E. coli SG13009(pQE60) were harvested, resuspended in 15 ml of Na MOPS buffer (0.5 mM; pH 7), and disrupted by four serial passages through a French pressure cell at 15,000 lb/in². The cell lysate was coupled to cyanogen bromide-activated Sepharose 4B (Pharmacia Biotech) as specified by the manufacturer, and the resulting affinity matrix was used to remove contaminating antibodies from the anti-ProQ serum. Fractions containing anti-ProQ antibodies were pooled and stored at 40°C.

Transport measurements. The radial streak test (27) was used to estimate the ProP activities of E. coli RM2, WG174, and WG703 (as proline analogue sensitivities). Cultures of E. coli RM2 and WG174 were prepared and transport measured by filtration assay essentially as described previously (26). MOPS minimal medium (28) was inoculated (0.5% [vol/vol]) with cells from an overnight culture in LB medium. MOPS medium contained NH₄Cl (9.5 mM) as a nitrogen source, glycerol (5 mg ml¹) as a carbon source, L-tryptophan (245 µM), and thiamine hydrochloride (1 µg ml¹). Upon reaching stationary phase, cells were subcultured in the same medium to achieve an optical density at 600 nm (OD₆₀₀) of 0.5. After growth to an OD of 1, cells were harvested by centrifugation and washed three times in unsupplemented MOPS medium (MOPS medium lacking phosphate, NH₄Cl, and organic nutrients). The optical density of the cells was adjusted to an OD₆₀₀ of 15.0, and amino acid uptake was measured in an assay mixture consisting of unsupplemented MOPS medium containing K₂HPO₄ (2.64 mM), glucose (2 mg ml¹), and NaCl as indicated. Uptake was initiated by the addition of the substrate L-proline (to a concentration of 200 µM; 10 Ci mol¹ [370 kBq µmol¹]), following a 3.0-min preincubation of the cells in the assay mixture. The preincubation time was adjusted for analysis of the kinetics of activation, as indicated. The assay mixture was sampled after 20, 40, and 60 s. All assays were done in triplicate, and all experiments were done at least twice. Each set of replicate assays was used to determine the rate of amino acid uptake over the 20- through 60-s interval. The rates are cited as means ± standard deviations.

Protein assays. Protein concentrations were determined by a bicinchoninic acid assay (34) with a kit obtained from Pierce (Rockford, Ill.), with dilutions of bovine serum albumin as the standard.

Nucleotide sequence accession number. The nucleotide sequence of proQ was submitted to GenBank and assigned accession no. L48409.

	RESULTS

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Isolation and sequencing of proQ. Allele proQ220::Tn5 was isolated by selecting bacteriophage Mu dII4042-derived recombinant plasmids (10, 40) isolated from E. coli WG174 (27), and the proQ sequence was determined (extending from nt 1913558 through 1912860 of the E. coli genome) (see Materials and Methods). The proQ ORF was predicted to encode a 232-amino-acid, basic, hydrophilic protein (molecular mass, 25,876 Da; calculated isoelectric point, 9.66; 32% D, E, R, or K; 54.5% polar amino acids) with no obvious N-terminal secretion signal sequence. The Tn5 insertion interrupted the sequence at codon E105.

View larger version (21K): [in this window] [in a new window]   FIG. 1.   ProQ is a hydrophilic protein with sequence similarities to E. coli FinO and other structural elements. The full-length sequence alignment of ProQ, FinO, HI1669, and HI1670 was created by the manual joining of local alignments identified by BlastP (1). +, conservative substitution.

ProQ regulates ProP posttranslationally. The recently published E. coli genome sequence facilitated the placement of the proQ ORF in relation to its neighbors (Fig. 2). Gene proQ (identical to the extended ORF yebJ; see GenBank accession no. AE000277) occurs at 41.2 centisomes in the segment of the E. coli chromosome flanked by loci cspC and holE (Table 2). ORF b1832, proQ, prc, and htpX constitute a block of genes known (or predicted) to be transcribed counterclockwise (in contrast to the flanking loci). E. coli strains defective in prc fail to grow at 42°C on solid hypotonic medium (1/2L medium; see Materials and Methods) and have morphologically elongated cells when grown at 42°C in the corresponding liquid medium (14, 33). The previous observation that insertion proQ220::Tn5 impairs ProP activity was attributed to its disruption of locus proQ (27, 36). Locus prc is downstream from proQ, and a putative prc promoter exists within the proQ ORF (downstream from the proQ220::Tn5 insertion [nt 1913173] at nt 1913074 to 1913048 [14]). It was therefore important to rule out the possibility that the proQ insertion exerted its effects on ProP by disrupting prc expression, either through polar effects within an operon including both proQ and prc or by directly disrupting transcription from a prc promoter located within proQ.

