Vol. 173, No. 19JOURNAL OF BACTERIOLOGY, Oct. 1991, p. 5975-59820021-9193/91/195975-08$02.00/0Copyright ©D 1991, American Society for Microbiology

Gel Electrophoretic Analysis of Zymomonas mobilis Glycolytic andFermentative Enzymes: Identification of Alcohol

Dehydrogenase II as a Stress ProteintHAEJUNG AN,' R. K. SCOPES,2 M. RODRIGUEZ,2 KYLIE F. KESHAV,1 AND L. 0. INGRAM'*Department of Microbiology and Cell Science, University of Florida, Gainesville, Florida 32611,1 and

Centre for Protein and Enzyme Technology, La Trobe University,Bundoora, Victoria, Australia 30832

Received 29 April 1991/Accepted 16 July 1991

The 13 major enzymes which compose the glycolytic and fermentative pathways in Zymomonas mobilis are

particularly abundant and represent one-half of the soluble protein in exponential-phase cells. One- andtwo-dimensional polyacrylamide gel electrophoresis maps were developed for 12 of these enzymes. Assignmentswere made by comigration with purified proteins, comparison with overexpressed genes in recombinantstrains, and Western blots (immunoblots). Although most glycolytic enzymes appeared resistant to turnoverand accumulated in stationary-phase cells, the protein levels of pyruvate kinase, alcohol dehydrogenase I, andglucokinase declined. Alcohol dehydrogenase II was identified as a major stress protein and was induced bothby exposure to ethanol and by elevated temperature (45°C). This enzyme, encoded by the adhB gene, isexpressed from tandem promoters which share partial sequence identity with the Escherichia coli consensus

sequence for heat shock proteins.

Zymomonas mobilis is an obligately fermentative, gram-negative bacterium of commercial interest (6, 34, 35, 54).Using an Entner-Doudoroff glycolytic pathway, each moleof glucose is metabolized to produce 2 mol of ethanol and ofcarbon dioxide plus a single mole of ATP. Despite this lowenergy yield from glycolysis, Z. mobilis is essentially pro-totrophic and grows rapidly with a doubling time of 90 min.Large amounts of glycolytic and fermentative enzymes arerequired to support the high rates of flux needed to generateATP. Together, the glycolytic plus fermentative enzymes(ethanologenic enzymes) compose one-half of soluble cellu-lar protein (1, 19, 33, 41, 48). Genes encoding most of theseenzymes have been cloned and sequenced (4, 10-13, 21, 38).Six are arranged in two polycistronic operons, and sevenappear to be monocistronic (4, 14, 28). Southern analyseshave confirmed that these glycolytic and ethanologenicgenes are present as single chromosomal copies. Two genet-ically unrelated alcohol dehydrogenases (ADHs) are present(21). All the major Z. mobilis ethanologenic enzymes havebeen highly purified and characterized (2, 7, 18, 22, 36, 37,42, 44 49, 53, 56).

In eukaryotes (26), three glycolytic enzymes have beenidentified as stress proteins: glyceraldehyde-3-phosphate de-hydrogenase (GAP), enolase (ENO), and phosphoglyceratekinase (PGK). Previous studies with Z. mobilis have cata-logued the stress proteins synthesized in response to ethanoland heat (31, 32). In some cases, these proteins wereabundant before stress treatment but increased in abundanceas a response to stress. In this study, we investigated thepossibility that one or more of the Z. mobilis stress proteinsis an abundant glycolytic or fermentative enzyme.

* Corresponding author.t Florida Agricultural Experiment Station Publication number

R01098.

MATERIALS AND METHODS

Organisms and growth. Table 1 describes the bacteria andplasmids used. Z. mobilis CP4 was grown with 10% glucoseat 30°C in complex medium (21). Escherichia coli strainswere grown at 37°C in Luria broth (27).

Protein extracts. Soluble extracts of Z. mobilis were pre-pared from exponential-phase cells (0.5 unit of optical den-sity at 550 nm) and from stationary-phase cells (72 h). Aftercentrifugation, cell pellets were washed in 10 mM sodiumphosphate buffer (pH 6.5) containing 10 mM mercaptoetha-nol, resuspended in an equal volume of this buffer, andbroken by passage through a French pressure cell at 12,000lb/in2. Membranes were removed by centrifugation, andnucleic acids were digested by adding CaCl2 (2 mM), 50 jigof DNase I per ml, and 100 ,ug of RNase A per ml (2 h, 22°C).

