Cloning, characterization, and nucleotide sequence analysis of a Zymomonas mobilis phosphoglucose isomerase gene that is subject to carbon source-dependent regulation

Abstract

The Zymomonas mobilis gene encoding phosphoglucose isomerase (pgi) was cloned by genetic complementation of an Escherichia coli pgi mutant. An enzyme assay and sodium dodecyl sulfate-polyacrylamide gel electrophoresis confirmed the presence of excess amounts of phosphoglucose isomerase in E. coli clones carrying the Z. mobilis pgi gene. The pgi gene is present in only one copy on the Z. mobilis genome. Nucleotide sequence analysis of the pgi region revealed an open reading frame of 1,524 bp preceded by a strong Shine-Dalgarno sequence. The pgi gene encodes a 507-amino-acid protein with a predicted molecular weight of 55,398. Z. mobilis phosphoglucose isomerase is between 38 and 43% identical to the enzyme from other species. Northern (RNA) blot analysis showed that the pgi transcript is 1.8 kb in length. The level of the pgi transcript was found to be influenced by the phase of growth and by the carbon and energy sources. Transcript levels increased with respect to total RNA during logarithmic growth and were threefold higher when grown on fructose than on glucose. These changes in transcript levels paralleled phosphoglucose isomerase activities in the cultures. Differential mRNA stability was not a factor, since the half-life of the pgi transcript was 6.3 min in glucose-grown cells and 6.0 min in fructose-grown cells. Thus, an increase in the rate of transcription appears to be at least partially responsible for the increased levels of phosphoglucose isomerase observed for Z. mobilis grown on fructose.