Vol. 165, No. 3JOURNAL OF BACTERIOLOGY, Mar. 1986, p. 1)040-10420021-9193/86/031040-03$02.00/0Copyright X) 1986, American Society for Microbiology

Effect of Ethanol and Heat Stresses on the Protein Pattern ofZymomonas mobilis

GERARD P. F. MICHEL AND JIRI STARKALaboratoire de Physiologie Microbienne, Universite d'Aix-Marseille, 13288 Marseille Cedex 9, France

Received 29 July 1985/Accepted 27 November 1985

Heat or ethanol shock of Zymomonas mobiis enhanced the labeling by [35S]methionine of severalpolypeptides and induced the synthesis of a new polypeptide (molecular weight, 18,500) associated with theenvelope fraction. These results indicate the existence of a typical heat-shock response in Z. mobilis. Thisresponse could be involved in the induction of increased ethanol tolerance in Z. mobilis cells.

Zymomonas mobilis converts glucose or fructose toethanol and CO2 in equimolar amounts. Hence it may beassumed that this organism should be particularly welladapted to withstand elevated alcohol concentrations accu-mulating in the growth medium. Indeed, its growth yield isunaffected by ethanol up to 7% (2). Ethanol tolerance iscorrelated with a characteristic membrane lipid composition(1, 3, 15, 16), but its mechanism appears to be more complex(2). In some organisms, ethanol has been shown to have aneffect similar to that of heat in stimulating the synthesis ofspecific heat-shock proteins controlled by the htpR gene (6,10, 17) and in the accumulation of alarmones (5). Of partic-ular interest is the simultaneous enhancement of ethanoltolerance and thermotolerance of Escherichia coli inducedby a short incubation at a supraoptimal temperature (10).Cross-resistance between ethanol and heat was also ob-served in Saccharomyces cerevisiae (13) and other eucary-otic organisms (7).We have reported specific changes in the membrane

protein composition of Z. mobilis induced by high ethanolconcentrations (9), but it was not clear whether they werecorrelated with the heat-shock response. A basic questionremained: does ethanol induce heat-shock proteins in anobligately fermentative organism? In this communication wepresent evidence for the existence of a typical heat-shockresponse induced by heat or ethanol and conferring in-creased heat and ethanol cross-resistance on Z. mobilis.

Induction of heat or ethanol resistance in Z. mobilis ZM4(1) was investigated by shifting bacteria grown at 30°C in acomplex MYPG medium (9) to higher temperatures or byadding increasing concentrations of alcohol. After plating onsolid MYPG medium, cell viability was estimated by count-ing colonies after 72 h of incubation at 30°C. Incubation at45°C for 10 min or addition of up to 7% (vol/vol) ethanolfollowed by incubation at 30°C for 30 min in the presence ofethanol had no effect on colony-forming ability. Highertemperatures or ethanol concentrations caused a rapid de-cline of the cell viability.As expected, sublethal ethanol or heat shock enhanced

cell resistance to subsequent heat and ethanol treatment.Indeed, when bacteria were shifted from 30 to 450C for 10min and then transferred to 50°C for 10 min before plating,96% of cells remained viable, whereas a control heated at50°C without pretreatment had only 42% survival. Similarly,95% of cells shifted to 500C for 10 min retained viabilitywhen they were preincubated at 30°C for 30 min in mediumwith 5% ethanol. Finally, 5% ethanol pretreatment for 20

min followed by 30 min of incubation in medium containing20% ethanol allowed survival of 0.73% of the population,whereas without pretreatment survival was only 0.0017%. Asimilar response was obtained when Z. mobilis was grown ina minimal medium containing 1% KH2PO4, 0.1% (NH4)2SO4,0.05% MgSO4 7H20, 0.0005% calcium pantothenate, and2% glucose and supplemented with 0.1% yeast extract (pH5). Thus, these experiments indicate that heat and ethanolpretreatment increases tolerance ofZ. mobilis to ethanol andheat.

