CHROMOSOMAL MUTANTS OF ESCHERICHIA COLI K-12 DEFECTIVE IN EXPRESSION OF F PLASMID CONJUGATION FUNCTIONS
MCEWEN, JOAN ELIZABETH
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Chromosomal mutants of Escherichia coli K-12 that are defective in expression of F plasmid-directed conjugation functions were isolated and characterized. The objective was to analyse regulatory and functional interactions that occur between chromosomal and F plasmid gene products during expression of conjugation functions. Chromosomal genes required for expression of conjugation functions were designated by the mnemonic cpx (conjugative plasmid expression).;Methods were developed for the specific isolation of chromosomal cpx mutants. Two independently-isolated, temperature-sensitive mutants were extensively characterized. Both exhibited identical pleiotropic defects in conjugation functions (Cpx('-) phenotype), isoleucine and valine biosynthesis (Ilv('-) phenotype) and envelope protein composition. Analysis of Ilv('+) revertants of the mutants demonstrated that, in each case, the Ilv('-), Cpx('-) and envelope defects were caused by the same mutations. The Ilv('-) property was therefore exploited to map the mutant genes responsible for the three mutant phenotypes.;Each mutant contains mutations in the same two genes: cpxA, located at 86.5 minutes on the E. coli K-12 map, and cpxB, located on 41 minutes. The cpxB mutation (cpxB1) was already present in the parent strain of the two mutants. It is cryptic in cpxA('+) strains. The cpxA mutations (cpxA1 and cpxA2) were independently induced during mutagenesis of the cpxA('+) cpxB1 parent strain. A cpxA mutation in a cpxB('+) strain causes partial expression of Cpx('-), Ilv('-) and envelope defects. Both a cpxA and a cpxB mutation are required for maximal expression of mutant phenotypes.;Hfr strains containing cpxA and cpxB mutations are temperature-sensitive for donor-specific bacteriophage sensitivity and conjugal DNA donor activity. These characteristics were attributed to the inability of mutant cells to elaborate F pili. Mutant cells are also defective in surface exclusion. This defect was attributed in part to a reduction in the amount of the F plasmid traT gene product in the outer membrane of mutant cells.;The temperature-sensitive Ilv('-) phenotype of cpxA('-) cpxB('-) cells was attributed to a temperature-sensitive defect in elaboration of active acetohydroxy acid synthase isozyme I, which catalyses the first reaction of both isoleucine and valine biosynthesis.;Mutants altered in the cpxA and cpxB genes exhibit striking changes in the protein composition of both the inner and the outer membrane. One of the proteins deficient in the outer membrane of mutant cells is murein lipoprotein. This deficiency appears to result from a defect in envelope assembly rather than in synthesis of murein lipoprotein.;The pleiotropic effect of the cpxA and cpxB mutations on F plasmid-directed conjugation functions, acetohydroxy acid synthase I production and envelope biogenesis indicates that the cpxA and cpxB genes are components of a cellular mechanism required for the synthesis or activity of several functionally unrelated proteins. The possibility that this mechanism is directly involved in the biogenesis of the cell envelope and the possible explanions for the mutant phenotypes from this perspective are discussed.