Purification of GAP and PGK. Protein extracts were pre-pared from overnight cultures of E. coli cells containingeither plasmid pLOI314 (gap) or plasmid pLOI322 (pgk). Z.mobilis GAP and PGK are present at high levels in theserecombinants and each represents the dominant proteinband observed in sodium dodecyl sulfate-polyacrylamide gelelectrophoresis (SDS-PAGE) gels (10, 12, 14). For purifica-tion, a 2-ml sample of extract containing 80 to 120 mg ofprotein was diluted to 38 ml with distilled water and then 2ml of BioLyte (pH 4 to 6 for GAP and pH 5 to 8 for PGK;Bio-Rad Laboratories, Richmond, Calif.) was added. Afterbeing loaded into the Rotofor (Bio-Rad), samples werefocussed at 12 W constant power (600 V) for 5.5 h (2°C).Fractions were analyzed for enzymatic activity and protein(41). Purity of the most active fractions was confirmed onisoelectric focussing PAGE gels (16, 17). The purest frac-tions were used for the identification of GAP and PGK.

Purification of other enzymes. Glucose-6-phosphate dehy-drogenase (GDH), ENO, pyruvate kinase (PYK), phospho-gluconate dehydratase (GDT), phosphoglyceromutase (PGM),and 6-phosphogluconolactonase (PGL) were purified as de-scribed elsewhere (2, 42, 45, 46). Phosphogluconate dehy-dratase was purified by an improved procedure based on

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TABLE 1. Strains and plasmids used in this study

Bacterial strain Relevant Source oror plasmid genotype reference

StrainsZ. mobilis CP4 Prototroph 41E. coli DH5a lacZ recA Bethesda Research

Laboratories

PlasmidspLOI193 (shuttle mob Cmr Tcr 9

vector)pUC18 lacI'Z' Apr Bethesda Research

LaboratoriespLOI314 Zm gap+ Apr 14pLOI322 Zm pgk+ Apr 14pLOI411 Zm adhB+ Cmr Tcr 13pLOI418 Zm pdc+ Cmr Tcr 28pTC120 Zm g1k+ Apr 4

that described earlier (47), using a dye-ligand adsorbentprepared from Procion Red H-E7B. Pyruvate decarboxylase(PDC) and ADHI were purified as previously described (11,27, 37).

One-dimensional (1-D) PAGE gels. Laemmli (25) SDS-PAGE gels (10% acrylamide) analogous to the second di-mension of O'Farrell gels (40) were used for the initialidentification of Z. mobilis enzymes.Two-dimensional (2-D) PAGE separation of soluble pro-

teins. Proteins were analyzed by a modification of theO'Farrell procedure (40). The isoelectric focussing gel con-tained 4% polyacrylamide, 9.2 M urea, 2% Nonidet P-40(vol/vol), and 2% (wt/vol) pH 3/10 BioLyte. A 20 mMsolution of sodium hydroxide was used as the cathodebuffer. For protein identifications, 80-,ug samples of Z.mobilis protein extract were combined with 30 ,ug of purifiedenzyme. Purified enzymes were also run individually forcomparison. The denaturing dimension was run in 10%polyacrylamide. Molecular weight markers (Pharmacia, Pis-cataway, N.J.) were included in a separate lane (p4osphor-ylase b, 94,000; albumin, 67,000; ovalbumin, 43,000; car-bonic anhydrase, 30,000; trypsin inhibitor, 20,100; anda-lactalbumin 14,400). Proteins were stained with Coo-massie blue R-250 or silver stain (30).

Induction of Z. mobilis stress proteins. Cultures (100 ml)were grown in rich medium at 30°C to 0.5 unit of opticaldensity at 550 nm. Aliquots of 10 ml were transferred tosterile 15-ml Corex centrifuge tubes for stress treatments andfor [35S]methionine incorporation. Heat shock proteins wereinduced by transferring the aliquots to 45°C for 10 min.Ethanol shock proteins were induced by the addition of 1.1ml of absolute ethanol and incubation at 30°C for 10 min.Ethanol-treated cells were harvested by centrifugation(6,000 x g, 3 min, ambient) and resuspended in conditionedmedium from a parallel untreated culture. Untreated con-trols, heat-shocked, and ethanol-shocked cells were labeledfor 10 min at 30°C with [35S]methionine (4 ,uCi/ml, 1,100Ci/mmol) and then incubated for 5 min in the presence of 2mM cold methionine to allow completion of nascent pro-teins. Alternatively, for measurements of induction ofADHII activity, cells were incubated for 15 min at 30°C inthe presence of 2 mM cold methionine and assayed asdescribed previously (27).

