Cloning and characterization of the Escherichia coli K-12 rfa-2 (rfaC) gene, a gene required for lipopolysaccharide inner core synthesis

Abstract

A genetically defined mutation, designated rfa-2, results in altered lipopolysaccharide (LPS) biosynthesis. rfa-2 mutants produce a core-defective LPS that contains lipid A and a single sugar moiety, 2-keto-3-deoxyoctulosonic acid, in the LPS core region. Such LPS core-defective or deep-rough (R) mutant structures were previously designated chemotype Re. Phenotypically, rfa-2 mutants exhibit increased permeability to a number of hydrophilic and hydrophobic agents. By restriction analyses and complementation studies, we clearly defined the rfa-2 gene on a 1,056-bp AluI-DraI fragment. The rfa-2 gene and the flanking rfa locus regions were completely sequenced. Additionally, the location of the rfa-2 gene on the physical map of the Escherichia coli chromosome was determined. The rfa-2 gene encodes a 36,000-dalton polypeptide in an in vivo expression system. N-terminal analysis of the purified rfa-2 gene product confirmed the first 24 amino acid residues as deduced from the nucleotide sequence of the rfa-2 gene coding region. By interspecies complementation, a Salmonella typhimurium rfaC mutant (LPS chemotype Re) is transformed with the E. coli rfa-2+ gene, and the transformant is characterized by wild-type sensitivity to novobiocin (i.e., uninhibited growth at 600 micrograms of novobiocin per ml) and restoration of the ability to synthesize wild-type LPS structures. On the basis of the identity and significant similarity of the rfa-2 gene sequence and its product to the recently defined (D. M. Sirisena, K. A. Brozek, P. R. MacLachlan, K. E. Sanderson, and C. R. H. Raetz, J. Biol. Chem. 267:18874-18884, 1992), the S. typhimurium rfaC gene sequence and its product (heptosyltransferase 1), the E. coli K-12 rfa-2 locus will be designated rfaC.