TEMPERATURE-SENSITIVE LESIONS IN THE FRANCISELLA-NOVICIDA VALA GENE CLONED INTO AN ESCHERICHIA-COLI MSBA LPXK MUTANT AFFECTING DEOXYCHOLATERESISTANCE AND LIPOPOLYSACCHARIDE ASSEMBLY AT THE RESTRICTIVE TEMPERATURE

The valAB locus of Francisella novicida has previously been found to b e highly similar at the deduced amino acid level to msbA lpxK of Esche richia coli, Both ValA and MsbA are members of the superfamily of ABC transporters, and they appear to have similar functions, In this study we describe the isolation of a temperature-sensitive valAB locus, DNA sequence analysis indicates that the only changes to the ValAB deduce d amino acid sequence are changes of S453 to an F and T458 to an I in ValA. E. coil strains defective in msbA and expressing temperature-sen sitive ValA rapidly ceased growth when shifted from a permissive tempe rature to a restrictive temperature, After 1 h at the restrictive temp erature, cells were much moire sensitive to deoxycholate treatment, To test the hypothesis that ValA is responsible for the transport or ass embly of lipopolysaccharide, we introduced gseA, a Kdo (3-deoxy-D-mann o-octulosonic acid) transferase from Chlamydia trachomatis, into a str ain with a temperature-sensitive valA allele and a nonfunctional msbA locus, These recombinants were defective in cell surface expression of the chlamydial genus-specific epitope within 15 min of a shift to the nonpermissive temperature. Also, there was enhanced association of th e epitope with the inner membrane after a shift to the nonpermissive t emperature. Thus, we propose that ValA is involved in the transport of lipopolysaccharide to the outer membrane.