[35S]methionine-labeled cells were harvested by centrifu-gation and washed twice in cold 10 mM Tris-HCl containing

CLUCUSECLI 4 (glk)

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Ir R LD (Od)CLYCENILDEuYSE-3-PESSPEtE PRBVATE

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IG ASH I (U&A)PCH |~(M ) ASN II (adi )

2-PEOUSPUILICEETE EJAL

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FIG. 1. Summary of Z. mobilis pathway for the fermentation ofglucose to ethanol and carbon dioxide.

2 mM methionine. Extracts containing soluble proteins wereprepared by resuspending pellets in 0.1 ml of lysis buffer (10mM Tris-HCl, 2% Nonidet P-40, 1 mg of egg lysozyme perml, 0.1 dig of DNase per ml, 2 mM methionine, 2 mMcalcium chloride, 10 mM P-mercaptoethanol; pH 8.5). Thesepreparations were stored on ice for 1 h and then incubatedfor 10 min at 45°C. Insoluble debris was removed by centrif-ugation at 10,000 x g for 10 min.

Gels were stained lightly with Coomassie blue beforeimpregnation with Enlightener (Dupont/NEN, Wilmington,Del.) and drying. Gels were fluorographed with KodakXAR-5 film at -70°C and subsequently photographed toallow identification of glycolytic and ethanologenic en-zymes.

Densitometry and quantitation of proteins. Video densitom-etry (Biomed Instruments Inc., Haywood, Calif.) was usedto estimate the relative amounts of proteins labeled with[35S]methionine after fluorography. Profiles of 1-D PAGEgels were also measured.

Antibodies and Western blotting (immunoblotting). In pre-vious studies, goat antibodies were prepared against PDC(11) and rabbit antibodies were prepared against ADHI andADHII (27). Rabbit antibodies to PGM, PGK, and ENOwere prepared in essentially the samie manner. 2-D PAGEgels were cut into sections and electroblotted to nitrocellu-lose membranes for Western analysis (51, 52). Proteins werestained with 0.5% ponceau S befdre development by usingalkaline phosphatase-conjugated second antibody (3, 5, 29).

RESULTS

Identification of Z. mobilis ethanologenic enzymes in 1-DSDS-PAGE gels. The main enzymes which form the Z.mobilis pathway from glucose to ethanol and carbon dioxideare shown in Fig. 1. NADH generated by glycolysis isoxidized during the production of ethanol. Apparent molec-ular weights for each of these enzymes and their subunits

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TABLE 2. Identification of glycolytic and fermentative enzymesfrom Z. mobilis on 2-D) and SDS-PAGE gels

Published App MWEnzyme ~ID AppEnzyme methoda MWb MW ID Reference

GLK A 34,700 33,000 G 49

GDH B 48,200 52,000 G 49

PGL F 25,800 26,600 G 46

GDT F 59,000 63,000 G 47

ALD 22,000 G 45

GAP C, D 40,300 41,000 G 4236,099 H 12

PGK C, D 41,500 44,000 G 4241,400 H 10

PGM B, E 28,600 26,000 G 42

ENO B 45,300 45,000 G 42

PYK B 54,500 57,000 G 42

PDC B, C, E 59,000 56,500 G 659,998 H 11

ADHI B 41,500 40,000 G 3634,000 I 1834,700 G 5636,096 H 21

ADHII C 38,000 38,000 G 3631,000 I 1831,100 G 5640,141 H 13

a Identification (ID) methods: A, overexpressed Z. mobilis gene in E. coli;B, purified Z. mobilis protein; C, overexpressed gene in Z. mobilis; D, purifiedprotein from recombinant E. coli; E, Western blot; F, tentative assignmentbased on comparison of appatent molecular weight with literature values; G,SDS-PAGE; H, translated seqtlence; I, gel filtration with SDS.

