Identification of a pKM101 region which confers a slow growth rate and interferes with susceptibility to quinolone in Escherichia coli AB1157.

The effect of plasmid pKM101 on the survival of Escherichia coli AB1157, growing in minimal medium, in the presence of a 4-quinolone DNA gyrase inhibitor was investigated. The presence of this plasmid decreased susceptibility to the quinolone ciprofloxacin, whereas mucAB genes present in a multicopy plasmid did not. The same effect of pKM101 was detected in a recA430 mutant, confirming that it was not really related to the SOS response. In contrast, when survival assays were performed under amino acid starvation conditions, pKM101 did not confer protection against ciprofloxacin. All of these results indicated that the synthesis of a product(s), different from MucAB, which was encoded by the plasmid pKM101 increased the rate of survival of the AB1157 strain in the presence of quinolone. To identify the gene(s) responsible for this phenotype, several plasmid derivatives carrying different portions of pKM101 were constructed. The 2.2-kb region containing korB, traL, korA, and traM genes was sufficient to decrease susceptibility to quinolone. This plasmidic fragment also made the AB1157 host strain grow more slowly (the Slo phenotype). Moreover, the suppression of the Slo phenotype by addition of adenine to the cultures abolished the decreased susceptibility to quinolone. These results are evidence that the protection against quinolone conferred by this region of pKM101 in strain AB1157 is a direct consequence of the slow growth rate.

Construction and characterization of two lexA mutants of Salmonella typhimurium with different UV sensitivities and UV mutabilities.

Salmonella typhimurium has a SOS regulon which resembles that of Escherichia coli. recA mutants of S. typhimurium have already been isolated, but no mutations in lexA have been described yet. In this work, two different lexA mutants of S. typhimurium LT2 have been constructed on a sulA background to prevent cell death and further characterized. The lexA552 and lexA11 alleles contain an insertion of the kanamycin resistance fragment into the carboxy- and amino-terminal regions of the lexA gene, respectively. SOS induction assays indicated that both lexA mutants exhibited a LexA(Def) phenotype, although SOS genes were apparently more derepressed in the lexA11 mutant than in the lexA552 mutant. Like lexA(Def) of E. coli, both lexA mutations only moderately increased the UV survival of S. typhimurium, and the lexA552 strain was as mutable as the lexA+ strain by UV in the presence of plasmids encoding MucAB or E. coli UmuDC (UmuDCEc). In contrast, a lexA11 strain carrying any of these plasmids was nonmutable by UV. This unexpected behavior was abolished when the lexA11 mutation was complemented in trans by the lexA gene of S. typhimurium. The results of UV mutagenesis correlated well with those of survival to UV irradiation, indicating that MucAB and UmuDCEc proteins participate in the error-prone repair of UV damage in lexA552 but not in lexA11. These intriguing differences between the mutagenic responses of lexA552 and lexA11 mutants to UV irradiation are discussed, taking into account the different degrees to which the SOS response is derepressed in these mutants.

Efficiency of MucAB and Escherichia coli UmuDC proteins in quinolone and UV mutagenesis in Salmonella typhimurium: effect of MucA and UmuD processing.

The role of MucAB and Escherichia coli UmuDC proteins in mutagenesis by 4-quinolone (4-Q) compared to that in UV mutagenesis has been studied in hisG428 Salmonella typhimurium strains. A low-copy plasmid carrying mucAB genes, but not umuDC, promotes reversion of the hisG428 mutation by the 4-Q ciprofloxacin. In contrast, a umuDC plasmid mediates the reversion of hisG428 by UV, although less efficiently than a mucAB one. In addition, a unique copy of mucAB genes is enough to promote UV mutagenesis, whereas, several copies of them are required to detect ciprofloxacin mutagenesis. Therefore, the mutagenic repair of quinolone damage by MucAB proteins is not a very efficient process. The presence of an umuD'C plasmid but not a mucA'B one, slightly increases the reversion of the hisG428 mutation by ciprofloxacin and this finding is further discussed. In contrast, MucA'B are still more active than UmuD'C proteins in UV mutagenesis. These results suggest that the enhanced processing of MucA compared to UmuD would not explain all functional differences between MucAB and UmuDC proteins in the error-prone DNA repair.

