Self-transmissible plasmid in Zymomonas mobilis carrying antibiotic resistance.

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.

Expression of a Lactose Transposon (Tn951) in Zymomonas mobilis.

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 of fermentable 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 degrees C but rapidly lost at 37 degrees C.

Lipid composition of Zymomonas mobilis: effects of ethanol and glucose.

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. O. Ingram, J. Bacteriol. 125:670-678, 1976). The fatty acid composition of Z. mobilis, an obligately ethanol-producing 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.

On the relationship between alcohol narcosis and membrane fluidity.

We have examined the relationship between membrane fluidity and the alcohol-induced loss of righting reflex at different temperatures using the fish Gambusia affinis. The potency of ethanol and hexanol increased dramatically with temperature. Ethanol-induced narcosis could be antagonized by a reduction in incubation temperature. Both an increase in temperature and the addition of ethanol caused an increase in membrane fluidity. However, membrane fluidity itself did not correlate with narcosis and was primarily determined by incubation temperature. Narcotic concentrations of ethanol caused a change in fluidity equivalent to less than that caused by a 2 degree increase in temperature while an 8 degree increase in temperature did not induce narcosis. From these studies, we conclude that the ethanol-induced increase in bulk membrane fluidity as measured by diphenyl-hexatriene is not the causal event for narcosis although the magnitude of this change does correlate with the alcohol sensitivity. We have also examined the effects of temperature adaptation on the sensitivity of these animals to ethanol. Summer animals contained higher levels of saturated fatty acids, exhibited a higher temperature range than winter animals and were more resistant to ethanol, providing further evidence for membrane structure as a determinant of alcohol sensitivity.