Vol. 171, No. 9JOURNAL OF BACTERIOLOGY, Sept. 1989, p. 4577-45820021-9193/89/094577-06$02.00/0

Expression of Zymomonas mobilis adhB (Encoding AlcoholDehydrogenase II) and adhB-lacZ Operon Fusions in

Recombinant Z. mobilistKYLIE F. MACKENZIE,1 TYRRELL CONWAY,2 H. C. ALDRICH,1 AND L. 0. INGRAM'*

Department of Microbiology and Cell Science, University of Florida, Gainesville, Florida 32611,' and Department ofBiology, University of Nebraska, Lincoln, Nebraska 685882

Received 14 March 1989/Accepted 31 May 1989

The Zymomonas mobilis alcohol dehydrogenase II gene (adhB) was overexpressed 7- to 14-fold on arecombinant plasmid, accompanied by a small decrease in growth rate. A fragment containing the truncatedgene with promoter reduced expression from the chromosomal gene as measured immunologically andenzymatically, consistent with the presence of a trans-active regulatory factor and positive regulatory control.Both the complete gene and the promoter fragment increased pyruvate decarboxylase and glucokinaseactivities, with no effect on alcohol dehydrogenase I or eight glycolytic enzymes. Tandem promoters from adhBexpressed ,-galactosidase at higher levels than did either promoter alone in operon fusions. Addition of 50 ,uMzinc sulfate in minimal medium reduced the expression of adhB and of the operon fusions. Abundant butinactive alcohol dehydrogenase II was produced in iron-limited cells. This inactive enzyme did not formintracellular aggregates, and no morphological changes were apparent by transmission electron microscopy.

Zymomonas mobilis is obligately ethanologenic and re-quires a fermentable sugar for energy generation and growtheven in rich medium (11, 24). Approximately 95% of fer-mentable sugars such as glucose are converted to ethanoland carbon dioxide, generating 1 mol of ATP per mol ofsugar metabolized. Unlike Saccharomyces spp., which arecapable of oxidative and fermentative growth, the pyruvate-to-ethanol pathway is indispensable in Z. mobilis for NADHoxidation. The ethanol pathway consists of a single pyruvatedecarboxylase and two isoenzymes of alcohol dehydroge-nase (ADH) (24). These three enzymes and all of theglycolytic enzymes are present at high levels in Z. mobilisand represent 30 to 50% of the soluble protein (14, 15).The ADHI and ADHII isoenzymes are quite distinct with

respect to pH optima, substrate ranges, and bound metals (6,7, 9, 12, 23, 26). The adhB gene, encoding ADHII, has beencloned and sequenced, and tandem promoter regions havebeen identified by primer extension (4). Southern blots ofgenomic digests indicate that adhB is present as a singlecopy and that it does not share appreciable nucleotidehomology with the gene encoding ADHI (T. Conway, C. K.Eddy, K. F. Mackenzie, J. P. Mejia, J. L. Pond, E. A. Utt,and L. 0. Ingram, Abstr. Annu. Meet. Am. Soc. Microbiol.1988, H138, p. 168). The N-terminal amino acid sequences ofthe two isoenzymes are completely different (12). ADHIIcontains 383 amino acids with a subunit molecular weight of40,141 based upon sequence analysis (4). The native enzymeis tetrameric and binds a single ferrous ion per subunit (12,23). ADHI is a zinc alcohol dehydrogenase with similarcharacteristics and substrate ranges to those found in yeastsand mammals (2, 8, 12, 26).

Z. mobilis ADHII, together with the new ADHIV ofSaccharomyces cerevisiae (5, 25) and propanediol oxidore-ductase of Escherichia coli (Conway et al., Abstr. Annu.Meet. Am. Soc. Microbiol. 1988), form a new group ofstructurally related enzymes that share considerable amino

* Corresponding author.t Florida Agricultural Experiment Station publication no. 9807.

acid homology. The bacterial enzymes are iron activated (12,22), whereas ADHIV requires zinc for activity (5).

