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

Summary

Several revertants to 5-methyltryptophan (MT) independence of the MT-dependent mutation trpA515 were altered in the trp promoter trpP1 (Callahan et al., 1978). This suggested the possibility that by screening MT-independent revertants for a class which would produce no prototrophic recombinants in spot transduction tests against the deletion supX38, which covers the trpP0 region but does not extend into the first structural gene of the trp operon (trpA), we could recover mutations of the trp promoter. Of two thousand revertants screened in this manner, ten carried deletions extending into trpP0. Nine of these extended into trpA and one, trpP0 (A?) 1351 apparently did not. These results show that genetic screening of MT-independent revertants of SO495 (trp A515) provides a means of isolating trp promoter mutations. The mutation trpP1443 which had been previously isolated from the MT-independent revertant trpP1443trpA515 (Callahan et al., 1978) was characterized genetically and biochemically and shown to have the attributes of a promoter mutation. It was found to map at the extreme operator end of the tryptophan operon. Strain SO443 (genotype trpP1443) showed “leaky” growth on medium not containing tryptophan, was subject to normal repression by tryptophan, but under derepression produced undetectable amounts of trpmRNA and the trp enzymes did not increase appreciably in level. The trpP1443 mutation is cis-dominant. Strain SO443 could revert to full prototrophy. A detailed study of the revertants thus obtained disclosed, in addition to true revertants at the trpP1443 site (class 1), three additional classes. Class 2 revertants were resistant to low concentrations of MT (10 μg/ml) and showed that they had retained the trpP1443 mutation in genetic crosses. The mutation conferring simultaneously full prototrophy and MT resistance was closely linked to trpP1443. Class 3 revertants were resistant to high concentrations (100 μg/ml) of MT and thus far have not shown the presence of trpP1443 in crosses. The mutation responsible for the reversion to prototrophy and resistance to the analogue is clearly within the early portion of the trp operon and could be at the trpP1443 site itself. Revertants of class 4 seem to be caused by mutations at an unlinked suppressor locus (or loci).

The recovery of four classes of prototrophic revertants of SO443 shows that a mutation of the promoter can be corrected not only by a true reversion to the wild type but in at least two additional ways. Reversions of class 2 can represent the creation of a new transcription start downstream from the promoter mutation itself and there is precedent for this class of event. Revertants of class 3 could represent a different event of this type, or a mutation at the trpP1443 site which simultaneously affects the operator function. Since there have been reports based on work in Escherichia coli that the trp promoter and operator overlap, this possibility should be seriously considered. Revertants of class 4 constitute an entirely novel and interesting class.