In other organisms, induction of thermotolerance is di-rectly correlated with induction of a specific set of heat-shock proteins (8). We therefore examined the effect ofinduced heat and ethanol tolerance on protein synthesis in Z.mobilis. Bacteria were preincubated for 10 min at 30°C inminimal medium containing 5% ethanol and then labeled inthe same medium with [35S]methionine (1,395 Ci mmol -1.0.8 jxCi ml-'). In a parallel experiment bacteria were ex-posed to a heat shock at 45°C and labeled. Samnples werewithdrawn at 2-min intervals and precipitated with cold 15%trichloroacetic acid. Precipitates collected on GF/C filters(Whatman Ltd.) were placed in vials containing Ready-SolvMP scintillant (Beckman Instruments, Inc.) and counted.Both heat- and ethanol-shocked cells displayed a transientbut severe slowing down of incorporation between thesecond and eighth minute of labeling. Then incorporationresumed, but at a lower rate than in the control (data notshown). The transient slow-labeling period observed in

TABLE 1. Labeling of polypeptides in heat- and ethanol-shockedZ. mobilisa

Mol mass Polypeptide % Radioactivity relative to control(kilodaltons) no. Ethanol shock Heat shock

66 3 137 17154 5 198 35438 10 133 12631 11 119 5318.5 14 100 41816.5 12 lOOb 5914 13 246 314

a Polypeptides were separated by two-dimensional gel electrophoresis.Gels were fluorographed, and [35S]methionine-labeled spots amplified byethanol or heat shock were cut out from the dnied gels. Radioactivity wasmeasured essentially as described by Pedersen and Reeh (12).

b Polypeptides 12 and 14 were hardly at all or not detected in the control, sovalues given are expressed in percentage relative to ethanol-treated cells.

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NOTES 1041

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treated Z. mobilis resembled the period in heat-shocked E.coli when the synthesis of most cell proteins is repressed andheat-shock proteins are induced (17).To determine the behavior of individual proteins in

ethanol- and heat-shocked Z. mobilis, a 6-min labeling wascarried out with 20 ,uCi of [35S]methionine per-ml. Proteinssolubilized and analyzed by two-dimensional electrophoresisby the method of O'Farrell (11) were detected byfluorography (14). Fluorogramis of labeled cell proteinsshowed several differences between the protein patterns ofstressed and unstressed cells (Fig. 1). Both heat and ethanolinduced the synthesis of polypeptide 14, which was notdetected in untreated cells, and stimulated synthesis of

-20.1A14

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-14.3FIG. 1. Two-dimensional gel electrophoresis of [35S] methionine-

labeled polypeptides of Z. mobilis. (A) Fluorograph of control cellspulse-labeled for 6 min; (B) cells pulse-labeled in medium containing5% ethanol as described in the text; (C) heat-shocked cells pulse-labeled at 45°C. Protein (50 ,ug) was loaded on isoelectric focusinggels; pH gradient was measured essentially as described by O'Far-rell (11). Positions of molecular mass markers are indicated in theright margin (in kilodaltons). Apparent spot intensity and measuredradioactivity shown in Table 1 are not always in agreement becausethe film was not preflashed (4).

polypeptides 3, 5, 10, 12, and 13. A slightly increasedsynthesis of polypeptide 11 was found only in ethanol-shocked cells. The level of polypeptide 5 was increasedthreefold in heat-shocked cells and twofold in ethanol-treated cells (Table 1). Polypeptide 14 was four times moreabundant in heat-shocked bacteria than in ethanol-shockedbacteria. Among the polypeptides whose synthesis wasstimulated by both shocks, 5 and 14 were found associatedwith the envelope fraction (data not shown).

In conclusion, our results clearly show that the stress of Z.mobilis by heat or ethanol increased the synthesis of severalpolypeptides of which at least six (i.e. polypeptides 3, 5, 10,12, 13, and 14) are heat-shock proteins. At present, thephysiological significance of these proteins is not known, butthere is reason to believe that they are involved in themechanism of tolerance of Z. mobilis and probably of othercells to heat and ethanol. Furthermore, our finding that someheat-shock proteins are localized in the envelope fraction ofZ. mobilis suggests the involvement of membranes in theethanol- and heat-shock response. We are currently studyingthe localization and fate of heat- and ethanol-shock proteinsin purified fractions of inner and outer membranes of Z.mobilis.

