Oxidative DNA Damage Defense Systems in Avoidance of Stationary-Phase Mutagenesis in Pseudomonas putida▿

Oxidative damage of DNA is a source of mutation in living cells. Although all organisms have evolved mechanisms of defense against oxidative damage, little is known about these mechanisms in nonenteric bacteria, including pseudomonads. Here we have studied the involvement of oxidized guanine (GO) repair enzymes and DNA-protecting enzyme Dps in the avoidance of mutations in starving Pseudomonas putida. Additionally, we examined possible connections between the oxidative damage of DNA and involvement of the error-prone DNA polymerase (Pol)V homologue RulAB in stationary-phase mutagenesis in P. putida. Our results demonstrated that the GO repair enzymes MutY, MutM, and MutT are involved in the prevention of base substitution mutations in carbon-starved P. putida. Interestingly, the antimutator effect of MutT was dependent on the growth phase of bacteria. Although the lack of MutT caused a strong mutator phenotype under carbon starvation conditions for bacteria, only a twofold increased effect on the frequency of mutations was observed for growing bacteria. This indicates that MutT has a backup system which efficiently complements the absence of this enzyme in actively growing cells. The knockout of MutM affected only the spectrum of mutations but did not change mutation frequency. Dps is known to protect DNA from oxidative damage. We found that dps-defective P. putida cells were more sensitive to sudden exposure to hydrogen peroxide than wild-type cells. At the same time, the absence of Dps did not affect the accumulation of mutations in populations of starved bacteria. Thus, it is possible that the protective role of Dps becomes essential for genome integrity only when bacteria are exposed to exogenous agents that lead to oxidative DNA damage but not under physiological conditions. Introduction of the Y family DNA polymerase PolV homologue rulAB into P. putida increased the proportion of A-to-C and A-to-G base substitutions among mutations, which occurred under starvation conditions. Since PolV is known to perform translesion synthesis past damaged bases in DNA (e.g., some oxidized forms of adenine), our results may imply that adenine oxidation products are also an important source of mutation in starving bacteria

Effect of incubation temperature on the frequency of MMS-induced Rif^R mutations in <i>P</i>. <i>putida</i> strains.

<p><i>P</i>. <i>putida</i> wild-type (<i>wt</i>), Δ<i>alkAtag</i> and Δ<i>imuCalkAtag</i> strains were incubated with 0.15 mM MMS overnight at 37°C or 30°C. The frequencies of MMS-induced mutagenesis were measured as described in Materials and Methods. Data represents the mean (±SE) values. Groups that have no common letter are significantly different at <i>P</i> < 0.0001, according to ANOVA followed by Bonferroni’s multiple comparisons test.</p

Effect of incubation temperature on the survival of MMS-treated <i>alkA</i>-deficient bacteria.

<p>The survival of <i>P</i>. <i>aeruginosa</i> Δ<i>alkA</i> (black) and Δ<i>imuCalkA</i> (grey) strains at 37°C or 30°C after treatment with 2.5 mM MMS for 45-min period is indicated. Data represents the mean (±SD) values. Columns with the pattern fill represent the initial CFU/ml. Letters indicate homogeneous groups according to ANOVA followed by Bonferroni’s multiple comparisons test (<i>P</i> < 0.05).</p

Effect of TLS polymerase deficiencies on the frequencies of MMS-induced Rif^R mutations in <i>P</i>. <i>putida</i> wild type and <i>alkA</i>- and <i>alkAtag</i>-deficient backgrounds.