View larger version (7K): [in this window] [in a new window]   FIG. 2.   Position and orientation of proQ on the E. coli genome. Arrows indicate positions and orientations of ORFs (see the text). Known and suggested functions of some of these loci are listed in Table 2.

prc3

View larger version (25K): [in this window] [in a new window]   FIG. 3.   Mutation proQ220::Tn5 does not alter the level of ProP. Membrane vesicles were prepared from E. coli cells grown in NaCl (0.3 M)-supplemented MOPS minimal medium. Membrane proteins (20 µg) were separated by SDS-PAGE, and Western blots were prepared with purified anti-ProP antibodies as described in Materials and Methods. Purified ProP(His)6 (1.3 µg) served as a control. The numbers to the left indicate the positions of molecular size markers (in kilodaltons).

ProQ is expressed as a soluble protein in wild-type E. coli. The proQ ORF was amplified with DNA from E. coli K-12 (proQ⁺) as a template and inserted in vector pQE60, yielding plasmid pJK1. This system was designed to amplify the expression of the wild-type proQ gene by placing it under the control of an IPTG (isopropyl--D-thiogalactopyranoside)-inducible bacteriophage T5 promoter system (Qiagen). An abundant protein with an apparent molecular mass of 30 kDa was present in cells of E. coli SG13009(pJK1) which were induced with IPTG but was not in those which were not (data not shown). This protein was contained in a fraction harvested by low-speed centrifugation from a French press lysate of these bacteria, suggesting that it was present as inclusion bodies. It was enriched by washing it with EDTA, deoxycholate, and lysozyme (29), further resolved from contaminants by SDS-PAGE, and eluted from the gel for antibody production. DNA sequence analysis revealed that the proQ locus in this plasmid contained mutation C64T, resulting in the predicted protein modification S22P.

View larger version (41K): [in this window] [in a new window]   FIG. 4.   Expression of proQ with vector pBAD24 yields a soluble protein with a molecular mass of 30 kDa. Shown are a Western blot, visualized with the BCIP/NBT reagent system, and an SDS-PAGE analysis of the soluble (S) and particulate (P) fractions derived from E. coli DH5(pDC77) with (I) or without (U) induction of protein expression by arabinose (2 mg/ml). These are compared with the 30-kDa protein overexpressed by E. coli SG13009(pJK1) (Q). The numbers indicate the positions of molecular size markers (in kilodaltons).

View larger version (65K): [in this window] [in a new window]   FIG. 5.   The ProQ protein is expressed by E. coli. A Western blot, visualized with the ECL reagent system, is shown. Soluble fractions derived from E. coli RM2 and WG174 and the 30-kDa protein overexpressed by E. coli SG13009(pJK1) (Q) were probed with antibodies raised against the latter protein. The arrow marks the 30-kDa immunoreactive protein that is present in E. coli RM2 carrying proQ+ but not in E. coli WG174 carrying proQ220::Tn5.

Mutation proQ220::Tn5 impairs activation of ProP. The rates of proline uptake via ProP in proQ⁺ and proQ220::Tn5 bacteria were measured as a function of the NaCl concentration employed to impose an osmotic upshift (Fig. 6). (The proline uptake observed under these conditions is attributable to transporter ProP, since it is absent from bacteria further defective in locus proP [27].) As previously determined, the optimal upshift for activation of ProP in wild-type bacteria was imposed with approximately 0.12 M NaCl. ProP activity was increased sevenfold to a maximum of 22 nmol/min/mg of protein. Activation of ProP in the proQ220::Tn5 strain occurred over a similar range of NaCl concentrations but was very limited (only a threefold increase to a maximum of 2.5 nmol/min/mg of protein). The kinetics of ProP activation after an osmotic upshift imposed with 0.12 M NaCl were determined for proQ⁺ and proQ220::Tn5 bacteria (Fig. 7). The data were fitted to a model describing an exponential increase in ProP activity, postshift (26; Fig. 7). The level of ProP activity approached by the proQ mutant bacteria (4.8 ± 0.2 nmol/min/mg of protein) was fivefold lower than that approached by the wild-type strain (22.7 ± 0.3 nmol/min/mg of protein). The rate constant (k) for activation of ProP was reduced 2.6-fold in the proQ mutant (from 0.75 ± 0.04 to 0.29 ± 0.04 s¹). Thus, insertion proQ220::Tn5 reduced both the rate and the extent of ProP activation by an osmotic upshift.

