JOURNAL OF BACTERIOLOGY, JUlY 1991, P. 4404-4410

Vol. 173, No. 14

0021-9193/91/144404-07$02.00/0 Copyright X 1991, American Society for Microbiology

Role of Heat Shock Protein DnaK in Osmotic Adaptation of Escherichia coli JEAN MEURY* AND MASAMICHI KOHIYAMA Institut Jacques Monod, Tour 43, 2 place Jussieu, F-75251 Paris Cedex, France Received 29 January 1991/Accepted 24 April 1991

Escherichia coli can adapt and recover growth at high osmolarity. Adaptation requires the deplasmolysis of previously plasmolyzed by the fast efflux of water promoted by osmotic upshift. Deplasmolysis is essentially ensured by a net osmo-dependent influx of K+. The cellular content of the heat shock protein DnaK is increased in response to osmotic upshift and does not decrease as long as osmolarity is high. The dnaK756(Ts) mutant, which fails to deplasmolyze and recover growth, does not take up K+ at high osmolarity; DnaK protein is required directly or indirectly for the maintenance of K+ transport at high osmolarity. The temperaturesensitive mutations dnaJ259 and grpE280 do not affect the osmoadaptation of E. coli at 30°C.

cells

Escherichia coli cells are able to adapt to high osmolarity, although growth is slowed in these conditions. When cells are shifted to a higher osmolarity, they transiently stop growing; then, after a lag, they resume growth with an increased doubling time. In a previous paper, we reported that, in minimal medium at 37°C, an osmotic upshift from 300 to 1,500 mosM triggered within a few minutes several metabolic disturbances which can be summarized (23) as follows. (i) Cell growth was stopped for 50 to 60 min: the greater the osmotic shift, the longer the duration of the lag before growth recovery. (ii) K+ transport by the Trk system was immediately turned on (24) so that cellular K+ content increased by 100% within 40 to 50 min. (iii) Net protein and DNA synthesis and cell division were transiently stopped for 40 to 50 min. The question arising from these results was whether an environmental stress factor such as osmotic upshift could induce a set of specific proteins, as do heat shock and oxidative stress. The response of different microorganisms to osmotic shift (e.g., downshift in halobacteria; upshift in cyanobacteria and gram-positive and gram-negative eubacteria) seems to involve modifications of protein synthesis, as indicated by bidimensional electrophoretic protein analysis. As yet, these responses have shown no clear communality. While halobacteria only increase the synthesis of several heat shock proteins when medium osmolarity is decreased (8), cyanobacteria increase the synthesis of several heat shock proteins and salt stress-specific proteins and repress the synthesis of some others in response to osmotic upshift (3). In Bacillus subtilis, synthesis of general stress proteins and specific proteins also has been shown to be stimulated by osmotic upshift (13). Three osmotic upshift-induced proteins have been detected in E. coli (7); they were considered to be neither heat shock proteins nor general stress proteins but possibly enzymes involved in oligosaccharide metabolism (16) and components of the betaine transport system encoded by the proU operon (2, 6). The present report focuses on the DnaK protein, a member of the heat shock group of proteins (12, 25), which is thought to modulate the heat shock response in E. coli (30) and may be involved in the (i) initiation of chromosome (28), X bacteriophage (1, 20, 32), and P1 plasmid (31) replication in *

E. coli and (ii) partition of the E. coli chromosome and P1 plasmid (5). Our results show that the DnaK content is increased by two- to threefold in response to osmotic shock; moreover, DnaK seems to be necessary for the maintenance of K+ transport at high osmolarity.

MATERIALS AND METHODS Strains and medium. The strains used in this work are listed in Table 1. Apart from strains ML30, obtained from Institut Pasteur, and TK2205, kindly supplied by W. Epstein, all other strains derive from the parent C600 and were kindly supplied by W. Messer. All strains were grown in a minimal medium containing, per liter, 100 mM sodium phosphate buffer (pH 7.2), 10 mM ammonium sulfate, 1 mM magnesium sulfate, 0.01 mM ferrous sulfate, 10 mg of thiamine, and 10 mM KCl (40 mM for strain TK2205). The carbon source was glucose, 4 g/liter. L-Leucine and L-threonine were added at 25- and 50-p,g/ml

concentrations, respectively. The A6. of the cell suspensions was measured.

