Temperature-Sensitive Cell Division Mutants of Escherichia coli with Thermolabile Penicillin-Binding Proteins

Abstract

The thermostability of the penicillin-binding proteins (PBPs) of 31 temperature-sensitive cell division mutants of Escherichia coli has been examined. Two independent cell division mutants have been found that have highly thermolabile PBP3. Binding of [ 14 C]benzylpenicillin to PBP3 (measured in envelopes prepared from cells grown at the permissive temperature) was about 30% of the normal level at 30°C, and the ability to bind [ 14 C]benzylpenicillin was rapidly lost on incubation at 42°C. The other PBPs were normal in both mutants. At 30°C both mutants were slightly longer than their parents and on shifting to 42°C they ceased dividing, but cell mass and deoxyribonucleic acid synthesis continued and long filaments were formed. At 42°C division slowly recommenced, but at 44°C this did not occur. The inhibition of division at 42°C was suppressed by 0.35 M sucrose, and in one of the mutants it was partially suppressed by 10 mM MgCl 2 . PBP3 was not stabilized in vitro at 42°C by these concentrations of sucrose or MgCl 2 . Revertants that grew as normal rods at 42°C regained both the normal level and the normal thermostability of PBP3. The results provide extremely strong evidence that the inactivation of PBP3 at 42°C in the mutants is the cause of the inhibition of cell division at this temperature and identify PBP3 as an essential component of the process of cell division in E. coli . It is the inactivation of this protein by penicillins and cephalosporins that results in the inhibition of division characteristic of low concentrations of many of these antibiotics.