<p>Bacteria were exposed to 0.15 mM MMS (A) or to 0.05 mM MMS (B) overnight. Data represents the mean (±SE). Letters indicate homogeneous groups. Groups that have no common letter are significantly different at <i>P</i> < 0.05, according to Kruskal-Wallis test followed by Dunn's multiple comparisons test (e.g., group with the letter ‘a’ is significantly different from the group with the letters ‘cb’, but not from the groups with the letters ‘ab’ or ‘ae’).</p

DNA Polymerases ImuC and DinB Are Involved in DNA Alkylation Damage Tolerance in <i>Pseudomonas aeruginosa</i> and <i>Pseudomonas putida</i>

<div><p>Translesion DNA synthesis (TLS), facilitated by low-fidelity polymerases, is an important DNA damage tolerance mechanism. Here, we investigated the role and biological function of TLS polymerase ImuC (former DnaE2), generally present in bacteria lacking DNA polymerase V, and TLS polymerase DinB in response to DNA alkylation damage in <i>Pseudomonas aeruginosa</i> and <i>P</i>. <i>putida</i>. We found that TLS DNA polymerases ImuC and DinB ensured a protective role against <i>N</i>- and <i>O</i>-methylation induced by N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) in both <i>P</i>. <i>aeruginosa</i> and <i>P</i>. <i>putida</i>. DinB also appeared to be important for the survival of <i>P</i>. <i>aeruginosa</i> and rapidly growing <i>P</i>. <i>putida</i> cells in the presence of methyl methanesulfonate (MMS). The role of ImuC in protection against MMS-induced damage was uncovered under DinB-deficient conditions. Apart from this, both ImuC and DinB were critical for the survival of bacteria with impaired base excision repair (BER) functions upon alkylation damage, lacking DNA glycosylases AlkA and/or Tag. Here, the increased sensitivity of <i>imuCdinB</i> double deficient strains in comparison to single mutants suggested that the specificity of alkylated DNA lesion bypass of DinB and ImuC might also be different. Moreover, our results demonstrated that mutagenesis induced by MMS in pseudomonads was largely ImuC-dependent. Unexpectedly, we discovered that the growth temperature of bacteria affected the efficiency of DinB and ImuC in ensuring cell survival upon alkylation damage. Taken together, the results of our study disclosed the involvement of ImuC in DNA alkylation damage tolerance, especially at low temperatures, and its possible contribution to the adaptation of pseudomonads upon DNA alkylation damage via increased mutagenesis.</p></div

Survival of exponentially growing <i>P</i>. <i>putida</i> TLS polymerase-deficient cells after MMS treatment.

<p>Survival was estimated at different concentrations of MMS after 45-min treatment period. Data represents the mean (±95%CI) values of three independent experiments performed in triplicate. (●) wild-type; (■) Δ<i>imuC</i>; (▲) Δ<i>dinB</i>; (▼) Δ<i>imuCdinB</i>. Asterisks indicate statistically significant difference (<i>P</i> < 0.05; two-way ANOVA followed by Tukey’s multiple comparison post hoc test) in sensitivity between the mutant and the wild-type strain, and between the Δ<i>dinB</i> and the Δ<i>imuCdinB</i> strains.</p

Effect of incubation temperature on transcription from the <i>P</i>. <i>putida lexA2</i> promoter.

<p>β-galactosidase activities expressed from the <i>lexA2</i> promoter-<i>lacZ</i> reporter were measured in the <i>P</i>. <i>putida</i> wild-type and in its <i>alkA-</i>deficient derivative incubated at 30°C (black) or 37°C (grey) for 1 hour (A) and overnight (B) in liquid LB medium supplemented with 0.3 mM MMS or not (control). Data represents the mean (±SE) values. Letters indicate homogeneous groups according to ANOVA followed by Bonferroni’s multiple comparisons test (<i>P</i> < 0.01).</p

Sensitivity of <i>P</i>. <i>putida</i> and <i>P</i>. <i>aeruginosa</i> wild-type and their DNA glycosylase-deficient derivatives to MMS and MNNG.

<p>Sensitivity was estimated by spotting 10-fold dilutions of overnight cultures of <i>P</i>. <i>putida</i> (A, B) and <i>P</i>. <i>aeruginosa</i> (C, D) onto LB plates containing different concentrations of MMS (A, C) and MNNG (B, D). Data represents the mean (±95%CI) values. (●) wild-type; (■) Δ<i>alkA</i>; (▲) Δ<i>alkAtag</i>. <i>P</i>. <i>putida</i> was incubated at 30°C and <i>P</i>. <i>aeruginosa</i> was incubated at 37°C.</p