Bacterial Cell Division Regulation: Characterization of the dnaH Locus of Escherichia coli

Abstract

The dnaH locus is the fourth gene to be identified as required for deoxyribonucleic acid polymerization in Escherichia coli. A temperature-sensitive mutant defective in this gene exhibited an abrupt decrease in rate of deoxyribonucleic acid synthesis when shifted to 42 C. The locus mapped in the proC-purE region of the chromosome by conjugation and was co-transducible with purE. dnaH+ is carried on the F',8 episome and is dominant over the dnaHmutation.Mutations which cause temperature sensitivity of deoxyribonucleic acid (DNA) replication in Bacillus subtilis and Escherichia coli (11) have been described. These mutations have been mapped at nine loci in B. subtilis (19) and at six loci in E. coli (36,37). In E. coli, the dnaA and dnaC gene products are required for replication initiation, dnaB, dnaE; and dnaG gene products are involved in polymerization (4,7,15,16,20,21), and dnaF (nrdA) is involved in precursor biosynthesis (8).We describe here an additional mutant of E. coli which is temperature sensitive for DNA synthesis and cell division. Upon a temperature shift to 42 C, rate of DNA synthesis and of cell division decreased immediately, but ribonucleic acid (RNA) synthesis and growth continued for two or three doublings. Viability decreased exponentially after a lag of about 20 min. The temperature sensitivity mutation was co-transducible with purE, and the locus is designated dnaH. Partial diploid strains which carry dnaH+ on an episome and dnaH on the chromosome were able to synthesize DNA, divide, and form colonies at 42 C; therefore, the wild-type dnaH allele is dominant. MATERIALS AND METHODSStrains. The principal bacterial strains used are listed in Table 1. Origins of the Hfr strains are shown in Fig. 1. Bacteriophages Plvir and f2 were obtained from J. L. Rosner and R. Johnston, respectively. Growth conditions. The yeast extract-tryptone medium of Howard-Flanders et al. (18) with 0.5% NaCl was used unless stated otherwise. Low-phosphate medium was the defined medium base (18) containing 0.1 M tris(hydroxymethyl)aminomethane (pH 7.2) in place of phosphate buffer and supple-mented with the following: glucose, 10 mg/ml; thiamine-hydrochloride, 5 ug/ml; Casamino Acids (Difco), 2.5 mg/ml; and phosphate, 0.2 gsmol/ml. Incubation was at 28 or 42 C with shaking. Temperature shifts were accomplished by transfer of cultures to previously equilibrated flasks.Phages f2 and Plvir were grown by the procedures of Loeb and Zinder (23) and Rosner (32), respectively.Isolation of the temperature-sensitive mutant AX642. Strain AX642, which carries the temperaturesensitive (ts) mutation ts20-16, was isolated during a process of isolating conditional cell division mutants. After mutagenesis of E. coli K-12 AB1157 (2) and screening of survivors for inability to form colonies at 42 C, temperature-sensitive clones were screened further for filament formation at 42 C. Mutant AX642 was discovered because it formed filaments which were three to four times the normal cell length during incubation at 42 C.Cell number, mass, and ...

Suppression of a DnaX temperature‐sensitive polymerization defect by mutation in the initiation gene, dnaA, requires functional oriC

“…The oriC+, dnaA+ dnaX(Ts) strain also stopped growth at 39°C after a 20‐fold absorbance increase over about 4 h (data not shown). At the more restrictive 42°C, a dnaX(Ts) mutant stopped DNA synthesis quickly with an average 17% increment and growth ceased after a fourfold mass increase over a 2 h period (Filip et al ., 1974).…”

Isolation and characterization of plaque-forming lambdadnaZ+ transducing bacteriophages

“…Position ofdnaZ on the E. coli map. Earlier mapping by P1 demonstrated that dnaZ is cotransducible with purE but did not determine the sequence of dnaZ with respect to other markers in this region (10). To determine this sequence, phage P1 was grown on strain AX727-(proC+ tsx+ dnaZts purE+) and used to transduce a proC tsx dnaZ + purE recipient (X156) to purE+ and proC+ at 300C.…”

“…The Escherichia coli dnaZ gene is one of the four known genes that code for products necessary for deoxyribonucleic acid (DNA) polymerization (10). In addition to participating in E. coli chromosome replication, the dnaZ product also is required for growth of phages M13, OX174, and A, but not for T7 or T4 growth (30,32).…”

“…Wickner and Hurwitz (36) demonstrated that the dnaZ protein participates in vitro in elongation of DNA during 4X174, M13, and ST-1 replicative-form synthesis from primed single-strand DNAs. Mapping studies demonstrated that dnaZ is cotransducible with purE, which is located near min 12 on the E. coli chromosome map (10).…”

A phage‐encoded inhibitor of Escherichia coli DNA replication targets the DNA polymerase clamp loader

“…2A), although not all of the cells were stained at the same intensity with ReAsH due to differences in permeabilization among individual cells. Furthermore, gp8-CCPGCC localization determined in a dnaXts strain, where the temperature sensitivity of the E. coli DNA polymerase III DnaX subunit results in a fast-stop replication elongation block at the non-permissive temperature (Filip et al, 1974), showed no localization at the non-permissive temperature while foci formed at the permissive temperature, indicating that: (i) focus formation was dependent on an active replisome ( Fig. 2A); and (ii) the formation of foci reflected an interaction with a component recruited to the replisome during replication elongation.…”