H-075. Mutations in the G-boxes in the Promoter Region of the Escherichia coli gnd Gene Lower gnd Gene Expression

R. N. Tolbert, E. M. Schlimm, T. T. Olungwe, A. J. Pease;
Villa Julie Coll., Stevenson, MD.

Previous research showed that the Escherichia coli gene gnd which encodes 6-phosphogluconate dehydrogenase (6PGD) is strongly growth rate regulated. G-boxes which are found in the gnd promoter region are suspected to control the growth rate regulation of the gnd gene. It has been found that gene expression in E. coli increases, stays the same, or decreases with growth rate. Growth rate-dependent regulation of these genes refers to the process that coordinates gene level expression with the cellular growth rate. Genes that are positively correlated with growth rate include the components of the translation apparatus including tRNA, rRNA, ribosomal proteins, elongation factors, and several genes encoding metabolic enzymes such as gnd. In the dam gene, there are 3 G-boxes in its promoter region that have been found to control the growth rate-dependent regulation of dam gene expression. The G-boxes in dam contain the sequence TTCAGTN20TGAG. The gnd promoter region has 3 very similar sequences in its promoter region. The purpose of the present study was to determine if the G-boxes in the gnd promoter region are responsible for growth rate-dependent regulation of gnd. The hypothesis was that mutations in the G-boxes would result in loss of growth rate-dependent regulation of gnd. All three G-boxes in the gnd promoter region were mutated. The mutations were designed to substitute the essential, conserved bases in the G-boxes of gnd. PCR fragments containing mutant and wild type promoter regions were subcloned into a single copy expression vector. The plasmids were transformed into a strain with an insertion in the chromosomal gnd gene which inactivates the gene. At a fast growth rate, it was found that the levels of 6PGD were 4-fold lower in cells harboring the plasmid with all three G-boxes mutated when compared to the plasmid with wild type gnd. Work is currently under way to determine if the effect on gnd gene expression is growth rate-dependent or not.