We previously reported that salicylate (SAL) decreases the LD50 of Cd++ in Escherichia coli by 3- to 4-fold. In order to determine the bacterial functions that are required for this synergy, we selected mutants (called Sci-) whose susceptibility to Cd++ was not enhanced by the presence of SAL. Surprisingly, 5/14 of these Sci- mutants were also cysteine auxotrophs. The mutations responsible for the auxotrophies were located in the cysB gene as determined by Hfr mapping and by complementation with a plasmid carrying wild-type cysB. The cysB mutations were shown to be responsible for the Sci- phenotypes since mutants that became Cys+ by recombination, complementation or reversion simultaneously became Sci+. Furthermore, cysB mutants that had previously been isolated solely on the basis of their requirement for cysteine were also found to be Sci-. Thus, a function of cysB is necessary for the potentiation of Cd++ susceptibility by SAL. Since CysB is a regulatory protein required for transcriptional activation of the several operons of the cysB regulon, cysB mutants do not express at least 12 other cys genes needed to synthesize cysteine. Mutations in 5 of these cys genes did not result in a Sci- phenotype. Thus, the Sci- phenotype is a property of only particular cys mutations and is not due simply to the inability to synthesize cysteine. So far, the only other cys mutations that render cells Sci- were found in cysE, a gene whose function is not under cysB control but is required for activation of the CysB protein. Since the transcription of cysB is regulated by supercoiling, we examined the sensitivity to Cd++ of topA mutants whose DNA is highly supercoiled. These cells were found to be hypersensitive to Cd++. Suppression of the topA mutation abolished the hypersensitivity. Cd++ resistant mutants were then isolated from the topA cells: 3/5 were cysB mutants and were Sci-. Thus, these effects of SAL and supercoiling have a common requirement for cysB. However, SAL did not affect the transcription of either cysB or cysE as shown by studies with appropriate cysB/E-lacZ fusion strains. Further studies are necessary to determine whether SAL affects the activity of CysB protein. We conclude that a function under cysB control (which may or may not be involved in cysteine biosynthesis) is required for the potentiation of Cd++ sensitivity by SAL and supercoiling.
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