In Z. mobilis, ADHII is the dominant enzyme duringfermentation, although both isoenzymes are expressed (7,12). ADHII is activated by ethanol accumulation, whereasADHI is inhibited. Iron-limited growth with chelators orwith 50 F.M zinc ions dramatically reduced ADHII activitybut not ADHI activity (9). Inactive ADHII protein persistedunder these conditions. However, transcriptional and trans-lational regulation were not eliminated as contributing to thedecline in ADHII activity (9).

In this study, we have continued to investigate the expres-sion of ADHII in Z. mobilis by using recombinant strainswhich contain multiple copies of the complete gene, thepromoter fragment, and adhB-lacZ operon fusions.

MATERIALS AND METHODS

Organism and growth conditions. Recombinants of Z.mobilis CP4 were maintained and grown with 10 mg oftetracycline per liter or 40 mg of chloramphenicol per liter asdescribed previously (9). For comparisons of glycolytic andethanologenic enzyme activities, cells were grown in com-plex medium containing 10% glucose. For experiments withmetal ion supplements, modified minimal medium containing5 FM zinc sulfate was used (9). Iron limitation experimentswith 1,10-phenanthroline as a chelator were carried out byusing complex medium containing 10% glucose and 5 g ofyeast extract per liter (half the normal level). Where indi-cated, media were supplemented with zinc sulfate or ferroussulfate (freshly prepared).

Culture tubes and stirred culture vessels were incubated ina water bath maintained at 30°C. Growth was monitoredspectrophotometrically at 550 nm with a Spectronic 70spectrophotometer (Bausch & Lomb, Inc., Rochester,N.Y.).

Genetic methods. Construction of shuttle vectors, diges-tion with restriction enzymes, transformation of E. coli, andconjugation of plasmids into Z. mobilis CP4 have beenpreviously described (3, 4).

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Enzyme assays. Enzyme activities in cells harvested duringexponential growth in medium containing 10% glucose (op-tical density at 550 nm of 0.5) or after 12 h of growth withchelator were determined. Cells were washed and suspendedin 50 mM K-MES buffer (pH 6.5) containing 0.2 mMmagnesium chloride and 0.2 mM ferrous ammonium sulfate,disrupted, and assayed for ADH activities immediately (9).Other glycolytic enzymes were assayed in cell pellets whichhad been stored at -20°C. Glucokinase and glucose-6-phosphate dehydrogenase were assayed as described byScopes et al. (17). Pyruvate decarboxylase was assayed asdescribed by Neale et al. (13). Remaining Z. mobilis glyco-lytic enzymes were assayed as described by Pauluk et al.(15). Recombinant Z. mobilis cells containing lacZ operonfusions were washed and assayed for 3-galactosidase activ-ity as described by Miller (10) with o-nitrophenol as astandard. Activities are expressed as international units permilligram of total cell protein and represent an average ofthree or more determinations.

Rocket immunoelectrophoresis. ADHII protein levels inrecombinants were compared by Laurel rocket immunoelec-trophoresis as previously described (9).

Gel electrophoresis. The Phast Gel System (Pharmacia,Inc., Piscataway, N.J.) was used to compare proteins inFrench press-disrupted cells (9). Proteins in native poly-acrylamide gradient gels (8 to 25% acrylamide [pH 8.8]), in12.5% polyacrylamide denaturing gels containing 2% sodiumdodecyl sulfate, and in isoelectric focusing gels (pH 3 to 9)were examined. Separations and staining conditions wereessentially those recommended by the manufacturer. Proteinstandards were obtained from Pharmacia and from SigmaChemical Co., St. Louis, Mo.DNA sequence analysis. DNA sequences of lacZ operon

fusion regions were determined by the dideoxy method asdescribed previously (4). Double-stranded plasmid DNAwas prepared for use as a template by the method of Ausubelet al. (1).

Electron microscopy. Cells were grown in minimal mediumwith or without a metal ion supplement (50 isM zinc or ferroussulfate) and in complex medium with 10% glucose. Fixationand microscopy were carried out as described previously (18).