Transduction of the dnaK+ allele in mutant WM1389. The dnaK+ allele was transduced by P1 vir phage to the mutant strain WM1389 (thr-J, tetracycline resistant) from the prototrophic strain GC527 (Hfr KL16). Transductants for the dnaK+ allele were first selected at 30°C on plates of minimal medium free of threonine but containing 100 ,ug of tetracycline per ml. Then thr+ colonies were transferred in the same medium and incubated at 30 and 43°C. The amount of thr+-dnaK+ cotransduction was 50%. The transductants were still tetracycline resistant. Potassium depletion. Ninety to 95% depletion of the total cell potassium was obtained by subjecting bacteria to sudden hypoosmotic shock. Bacteria were pelleted twice and then resuspended in distilled water (22). Measurement of intracellular K+. Samples of bacteria were filtered through Millipore nitrocellulose membrane filters (type HA, 0.45-,um pore size) and washed twice with 2.5 ml of hyperosmotic NaCl solution (0.8 M). Hyperosmotic washing has been shown to minimize the leakage of K+ and metabolites triggered by hypoosmotic media (22). Filters were placed in tubes with 5 ml of 0.1 N HNO3; after 5 min, K+ was determined with an atomic absorption spectrophotometer (Pye Unicam SP9). Results were expressed as

Corresponding author. 4404

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Strain C600 WM1389 WM1390 WM1289 C600 grpE TK2205

ROLE OF DnaK IN OSMOTIC ADAPTATION OF E. COLI

TABLE 1. Bacterial strains Genotype X- leuB6 thi-l thr-l supE44 A- leuB6 thi-1 thr-l supE44 lacYl TnlO dnaK756(Ts) WM1389-plasmid pMOB45:dnaK+ A- IeuB6 thi-l thr-l supE44 lacYl dnaJ259(Ts) X-leuB6 thi-l thr-l thr-l supE44 grpE280 F- thi rha lacZ nagA kdpABCS trkA405 trkDI

millimoles of K+ per gram of total proteins. A sample of cells pelleted in an Eppendorf centrifuge for protein assay. Then cells were resuspended in 0.1 N NaOH and kept overnight at room temperature. Proteins were determined on aliquots by the method of Lowry et al. (21). Preparation of cell lysates for DnaK assay. A 10-ml portion of cells at an optical density of 0.4 at 600 nm was pelleted at 6,000 x g (10 min at 4°C). The pellets were resuspended in 200 ,ul of cold buffer containing 20 mM Tris HCI (pH 7.5), 10 mM magnesium acetate, 0.25 M KCI, and 0.2 mM phenylmethylsulfonyl fluoride. Suspensions were frozen in liquid nitrogen and then thawed at 4°C, and 80 ,u1 of the same buffer containing 60 ,ug of DNase per ml, 300 ,ug of RNase per ml, 1.2 mg of lysozyme per ml, and 0.5 mM freshly prepared phenylmethylsulfonyl fluoride was added. The suspensions were vortexed and incubated on ice for 30 min; they were then frozen and thawed again at 4AC and received 80 ,ul of Tris buffer containing 4% Triton X-100 and'3 M KCl. After a new cycle of freezing and thawing, the lysates were centrifuged at 12,000 x g for 10 min; supernatants were collected, and their volumes were determined. Protein concentration of the supernatants was estimated by the method of Bradford, using bovine serum albumin as a standard (4). Sodium dodecyl sulfate-polyacrylamide gel electrophoresis, transfer to nitrocellulose, and immunoblotting. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis was carried out by the method of Landoulsi et al. (19). A 40-pI amount of each lysate containing 10 ,ug of proteins was charged onto a 10% polyacrylamide gel along with samples of purified DnaK protein ranging from 3 to 12 ng. After electrophoresis, the gel was equilibrated for 1 h in transfer buffer (39 mM glycine, 48 mM Tris, pH 8.2). The gel was then placed on a sheet of nitrocellulose (0.45-p.m pore size, type HA; Millipore) equilibrated with transfer buffer. Electrical transfer was carried out by using an LKB electrophoretic graphite transfer unit for 75 min. The nitrocellulose membrane was then saturated for 1 h at room temperature with Tris-buffered saline (TBS; 50 mM Tris HCI [pH 7.4], 0.2 M NaCl, 5% powdered skimmed milk). Saturated membrane was incubated overnight at 4°C in a cellophane bag with anti-DnaK protein antiserum diluted 1,000-fold in TBS-milk buffer. Then the membrane was washed three times for 5 min each with TBS-milk buffer and incubated for 2 h with rabbit anti-immunoglobulin G-peroxidase (Miles Scientific) diluted 1,000-fold in TBS buffer containing 1% powdered skimmed milk. At the end, the membrane was washed for three 15-min periods with TBS alone, and the DnaK-immunoglobulin G-peroxidase complex was revealed by using the 4-chloro-l-naphthol-H202 development method (15). Color was developed within 60 s, and the reaction was stopped by repeated washes with distilled water. The blots were photographed and densitomwas

