Genetics, Vol. 166, 681-692, February 2004, Copyright © 2004
Distinct Signatures for Mutator Sensitivity of lacZ Reversions and for the Spectrum of lacI/lacO Forward Mutations on the Chromosome of Nondividing Escherichia coli
Shanti M. Bharatana,b,
Manjula Reddya, and
J. Gowrishankarb
a Centre for Cellular and Molecular Biology, Hyderabad 500 007, India
b Laboratory of Bacterial Genetics, Centre for DNA Fingerprinting and Diagnostics, Hyderabad 500 076, India
Corresponding author:
J. Gowrishankar, Centre for DNA Fingerprinting and Diagnostics, ECIL Rd., Nacharam, Hyderabad 500 076, India., shankar{at}cdfd.org.in (E-mail)
Communicating editor: S. T. LOVETT
A conditional lethal galE(Ts)-based strategy was employed in Escherichia coli, first to eliminate all growth-associated chromosomal reversions in lacZ or forward mutations in lacI/lacO by incubation at the restrictive temperature and subsequently to recover (as papillae) spontaneous mutations that had arisen in the population of nondividing cells after shift to the permissive temperature. Data from lacZ reversion studies in mutator strains indicated that the products of all genes for mismatch repair (mutHLS, dam, uvrD), of some for oxidative damage repair (mutMT), and of that for polymerase proofreading (dnaQ) are required in dividing cells; some others for oxidative damage repair (mutY, nth nei) are required in both dividing and nondividing cells; and those for alkylation damage repair (ada ogt) are required in nondividing cells. The spectrum of lacI/lacO mutations in nondividing cells was distinguished both by lower frequencies of deletions and IS1 insertions and by the unique occurrence of GC-to-AT transitions at lacO +5. In the second approach to study mutations that had occurred in nondividing cells, lacI/lacO mutants were selected as late-arising papillae from the lawn of a galE+ strain; once again, transitions at lacO +5 were detected among the mutants that had been obtained from populations initially grown on poor carbon sources such as acetate, palmitate, or succinate. Our results indicate that the lacO +5 site is mutable only in nondividing cells, one possible mechanism for which might be that random endogenous alkylation (or oxidative) damage to DNA in these cells is efficiently corrected by the Ada Ogt (or Nth Nei) repair enzymes at most sites but not at lacO +5. Furthermore, the late-arising papillae from the second approach were composed almost exclusively of dominant lacI/lacO mutants. This finding lends support to "instantaneous gratification" models in which a spontaneous lesion, occurring at a random site in DNA of a nondividing cell, is most likely to be fixed as a mutation if it allows the cell to immediately exit the nondividing state.