RESULTS

Construction of shuttle vectors containing adhB. Four shut-tle vectors were prepared which contained either the com-plete adhB gene or a promoter fragment with the truncatedgene in both orientations as follows. A PstI linker wasinserted into the Klenow-treated BstEII site of pLOI287 (4)to form pLOI410 (Fig. 1A). The adhB gene, including thepromoter, was then removed from pLOI410 as a 2.5-kilobase(kb) PstI fragment and inserted into the PstI site of pLOI193in both orientations (pLOI411 [Fig. IC]). The construct withadhB transcription in the same direction as tet was labeled R(right); the opposite orientation was labeled L (left). Most ofthe adhB gene was removed from pLOI410 by HincIl diges-tion and ligation (Fig. 1B). In this construct (pLOI412), onlythe promoter and N-terminal 11 codons of adhB remained,flanked by PstI sites. This adhB promoter region was re-moved from pLOI412 on a 0.45-kb PstI fragment and insertedinto pLOI193 to produce pLOI413R and pLOI413L (Fig. 1C).

Glycolytic and ethanologenic enzyme activities in recombi-nant strains of Z. mobilis. Recombinants with the completeadhB gene contained sevenfold-higher ADHII activity thandid strain CP4 with vector alone in complex medium (Table1). The activities of the left and right constructs were essen-

tially the same, although the right constructs were more stablymaintained. Overexpression of ADHII in CP4(pLOI411) wasaccompanied by a 10% increase in generation time relative tothe other recombinant strains. ADHII activities in the recom-binants containing the promoter alone were consistentlylower than that in the control, strain CP4(pLOI193).ADHI activities remained unchanged in all recombinants.

Pyruvate decarboxylase and glucokinase activities wereelevated in strains containing the promoter fragment(pLO1413) and the complete gene as compared withCP4(pLOI193). Glyceraldehyde-3-phosphate dehydrogenaseactivity was also elevated in recombinants containing thecomplete gene (pLOI411) but not the promoter. The activi-ties of the remaining five enzymes, glucose-6-phosphatedehydrogenase, phosphoglycerate kinase, phosphoglycero-mutase, enolase, and pyruvate kinase, were not affected bymultiple copies of adhB or the promoter fragment.

Effect of metal ion availability on ADH activities. Previousstudies have shown that iron starvation with chelators incomplex medium eliminated ADHII activity but did notblock the synthesis or accumulation of the inactive protein(9). The addition of high levels of zinc to minimal mediumhad a similar effect on ADHII and increased the ironrequirement for the synthesis of active ADHII (9).The addition of 1,10-phenanthroline to recombinants

growing in complex medium resulted in iron starvation, asevidenced by iron-reversible inhibition of growth (data notshown). This iron starvation eliminated ADHII activity inCP4(pLOI193) and CP4(pLOI413) and greatly reduced thelevel of this activity in CP4(pLOI411) containing the com-plete gene (Table 2). ADHI activity did not increase tocompensate for the loss of ADHII activity.

In minimal medium, ADHII activity was 14-fold higher inCP4(pLOI411) than with vector alone (Table 3). The pro-moter constructs CP4(pLOI413R) and CP4(pLOI413L), re-spectively, exhibited similar ADHII activity to and slightlylower ADHII activity than CP4(pLOI193). The addition ofzinc sulfate decreased the activities of ADHII in all con-structs and had a lesser effect on ADHI activity. Theactivities of recombinants containing the complete gene orthe promoter fragment decreased by approximately two-thirds, whereas these activities decreased by half inCP4(pLOI193). The addition of ferrous sulfate increased theactivity of ADHII in all constructs.

Immunological detection of ADHII protein. Rocket immu-noelectrophoresis was used to compare the relative amountsof ADHII protein present in recombinants grown underdifferent conditions, independent of activity (Fig. 2). ADHIIprotein was abundant in iron-starved cells grown in complexmedium (Fig. 2A), although enzyme activity was nearlyeliminated (Table 2). Again, CP4(pLOI411) contained ele-vated amounts of this protein compared with CP4(pLOI193).ADHII protein levels were lower in CP4(pLOI413) contain-ing the promoter than in vector alone.

