Inhibition of lipopolysaccharide biosynthesis and cell growth following inactivation of the kdtA gene in Escherichia coli.
The enzyme 3-deoxy-D-manno-octulosonic acid (Kdo) transferase is encoded by the kdtA gene in Escherichia coli. The enzyme is a single polypeptide that catalyzes the transfer of two Kdo residues to a tetraacyldisaccharide-1,4'-bisphosphate precursor of lipid A, designated lipid IVA (Belunis, C.J., and Raetz, C.R.H. (1992) J. Biol. Chem. 267, 9988-9997). To determine if Kdo transfer to lipid IVA is required for growth, we constructed a strain of E. coli with a chromosomal kdtA::kan insertion mutation. In mutants carrying the kdtA::kan allele on the chromosome, cell growth and Kdo transferase activity were dependent upon a copy of the intact kdtA gene on a plasmid. When the kdtA-bearing plasmid was itself temperature sensitive for replication, the growth of these strains was inhibited after several hours at 44 degrees C, and Kdo transferase activity in extracts became undetectable. Concomitantly, the cells accumulated massive amounts of lipid IVA, the precursor of (Kdo)2-lipid IVA. The kdtA::kan mutation could also be complemented by hybrid plasmids bearing the gseA gene of Chlamydia trachomatis. gseA specifies a distinct Kdo transferase that adds three Kdo moieties to lipid IVA. Lipopolysaccharide from E. coli kdtA::kan constructs complemented by gseA reacts strongly with antibodies directed against the genus-specific epitope of Chlamydia, whereas lipopolysaccharide from parental E. coli K-12 does not. Our studies prove that Kdo attachment during lipid A biosynthesis is essential for cell growth and accounts for the conditional lethality associated with mutations in Kdo biosynthesis.
D002236:Carbohydrate Conformation; D002240:Carbohydrate Sequence; D002876:Chromosomes, Bacterial; D004926:Escherichia coli / Q000235:genetics / Q000502:physiology*; D005798:Genes, Bacterial; D005816:Genetic Complementation Test; D005838:Genotype; D007700:Kinetics; D008070:Lipopolysaccharides / Q000037:antagonists & inhibitors / Q000096:biosynthesis*; D008969:Molecular Sequence Data; D016254:Mutagenesis, Insertional; D010957:Plasmids; D012486:Salmonella typhimurium / Q000235:genetics; D013045:Species Specificity; D013696:Temperature; D013997:Time Factors; D014166:Transferases / Q000235:genetics* / Q000378:metabolism
D008070:Lipopolysaccharides; D014166:Transferases; C019917:3-deoxy-D-manno-octulosonate transferase