DNA Polymerases ImuC and DinB Are Involved in DNA Alkylation Damage Tolerance in Pseudomonas aeruginosa and Pseudomonas putida

Abstract

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 Pseudomonas aeruginosa and P. putida. We found that TLS DNA polymerases ImuC and DinB ensured a protective role against N- and O-methylation induced by N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) in both P. aeruginosa and P. putida. DinB also appeared to be important for the survival of P. aeruginosa and rapidly growing P. putida 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 imuCdinB 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.

Fig 1. Sensitivity of P. aeruginosa and P. putida wild-type and their TLS polymerase-deficient derivatives to MMS and MNNG.

Sensitivity was estimated by spotting 10-fold dilutions of overnight cultures of P. aeruginosa (A, B) and P. putida (C, D) onto LB plates containing MMS (A, C) or MNNG (B, D). P. aeruginosa was incubated at 37°C for 24 h (A, B). P. putida was incubated at 30°C on MMS-containing plates for 48 h (C) and on MNNG for 24 h (D). Data represents the mean (±95%CI) values. (●) wild-type; (■) ΔimuC; (▲) ΔdinB; (▼) ΔimuCdinB. Asterisks indicate statistically significant difference (maximum value P < 0.05; two-way ANOVA followed by Tukey’s multiple comparison post hoc test) in sensitivity between the mutant and the wild-type strain or between the ΔdinB and the ΔimuCdinB (A, D).

Fig 2. Survival of exponentially growing P. putida TLS polymerase-deficient cells after MMS treatment.

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; (■) ΔimuC; (▲) ΔdinB; (▼) ΔimuCdinB. Asterisks indicate statistically significant difference (P < 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 ΔdinB and the ΔimuCdinB strains.

Fig 3. Sensitivity of P. putida and P. aeruginosa wild-type and their DNA glycosylase-deficient derivatives to MMS and MNNG.

Sensitivity was estimated by spotting 10-fold dilutions of overnight cultures of P. putida (A, B) and P. aeruginosa (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; (■) ΔalkA; (▲) ΔalkAtag. P. putida was incubated at 30°C and P. aeruginosa was incubated at 37°C.

Fig 4. Sensitivity of P. putida ΔalkA and ΔalkAtag strains and their different TLS polymerase-deficient derivatives to MMS and MNNG.

Sensitivity was estimated by spotting 10-fold dilutions of overnight cultures of P. putida ΔalkA (A, B) and ΔalkAtag (C, D) strains onto LB plates containing different concentrations MMS (A, C) and MNNG (B, D) and incubated at 30°C for 24 h. Data represents the mean (±95%CI) values. (●) ΔalkA; (■) ΔimuCalkA; (▲) ΔdinBalkA; (▼) ΔimuCdinBalkA (A, B); (●) ΔalkAtag; (■) ΔimuCalkAtag; (▲) ΔdinBalkAtag; (▼) ΔimuCdinBalkAtag (C, D). Asterisks indicate statistically significant difference (P < 0.05; two-way ANOVA followed by Tukey’s multiple comparison post hoc test) in the sensitivity of the ΔalkA or the ΔalkAtag mutant in comparison with the other deletion mutants. In addition, significant difference in sensitivity between the ΔdinBalkA or ΔimuCalkA and the ΔimuCdinBalkA (A); ΔdinBalkAtag or ΔimuCalkAtag and the ΔimuCdinBalkAtag strains (D) is indicated.

Fig 5. Sensitivity of P. aeruginosa ΔalkA strain and its TLS polymerase-deficient derivatives to MMS and MNNG.

Sensitivity was estimated by spotting 10-fold dilutions of overnight cultures onto LB plates containing different concentrations of MMS (A) and MNNG (B), and incubated at 37°C for 24 h. Data represents the mean (±95%CI) values. (●) ΔalkA; (■) ΔimuCalkA; (▲) ΔdinBalkA; (▼) ΔimuCdinBalkA. Asterisks indicate statistically significant difference (P < 0.05; two-way ANOVA followed by Tukey’s multiple comparison post hoc test) in the sensitivity of the ΔalkA mutant in comparison with the other deletion mutants. In addition, significant difference in sensitivity between the strains ΔimuCalkA or ΔdinBalkA and the ΔimuCdinBalkA (A) is indicated.

Fig 6. Study of the requirement of imuA and imuB for the alkyl damage tolerance.

Sensitivity of P. aeruginosa (A) and P. putida (B) ΔalkA strains and their imuA-, imuB-, imuC- and imuABC-deficient derivatives to MMS was compared. Sensitivity was estimated by spotting 10-fold dilutions of overnight cultures of P. aeruginosa or P. putida onto LB plates supplemented with 0.5 mM MMS and incubated at 37°C or 30°C for 24 h, respectively.

Fig 7. Effect of TLS polymerase deficiencies on the frequencies of MMS-induced Rif^R mutations in P. putida wild type and alkA- and alkAtag-deficient backgrounds.

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 P < 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’).

Fig 8. Effect of the ImuC and DinB deficiencies on the frequencies of MMS-induced Rif^R mutations in P. aeruginosa alkA-deficient bacteria.

Bacteria were exposed to 0.15 mM MMS overnight. Data represents the mean (±SE). Groups that have no common letter are significantly different at P < 0.05, according to Kruskal-Wallis test followed by Dunn's multiple comparisons test.

Fig 9. Effect of incubation temperature on the sensitivity of P. aeruginosa and P. putida TLS polymerase-deficient strains to MMS.

10-fold dilutions of overnight cultures of P. aeruginosa (A, B) and P. putida (C) were spotted in parallel onto LB plates containing different concentrations of MMS and incubated at 37°C and 30°C. Sensitivity of P. aeruginosa TLS polymerase-deficient strains to 2.5 mM MMS (A); sensitivity of P. aeruginosa TLS polymerase-deficient ΔalkA mutants to 0.6 mM MMS (B); sensitivity of P. putida TLS polymerase-deficient ΔalkA mutants to 0.7 mM MMS (C) is demonstrated. P. aeruginosa strains (A, B) were incubated at 37°C for 24 h and at 30°C for 48 h; P. putida (C) was incubated at 37°C and 30°C for 24 h. Data represents the mean (±95%CI) values. Letters indicate homogeneous groups according to ANOVA followed by Bonferroni’s multiple comparisons test (P < 0.05).

Fig 10. Effect of incubation temperature on the survival of MMS-treated alkA-deficient bacteria.

The survival of P. aeruginosa ΔalkA (black) and ΔimuCalkA (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 (P < 0.05).

Fig 11. Effect of incubation temperature on transcription from the P. aeruginosa dinB promoter.

β-galactosidase activity expressed from the dinB promoter-lacZ reporter was measured in the P. aerugionsa wild-type (A) and in its alkA-deficient derivative (B) incubated at 37°C (black) and 30°C (grey) overnight in liquid LB medium supplemented with 0.5 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 (P < 0.05).

Fig 12. Effect of incubation temperature on the frequency of MMS-induced Rif^R mutations in P. putida strains.

P. putida wild-type (wt), ΔalkAtag and ΔimuCalkAtag 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 P < 0.0001, according to ANOVA followed by Bonferroni’s multiple comparisons test.

Fig 13. Effect of incubation temperature on transcription from the P. putida lexA2 promoter.

β-galactosidase activities expressed from the lexA2 promoter-lacZ reporter were measured in the P. putida wild-type and in its alkA-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 (P < 0.01).