Analysis of the ciprofloxacin-induced mutations in Salmonella typhimurium.

The mutagenic events induced by ciprofloxacin, a potent antimicrobial agent, have been characterized. For this, a battery of His mutants of Salmonella typhimurium (hisG428, his G46, His C9070, and his G1775 targets) that detects the six possible transitions and transversions [Levin and Ames (1986): Environ Mutagen 8:9-28] and two additional His strains (hisC3076 and his D3052 targets) carrying frameshift mutations have been used. Our results indicate that GC-TA transversions are the major base-pair substitution induced by ciprofloxacin and that GC-At transitions are also produced, but to a lesser degree. However, we cannot discard the fact that At-Ta transversions are also induced. In addition, the data indicate that the mutational specificity of ciprofloxacin depends on the location of the target. Intragenic base-pair substitutions are the most frequent mutations at the hisG428 target when it is on the chromosome, whereas 3 or 6 base-pair deletions are the major mutagenic events when this target is on the plasmid pAQ1. We have shown that ciprofloxacin also induces deletions/insertions at the hisC3076 and hisD3052 frameshift targets. Therefore, this inhibitor of DNA gyrase promotes a wide pattern of mutations including different kinds of base-pair substitutions, 3 or 6 base-pair deletions, and insertions/deletions resulting in frameshifts. All of these mutagenic events require the MucAb proteins involved in the error-prone repair, with the exception of base-pair insertions/deletions at the hisD3052 target, which are independent of the presence of plasmid pKM101.

The role of the excision and error-prone repair systems in mutagenesis by fluorinated quinolones in Salmonella typhimurium.

Patterns of reversion produced by ciprofloxacin, enoxacin and ofloxacin in Salmonella typhimurium strains carrying the hisG428 ochre mutation have been studied. These fluorinated quinolones produce a significant increase in reversion of this mutation, even when it is located on the chromosome. Nevertheless, reversion is higher when the hisG428 mutation is on the multicopy plasmid pAQ1 than when it is on the chromosome. Reversion of hisG428 induced by fluorinated quinolones is abolished both in a uvrB genetic background and in the absence of the plasmid pKM101. Therefore, mutagenesis produced by fluorinated quinolones in the Salmonella mutagenicity assay is significantly affected by both the excision repair and the error-prone repair systems. Furthermore, fluorinated quinolones are also detected as moderate mutagens with the base substitution hisG46 mutation when both repair systems are functional in the tester strain.

Induction of SOS genes in Escherichia coli and mutagenesis in Salmonella typhimurium by fluoroquinolones.

The induction of several SOS genes of Escherichia coli by fluoroquinolones has been studied. Three different SOS gene fusions (recA::lacZ, umuC::lacZ and sulA::lacZ) have been introduced into the E.coli MC1061 strain to study the induction of these SOS genes in the same genetic background. Data on the basal level of expression of these fusions, as well as their induction by mitomycin C and N-methyl-N'-nitro-N-nitrosoguanidine are presented. Using these strains, we have found that, like nalidixic acid, ofloxacin, enoxacin and ciprofloxacin are strong inducers of the SOS genes tested, umuC gene expression being the highest. Furthermore, fluoroquinolones produced a significant increase in the reversion of the base substitution hisG428 mutation in the TA102 Salmonella tester strain, while no effect was found in strains TA98, TA100, TA1537 and TA1535. These data indicate that the error-prone repair pathway can participate in mutagenesis induced by fluoroquinolones and also that the damage produced by these chemicals may be similar to that produced by nalidixic acid.