4405

etry was carried out on negatives, using a zig-zag program on a Shimadzu dual-wavelength thin-layer chromatographic

density scanner. RESULTS As reported previously (23), absorbance variations triggered by osmotic upshock show three successive phases, as shown for strain C600 in Fig. 1. An immediate increase of absorbance due to a very fast efflux of water (cell plasmolysis) and to cell shrinkage reaches a maximum within 2 to 3 min (17). This is followed by a phase of partial deplasmolysis, characterized by a decrease of absorbance which can be attributed to osmo-dependent K+ uptake (11, 23) and synthesis of trehalose (9). In the third phase, absorbance increases again as a consequence of growth recovery. The dnaK+ gene product is involved in osmotic adaptation. Figure 1 shows the variations of absorbance triggered by raising the medium osmolarity from 300 to 1,500 mosM, at 30°C, in cultures of two bacterial strains: the C600 parent strain and the dnaK756(Ts) mutant WM1389. Immediately after addition of 0.6 M NaCl, all cells started to plasmolyze and the absorbance increased by 40 to 50% in the two strains. In contrast, further deplasmolysis depended on the strain. Reproducibly, the duration of deplasmolysis was found to be 60 to 70 min in the parent C600, while the absorbance decreased by 20 to 25%. Conspicuously, in the dnaK mutant WM1389, the mean duration of deplasmolysis was 120 to 130 min and absorbance never decreased by more than 5 to 10%. When the deplasmolysis phase ended, the C600 strain could recover growth, with a doubling time of 290 min versus 90 min at low osmolarity. The dnaK mutant WM1389 could not recover significant growth even after 5 h, although >90% of cells were still viable when plated at low osmolarity on solid medium (data not shown). Transductants of WM1389 for the dnaK+ allele by P1 phage could deplasmolyze and recover growth, as did strain C600 (data not shown). When cells of strain WM1390, which overproduces DnaK protein by 20-fold (21a; unpublished results), were shifted to 1,500 mosM (Fig. 1), the duration of deplasmolysis (40 to 50 min) was shorter than in the parent strain C600 (60 to 70 min); the doubling time of recovered growth (180 min) was significantly shorter than that of C600 (290 min). DnaK content increases in- response to osmotic upshift. The wild-type strain C600 was assayed for DnaK content by immunoblotting with anti-DnaK antibodies. Figure 2a and b shows that, within 5 min after shift, C600 cells had increased their DnaK content as determined by immunoblotting and densitometric analysis. The mean increase was 2.8-fold. This level was maintained for 45 min, when cells started to resynthesize proteins and recover growth. The same experiment was repeated three times, and the mean DnaK increases all were two- to threefold. The same results were obtained with another wild-type strain, 'ML30 (data not

shown). The dnaK756 mutant is deficient in K+ transport at high osmolarity. In E. coli cells grown in a medium containing >5 mM KCI, the only expressed K+ transport system is the Trk+ system (10). As shown elsewhere (24), a sudden elevation of external osmolarity immediately turns on the Trk pump; the resulting influx of K+ ions triggers a fast increase of cellular K+ content. Figure 3 shows that cells of strain C600, grown at 30°C in a medium containing 10 mM KCI, increased their K+ content by 100% within 40 to 50 min after the shift from 300 to

4406

MEURY AND KOHIYAMA

J. BACTERIOL.

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TIME (hr) FIG. 1. Effect of dnaK756 allele on osmotic adaptation. All strains were grown at 30°C in 300 mosM minimal medium (containing 10 mM KCl) up to an optical density of 0.2 to 0.3 at 600 nm, when 0.6 M NaCl was added (shown by an arrow). The A6. was measured. Symbols: strain WM1389 at 300 (O) and 1,500 (U) mosM; strain WM1390 at 300 (A) and 1,500 (A) mosM; parent strain C600 at 300 (0) and 1,500 (0) mosM.

