Expression of Wild-Type rcsB Gene Under the pBAD Promoter Restores Capsule Production in Escherichia coli rcsB Mutant and Permits Normal Cell Morphology

Abstract

Previous studies have been inconclusive whether increased capsule size directly confers antibiotic resistance in Escherichia coli. In this study, rcs B gene expression was upregulated using the vector araBAD promoter and induction with varying concentrations of L-arabinose to determine the degree of rcs B involvement in determining capsule size. The rcs B gene was amplified by PCR and cloned into the pBAD-TOPO construct. Restriction digest analysis confirmed that rcs B was present in the correct orientation. Capsule visualization which followed subcloning of the constructs into an rcs B deficient E. coli strain showed restoration of a capsule, but showed little increase in size over wild-type at 2% L-arabinose induction. When the empty pBAD-TOPO vector was transformed into the same rcs B deficient E. coli strain, the L-arabinose induction yielded elongated tubular poles and additional changes in cell morphology. Normal cell morphology was observed for cells containing the pBADrcs B construct. Our results indicate that overexpression of rcs B gene does not greatly enhance capsule formation in the absence of a stimulant and overexpression of empty vectors would not be a suitable control for assessing the role of a cloned gene.