On the process of cellular division in Escherichia coli: II. Membrane protein alterations associated with mutations affecting the initiation of DNA synthesis

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A procedure for analysis of the protein composition of bacterial membrane preparations is presented, utilizing radioactive labeling with either [ 3H]leucine or [ 14C]leucine during growth, subsequent purification of membranes, and disaggregation of the membrane in sodium dodecyl sulfate, followed by electrophoresis and scintillation counting of the membrane protein components. Using this procedure, an analysis of a temperature-sensitive mutation blocking the initiation of DNA synthesis was made. The results indicate that two mutations of the DnaA locus lead to the same alterations in membrane proteins. A protein component of molecular weight approximately 60,000 is deficient in isogenic strains carrying either the T46 or T83 DnaA mutations when cells are incubated under restrictive growth conditions (41 °C). Another protein component, of molecular weight approximately 32,000, is increased in the mutant relative to the wild-type strain, under permissive conditions (30 °C) as well as restrictive conditions (41 °C); the difference is magnified, however, under restrictive growth conditions. The expression of the mutation seems to be strain dependent, because the T46 mutation, while exhibiting the absence of a protein in two strains examined, did not show the relative increase in the 32,000 molecular weight component in both strains. Thus, DnaA mutants exhibit associated membrane protein alterations; whether these are direct or secondary effects is still unclear.