A gene that specifies production of Escherichia coli DNA topoisomerase I (w protein) was identified with the aid of a radioimmunoassay for this protein. E. coli DNA topoisomerase I was produced by Salmonella typhimurium merodiploids that harbored E. coli plasmid F' 123, but not by strains that lost this plasmid. Analysis of strains with spontaneous deletions of F' 123 showed that the gene, topA, required for production of the E. coli w protein was between the trp operon and the cysB gene. Deletions that eliminated topA also eliminated the supX gene. We suggest that topA is the structural gene ofE. coli DNA (5)]. In the absence of high-energy cofactors, this enzyme catalyzes several reactions in vitro, including the relaxation of supercoiled DNA (4), the knotting and unknotting ofsingle-stranded DNA rings (6), and the intertwining ofsinglestranded rings of complementary sequences (7).Identifying and locating the structural gene of DNA topoisomerase I is basic to any understanding of its physiological role. We have approached this problem in the absence of mutants by utilizing the natural differences between E. coli DNA topoisomerase I and the homologous enzyme of Salmonella typhimurium. Similar techniques have been used to map several other genes (8-11). We report here the identification and location of an E. coli gene that is required for the production of E. coli DNA topoisomerase I in merodiploid strains of S. typhimurium.
MATERIALS AND METHODSBacterial Strains. The strains of E. coli K-12 and S. typhimurium LT2 used are described in Table 1. The presence of F' plasmids was verified by curing in the presence of acridine orange with concomitant acquisition of appropriate auxotrophic requirements or by conjugation (16). Transductions mediated by phage P22 were performed as described (12).Enzymes. E. coli DNA topoisomerase I was purified from strain RY13 as described (17). S. typhimurium DNA topoisomerase I was purified from strain RED10 by a similar procedure. Both enzyme preparations were at least 90% homogeneous on sodium dodecyl sulfate/polyacrylamide gels.
Mutants of bacteriophage T4D which fail to induce the deoxyribonucleotidespecific T4 3'-phosphatase have been isolated. These mutants (T4pseT) grow as well as wild-type T4 in most strains of Escherichia coli, but not in the T4-sensitive "Hospital Strain," CT196, or in a derivative strain, CTr5x. Both the formation of infectious centers and the final yield of phage are reduced by 98% when CTr5x is infected by T4pseT mutants. The growth defects are accompanied by a 50% reduction in the rate of T4 DNA synthesis, a decrease in the singlestrand length of the DNA product to about one-half the mature length, and greatly reduced packaging of DNA into phage particles. Introduction of an extracistronic suppressor mutation (stp) into T4pseT eliminates both the requirement for the T4 3'-phosphatase in infected CTr5x and the other observed effects of the pseT mutations. The pseT gene lies between genes 63 and 31. The stp gene lies in the nonessential region between rIIB and ac. Our results suggest that 3-phosphoryl termini can disrupt T4 DNA replication to the extent that T4 3'-phosphatase becomes required for phage production. formation of 3-phosphoryl termini on T4 DNA. A preliminary report of this work has been presented (R. E. Depew and N. R. Cozzarelli, Abstr. Annu. Meet. Amer. Soc. Microbiol. 1973, p. 234). MATERIALS AND METHODS Phage strains. The wild-type strain was T4D. T4D amber and temperature-sensitive mutants were originally from the collection of R. S. Edgar. Amber mutants N54 (gene 31), N81 (gene 41), N122 (gene 888 on September 28, 2020 by guest http://jvi.asm.org/ Downloaded from T4 3'-PHOSPHATASE DNA METABOLISM 42), N82 (gene 44), N130 (gene 46), and the temperature-sensitive mutant A80 (gene 30) were obtained from the collection of the
A transposon TnWO 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::TnlO 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 toiC. 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.
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