The gene which encodes alcohol dehydrogenase II (adhB) from Zymomonas mobilis was cloned in Escherichia coli as a 1.4-kilobase DNA fragment by using a novel indicator plate which directly detects the expression of this activity by recombinant colonies. The DNA sequence for this clone contained an open reading frame encoding a polypeptide of 383 amino acids, with a molecular weight of 40,141. Although this protein exhibited very little homology with other known alcohol dehydrogenases, the predicted amino acid composition was in excellent agreement with that reported for the purified alcohol dehydrogenase II protein from Z. mobilis. In Z. mobilis, the adhB gene was transcribed from tandem promoters which were separated by 100 base pairs and ended with a transcriptional terminator (13-base-pair palindrome). In Escherichia coli, only one of the Z. mobilis promoters was used, despite apparent similarity to the enteric consensus promoter. The adhB gene was transcribed at low levels in E. coli from the P2 promoter of Z. mobilis but was expressed well in E. coli under control of the lac promoter (approximately 0.25% of the total cell protein).
DNA sequence analysis showed that pyruvate decarboxylase (one of the most abundant proteins in Zymomonas mobiis) contains 559 amino acids. The promoter for the gene encoding pyruvate decarboxylase was not recognized by Escherichia coli, although the cloned gene was expressed at relatively high levels under the control of alternative promoters. The promoter region did not contain sequences which could be identified as being homologous to the generalized promoter structure for E. coli. Hydropathy plots for the amino acid sequence indicated that pyruvate decarboxylase contains a large number of hydrophobic domains which may contribute to the thermal stability of this enzyme.The regeneration of NAD+ in the obligately fermentative bacterium Zymomonas mobilis is carried out with two enzymes, pyruvate decarboxylase (EC 4.1.1.1) and alcohol dehydrogenase (EC 1.1.1.1) (23,30). Although this simple regeneration system is widely utilized by eucaryotes such as the yeasts, fungi, and higher plants (31), it is relatively rare among the bacteria (10, 29). Very few bacterial genera have been shown to contain pyruvate decarboxylase, the key enzyme catalyzing the nonoxidative decarboxylation of pyruvate to produce acetaldehyde plus CO2 In Z. mobilis, this enzyme is reported to be a tetramer of identical subunits with a molecular weight of approximately 55,000 + 5,000 (13). Recent studies by Brau and Sahm (5) have described the cloning of pyruvate decarboxylase from Z. mobilis. These studies indicated that the gene encoding pyruvate decarboxylase is contained on a 4.6-kilobase-pair (kb) SphI fragment.The pyruvate decarboxylase gene is normally expressed at high levels in Z. mobilis and constitutes up to 5% of the total soluble protein (2). Brau and Sahm (5) have also reported a high level of expression of this Z. mobilis gene in Escherichia coli. Previous studies in our laboratory (6; T. Conway and L. 0. Ingram, Abstr. Annu. Meet. Am. Soc. Microbiol. 1986, H62, p. 137; T. Conway and L. 0. Ingram, submitted for publication) defined sequences of DNA fragments from Z. mobilis which exhibited promoter activity in both E. coli and Z. mobilis. These studies indicated that similar regions of DNA were recognized as promoters by both organisms, although the dominant sites of transcriptional initiation were usually different. In this study, we examined the sequence of the pyruvate decarboxylase gene from Z. mobilis and its promoter and identified the sites of transcriptional initiation in both Z. mobilis and E. coli. Our studies indicate that the high level of expression of this gene in E. coli is not due to the recognition of the native Z. mobilis promoter. MATERIAL AND METHODSBacterial strains, plasmids, and growth conditions. The strains and plasmids used in this study are summarized in (20,000 lb/in2). Cell debris was removed by centrifugation for 30 min at 20,000 x g at 4°C. Pyruvate decarboxylase (120 mg) was purified from the resulting soluble protein fraction essentially as described by Hoppner and Doelle (13). The final yields ...
The rate at which Z. mobilis (Entner-Doudoroff pathway) converts
Accumulation of alcohol during fermentation is accompanied by a progressive decrease in the rate of sugar conversion to ethanol. In this study, we provided evidence that inhibition of fermentation by ethanol can be attributed to an indirect effect of ethanol on the enzymes of glycolysis involving the plasma membrane. Ethanol decreased the effectiveness of the plasma membrane as a semipermeable barrier, allowing leakage of essential cofactors and coenzymes. This leakage of cofactors and coenzymes, coupled with possible additional leakage of intermediary metabolites en route to ethanol formation, is sufficient to explain the inhibitory effects of ethanol on fermentation in Zymomonas mobilis.
We have described a procedure for the isolation of lacZ' fusion genes which contain anchor sequences conferring membrane association. This method was used to isolate fragments of DNA from Zymomonas mobilis which contain promoter activity and amino-terminal sequences. The sequences and transcriptional initiation sites of three of these were compared. Both Escherichia coli and Z. mobilis recognized similar regions of DNA for transcriptional initiation. Five to eight consecutive hydrophobic amino acids in the amino terminus served to anchor these hybrid proteins to the membrane in both E. coli and Z. mobiis. General features observed in the Z. mobilis fragments included partial sequence homology with the -35 region sequence of E. coli, repetitive and palindromic A+T-rich regions preceding and adjoining the -10 region, a sequence resembling the consensus sequence of E. coil in the -10 region, and a potential ribosomal-binding site (AGGA) 8 to 12 bases upstream from an in-frame start codon. The level of expression of fusion proteins was generally higher in E. coil than in Z. mobiis. This higher level of expression in E. coli may result from multiple sites of transcriptional initiation and higher plasmid copy number.Zymomonas mobilis is an unusual gram-negative bacterium of uncertain taxonomic position (23). This organism, alone, is obligately fermentative and utilizes an EntnerDoudoroff pathway for glycolysis (39). It exhibits an ethanol tolerance equivalent to that of yeasts and is capable of rapid ethanol production (29), making it potentially useful for the commercial production of fuel ethanol.Little is known about promoter structure and gene expression in this organism. Previous studies have shown that the tetracycline gene contained in broad-host-range plasmids such as RP4, pGC91.14, pRK290, and pCVD305 are expressed at sufficient levels to allow selection (7,37,38). Other studies have shown that the lacZ gene on transposon Tn951 is expressed poorly even in the presence of inducer (7). Recent studies in our laboratory have indicated that the chloramphenicol resistance gene is expressed in Z. mobilis (T. Conway and L. 0. Ingram, Abstr. Annu. Meet. Am. Soc. Microbiol. 1986, H62, p. 137) under the control of the Escherichia coli consensus promoter (31). No studies have been reported concerning the sequence requirements for membrane localization or protein export in Z. mobilis.In this study, we investigated promoter structure in Z. mobilis by isolating lacZ gene fusions in which the promoter, translational start sequence, and N-terminal peptide sequence are provided by fragments of Z. mobilis DNA. High levels of 0-galactosidase have been reported previously to confer a lactose-positive phenotype in the absence of functional lactose permease (33), and this feature was used to bias our selection for hybrids which exhibited high levels of ,-galactosidase activity, i.e., Z. mobilis fragments with strong promoter activity. Over half of the hybrids selected in this way were found to be associated with the cell membrane....
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