In minimal medium (Fig. 2B), the recombinant containingthe promoter fragment contained less ADHII protein thandid the control strain, CP4(pLOI193). ADHII was overex-pressed in the recombinant with the complete gene. Seven-fold more protein was added to the wells containing recom-binants with pLOI193 or pLOI413 than to the wellscontaining CP4(pLOI411). The addition of zinc sulfate re-sulted in a decrease in ADHII protein levels that was lowerthan the zinc-induced reduction of ADHII activity (Table 3).The addition of ferrous sulfate had no effect on ADHIIprotein levels in the control or in the recombinant containingthe complete gene.

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FIG. 1. Plasmid constructs with Z. mobilis adhB. (A) pLOI410; the promoter (Prom.) and coding region for adhB are marked. (B)pLOI412, a HinclI deletion of pLOI410. (C) Construction of shuttle vectrs by inserting PstI fragments containing adhB (pLOI411) or theadhB promoter (pLOI413) from pLOI410 and pLOI412, respectively, into the PstI site of pLOI193. (D) Construction of adhB-lacZ operonfusions. Promoter fragments were inserted into the SmaI site of pLO1262. The resulting operon fusions (including the upstream E. coli rnnBlterminator [trm]) were removed as a SalI-PstI fragment and used to replace the smaller SalI-PstI fragment of pLOI193 (panel C). These shuttlevectors were designated pLOI501 (promoter P1 only), pLOI502 (promoter P2 only), pLOI503 (both adhB promoters), and pLOI500(promoterless control). The native lacZ Shine-Dalgarno region is denoted by S.D.

HindIllPstI

SalIHinclI

:i. pUCci

Detection of ADHII by gel electrophoresis. To examine thepossibility that the zinc-induced reduction in ADHII activityresulted from modification of the ADHII protein, we exam-ined cell extracts by gel electrophoresis (Fig. 3). ADHII wasclearly evident in extracts of CP4(pLOI411) as an overpro-duced protein. Although this band may include other pro-teins, the levels of these did not change in CP4(pLOI193) orCP4(pLOI413). The ADHII protein ran with an apparentmolecular weight of 36,000 in a sodium dodecyl sulfate-

polyacrylamide gel, with an isoelectric point of 4.9 in anisoelectric focusing gel, and with an apparent molecular weightof 120,000 in native gels. Although growth in minimal mediumcontaining 50 ,uM zinc sulfate caused a 70% reduction inADHII activity in CP4(pLOI411), the bands identified asADHII protein appeared unchanged in all three types of gels.Thus, the active and inactive ADHII proteins appeared phys-ically identical in terms of apparent native molecular weight,apparent subunit molecular weight, and isoelectric point.

BPstl

(kb) 0

P2 (codon 11)

------

------ --

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TABLE 1. Activities of glycolytic and ethanologenic enzymesin recombinant Z. mobilis grown on complex

medium with 10% glucose

Sp act (lU/mg of total cell protein) (SD)Enzyme

pLOI193 pLO1411 pLO1413

ADHI 0.9 (0.1) 1.0 (0.2) 0.9 (0.1)ADHII 6.0 (0.9) 39 (8.9) 4.3 (0.5)Pyruvate decarboxylase 2.8 (0.2) 3.2 (0.2) 3.7 (0.2)Glucokinase 0.26 (0.02) 0.42 (0.04) 0.40 (0.05)Glucose-6-phosphate 1.3 (0.1) 1.5 (0.1) 1.6 (0.2)dehydrogenase

Glyceraldehyde-3-phosphate 2.0 (0.1) 3.2 (0.5) 2.1 (0.4)dehydrogenase

Phosphoglycerate kinase 3.2 (0.4) 3.4 (0.1) 3.2 (0.4)Phosphoglyceromutase 1.5 (0.3) 1.6 (0.2) 1.6 (0.1)Enolase 3.4 (0.5) 3.1 (0.1) 3.8 (0.4)Pyruvate kinase 3.1 (1.2) 2.4 (0.2) 2.1 (0.1)

Electron microscopy. CP4 recombinants were comparedby transmission electron microscopy (data not shown). Nomorphological differences were observed between recombi-nant cells which overexpressed ADHII (pLOI411) and thecontrol with vector alone (pLOI193). Strain CP4(pLOI411)grown with added zinc sulfate (50 ,uM) contained abundantbut inactive ADHII protein. This inactive protein did notform observable cytoplasmic aggregates; cells appeared sim-ilar to the control strain, CP4(pLOI193).

