Physical mapping of the exuT and uxaC operators by use of exu plasmids and generation of deletion mutants in vitro

Abstract

Operons uxaCA and exuT of the hexuronate system are very closely linked on the Escherichia coli genetic map. Using plasmid vectors constructed by Casadaban et al. (J. Bacteriol. 143:971-980, 1980), we formed exuT-lacZ and uxaA-lacZ fusions in vitro. The phenotypic properties of the new plasmids allowed us to confirm that the exuT and uxaCA operons are divergently transcribed. An analysis of these fusion plasmids and derivatives in the presence of multiple copies of the exuR regulatory gene demonstrated that the two operons possess separate control regions. The precise location of the operator site relative to endonuclease restriction sites was determined. In addition, deletions of different lengths were generated on exu plasmids by restriction enzymes and were recombined into the chromosome. The expression of the exu regulon genes in the resulting deletion mutants is in agreement with the postulated location of the exuT and uxaCA operators in the fusion plasmids.