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A mutation to 5-methyltryptophan dependence in the tryptophan (trp) operon of Salmonella typhimurium

II. Studies of 5-methyltryptophan-dependent mutants and their revertants

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Summary

Mutants of S. typhimurium with a defect in the first structural gene of the trp operon can utilize anthranilic acid (AA) as a growth factor. Among a group of 5-methyltryptophan (MT) resistant derivatives of trpA mutants we encountered several with a novel phenotype: they actually grew better in the presence of MT than in its absence. Normally MT inhibits growth of S. typhimurium at the concentration we employed due to its ability to act as co-repressor of the trp operon, and as a feedback inhibitor of anthranilate synthetase (AS) the first enzyme for tryptophan biosynthesis. Mutations to MT-dependence were only found in strains carrying extremely polar trpA mutations. In all cases analyzed, mutations causing MT-dependence mapped at the extreme operator distal end of trpA. The mutation trpA515 responsible for MT-dependence in strain SO61 (genotype trpA49trpA515) was, recombined away from the polar mutation. The strain thus obtained, SO495 was totally dependent on MT for growth on AA supplement. Strain SO495 lacks AS and under repressing grwoth conditions synthesizes the trp enzymes constitutively at 2–3 times the basal level. Under derepression, while the levels of the distal enzymes, as represented by tryptophan synthetase-β subunit (TSβ), did not increase there was a marked drop in the activity of anthranilate-PRPP phosphoribosyltransferase, (PRT) the enzyme catalyzing the second step of tryptophan biosynthesis. trpA515 was found to revert to prototrophy at a low frequency (about 10−8) which was not increased by chemical mutagens or ultraviolet radiation. In contrast, it was found to revert to MT-independence (growth on AA in the absence of MT) at a fairly high spontaneous frequency (about 10−6) and this frequency could be increased approximately tenfold by mutagens causing base substitutions or deletions but not by frameshift mutagens. About one hundred MT-independent revertants of trp A515 were mapped and found to fall into three general classes: (A) mutations at or near the trpA515 site (B) secondary mutations located upstream from trpA515, (C) deletions of various sizes. Based on a detailed genetic and physiologocal study of twelve representative MT-independent revertants, it appears that trpA515 may be caused by the insertion of a piece of DNA with some of the properties described for the IS elements found in Escherichia coli. The trpA515 insertion should contain (in this order), a transcription terminator, a low efficiency promoter and, probably, a translation start signal.

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Author notes

  1. Robert Callahan III

    Present address: Meloy Laboratories, National Cancer Institute, Electronics Drive, 22151, Springfield, VA

  2. M. Margaret Dooley

    Present address: Department of Biochemistry and Biophysics, University of California, 95616, Davis, CA

Authors and Affiliations

  1. Department of Biology, Syracuse University, 13210, Syracuse, NY, USA

    Robert Callahan III & M. Margaret Dooley

  2. AMC Cancer Research Center and Hospital, 80214, Lakewood, CO, USA

    Elias Balbinder

Authors
  1. Robert Callahan III
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  2. M. Margaret Dooley
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  3. Elias Balbinder
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Additional information

Communicated by T. Yura

Paper # 1 of this series is by Callahan, R., and Balbinder, E.: Science, 168, 1586–1589 (1970)

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Callahan, R., Margaret Dooley, M. & Balbinder, E. A mutation to 5-methyltryptophan dependence in the tryptophan (trp) operon of Salmonella typhimurium . Molec. Gen. Genet. 165, 129–143 (1978). https://doi.org/10.1007/BF00269901

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