1,500 mosM. Whereas in cells of the dnaK756 mutant WM1389 the K+ increment was never higher than 30 to 40% of the initial content, the WM1389 cells transduced for the wild dnaK+ allele and cells of the DnaK-overproducing strain WM1390 increased their K+ content by 100%, as in C600. The question that arose, therefore, was whether the dnaK756 mutant WM1389 could be impaired in K+ transport as a consequence of the dnaK756 mutation. The ability of the mutant strain to accumulate K+ via the Trk system was tested. Cells grown in a medium containing 10 mM KCl were depleted of K+ (22) and then allowed to take up K+ at different osmolarities in an Na+ phosphate buffer (0.1 M; pH 7) with glucose as the carbon source. The kinetics of K+ uptake at 300 and 1,500 mosM in strains C600 and WM1389 can be seen in Fig. 4. At low osmolarity, the two strains had the same rate of net K+ uptake and the same accumulation level. At high osmolarity, unlike C600, the dnaK mutant WM1389 was unable to take up K+. Moreover, more K+ was lost with respect to the K+-depleted control cells, although 10 mM KCl was present in the medium. When the medium was free of glucose, which permits K+ to be taken up but turns off the K+ efflux, the same result was obtained, as if only the K+ influx was changed as a consequence of the dnaK756 mutation. Whatever the osmolarity, K+ uptake in strain WM1390 looked like that in C600 (data not shown). K+ uptake was also measured at 42°C, the restrictive temperature, in a 300 mosM medium. Interestingly, what-

the strain, C600 or the dnaK mutant, it was not significantly different from that at 30°C. Figure 5 shows the K+ content as a function of external osmolarity. In the dnaK756 mutant WM1389, the higher the external osmolarity, the higher the impairment of K+ transport and, therefore, the lower the final K+ content. In C600 and the DnaK-overproducing strain, the higher the external osmolarity, the higher the cellular K+ content, even though the K+ content decreased slightly when the osmolarity exceeded 1,300 mosM; the same result could be obtained with all other strains carrying the wild dnaK+ allele, such as AB1157 and ML30. The results support the idea that the DnaK protein is required to maintain the activity of the Trk pump at high osmolarity. A high external K+ concentration overcomes the DnaK defect. When mutant TK2205, which lacks both K+ transport systems Kdp and Trk (27) and therefore requires high external K+ for growth, was grown in minimal medium containing 40 mM KCl and then was shifted from 300 to 1,500 mosM with 0.6 M KCl, its K+ content increased by 2.5-fold within 30 min (data not shown). Obviously, in these conditions, K+ had entered the cell by a pathway different from K+ transport systems (27). When cells of strain C600 were shifted from 300 to 1,500 mosM by addition of 0.6 M KCl in place of 0.6 M NaCl, the increase of internal K+ content was both faster and higher. Deplasmolysis was also found to be faster and the recovered ever

ROLE OF DnaK IN OSMOTIC ADAPTATION OF E. COLI

VOL. 173, 1991

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FIG. 2. Immunoblot and corresponding densitometric analysis of the variations of DnaK in wild-type strain C600, in response to osmotic upshift, by using anti-DnaK antibodies. Immunoblotting and densitometric analysis were carried out as described in Materials and Methods. (a) Lanes 1 and 2, controls at 300 mosM; lanes 3 to 7, 1, 5, 15, 25, and 35 min after the shift, respectively; lanes 8 to 11, immunoblot of purified DnaK: 3, 6, 9, and 12 ng, respectively. (b) Peak areas (@) from densitometric analysis and A6. (A) are plotted versus time. (c) Peak areas (A) from densitometric analysis of purified DnaK immunoblot are plotted versus the amount of DnaK. Areas are expressed in arbitrary units.

