New cysE-pyrE-linked rfa mutation in Escherichia coli K-12 that results in a heptoseless lipopolysaccharide

Abstract

A new novobiocin-supersensitive mutant of Escherichia coli K-12 has been characterized biochemically and genetically. Lipopolysaccharide prepared from this mutant strain is truncated and contains 2-keto-3-deoxyoctulosonic acid as its only core sugar. This new core-defective mutation, designated rfa-2, results in increased sensitivity to several hydrophobic and some hydrophilic agents. Genetic analysis of the rfa mutant indicated that the rfa-2 locus is located at 81 min on the chromosome. The order of the genes in this region based on transduction analysis is xyl cysE rfa-2 rfaD70 pyrE. P1 transduction analyses indicate that the rfa-2 marker is nonallelic with the recently described cysE-pyrE-linked rfaD70 locus. Plasmids carrying the wild-type rfaD70+ allele failed to abolish the rfa-2 phenotypes. Further, the rfaD gene product, ADP-L-glycero-D-mannoheptose-6-epimerase, was detected in crude extracts of a rfa-2 mutant strain, CL609, and was absent in the rfaD70 mutant. The wild-type rfa-2 allele codes either for a specific heptose biosynthetic enzyme (different from the rfaD gene product) or an enzymatic activity required for the addition of heptose to the lipid A-2-keto-3-deoxyoctulosonic acid acceptor.