On the process of cellular division in Escherichia coli: IV. Altered protein composition and turnover of the membranes of thermosensitive mutants defective in chromosomal replication

Abstract

Temperature-sensitive mutants of Escherichia coli which are impaired in the process of chromosomal replication at 40 °C have alterations in their membrane composition at the restrictive temperature. Membrane preparations from three distinct mutants of the DnaB locus were found to have a relative deficiency of a protein component of molecular weight 60,000, and a relative increase in a component of molecular weight 30,000, in comparison with isogenic wild-type strains, when the membranes were disaggregated at 40 °C in 1% sodium dodecyl sulfate. These changes seen with DnaB mutants are qualitatively similar to those seen in the DnaA (“initiator”) mutants previously examined (Shapiro, Siccardi, Hirota & Jacob, 1970). However, when harsher conditions of disaggregation are used, by heating at 100 °C, the membrane preparations disaggregate further, and the DnaA and DnaB mutant preparations may be differentiated. The DnaA mutant now shows a relative deficiency of a component of molecular weight 35,000, and a relative increase of components of molecular weight below 25,000, suggesting that the altered protein complexes have dissociated to smaller molecular weight units. On the other hand, when the DnaB mutant is disaggregated at 100 °C the previously noted difference is almost obliterated, suggesting that the difference between mutant and wild type DnaB membrane preparations seen with disaggregation at 40 °C is due to a difference in the ability of the preparations to disaggregate. The results are compatible with the hypothesis that the membrane expression of the DnaA defect is in an alteration of the protein composition of the membranes, whereas the DnaB mutation affects the strength of the bonds holding certain membrane proteins together. The turnover of proteins in the membranes of the DnaB mutants, as measured under conditions of incomplete disaggregation (40 °C), was different from that of the wild type, with the major alteration being the slower turnover of the 60,000 molecular weight component. This difference was not seen in the presence of chloramphenicol, suggesting that protein synthesis is needed for the alteration to occur. The membrane changes seen with the DnaB mutants are primary effects of the mutation. That these were not secondary to the cessation of growth or DNA synthesis, or to cell death at the restrictive temperature, was shown by phenotypic suppression of the mutation by salt. The presence of 2% NaCl, previously shown (Ricard & Hirota, 1969) to permit normal growth of mutant DnaB-BT313 at 40 °C, does not reverse the appearance of the membrane protein alterations. Thus, the DnaB mutation, which is associated with a defect in chromosomal replication, appears to have its primary effect in altering the structure of the bacterial membrane; it is still not clear whether the DnaA mutation, which is associated with a defect in initiation of DNA synthesis, leads to primary or secondary membrane changes.