b App MW, apparent molecular weight.

have been reported previously (Table 2). To aid in theidentification of ethanologenic enzymes on 2-D gels, bandswhich included 12 of these proteins were first identified in1-D SDS-PAGE gels run under conditions analogous to thesecond dimension (Fig. 2). Identifications were based oncomparisons with purified Z. mobilis enzymes, recombinantenzymes, and Western blots (Table 2). Apparent molecularweights ranged from 59,000 for PDC to 22,600 for 2-keto-3-deoxy-6-phosphogluconate aldolase (ALD) under our condi-tions. A tentative assignment Was made for ALD based onabundance and molecular weight. PGM, glucokinase (GLK),and ENO appeared particularly well resolved.

Identification of Z. mobilis enzymes in 2-D PAGE gels. Over500 spots were resolved on 2-D gels of protein extracts(French press) stained with silver reagent (data not shown),while only 130 spots were evident on gels stained withCoomassie blue (Fig. 3A). In preliminary experiments, avariety of ampholines and isoelectric focussing conditionswere investigated to optimize protein separations. Fifteenprominent protein spots were well separated under theconditions selected and were presumed to include the abun-

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30 43 67APPARENT MOLECULAR WEIGHT

FIG. 2. SDS-PAGE separation of soluble proteins from log-phase Z. mobilis. Top: Densitometric scan indicating bands contain-ing glycolytic and fermentative enzymes. ALD is shown in paren-theses and represents a tentative assignment based on literaturereports of apparent molecular weight. Other assignments are basedon comigration with purified proteins, overexpression of clonedgenes in recombinants, or Western blots. Bottom: SDS-PAGE ofsoluble proteins from Z. mobilis.

dant enzymes of glycolysis and fermentation. In many cases,these abundant proteins formed charge chains or exhibitedmicroheterogeneity analogous to abundant proteins in othersystems (20, 39, 43). Loading the isoelectric focussing di-mension from the acidic end would be expected to minimizeor alter chemical reactions and protein associations. Gelsrun in this fashion exhibited reduced heterogeneity. How-ever, such gels were unsatisfactory for further investigationsbecause of the failure of much of the protein to remainsoluble and migrate to equilibrium in the isoelectric focus-sing dimension.Using both the 1-D map as a guide and the methods

reported in Table 2, unambiguous assignments were madefor 12 of the ethanologenic enzymes (Fig. 3B). ALD, thesmallest of the ethanologenic enzymes, was tentativelyidentified. Two prominent spots were observed with purePGM corresponding to those identified in protein extracts.These two prominent spots may represent products of post-transcriptional modification (55, 57) or closely related isoen-zymes ofPGM. No Z. mobilis genes have been reported thatencode PGM, although subcloning and sequencing of thisgene is currently under way in our laboratory. Among theglycolytic and ethanologenic enzymes examined, ENO ex-hibited the least tendency to form chains of spots.

Effect of growth phase on relative abundance of ethanolo-genic enzymes. The phase of growth had a pronounced effecton the abundance of soluble proteins in Z. mobilis extracts,although glycolytic and ethanologenic enzymes remained asthe dominant protein spots in soluble extracts from expo-nential- and stationary-phase cells (Fig. 3 and 4). Approxi-mately one-half of the proteins observed in extracts fromlog-phase cells were absent in extracts from stationary-phasecells. In stationary cultures, the relative abundance of fiveethanologenic enzymes increased (ENO, PGK, GAP,ADHII, and PGM). GDH, GDT, and PDC declined inabundance. PYK, ADHI, and GLK were essentially absent(represented by diamonds in Fig. 4B) in protein extractsfrom the stationary phase.

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5978 AN ETAL.J.BCEIL

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FIG. 3. Identification of glycolytic anid ethanologenic enzymes in 2-D PAGE gels of soluble proteins from log-phase Z. mobilis. (A) Frenchpress extract from exponential-phase cells. (B) Photoc-opy of panel A which is labeled to serve as a composite map of glycolytic'andfermentative 'enzymes. Assignments are based on comigraition with purified proteins, overexpression' of cloned genes in recombinants, orWestern blots (Table 2). ALD is a tentative assignmenit based on its presence as an abundant protein corresponding in size to literature reportsof apparent molecular weight. Selected stress proteins are circled. The approximate pH range of the gels was, from left to right, 8 to 4.