Construction of lacZ' operon fusions. A 3-galactosidaseoperon fusion vector was constructed from two plasmids,pMC1871 (19) and pRS415 (21). The 4.2-kb Sacl-Sall frag-ment in pRS415 was replaced by a 1.2-kb Sacl-Sall fragmentfrom pMC1871, eliminating lacYA (Fig. 1). This plasmid(pLOI262) contains a strong upstream terminator (four re-peats of the E. coli rrnBl terminator), a unique SmaI site forthe insertion of promoters, and the full coding region forlacZ with the native ribosomal binding site.A series of adhB-lacZ operon fusions were constructed by

inserting blunt-ended DNA fragments from pLOI412 (Fig.1B) containing adhB promoters into the SmaI site ofpLOI262 (Fig. 1D). The 0.45-kb PstI fragment containingboth promoters (P1 and P2) was purified by using agarose gelelectrophoresis. Digestion with HaeIII cleaved this PstIfragment into two pieces containing the separate promoters.After treatment with the Klenow fragment of E. coli DNApolymerase to generate blunt ends, three constructs wereprepared which contained P1 alone (160 base pairs), P2 alone

TABLE 2. Effects of 1,10-phenanthroline (iron chelator) on ADHactivities in strain CP4 recombinants

Phenanthroline Sp act (lU/mg of cell protein)Plasmid (30 ,uM) added Total (SD) ADHI ADHII

pLOI193 - 4.6 (0.3) 0.9 3.7+ 1.2 (0.1) 1.2 0

pLOI411R - 26.6 (0.6) 1.1 25.5+ 2.4 (0.3) 1.6 0.8

pLOI413R - 4.5 (0.3) 1.5 3.1+ 1.0 (0.1) 1.0 0

Cells were grown in complex medium containing half the normal level ofyeast extract. Iron-starved cells were grown in the presence of 1,10-phenan-throline for 12 h and compared with exponentially growing cells without thisaddition.

TABLE 3. Effects of zinc and ferrous ions on ADH activities inCP4 recombinants during growth on minimal medium

Sp act (lU/mg of total cell protein) with following metal:

Plasmid None 50 ,uM Zn2+ 50 ,uM Fe2+

ADHI ADHII ADHI ADH II(SD) (SD) (SD)

pLOI193 1.8 1.9 (0.2) 1.4 0.9 (0.2) 1.2 2.6 (0.6)pLO1411L 1.6 28 (3.4) 1.2 8.2 (1.2) 1.2 31(5.8)pLO1411R 1.7 31 (2.5) 1.8 9.6 (1.4) 1.7 35 (7.4)pLOI413L 1.7 1.4 (0.2) 1.8 0.4 (0.1) 1.4 2.5 (0.5)pLO1413R 1.8 2.0 (0.1) 1.3 0.3 (0.1) 1.5 2.2 (0.4)

(290 base pairs), and both promoters (450 base pairs).Orientation and identity were established by DNA sequenceanalysis. The HinclI deletion of pLOI410 restored the Sallsite at the junction with the polylinker.Each of the adhB-la(Z operon fusions and a promoterless

control fragment were removed as PstI-SaiI fragments andused to replace the analogous fragment in the tet gene of ourshuttle vector, pLOI193. Partial digestion with Sall wasrequired to isolate the operon fusion with both promotersand with the P2 promoter owing to the presence of a secondSall site. The resulting constructions (Fig. ID) were desig-nated pLOI500 (promoterless), pLOI501 (P1), pLOI502 (P2),and pLOI503 (both promoters). Constructs with operonfusions were resistant to chloramphenicol and sensitive totetracycline and expressed f3-galactosidase activity on indi-cator plates. All constructs retained the upstream transcrip-tional terminator to minimize background expression. TheadhB ribosomal binding site and 11 N-terminal codons werepresent in pLOI502 and pLOI503.

