Genetic analysis of mutations that compensate for loss of Escherichia coli DNA topoisomerase I.

A transposon Tn10 insertion in topA, the structural gene of Escherichia coli DNA topoisomerase I, behaves as an excluded marker in genetic crosses with many strains of E. coli. However, derivative strains that accept this mutant topA allele are readily selected. We show that many of these topA mutant strains contain additional mutations that compensate for the loss of DNA topoisomerase I. Genetic methods for mapping and manipulating such compensatory mutations are described. These methods include a plate-mating test for the ability of strains to accept a topA::Tn10 allele and a powerful indirect selection for transferring compensatory mutations from male strains into non-compensatory female strains. One collection of spontaneous compensatory mutants is analyzed in detail and is shown to include compensatory mutations at three distinct loci: gyrA and gyrB, the genes that encode the subunits of DNA gyrase, and a previously unidentified locus near tolC. Mutations at this third locus, referred to as toc (topoisomerase one compensatory) mutations, do not behave as point mutations in transductional crosses and do not result in lowered DNA gyrase activity. These results show that wild-type strains of E. coli require DNA topoisomerase I, and at least one class of compensatory mutations can relieve this requirement by a mechanism other than reduction of DNA gyrase activity.

Escherichia coli and Salmonella typhimurium supX genes specify deoxyribonucleic acid topoisomerase I.

Mutations of the Escherichia coli or Salmonella typhimurium supX genes eliminated deoxyribonucleic acid topoisomerase I. Suppression of a supX amber mutation partially restored the topoisomerase. Multicopy plasmids carrying supX+ caused overproduction of topoisomerase. Thus, these supX genes were identified as topA genes which specify deoxyribonucleic acid topoisomerase I.

Salmonella typhimurium mutants with reduced levels of transfer ribonucleic acid-inhibitable endodeoxyribonucleolytic activity.

Two methods of mutagenesis, chemical alkylation and insertion of the tetracycline resistance transposon Tn10, were used to generate mutants of Salmonella typhimurium which had reduced levels of endodeoxyribonucleolytic activity. The chemically induced mutations defined a locus, endA, which was cotransduced with serA at a low frequency and with metK at a high frequency. Three-factor crosses revealed that metK was between serA and endA. The major endodeoxyribonucleolytic activity in crude extracts of s. typhimurium was similar to the activity of Escherichia coli endonuclease I. A Tn10 insertion mutation of endA resulted in the most severe loss of endodeoxyribonucleolytic activity among the endA alleles studied. Two of the chemically induced mutations resulted in activities which were more thermolabile than the wild-type activity.