The discovery and development of antibiotics revolutionized medicine, providing easy cures for previously untreatable diseases. However, for every significant infectious disease caused by bacteria, strains resistant to all available antibiotics have been reported [54
]. We are interested in understanding how bacteria evolve resistance, and have first focused on the antibiotic ciprofloxacin. Ciprofloxacin and the other quinolones are perhaps the most important antibiotics currently available [16
], partly because of the low levels of resistance currently observed with these newer synthetic drugs. However, clinical resistance to the quinolones is evolving at an alarming rate due to mutations in gyrase, topoisomerase IV, and efflux pumps or their regulators [1
]. In this study we have shown, in vivo, that preventing LexA cleavage renders bacteria unable to evolve resistance to either ciprofloxacin or rifampicin in a mouse thigh infection model. In vitro, the ability of bacteria to induce mutation and evolve resistance to ciprofloxacin is also dramatically reduced by rendering LexA uncleavable. Thus, our results indicate that the mutations that confer resistance to ciprofloxacin and rifampicin are not simply the result of unavoidable errors accumulated during genome replication, but rather are induced via the derepression of genes whose protein products act to significantly increase mutation rates.
In principle, part of the observed increase in mutation rate after exposure to ciprofloxacin could result from selection against resistant mutants during the pre-exposure growth in liquid media (thus underestimating the pre-exposure mutation rates). However, the impact of selection is unlikely to be significant, as gyrA mutations are tolerated without a significant increase in doubling time (). Further evidence that the resistance-conferring mutations are induced is provided by the fact that deletion or mutation of several genes, including lexA, renders cells unable to evolve significant levels of resistance.
The data suggest that the increase in mutation rate is caused by recombination pathways that are induced to repair antibiotic-mediated DNA damage. A mechanism consistent with our results is illustrated in . Three recombinational repair pathways appear to be involved that are distinguished by the type of damage they repair and the type of mutation they induce. One pathway is IR-mediated repair of free DSBs where the protein has dissociated from the DNA (pathway A, ). We suggest that this pathway is responsible for the observed codon deletions. The induction of small nucleotide deletions has been observed in bacteria [55–57
], and in eukaryotes, it has been suggested that small deletions (including codon deletions) arise during IR after an inhibited topoisomerase aberrantly releases free DSBs, the ends of which are processed by exonucleases and polymerases before being rejoined [58
]. While we may have observed a similar phenomenon in our in vitro studies, the codon deletion mutants are unlikely to be of much clinical significance on their own, as they have relatively low ciprofloxacin MICs () and have never been observed in the clinic [1
]. Isolation of these mutants in the current study was most likely due to the permissive drug concentrations employed.
Proposed Response to Ciprofloxacin
In addition to IR, RDR and replication fork repair are also induced to repair DSBs in cases where the topoisomerase has dissociated from, or remains bound to, the DNA, respectively (pathways B and C, ). These pathways may be more relevant to clinical resistance as they induce the substitution mutations ubiquitously found in clinically resistant strains. In both pathways, the RecBCD nuclease/helicase loads at DSEs generated (directly or indirectly) by ciprofloxacin and simultaneously degrades and unwinds the duplex while loading RecA onto the ssDNA of the nascent 3′-overhang. (For replication fork repair, RecG and RuvABC are required to prepare the DSE [50
].) RecA forms filaments that promote strand invasion of the ssDNA into a homologous sequence, resulting in the formation of an intermediate known as a displacement-loop structure (D-loop). The invading strand may then prime DNA synthesis, using the homologous sequence as a template, ultimately restoring the genetic information disrupted by the DSE [48
]. In the case of replication fork repair, the covalently bound topoisomerase must still be displaced from DNA in order to reinitiate processive synthesis. We propose that the topoisomerase is displaced by RuvAB, which has recently been shown to branch migrate D-loop-like structures and simultaneously displace covalently-bound ciprofloxacin-topoisomerase IV complexes [61
], or perhaps by Rep or UvrD helicase, which have both been shown to displace bound proteins from duplex DNA [62
]. Following PriA recognition and binding, a processive replication fork is reestablished. However, with the continued presence of ciprofloxacin these processes will continue, resulting in the persistence of the RecA-ssDNA filaments, which eventually degrade enough LexA to derepress the error-prone, SOS-regulated polymerases (pathway D, ).
The data suggest that the induction of substitution mutations requires the derepression of all three SOS-regulated polymerases, Pol II, Pol IV, and Pol V. While the evolution of resistance to ciprofloxacin by substitution mutation is to our knowledge the first process found to require all three of the E. coli
inducible polymerases, this observation is consistent with previous studies showing that multiple polymerases are required for some mutations [9
]. It is also consistent with the two-step model of translesion synthesis, wherein one specialized polymerase is required for dNTP misinsertion and another for continued synthesis (mispair extension) [9
]. We propose that the induced mutations conferring antibiotic resistance in vitro and in vivo are the result of Pol V mispair synthesis [66
] and Pol IV mispair extension [10
], while Pol II may be required to initiate replication restart [41
] (after which it may be replaced by Pol V and then Pol IV), or to fix the nascent mutation by extending the primer terminus sufficiently to avoid exonucleolytic proofreading upon reloading of Pol III [69
]. This process continues until mutations are made that allow for the resumption of normal DNA synthesis.
The key signal that links the cellular response to the antibiotic with the evolution of resistance appears to be the RecA-ssDNA filaments that are formed to facilitate the repair of antibiotic-mediated DNA damage. These RecA-ssDNA filaments also induce LexA cleavage and derepression of the mutagenic polymerases. We suggest that a similar mechanism might also serve to induce mutation and evolution in response to other antibiotics, or other forms of cellular stress, where DNA damage per se is not involved. For example, the ratio of ATP to ADP determines the level of supercoiling in the bacterial genome [17
], and both increased and decreased levels of supercoiling inhibit replication fork progression [70
]. Thus, different stresses that perturb metabolism (i.e., alter ATP/ADP ratios) might also alter DNA topology and result in stalled replisomes; recombination-based rescue and RecA-ssDNA filament formation; and the induction of mutations required to reestablish a normal cellular environment. Interestingly, it has recently been shown that β-lactams can induce the SOS response via a two-component signal transduction system [71
The traditional paradigms of DNA replication and mutation suggest that resistance-conferring mutations are the inevitable consequence of polymerase errors, and offer no obvious means for intervention. In stark contrast, the model described above suggests that bacteria play an active role in the mutation of their own genomes by inducing the production of proteins that facilitate mutation, including Pol IV and Pol V, as has been suggested with other forms of mutation [7–15
]. In turn, this suggests that inhibition of these proteins, or the prevention of their derepression by inhibition of LexA cleavage, with suitably designed drugs, might represent a fundamentally new approach to combating the emerging threat of antibiotic-resistant bacteria. Future efforts will focus on determining the generality of the observations, in terms of both other pathogenic bacteria and other antibiotics.