View larger version (22K): [in this window] [in a new window]   FIG. 6.   Mutation proQ220::Tn5 impairs ProP activation. E. coli RM2 carrying proQ+ (white circles) and WG174 carrying proQ220::Tn5 (black circles) were cultivated in MOPS medium, and proline uptake rates were measured as described in Materials and Methods. Supplementary NaCl was added to the transport assay mixtures at the indicated levels. Error bars indicate standard deviations.

View larger version (14K): [in this window] [in a new window]   FIG. 7.   Mutation proQ220::Tn5 reduces the rate and extent of ProP activation. E. coli RM2 carrying proQ+ (white circles) and WG174 carrying proQ220::Tn5 (black circles) were cultivated in MOPS medium, and proline uptake rates were measured as described in Materials and Methods. Cells were incubated in NaCl (0.12 M)-supplemented uptake assay mixtures for the indicated periods, before radiolabelled proline was added to initiate the uptake assay. Solid lines represent the outcome of nonlinear regression analyses performed as described by Milner et al. (26). Error bars indicate standard deviations.

	DISCUSSION

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Cells from diverse organisms can accumulate similar arrays of organic compounds, all known to be compatible with and/or to stabilize protein structure, when challenged by hypertonic environments (35). The machinery of compatible solute accumulation has been described for some organisms (e.g., E. coli [3, 4, 42]), but its regulation is not well understood. Although proteins Fis and CAP are involved, no trans-acting transcriptional regulatory element specific to locus proP has been implicated in the impressive modulation of its transcription by osmotic stress. Transporter ProP is activated, in the absence of protein synthesis, when whole bacteria (11), cytoplasmic membrane vesicles (26), or proteoliposomes incorporating purified ProP (30) are subjected to an osmotic upshift with a membrane-impermeant osmolyte. Our research is designed to elucidate the mechanisms by which ProP senses osmolality changes and mounts its osmoregulatory response. Since ProP activity is impaired by insertion proQ220::Tn5 (27, 36), we are exploring the structure and function of proQ as well as its relationship to ProP.

In this study we establish that the effects of the insertion on ProP are due to the altered expression of locus proQ and not to polar effects on downstream locus prc (see the text and Fig. 2 and 5). The previous conclusion that the Tn5 insertion in proQ does not influence proP transcription was confirmed. Database analysis identified two proteins with weak sequence similarities to ProQ. Within this group of homologues, the relationship that appeared most interesting was the weak similarity with translational regulator FinO. This raised the possibility that ProQ could be acting at a translational level to alter the levels of ProP protein. However, this study has shown that the level of ProP protein present in either whole cells (data not shown) or membrane vesicles is not altered by the Tn5 insertion in locus proQ (Fig. 3).

This study has further shown that the rate and extent of ProP activation are significantly reduced in a proQ220::Tn5 strain of E. coli. These reports are significant in documenting the only trans-acting factor which is known to influence the osmotic activation of ProP. Gene proQ is predicted to encode a 232-amino-acid protein that is both basic and hydrophilic in nature. SDS-PAGE and Western blot analysis indicate that the overexpressed ProQ protein is soluble, as predicted (Fig. 4). The subcellular location of the protein in wild-type bacteria remains to be determined, however.

ProP activity is observed in cytoplasmic membrane vesicles (26) and proteoliposomes prepared with purified ProP (30). There are some significant differences between the ProP activities of these vesicle systems and those of whole cells, however. The hyperosmotic shift which gives maximal ProP activity in cells (0.2 osM) is lower than that required in membrane vesicles (0.8 osM) (22). As well, ProP is active in whole cells even without an osmotic shift, whereas ProP activities in proteoliposomes and membrane vesicles absolutely require a hyperosmotic shift (22, 30). Given these contrasting features of ProP activities in cells and vesicle systems, we now believe that ProQ is a structural element which influences the osmotic activation of ProP at a posttranslational level.