growth rate was slightly improved (Fig. 6a), as if a part of the accumulated K+ had bypassed the K+ pump. When cells of the dnaK756 mutant WM1389 were shifted at 30°C from 300 to 1,500 mosM by addition of 0.6 M KCl, the pattern of deplasmolysis and subsequent growth looked like that of the wild-type parent instead of what happens when 0.6 M NaCl is used (Fig. 6b). Internal K+ content also increased as fast as in the parent C600. Effect of a dnaJ or grpE mutation on osmotic adaptation. Since there is evidence that the DnaK, DnaJ, and GrpE proteins function together in the initiation of E. coli and A phage DNA replication, we looked for a possible role for these products in osmotic adaptation. The responses, at 30°C, of mutants WM1289 dnaJ259(Ts) and C600 grpE280 (Ts) to osmotic upshift were not different from that of their wild-type parent. In particular, deplasmolysis was achieved in all strains by a 100% increase in K+ content. DISCUSSION Our results suggest that the heat shock protein DnaK is involved in the response of E. coli to osmotic stress. Clearly, cells carrying the mutation dnaK756 cannot deplasmolyze upon osmotic upshock and, as a consequence, cannot recover growth as does the wild-type parent C600. That deplasmolysis cannot be achieved in the dnaK756 mutant seems to be accounted for by the inability of these cells to perform a sufficient osmo-dependent K+ uptake. Transducing the wild allele dnaK+ in the dnaK756 mutant restores both the ability to grow at 42°C in a low-osmolarity

medium and the ability to deplasmolyze upon shock; morethe osmo-dependent K+ influx is also recovered. Evidently, the mutant DnaK protein cannot substitute for the wild-type protein, suggesting that the DnaK+ protein is necessary for cells to take up K+ and deplasmolyze when osmotically shifted up. The same conclusion can be drawn from the experiments in which a DnaK overproducer strain was osmotically shocked. Clearly, the possibility that mutations other than dnaK756 could be responsible for the K+ transport deficiency has been ruled out since the presence of the plasmid carrying dnaK+ in the mutant restores K+ over,

transport.

The question now arises, how is DnaK protein involved in the transport of K+ at high osmolarity? It cannot be definitively excluded that the impairment of K+ transport observed at high osmolarity results from a nonspecific defect induced by the dnaK756 mutation. A defect of membrane energization is a possibility, but when the initial rates of uptake of K+ and other substrates such as maltose or proline were compared in both dnaK+ and dnaK756 strains, no significant difference was revealed at 300 mosM (data not shown). The data can also be interpreted as a specific impairment of K+ transport, which seems to be essential for the reversion of plasmolysis and the recovery of membrane functions. In this context, E. coli DnaK protein could play a role in relation to its general "chaperoning" function in protein folding or protection of denatured polypeptides (14, 26). By this view, DnaK could aid in the translocation of a possible periplasmic component of the Trk pump. Another concep-

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FIG. 3. Effect of osmotic upshift on K+ content in a strain carrying the dnaK756 allele. Cells were grown at 30°C in 300 mosM minimal medium containing 10 mM KCI. Then the osmolarity of the medium was raised to 1,500 mosM by addition of 0.6 M NaCl (indicated by an arrow). The K+ content of strain WM1389 (U) was compared with those of the wild-type parent C600 (0) and strain WM1390 harboring a multicopy plasmid carrying the wild allele dnaK+ (l).

tually related possibility can also be considered: partially denatured proteins with exposed hydrophobic groups could interact to form insoluble aggregates. This could result from the removal of water from the cytoplasm as an immediate consequence of osmotic upshift (29). Conceivably, at high osmolarity DnaK, which can also be localized in the membrane fraction (18), could prevent, specifically or nonspecifically, the denaturation of some component of the Trk system. An alternative possibility can be inferred from the suggestion that DnaK could turn off the synthesis of heat shock proteins (30). Some proteins induced by osmotic upshift could exert a negative effect on K+ transport via Trk. This control could be exerted as long as the DnaK content has not reached a higher level; therefore, it could be lengthened in the dnaK756 mutant and shortened in the DnaK-overproducing strain. However, the high internal K+ content elicited in dnaK756 cells by a 0.6 M KCl shift could overcome the dnaK756 defect, which would seem to contradict such a view. DnaK content is increased by two- to threefold upon osmotic upshock. Similar increases in cellular DnaK protein have been observed under other stressful growth conditions such as heat shock. However, at the present time, it is not known whether these increases in DnaK are necessary for cell survival under stressful conditions such as osmotic shock. Improved recovery from osmotic shock was observed, however, in a C600-derived strain overproducing DnaK. In contrast to the requirement for DnaK, the heat shock proteins DnaJ and GrpE do not seem to be essential for osmotic adaptation, although they may be required for replication of both E. coli and X phage chromosomes (32).

