Modulation of RPOS Expression by an Inducible RPOS Sense and Antisense on a High-Copy Plasmid and as a Single Copy in the Escherichia Coli Chromosome

Abstract

Escherichia coli and several other bacteria express a stationary phase sigma factor, RpoS, for RNA polymerase that is responsible for inducing the expression of stress response genes. rpoS expression is induced during early exponential phase and the concentration of RpoS dramatically increases during the transition from log phase to stationary phase. The goal of this study was to test whether rpoS expression could be modulated using an inducible rpoS antisense and rpoS sense. In the first part of this study, a rpoS antisense under the control of an IPTG-inducible promoter was tested for its efficiency for modulating the expression of the RpoS regulon. RpoS-dependent and RpoS-independent lacZ fusions were utilized to quantify the effect of rpoS antisense expression on rpoS translation. Unlike an earlier study, the results of this study suggest that the rpoS antisense was not induced and/or that it was not inhibiting the expression of RpoS-dependent genes. In the second part of this study, an IPTG-inducible rpoS sense was used to test whether expression of certain members of the RpoS regulon were solely dependent on RpoS and not additional factors present in stationary phase. Thus, their expression could be induced in exponential phase. The rpoS sense was integrated into the E. coli chromosome at the ebg locus by homologous recombination utilizing a one-step PCR method. Some putative rpoS sense recombinants showed an increase in catalase expression, which is known to be RpoS-dependent. However, upon further verification, the 5kb PCR fragment encoding the inducible rpoS sense did not appear to have integrated to the intended site.