Temperature-sensitive mutants in rfaI and rfaJ, genes for galactosyltransferase I and glucosyltransferase II, for synthesis of lipopolysaccharide in Salmonella typhimurium

Abstract

Certain rough mutants of Salmonella typhimurium LT2 were shown to be temperature sensitive for the production of lipopolysaccharide (LPS). When grown at the restrictive temperature (42 or 45.degree. C), the cells contained LPS deficient in O (somatic) side chains, based on phage-sensitivity data and gel electrophoresis of the LPS. Cells grown at the permissive temperature, 30.degree. C, made LPS resembling that of smooth cells. The mobility of the LPS in gels, the phage sensitivity patterns, and gas chromatographic analysis indicate that LPS of 45.degree. C-grown cells of SA126 (rfaJ3012) is of chemotype Rb2, with one glucose and two galactose units (and thus inferred to be due to a mutation in rfaJ), and LPS of 45.degree. C-grown cells of SA134 (rfaI3020) is of chemotype Rb3, with one glucose and one galactose unit (inferred to be rfaI). These inferences were confirmed, for pKZ26 (pBR322-rfaGBIJ) and pKZ27 (pBR322-rfaGBI) both complement rfaI3020, but only pKZ26 complemented rfaJ3012. In addition, pKZ26 carrying a Tn5 insertion resulting in loss of complementation of a known rfaJ mutation, but not of rfaG, B, or I, also resulted in loss of rfaJ3012 complementation. Based on gel analysis, there is a small amount of the LPS containing smooth side chains in cells of SA126 grown at 45.degree. C; following a switch to 30.degree. C, the amount of LPS with O side chains gradually increased, and the amount of core LPS was reduced, though even after 3 h the LPS does not fully resemble that of smooth strains. Cells grown at 42.degree. C show limited capacity to adsorb a smooth-specific phage, P22, but the capacity to adsorb the phage increased fourfold after 5 min growth at 30.degree. C, and by 60 min at 30.degree. C adsorption resembled that seen by smooth cells. These data indicate that synthesis of the smooth LPS begins less than 5 min after shift to the permissive temperature, and that phage adsorption is a more sensitive test for the appearance of smooth side chains than the use of polyacrylamide gels with silver stains.