Zymomonas mobilis is an alcohol-tolerant microorganism which is potentially useful for the commercial production of ethanol. This organism was found to contain cardiolipin, phosphatidylethanolamine, phosphatidylglycerol, and phosphatidylcholine as major phospholipids. Vaccenic acid was the most abundant fatty acid, with lesser amounts of myristic, palmitic, and palmitoleic acids. No branched-chain or cyclopropane fatty acids were found. Previous studies in our laboratory have shown that ethanol induces the synthesis of phospholipids enriched in vaccenic acid in Escherichia coli (L. 0. Ingram, J. Bacteriol. 125:670-678, 1976). The fatty acid composition of Z. mobilis, an obligately ethanolproducing microorganism, represents an extreme of the trend observed in E. coli. In Z. mobilis, vaccenic acid represents over 75% of the acyl chains in the polar membrane lipids. Glucose and ethanol had no major effect on.the fatty acid composition of Z. mobilis. However, both glucose and ethanol caused a decrease in phosphatidylethanolamine and phosphatidylglycerol and an increase in cardiolipin and phosphatidylcholine. Ethanol also caused a dose-dependent reduction in the lipid-to-protein ratios of crude membranes. The lipid composition of Z. mobilis may represent an evolutionary adaptation for survival in the presence of ethanol. Zymomonas mobilis is a gram-negative microorganism of uncertain taxonomic position (7, 10, 41-44). This organism is obligately fementative, utilizing glucose by the Entner-Doudoroff pathway (8, 13). Z. mobilis is commonly found as a spoilage organism in ciders and beers (7). It is present in ripening honey (36) and is used in the fermentation of cacao beans to make cocoa and chocolate (31). Zymomonas is used in the fermentation of palm wines (30) and plant saps (7, 42) to produce alcoholic beverages and has yet to be exploited for large-scale commercial alcohol production (34, 42). Z. mobilis is capable of producing up to 1.9 mol of ethanol per mol of glucose fermented (12, 13, 24, 42). It is reported to exhibit higher ethanol tolerance (42), higher glucose tolerance (33, 34, 42), and more rapid fermentation than Saccharomyces cerevisiae (26, 33, 34), the organism used for ethanol production in the United States. Growth in the presence of ethanol has been shown to cause changes in the lipid composition of many organisms. In Escherichia coli (15-18, 20) and in yeasts (3, 35), alcohol induces an increase in the proportion of 18:1 fatty acid at the expense of 16:0. This change has been t Florida Agricultural Experiment Station publication no. 4377.

The potential utility of Zymomonas mobilis as an organism for the commercial production of ethanol would be greatly enhanced by the addition of foreign genes which expand its range offermentable substrates. We tested various plasmids and mobilizing factors for their ability to act as vectors and introduce foreign genes into Z. mobilis CP4. Plasmid pGC91.14, a derivative of RP1, was found to be transferred from Escherichia coli to Z. mobilis at a higher frequency than previously reported for any other plasmids. Both tetracycline resistance and the lactose operon from this plasmid were expressed in Z. mobilis CP4. Plasmid pGC91.14 was stably maintained in Z. mobilis at 30°C but rapidly lost at 37°C.

The cryptic plasmid pRUT41 from Zymomonas mobilis was examined for its biological properties. This plasmid was found to be conjugally transferred from Z. mobilis CP4 to Escherichia coli BM21 and to carry genes for antibiotic resistance (gentamicin, kanamycin, and streptomycin). Covalently closed circular plasmid DNA was isolated from eight transconjugants of E. coli BM21. These plasmids were identical in mobility on agarose gels and exhibited the same restriction patterns as the native pRUT41 plasmid isolated from Z. mobilis. The plasmid location of the antibiotic resistance genes was further confirmed by transforming E. coli BM21 with isolated pRUT41 plasmid from strain CP4 and with plasmids from the transconjugants of BM21. Resistance to streptomycin, kanamycin, and gentamicin was tightly linked and transferred together in all cases.

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