In a growth-restricting environment (for example, during starvation), mutants arise that are able to take over bacterial populations by a process known as stress-induced mutagenesis, stationary-phase mutagenesis, or adaptive mutation. The ability of bacteria of the genus Pseudomonas to mutate rapidly is relevant to understanding the evolution of novel metabolic activities in microbes under starvation conditions but also to understanding the development of antibiotic-resistance mutations, colonization of new bacterial hosts, and pathogenesis. We use Pseudomonas putida as a model organism to study molecular mechanisms of mutagenic processes in bacteria, with emphasis on the various DNA polymerases and DNA repair systems involved in mutagenesis under stressful conditions.
Role of ImuB and DnaE2 in Stationary-Phase Mutagenesis in Pseudomonas
Several bacterial species carry in their genomes a so-called “mutagenesis” gene cluster encoding ImuB, which is similar to Y-family DNA polymerases, and DnaE2, which is related to the catalytic subunit DnaE of Pol III. Y-family DNA polymerases are known to be involved in stationary-phase mutagenesis, and the DnaE2 homologs characterized so far have expressed a mutator phenotype. We investigated the role of ImuB and DnaE2 in stationary-phase mutagenesis in P. putida, and our results show that these proteins have opposite roles in stationary-phase mutagenesis. ImuB augments the accumulation of stationary-phase mutants up to twofold. Our previous data revealed that the Pol IV homolog DinB is essential for frameshift mutations in long-term starving P. putida. Interestingly, the absence of ImuB also reduces the frameshift mutation frequency. However, the frequency of frameshift mutations measured in a DinB-ImuB double-knockout strain is higher than that detected in strains lacking only DinB. These data indicate that ImuB influences DNA polymerase traffic. It is possible that, in the absence of ImuB and Pol IV, the access of a different DNA polymerase to the replication apparatus is favored, thereby facilitating frameshift mutations in starving bacteria. Studies are in progress to investigate this possibility.
In contrast to ImuB, DnaE2 has no significant effect on the emergence of frameshift mutants. Moreover, it acts as an antimutator by suppressing the accumulation of base substitution mutants in starving bacteria. We found similar antagonistic effects of DnaE2 and ImuB on mutagenesis when we looked at UV mutagenesis. These data distinguish the DnaE2 of P. putida from homologs studied in other organisms. Thus, a study of P. putida DnaE2 would help elucidate mechanisms underlying the mutator phenotype of certain DnaE2 homologs.
Role of DNA Repair in Mutagenic Processes
Nucleotide excision repair (NER) is one of the most important repair systems to counteract various forms of DNA damage induced by chemicals or irradiation. However, relatively little is known about the functions of NER in the repair of DNA that is not exposed to exogenous DNA-damaging agents. We have investigated the role of NER in mutagenesis in P. putida. In addition to the NER genes uvrA and uvrB, the genome of this organism contains the NER gene uvrA2. Genetic studies on the effects of uvrA, uvrA2, and uvrB on mutagenic processes revealed that all these genes are responsible for repairing UV-induced DNA damage in P. putida. However, uvrA plays a more important role in this process than uvrA2, as deletion of the uvrA2 gene has only a minor effect on the UV tolerance of bacteria. Interestingly, the lack of a functional uvrB or uvrA2 gene reduces the frequency of stationary-phase mutations. The contribution of uvrA2 and uvrB to mutagenesis appears to be most significant in the case of single-base-pair deletions whose emergence depends on error-prone DNA polymerase Pol IV. These data imply that NER has a dual role in mutagenesis in P. putida—besides functioning in repairing damaged DNA, NER is also important for generating mutations. We hypothesize that NER enzymes may initiate spontaneous DNA repair and that the ensuing DNA repair synthesis might be mutagenic.
Our previous studies revealed that a lack of the DNA mismatch repair (MMR) proteins MutS and MutL had less influence on the rate of emergence of Pol IV–dependent frameshift mutants than on emergence of base substitution mutants. Although our results indicated that MMR is still functional in long-term starved bacteria, we detected no significant differences in the accumulation of single-base-pair deletion mutants in wild-type and MMR-defective bacteria. It is possible that Pol IV is recruited to the DNA replication machinery preferentially in DNA repair synthesis initiated by NER and MMR. If both NER and MMR facilitate Pol IV–dependent stationary-phase mutation, this could explain why we observed stronger antimutator effects on frameshift mutations than on base substitution mutations when both MutS and UvrB are inactivated in P. putida. We are performing more detailed studies of the interactions between various components of DNA repair and DNA polymerases on mutagenesis in Pseudomonas.
Role of Oxidative Damage in Mutagenesis in P. putidaOxidative damage of DNA is a source of mutations in living cells. We have studied the relationship of DNA oxidative damage to mutagenesis in stationary-phase bacteria. The enzymes MutY, MutM, and MutT, which repair oxidized guanine, are involved in suppressing base substitution mutations in carbon-starved P. putida. Interestingly, although the lack of MutT causes a strong mutator phenotype under starvation conditions of bacteria, we observed no such effect in growing bacteria, which indicates that MutT has a backup system that is functional only in actively growing cells. The knockout of MutM affected the spectrum of mutations but did not change the mutation frequency in this bacterium. The protein Dps is known to protect DNA from oxidative damage. We found that cells of dps-defective P. putida are more sensitive to sudden exposure to hydrogen peroxide than are wild-type cells and that the absence of Dps does not affect the accumulation of mutations in populations of starved bacteria. Thus, it is possible that the protective role of Dps on genome integrity becomes essential only when bacteria are exposed to exogenous agents leading to oxidative damage of DNA.
Last updated September 2009