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TIME (min) FIG. 4. K+ transport is impaired in a strain carrying the dnaK756 allele. Cells of strains WM1389 and C600 grown at 30°C in 300 mosM minimal medium containing 10 mM KCI were depleted of K+ by washing twice with distilled water. Cells were resuspended in the presence of glucose at two osmolarities: 300 mosM in 0.1 M sodium phosphate buffer (pH 7) and 1,500 mosM in the same buffer supplemented with 0.6 M NaCl. K+ uptake was triggered by the addition of 10 mM KCI to the medium. Symbols: strain C600 at 300 (0) and 1,500 (-) mosM; strain WM1389 at 300 mosM (A) and 1,500 (A) mosM.

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FIG. 5. Dependence of K+ content on medium osmolarity in the dnaK756 mutant compared with that of the wild-type parent C600 and the DnaK-overproducing strain WM1390. Cells grown at 30°C in 300 mosM minimal medium were depleted of K+ by washing twice with distilled water, and their residual K+ content was measured. Cells were then resuspended in media of osmolarities of between 300 and 1,500 mosM. Resuspension medium at 300 mosM contained 0.1 M sodium phosphate buffer (pH 7) and glucose. Osmolarities higher than 300 mosM were built up with increasing concentrations of NaCl. K+ uptake was started by the addition of 10 mM KCI; K+ content was determined at steady state. Symbols: strain WM1389 (A); strain C600 (0); DnaK-overproducing strain WM1390 (O).

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FIG. 6. A high external K+ concentration overcomes the DnaK defect in a strain carrying the dnaK756 allele. Wild-type strain C600 (a) and mutant strain WM1389 (b) were grown at 30°C in 300 mosM minimal medium. Then the osmolarity of culture samples was increased with either 0.6 M NaCl or 0.6 M KCI. The Awo and K+ content were measured. Symbols: A6. (open symbols) and K+ content (closed symbols) at 300 mosM (O and 0) and then at 1,500 mosM with 0.6 M NaCl (EL and *) or 0.6 M KCI (A and A).

4410

MEURY AND KOHIYAMA ACKNOWLEDGMENTS

We are indebted to A. Malki, who purified DnaK and kindly supplied the anti-DnaK antibodies; to R. D'Ari and P. Hughes for help in improving the manuscript; and to Annie Bracone for technical assistance. This work was partly supported by a grant from the Ligue Nationale Contre le Cancer.

REFERENCES 1. Alfano, C., and R. McMaken. 1989. Heat-shock protein-mediated disassembly of nucleo-protein structures is required for the initiation of bacteriophage A DNA replication. J. Biol. Chem. 264:10709-10718. 2. Barron, A., G. May, E. Bremer, and M. Villarejo. 1986. Regulation of envelope protein composition during adaptation to osmotic stress in Escherichia coli. J. Bacteriol. 167:433-438. 3. Bhagwat, A. A., and S. K. Apte. 1989. Comparative analysis of proteins induced by heat shock, salinity, and osmotic stress in the nitrogen-fixing cyanobacterium Anabaena sp. strain L-31. J. Bacteriol. 171:5187-5189. 4. Bradford, M. M. 1976. A rapid and sensitive method for the quantitation of microgram quantities of protein using the principle of protein-dye binding. Anal. Biochem. 72:248-254. 5. Bukau, B., and G. Walker. 1989. AdnaK52 mutants of Escherichia coli have defects in chromosome segregation and plasmid maintainance at normal growth temperature. J. Bacteriol. 171: 6030-6038. 6. Cairney, J., I. R. Booth, and C. F. Higgins. 1985. Osmoregulation of gene expression in Salmonella typhimurium: proU encodes an osmotically induced betaine transport system. J. Bacteriol. 164:1224-1232. 7. Clark, D., and J. Parker. 1984. Proteins induced by high osmotic pressure in Escherichia coli. FEMS Microbiol. Lett. 25:81-83. 8. Daniels, C. J., A. H. Z. McKee, and W. F. Doolittle. 1984. Archaebacterial heat-shock proteins. EMBO J. 3:745-749. 9. Dinnbier, U., E. Limpinsel, R. Schmidt, and E. P. Bakker. 1988. Transient accumulation of potassium glutamate and its replacement by trehalose during adaptation of growing cells of Escherichia coli K-12 to elevated sodium chloride concentrations. Arch. Microbiol. 150:348-357. 10. Epstein, W. 1985. The Kdp system: a bacterial K+-ATPase. Curr. Top. Membr. Transp. 23:153-175. 11. Epstein, W., and S. Schultz. 1965. Cation transport in Escherichia coli. V. Regulation of cation content. J. Gen. Physiol. 49:221-234.