-43

Induction of stress proteins.; Figutes 5A, B, and D providea. comparison of proteins from detetgent extracts of un-treated and stressed cells labeled with [3Smethio-nine. Toidentify radioactive spots corresponding to glycol'ytic andethanologenic enzymes, we also stained gels with Coomassieblue (Fig. 5C and F) and compared them with the map ofFrench press extracts (Fig. 3B). All glycolytic en'zymnes and

ethanologenic enzymes were labeled with [35S]methion'ineexcept PGM (Fig. 5A), which is typically low in methionin'e(55).. Although the resolution of detergent extracts wasinferior to that of French press extracts, detergent extractionprovided a safer method for .processing. Stained proteins ihnthe dried, radioactive gels served as internal marke'rs for theaissignment of stress proteins.

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Photocopy of panel A which has been labeled as a map. Dashes denote proteins which are present but not clearly seen in the photocopy.Di'amonds indicate the near absence of three proteins (PYK, ADHI, and GLK). Selected stress proteins are circled. The apiproximate pHrange of the gel, from left to right, was 8.0 to 4.0.

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FIG. 5. 2-D PAGE gels and fluorographs of soluble proteins from log-phase cells after heat stress and ethanol stress. Molecular weights(MW) are indicated for gels A and D on the left. (A) Fluorograph of untreated control cells. (B) Fluorograph of ethanol-stressed cells. (C)Dried gel shown in panel B stained lightly with Coomassie blue. Ethanologenic enzymes serve as internal references for comparison withfluorograph. (D) Fluorograph of heat-shocked cells. (E) Photocopy of panel D labeled to serve as a map for proteins induced by heat shock.(F) Dried gel shown in panel D stained with Coomassie blue. Ethanologenic enzymes are marked on the Coomassie-stained gels and serveas internal references for fluorographs. Numbers indicate apparent molecular masses of stress proteins, which are circled. The approximatepH range of the gels, from left to right, was 8.0 to 4.0.

After ethanol stress, incorporation into glycolytic en-

zymes was severely reduced, with only two major proteinsbeing labeled. ENO was the most abundant protein incontrol cells, and incorporation into this protein was used as

a reference protein to estimate relative induction of the twodominant stress proteins (Table 3). The larger ethanol stressprotein with an apparent mass of 58,000 (SP58) was alsoabundant in unstressed cells (Fig. 3B and 5C). The smallerprotein with an apparent mass of 38,000 (SP38) was identi-fied as ADHII.

After heat stress, SP58 and ADHII (SP38) were stronglylabeled with [35S]methionine (Fig. 5D and E; Table 3).Incorporation into five additional proteins also increased

after heat treatment. Although these stress proteins rangedfrom an apparent molecular weight of 18,000 to 70,000, nonecorresponded to glycolytic or ethanologenic enzymes. Allseven stress proteins were evident in unstressed cells (Fig. 3and SA). It is interesting that ADHII, SP58, SP60, and SP70remained as abundant proteins in the stationary phase (Fig.4).The induction of adhB in response to stress was further

confirmed by direct measurements of ADHII activity. Thespecific activity of this enzyme increased in stressed cells by10% and by 30% after heat stress and ethanol stress,respectively, in comparison with control cells.Comparison of adhB promoters with E. coli stress promot-

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TABLE 3. Synthesis of ADHII (SP38) and SP58 afterheat and ethanol stress

Relative area (units)' Induction ratioTreatment (treated/untreated)

ADHII SP58 ENO ADHII SP58

Untreated 0.23 0.41 1.010%o ethanol 5.4 24 1.0 23 59450C 1.4 3.0 1.0 6.1 7.3

a Area units were normalized to that of ENO to allow comparison amongdifferent gels.

ers. As shown in Table 4, the P1 and P2 promoters of Z.mobilis adhB share similarity with the E. coli consensussequence for stress promoters (15, 39). This similarity wasparticularly evident between the P1 promoter of the E. colistress gene DnaK and the P2 promoter of adhB. Nineconsecutive bases were identical in the -35 region, followedby a spacing of 14 bases and four matching bases in the -10region.

