Genetic analysis of mutations causing borrelidin resistance by overproduction of threonyl-transfer ribonucleic acid synthetase.

Mutations leading to borrelidin resistance in Escherichia coli by overproduction of threonyl-transfer ribonucleic acid synthetase were anaylzed genetically. The regulatory mutations were closely linked to the treonyl-transfer ribonucleic acid synthetase structural gene (thrS), located clockwise to it. The mutation that causes the threefold-increased enzyme level was more distant from thrS than the mutation responsible for the ninefold overproduction. Both mutations were cis dominant in merodiploid strains, indicating that they affected promoter-operator-like control elements. Overproduction was restricted to threonyl-transfer ribonucleic acid synthetase and was not observed for the products of genes neighboring thrS (e.g., infC, pheS, pheT, and argS), providing evidence that thrS is transcribed singly and that gene amplificationis not a likely basis for increased thrS experession.

Escherichia coli mutants overproducing phenylalanyl- and threonyl-tRNA synthetase.

The structural genes for threonyl-tRNA synthetase (ThrRS) and phenylalanyl-tRNA synthetase (PheRS) are closely linked on the Escherichia coli chromosome. To study whether these enzymes share a common regulatory element, we have investigated their synthesis in mutants which were selected for overproduction of either ThrRS or PheRS. It was found that mutants isolated previously for overproduction of ThrRS as strains resistant to the antibiotic borrelidin (strains Bor Res 3 and Bor Res 15) did not show an elevated level of PheRS. PheRS-overproducing strains were then isolated as revertants of strains with structurally altered enzymes. Strain S1 is a temperature-resistant derivative of a temperature-sensitive PheRS mutant, and strain G118 is a prototrophic derivative of a PheRS mutant which shows phenylalanine auxotrophy as a consequence of an altered K(m) of this enzyme for the amino acid. In both kinds of revertants, S1 and G118, the concentration of PheRS and ThrRS was increased by factors of about 2.5 and 1.8, respectively, whereas the level of other aminoacyl-tRNA synthetases was not affected by the mutations. Genetic studies showed that the simultaneous overproduction of PheRS and ThrRS in revertants G118 and S1 is based upon gene amplification, since this property was easily lost after growing the cells in the absence of the selective stimulus, and since this loss could be prevented by the presence of the recA allele. By similar criteria, the four- and eightfold overproduction of ThrRS in strains Bor Res 3 and Bor Res 15, respectively, was very stable genetically, indicating that it is caused by a mutational event other than gene amplification. From these results, we conclude that the concomitant increase of PheRS and ThrRS in strains G118 and S1 is an expression of gene duplication and not of a joint regulation of these two aminoacyl-tRNA synthetases. This conclusion is further supported by the result that, in mutant G118 as well as in its parental strain G1, growth in minimal medium lacking phenylalanine led to an additional twofold increase of their PheRS concentration. This increase was restricted to the PheRS, since the level of other aminoacyl-tRNA synthetases, including the ThrRS, stayed unchanged.