Effect of metal ion availability on expression of adhB-1acZoperon fusions. The expression of 3-galactosidase in recom-binants was examined (Table 4). Strain CP4 containing apromoterless lacZ was compared with homologous con-structs containing the P1 promoter, the P2 promoter, andboth promoters. Negligible activity was observed without apromoter. Constructs with both promoters contained higheractivities than did constructs with either promoter alone.

A

FIG. 2. Detection of ADHII by rocket immunoelectrophoresis.(A) Iron-starved cells grown for 12 h in complex medium containing30 p.M 1,10-phenanthroline. Lane 1 contained 2 ,ug of protein; lanes2 and 3 contained 20 jig of protein. Lanes: 1, CP4(pLOI411); 2,CP4(pLO1413); 3, CP4(pLOI193). (B) Effects of metal ion supple-ments and recombinant plasmids on ADHII expression in minimalmedium. Lanes 1 through 6 contained 20 pLg of protein; lanes 7through 9 contained 3 ,ug of protein. Lanes: 1, CP4(pLOI193) noadditions; 2, CP4(pLOI193) with 50 ,uM Zn; 3, CP4(pLOI193) with50 piM Fe; 4, CP4(pLOI413) with no additions; 5, CP4(pLOI413)with 50 ,uM Zn; 6, CP4(pLO1413) with 50 F.M Fe; 7, CP4(pLOI411)with no additions; 8, CP4(pLO1411) with 50 F.M Zn; 9.CP4(pLO1411) with 50 F.M Fe.

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FIG. 3. Comparison of proteins in recombinants grown in minimal medium with and without 50 KM zinc by polyacrylamide gelelectrophoresis. Odd-numbered lanes are controls, and even-numbered lanes contain extracts from cells grown with 50 ,uM Zn. The ADHIIregion is marked by arrows on the right side of each figure. Isoelectric points (pl) and molecular masses (kilodaltons [kD]) are listed at theleft. Lanes: 1 and 2, CP4(pLOI193); 3 and 4, CP4(pLOI413); 5 and 6, CP4(pLOI411). (A) Isoelectric focusing (pH 3 to 9). Lanes were loadedwith approximately 8 Kg of protein each. (B) Sodium dodecyl sulfate (2%) gels with 12.5% polyacrylamide (4 Kg of protein per lane). (C)Native polyacrylamide gradient gels (8 to 25%; pH 8.8).

The P2 promoter was severalfold more active than the P1promoter. Higher levels of expression were observed inminimal medium than in complex medium. Iron starvationby the addition of phenanthroline as a chelator did notdecrease expression. The addition of zinc sulfate decreasedexpression by 25% in all three operon fusions relative to theminimal medium controls. The addition of 50 F.M ferroussulfate did not enhance expression but caused a smalldecrease in 3-galactosidase activity.

DISCUSSION

Present studies indicate that the overexpression of ADHIIin Z. mobilis from a multicopy plasmid is not highly detri-mental to growth. Depending upon the growth medium,7-fold (complex medium)- to 14-fold (minimal medium)-higher activities of this enzyme were produced with only a

small reduction in growth rate as compared with controlscontaining vector alone. Previous studies in our laboratoryhave estimated the copy number of this vector to be between15 and 30 in Z. mobilis in complex medium (3). Thus, incomplex medium, overexpression appears to fall short ofpotential levels based upon considerations of gene dosagealone. Under many conditions, CP4 recombinants contain-

ing the adhB promoter fragment and truncated adhB geneproduced lower levels of ADHII activity and ADHII proteinfrom the chromosomal adhB gene than did recombinantswith the vector alone. Both of these observations are con-

sistent with the hypothesis that a trans-active positive ef-fector limits the expression of adhB.

trans-active factors may be involved in the expression ofother glycolytic enzymes. The presence of multiple copies ofboth the complete gene and the promoter fragment increasedglucokinase activity and pyruvate decarboxylase activity.Glyceraldehyde-3-phosphate dehydrogenase activity was

also elevated by overexpression of ADHII. In contrast to thechanges in biosynthetic enzymes, these changes were smallin terms of a percentage of the basal level. However, thesechanges represent large amounts of protein relative to thelow levels of biosynthetic enzymes. Only modest changesmay be allowed in these essential enzymatic activities. Suchchanges may be important to optimize glycolysis and ethanolproduction. The expression of ADHI and many other glyco-lytic enzymes was not affected.The addition of 50 F.M zinc sulfate caused a large reduc-

tion in ADHII activity during growth in minimal medium,with a lesser decrease in ADHII protein as measured by