DISCUSSION

Comparative stability of ethanologenic enzymes. Previousstudies by Osman et al. (41) indicated that the specificactivities of all but two glycolytic enzymes continued toincrease until maximum cell density was achieved. Thisincrease in specific activity has now been confirmed in partas an increase in abundance of glycolytic and ethanologenicenzymes in 2-D PAGE gels of soluble proteins. GLK andGAP were exceptional in that the specific activities of thesetwo enzymes began to decline near the end of the log phase.GLK appears to be degraded and is absent from protein gelsof stationary-phase cells. However, GAP appears to be quiteresistant to turnover despite the apparent loss of activity(41). A similar accumulation of abundant but inactive ADHIIhas been reported previously in recombinant strains of Z.mobilis (27). Both of these enzymes are easily inactivatedduring purification (36, 42), and it is likely that the abun-dance of these proteins reflects the resistance of an inactiveform to proteolysis and turnover.

Five ethanologenic enzymes (GAP, PGK, ADHII, ENO,and PGM) remained abundant even in the stationary phase,indicating resistance to proteolysis. It is likely that thisresistance is a contributing factor in the requisite productionof high levels needed for ATP generation. In contrast, GLK,PYK, and ADHI were absent or greatly reduced in proteinextracts from stationary-phase cells. These three enzymesappear to have been degraded relative to the other glycolyticand ethanologenic enzymes. Since all glycolytic enzymes areessential for ATP generation, the proteolysis of PYK and

TABLE 4. Comparison of Z. mobilis adhB promoter sequenceswith those of heat shock genes from E. coli (15)

Organism Promoter -35 region Spacing -10 region

Z. mobilis adhB P1 AAGCAGCCTTGCT 13 GCGAGTAGA

E. coli dnaK P1

Z. mobilis adhB P2

TCTCCCCCTTGAT

CGAACCCCTTGAT

14 CCCCATTTA

14 AGACATATT

E. coli Consensus TCTC-CCCTTGAA 13-15 CCCCAT-TA

GLK may be a primary factor leading to decreased flux (andethanol production [41]) and loss of viability in older cul-tures.

It has been suggested that GLK has a high controlcoefficient for the overall pathway, being the first enzymeand the enzyme present at the lowest level based on in vitromeasurements of activity (1, 48). Degradation of this enzymeat a stage when growth is slowing could prevent excessiveATP production and facilitate survival as nutrients becomelimiting.

Stress proteins in Z. mobilis. Our results demonstrate thatthe fermentative enzyme ADHII (adhB) is a prominentstress protein (SP38) in Z. mobilis. To the best of ourknowledge, an ADH has not been previously reported as astress protein in bacteria. The physiological role of thisenzyme with respect to stress is unknown. Perhaps thisenzyme could serve to reestablish an NADH-NAD balanceafter exposure to stress conditions or act as a chaperonin tostabilize other essential enzymes.The adhB gene is expressed from tandem promoters (13).

Both promoters have been confirmed as functional in Z.mobilis by operon fusions with lacZ (27). The P2 promoter isfive times as active as the P1 promoter and is very similar toheat shock promoters of E. coli (15, 50). No other sequencesresembling the enteric heat shock consensus promoter arepresent in the promoter regions of other known genesencoding Z. mobilis glycolytic and ethanologenic enzymes.A total of seven putative heat stress proteins were identi-

fied in Z. mobilis. SP70, SP58, SP38 (ADHII), and SP18appear to correspond to those reported previously by Micheland Starka (31, 32). All stress-induced proteins were evidentin control cells, albeit at low levels in some cases. It is likelythat stress proteins are at least partially induced by theobligate production of ethanol as a fermentation product. Itis interesting that three abundant acidic stress proteins,SP70, SP58, and SP18, correspond in approximate sizes andpositions on 2-D gels to those identified in E. coli as DnaK,GroEL, and GroES, respectively (23, 24, 50). In E. coli,these proteins are also very abundant during normal growthbut increase in abundance after exposure to thermal orethanol stress.

ACKNOWLEDGMENTSThese studies were supported in part by the Florida Agricultural

Experiment Station, by grant FG05-86ER3574 from the Office ofBasic Energy Science, U.S. Department of Energy, and by grant88-37233-3987 from the Alcohol Fuels Program, U.S. Department ofAgriculture. Grants from the Australian Research Council and theAustralian Universities International Development Plan are grate-fully acknowledged.

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