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 rfia-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 rfia-2 gene coding region. By interspecies complementation, a Salmonefla 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 pg 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 rfia-2 gene sequence and its product to the recently defined

Deletion of the Heptosyltransferase Genes rfaC and rfaF in Escherichia Coli K‐12 Results in an Re‐Type Lipopolysaccharide with a High Degree of 2‐Aminoethanol Phosphate Substitution

“…The definition of such strains was mostly based on the finding that the core region contained only Kdo, and they were classified as Re chemotype according to the nomenclature of Sulmoneltu. However, only in S. enterica serovar Typhimurium (Sanderson et al, 1974;Chatterjee et al, 1976) and E. coli K-12 (Chen and Coleman, 1993) strains have been successfully applied in interspecies complemention experiments (Chen and Coleman, 1993;Zhou et al, 1994), they were not suitable for analysis of the immediate reaction products of the recombinant enzymes. However, conclusive in vitro assays for RfaC must contain pure preparations of ADPHep and (Kdo),-lipid A acceptor, the last of which should be labeled radioactively to improve detection.…”

“…The genes rfuC and rfaF are directly linked within an operon of the rfa locus of E. coli K-12 ( Fig. 2A, and Chen and Coleman, 1993). Thus, a 849-bp SrnalNruI fragment including parts of both genes was deleted in one step from the chomosomal region of strain W3110 which had been subcloned into a plasmid (see Experimental Procedures).…”

Amplification of a novel gene, sanA, abolishes a vancomycin-sensitive defect in Escherichia coli

“…The vancomycin-sensitive phenotype of mutant 87. 6 transformed with different plasmids was tested at 37ЊC on LB plates containing 100 g of vancomycin per ml and 50 or 100 g of ampicillin per ml. For the determination of the MIC, LB plates were prepared with concentrations of vancomycin of 25,50,100,150,200,250,300,350, and 400 g/ml.…”

A novel strategy to isolate cell-envelope mutants resistant to phage infection: bacteriophage mEp213 requires lipopolysaccharides in addition to FhuA to enter Escherichia coli K-12

“…1). The waaC gene has 960 bp and encodes a peptide of 319 aa, ADP-heptose : LPS heptosyltransferase I (Chen & Coleman, 1993;Kadrmas & Raetz, 1998; GenBank accession no. BAE77671).…”