Mutational Consequences of Ciprofloxacin in Escherichia coli

Abstract

We examined the mutagenic specificity of the widely used antibiotic ciprofloxacin (CPR), which displays weak to moderate mutagenic activity in several bacteria and generates short in-frame deletions in rpoB in Staphylococcus aureus To determine the spectrum of mutations in a system where any gene knockout would result in a recovered mutant, including frameshifts and both short and long deletions, we examined CPR-induced mutations in the thymidylate synthase-encoding thyA gene. Here, any mutation resulting in loss of thymidylate synthase activity generates trimethoprim (Trm) resistance. We found that deletions and insertions in all three reading frames predominated in the spectrum. They tend to be short deletions and cluster in two regions, one being a GC-rich region with potential extensive secondary structures. We also exploited the well-characterized rpoB-Rif(r) system in Escherichia coli to determine that cells grown in the presence of sublethal doses of CPR not only induced short in-frame deletions in rpoB, but also generated base substitution mutations resulting from induction of the SOS system. Some of the specific point mutations prominent in the spectrum of a strain that overproduces the dinB-encoded Pol IV were also present after growth in CPR. However, these mutations disappeared in CPR-treated dinB mutants, whereas the deletions remained. Moreover, CPR-induced deletions also occurred in a strain lacking all three SOS-induced polymerases. We discuss the implications of these findings for the consequences of overuse of CPR and other antibiotics.

FIG 1

Sequence changes in ropB. The locations of spontaneous mutations in the two wild-type backgrounds are shown above the DNA sequence. The letters represent base substitutions. The locations of CPR-derived mutations in the wild-type CC107 background and a dinB-deficient strain are shown below. The solid lines are aligned with deleted base pairs; the arrows indicate locations of insertions. The numbers with plus signs in parentheses show the number of additional mutations at any given site that appeared more than once in a sample of four from the same culture. Mutants from three strains are shown in the following colors: green, BW25113 background; black, CC107 background; red, a dinB derivative of BW25113.

FIG 2

Sequence changes in thyA. The locations of spontaneous mutations in the CC107 wild-type background are shown above the DNA sequence (black); the locations of CPR-derived mutations in the wild-type BW25113 background (red) and the CC107 background (black) are shown below. The letters represent base substitutions, the circles indicate the A·T→T·A base substitution at the hot spot at bp 131, the diamonds indicate deletions of a single base pair, the triangles indicate insertions of a single base pair, the solid lines are aligned with deleted base pairs, and the arrows indicate the locations of insertions. The numbers with plus signs in parentheses show the number of additional mutations at any given site that appeared more than once in a sample of four from the same culture.

FIG 3

Potential single-strand secondary structure in thyA. Position 298 is centered within a region where a cluster of deletions of various lengths are observed. The Mfold program revealed a putative secondary structure between bp 261 and 320 (−16.4 kcal).