Abstract
Zinc inhibits the virulence of diarrheagenic E. coli by inducing the envelope stress response and inhibiting the SOS response. The SOS response is triggered by damage to bacterial DNA. In Shiga-toxigenic E. coli, the SOS response strongly induces the production of Shiga toxins (Stx) and of the bacteriophages that encode the Stx genes. In E. coli, induction of the SOS response is accompanied by a higher mutation rate, called the mutator response, caused by a shift to error-prone DNA polymerases when DNA damage is too severe to be repaired by canonical DNA polymerases. Since zinc inhibited the other aspects of the SOS response, we hypothesized that zinc would also inhibit the mutator response, also known as hypermutation. We explored various different experimental paradigms to induce hypermutation triggered by the SOS response, and found that hypermutation was induced not just by classical inducers such as mitomycin C and the quinolone antibiotics, but also by antiviral drugs such as zidovudine and anti-cancer drugs such as 5-fluorouracil, 6-mercaptopurine, and azacytidine. Zinc salts inhibited the SOS response and the hypermutator phenomenon in E. coli as well as in Klebsiella pneumoniae, and was more effective in inhibiting the SOS response than other metals. We then attempted to determine the mechanism by which zinc, applied externally in the medium, inhibits hypermutation. Our results show that zinc interferes with the actions of RecA, and protects LexA from RecA-mediated cleavage, an early step in initiation of the SOS response. The SOS response may play a role in the development of antibiotic resistance and the effect of zinc suggests ways to prevent it.
Highlights
Our laboratories became interested in the E. coli “Save Our Ship” (SOS) response to DNA damage because of role of the SOS responses in inducing Shiga toxin (Stx) from Shiga-toxigenic E. coli (STEC) [1, 2]
Ciprofloxacin-induced recA expression was inhibited by zinc acetate, as we expected based on the known inhibitory effect of zinc on Stx toxin production
Faint LexA cleavage products could be seen in some lanes, but these were too faint for accurate quantitation, so we focused on quantitation of the intact, uncleaved LexA
Summary
Our laboratories became interested in the E. coli SOS response to DNA damage because of role of the SOS responses in inducing Shiga toxin (Stx) from Shiga-toxigenic E. coli (STEC) [1, 2]. In addition to Stx production, the SOS response triggers a myriad of bacterial cell responses, including DNA repair, elongation of bacterial cells, induction of error-prone DNA polymerases [3], induction of latent bacteriophage, and inhibition of cell division. The SOS response is strongly induced by treatments that damage bacterial DNA, including UV light, quinolone antibiotics, and mitomycin C. Kim et al recently reported that tetracycline antibiotics commonly used for growth promotion in cattle were surprisingly strong inducers of Stx and of the SOS response in STEC [8], contrary to previous predictions [9]. We initially measured recA expression by quantitative RT-PCR in response to ciprofloxacin and mitomycin C in order to be able to optimize drug concentrations and time courses needed to observe maximal activation of the SOS, and to confirm that zinc blocked recA expression. In addition to classical antibiotic inducers of SOS response, such as ciprofloxacin and mitomycin C, we tested drugs such as 5-fluorouracil, zidovudine, and other antivirals and anti-cancer drugs, since these have been reported to induce the SOS response as well [13, 14], and demonstrated that zinc’s ability to inhibit the SOS response was not shared by most other transition metals
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