TABLE 4. Effects of metal ion availability on the specific activities of P-galactosidase in CP4 containing adhB-lacZ operon fusions

Plasmid Sp act (IU/mg of total cell protein) (SD) with following addition:(promoter) Mediumo

+2(promoter) Medium" None Phe 50 p.M Zn2+ 50 ,uM Fe2+pLOI500 (none) Complex 0.004 (0.002) 0.004 (0.002)pLOI1501 (P1) Complex 0.032 (0.002) 0.070 (0.001)pLOI502 (P2) Complex 0.287 (0.003) 0.350 (0.002)pLOI503 (P1 + P2) Complex 0.385 (0.005) 0.349 (0.006)

pLOI500 (none) Minimal 0.002 (0.002) 0.002 (0.002) 0.002 (0.002)pLOI501 (P1) Minimal 0.111 (0.003) 0.084 (0.001) 0.111 (0.004)pLOI502 (P2) Minimal 0.508 (0.009) 0.385 (0.006) 0.393 (0.007)pLOI503 (P1 + P2) Minimal 1.103 (0.040) 0.821 (0.028) 0.988 (0.008)

a Cells were grown in complex medium containing half the normal level of yeast extract; iron-starved cells were grown in the presence of 1,10-phenanthroline(Phe) for 12 h and compared with exponential-phase cells without this addition. Alternatively, cells were grow in minimal medium supplemented with ferroussulfate or zinc sulfate and compared in the exponential phase.

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rocket immunoelectrophoresis. Z. mobilis ADHII requiresiron for activity but is inactive with zinc as the bound metal(12, 23). The decrease in activity with added zinc may reflectcompetition between the two metals during the folding of thenascent polypeptide, resulting in an inactive form. A zinc-induced decrease in 3-galactosidase was observed in adhB-lacZ operon fusions containing the tandem adhB promotersand with each promoter individually, confirming transcrip-tional control. The addition of iron did not increase the levelof ADHII protein in CP4(pLOI193) or ,B-galactosidase inoperon fusions.A number of factors may contribute to the high level of

expression of Z. mobilis genes such as adhB. This genecontains tandem promoters, each of which drives high levelsof expression of f-galactosidase in operon fusions. Thehighest level of 3-galactosidase (1.1 IU) was observed withthe tandem adhB promoters. This is equivalent to approxi-mately 3,300 Miller units of activity, threefold fully inducedE. coli (20). Based upon a specific activity of 450 IU for pure3-galactosidase (20), this represents approximately 0.2% of

cellular protein. Recombinants containing the completeadhB gene (with promoter) or vector alone contained 40 and6 IU of ADHII activity, respectively, when grown undersimilar conditions. The 36 IU attributable to multiple genecopies represents approximately 3.6% of total cellular proteinbased upon the specific activity of pure enzyme, 950 IU (12).

It is unlikely that the 18-fold difference in expression ofadhB and lacZ under the control of the adhB promoterresults from differences in the ribosomal binding sites. Bothconstructs have four-base, canonical Shine-Dalgarno(AGGA, GAGG) regions with similarly spaced start codons.Differences in codon usage represent the most probablecause of this disparate expression. The adhB gene is highlybiased in codon usage as compared with lacZ. Thirteencodons are unused in adhB, compared with three unused inlacZ. In specifying 9 of 20 amino acids, the most frequentlyused codons in adhB differed from those of lacZ.

ACKNOWLEDGMENTS

K.F.M. thanks K. K. Singh for his encouragement during thiswork.

This study was supported in part by the Florida AgriculturalExperiment Station and 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.

LITERATURE CITED1. Ausubel, F. M., R. Brent, R. E. Kingston, D. D. Moore, J. G.

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