12. Georgopoulos, C. P., K. Tilly, D. Drahos, and R. Hendrix. 1982. The B66.0 protein of Escherichia coli is the product of the dnaK+ gene. J. Bacteriol. 149:1175-1177. 13. Hecker, M., C. Heim, U. Volker, and L. Wolfel. 1988. Induction of stress proteins by sodium chloride treatment in Bacillus subtilis. Arch. Microbiol. 150:564-566. 14. Hemmingsen, S. M., C. Woodford, S. M. van der Vies, K. Tilly, D. T. Dennis, C. P. Georgeopoulos, R. W. Hendrix, and R. Ellis. 1988. Homologous plant and bacterial proteins chaperone oligomeric protein assembly. Nature (London) 333:330-334. 15. Howe, J. G., and J. W. B. Hershey. 1981. A sensitive immuno-

J. BACTERIOL.

blotting method for measuring protein synthesis initiation factor levels in lysates of Escherichia coli. J. Biol. Chem. 256:1283612839. 16. Kennedy, E. P. 1982. Osmotic regulation and the biosynthesis of membrane-derived oligosaccharides. Proc. Natl. Acad. Sci. USA 79:1092-1095. 17. Koch, A. 1984. Shrinkage of growing Escherichia coli cells by osmotic challenge. J. Bacteriol. 159:919-924. 18. Kostyal, D. A., M. Farrell, A. McCabe, Z. Mei, and W. Firshein. 1989. Replication of an RK2 miniplasmid derivative in vitro by a DNA/membrane complex extracted from Escherichia coli. Involvement of the DnaA but not DnaK host proteins and association of these and plasmid-encoded proteins with the inner membrane. Plasmid 21:226-237. 19. Landoulsi, A., P. Hughes, R. Kern, and M. Kohiyama. 1989. dam methylation and the initiation of DNA replication on oriC plasmid. Mol. Gen. Genet. 216:217-223. 20. Liberek, K., C. Georgopoulos, and M. Zylicz. 1988. Role of the Escherichia coli DnaK and DnaJ heat-shock proteins in the initiation of bacteriophage X DNA replication. Proc. Natl. Acad. Sci. USA 85:6632-6636. 21. Lowry, 0. H., N. J. Rosebrough, A. L. Farr, and R. J. Randall. 1951. Protein measurement with the Folin phenol reagent. J. Biol. Chem. 193:265-275. 21a.Messer, W. Unpublished data. 22. Meury, J. 1976. Potassium transport in Escherichia coli. Thesis. Universite de Paris, Paris. 23. Meury, J. 1988. Glycine betaine reverses the effects of osmotic stress on DNA replication and cellular division in Escherichia coli. Arch. Microbiol. 149:232-239. 24. Meury, J., A. Robin, and P. Monier-Champeix. 1985. Turgorcontrolled fluxes and their pathways in Escherichia coli. Eur. J. Biochem. 151:613-619. 25. Neidhardt, F. C., and R. A. Van Bogelen. 1981. Positive regulatory gene for temperature-controlled proteins in Escherichia coli. Biochem. Biophys. Res. Commun. 100:894-900. 26. Pelham, H. R. B. 1986. Speculations on the functions of the major heat-shock and glucose-regulated proteins. Cell 46:959961. 27. Roads, D., F. B. Waters, and W. Epstein. 1976. Cation transport in Escherichia coli. VIII. Potassium transport mutants. J. Gen. Physiol. 67:325-341. 28. Sakakibara, Y. 1988. The dnaK gene of Escherichia coli functions in initiation of chromosome replication. J. Bacteriol. 170:972-979. 29. Schobert, B. 1977. Is there an osmotic regulatory mechanism in algae and higher plants? J. Theor. Biol. 68:17-26. 30. Tilly, K., N. McKittrick, M. Zylicz, and C. Georgopoulos. 1983. The DnaK protein modulates the heat-shock response of Escherichia coli. Cell 34:641-646. 31. Tilly, K., and M. Yarmolinsky. 1989. Participation of Escherichia coli heat shock proteins DnaJ, DnaK, and GrpE in P1 plasmid replication. J. Bacteriol. 171:6025-6029. 32. Zylicz, M., D. Ang, K. Liberek, and C. Georgopoulos. 1989. Initiation of X DNA replication with purified-host and bacteriophage-encoded proteins: the role of the DnaK, DnaJ and GrpE heat-shock proteins. EMBO J. 8:1601-1608.