Suppression of Temperature-Sensitive Chromosome Replication of an Escherichia coli dnaX (Ts) Mutant by Reduction of Initiation Efficiency

Abstract

ABSTRACT Temperature sensitivity of DNA polymerization and growth of a dnaX (Ts) mutant is suppressible at 39 to 40°C by mutations in the initiator gene, dnaA . These suppressor mutations concomitantly cause initiation inhibition at 20°C and have been designated Cs,Sx to indicate both phenotypic characteristics of cold-sensitive initiation and suppression of dnaX (Ts) . One dnaA (Cs,Sx) mutant, A213D, has reduced affinity for ATP, and two mutants, R432L and T435K, have eliminated detectable DnaA box binding in vitro. Two models have explained dnaA (Cs,Sx) suppression of dnaX , which codes for both the τ and γ subunits of DNA polymerase III. The initiation deficiency model assumes that reducing initiation efficiency allows survival of the dnaX (Ts) mutant at the somewhat intermediate temperature of 39 to 40°C by reducing chromosome content per cell, thus allowing partially active DNA polymerase III to complete replication of enough chromosomes for the organism to survive. The stabilization model is based on the idea that DnaA interacts, directly or indirectly, with polymerization factors during replication. We present five lines of evidence consistent with the initiation deficiency model. First, a dnaA (Cs,Sx) mutation reduced initiation frequency and chromosome content (measured by flow cytometry) and origin/terminus ratios (measured by real-time PCR) in both wild-type and dnaX (Ts) strains growing at 39 and 34°C. These effects were shown to result specifically from the Cs,Sx mutations, because the dnaX (Ts) mutant is not defective in initiation. Second, reduction of the number of origins and chromosome content per cell was common to all three known suppressor mutations. Third, growing the dnaA (Cs,Sx) dnaX (Ts) strain on glycerol-containing medium reduced its chromosome content to one per cell and eliminated suppression at 39°C, as would be expected if the combination of poor carbon source, the Cs,Sx mutation, the Ts mutation, and the 39°C incubation reduced replication to the point that growth (and, therefore, suppression) was not possible. However, suppression was possible on glycerol medium at 38°C. Fourth, the dnaX (Ts) mutation can be suppressed also by introduction of oriC mutations, which reduced initiation efficiency and chromosome number per cell, and the degree of suppression was proportional to the level of initiation defect. Fifth, introducing a dnaA (Cos) allele, which causes overinitiation, into the dnaX (Ts) mutant exacerbated its temperature sensitivity.