We thank J. P. Bouche and J. M. Pages for their advice andZdenka Starka for excellent technical assistance.

This work was supported by grant AIP 5012 from the ProgrammeInterdisciplinaire de Recherches sur les Sciences pour l'Energie etMatibres Premitres-Agence Frangaise pour la Maitrise de l'Energie.

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1042 NOTES

LITERATURE CITED

1. Carey, V. C., and L. 0. Ingram. 1983. Lipid composition ofZymomonas mobilis: effects of ethanol and glucose. J. Bacte-riol. 154:1291-1300.

2. Ingram, L. O., and T. M. Buttke. 1984. Effects of alcohols onmicro-organisms. Adv. Microbiol. Physiol. 25:254-290.

3. Ingram, L. 0. and N. S. Vreeland. 1980. Differential effects ofethanol and hexanol on the Escherichia coli cell envelope. J.Bacteriol. 144:481-488.

4. Laskey, R. A., and A. D. Mills. 1975. Quantitative film detectionof 3H and 4C in polyacrylamide gels by fluorography. Eur. J.Biochem. 56:335-341.

5. Lee, P. C., B. R. Bochner, and B. N. Ames. 1983. AppppA,heat-shock stress and cell oxidation. Proc. Natl. Acad. Sci.USA 80:7496-7500.

6. Lemaux, P. G., S. L. Herendeen, P. L. Bloch, and F. C.Neidhardt. 1978. Transient rates of synthesis of individualpolypeptides in E. coli following temperature shifts. Cell13:427-434.

7. Li, G. C., and G. M. Hahn. 1978. Ethanol-induced tolerance toheat and to adriamycin. Nature (London) 274:699-701.

8. McAlister, L, and D. B. Finkelstein. 1980. Heat shock proteinsand thermal resistance in yeast. Biochem. Biophys. Res. Com-mun. 93:819-824.

9. Michel, G. P. F., T. Azoulay, and J. Starka. 1985. Ethanol effecton the membrane protein pattern of Zymomonas mobilis. Ann.Inst. Pasteur Microbiol. 136:173-179.

10. Neidhardt, F. C., R. A. Van Bogelen, and V. Vaughn. 1984. Thegenetics and regulation of heat shock proteins. Annu. Rev.Genet. 18:295-329.

11. O'Farreil, P. H. 1975. High resolution two-dimensional electro-phoresis of proteins. J. Biol. Chem. 250:4007-4021.

12. Pedersen, S., and S. V. Reeh. 1976. Analysis of the proteinssynthesized in ultraviolet light-irradiated Escherichia coli fol-lowing infection with bacteriophages Xdrif' 18 and Xdfus-3. Mol.Gen. Genet. 144:339-343.

13. Plesset, J., C. Palm, and C. S. McLaughlin. 1982. Induction ofheat shock proteins and thermotolerance by ethanol in Saccha-romyces cerevisiae. Biochem. Biophys. Res. Commun.108:1340-1345.

14. Skinner, M. K., and M. D. Griswold. 1983. Fluorographicdetection of radioactivity in polyacrylamide gels with 2,5-diphenyloxazole in acetic acid and its comparison with existingprocedures. Biochem. J. 209:281-284.

15. Thomas, D. S., J. A. Hossack, and A. H. Rose. 1978. Plasma-membrane lipid composition and ethanol tolerance in Saccha-romyces cerevisiae. Arch. Microbiol. 117:239-245.

16. Thomas, D. S., and A. H. Rose. 1979. Inhibitory effect of ethanolon growth and solute accumulation by Saccharomyces cerevi-siae as affected by plasma-membrane lipid composition. Arch.Microbiol. 122:49-55.

17. Yamamori, T., K. Ito, Y. Nakamura, and T. Yura. 1978.Transient regulation of protein synthesis in Escherichia coliupon shift-up of growth temperature. J. Bacteriol. 134: 1133-1140.

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