Role of Pseudomonas aeruginosa dinB-encoded DNA polymerase IV in mutagenesis

Abstract

Pseudomonas aeruginosa is a human opportunistic pathogen that chronically infects the lungs of cystic fibrosis patients and is the leading cause of morbidity and mortality of people afflicted with this disease. A striking correlation between mutagenesis and the persistence of P. aeruginosa has been reported. In other well-studied organisms, error-prone replication by Y family DNA polymerases contributes significantly to mutagenesis. Based on an analysis of the PAO1 genome sequence, P. aeruginosa contains a single Y family DNA polymerase encoded by the dinB gene. As part of an effort to understand the mechanisms of mutagenesis in P. aeruginosa, we have cloned the dinB gene of P. aeruginosa and utilized a combination of genetic and biochemical approaches to characterize the activity and regulation of the P. aeruginosa DinB protein (DinB(Pa)). Our results indicate that DinB(Pa) is a distributive DNA polymerase that lacks intrinsic proofreading activity in vitro. Modest overexpression of DinB(Pa) from a plasmid conferred a mutator phenotype in both Escherichia coli and P. aeruginosa. An examination of this mutator phenotype indicated that DinB(Pa) has a propensity to promote C-->A transversions and -1 frameshift mutations within poly(dGMP) and poly(dAMP) runs. The characterization of lexA+ and DeltalexA::aacC1 P. aeruginosa strains, together with in vitro DNA binding assays utilizing cell extracts or purified P. aeruginosa LexA protein (LexA(Pa)), indicated that the transcription of the dinB gene is regulated as part of an SOS-like response. The deletion of the dinB(Pa) gene sensitized P. aeruginosa to nitrofurazone and 4-nitroquinoline-1-oxide, consistent with a role for DinB(Pa) in translesion DNA synthesis over N2-dG adducts. Finally, P. aeruginosa exhibited a UV-inducible mutator phenotype that was independent of dinB(Pa) function and instead required polA and polC, which encode DNA polymerase I and the second DNA polymerase III enzyme, respectively. Possible roles of the P. aeruginosa dinB, polA, and polC gene products in mutagenesis are discussed.

FIG. 1.

In vitro DinB_Pa DNA polymerase activity. (A) In vitro primer extension assays were performed as described in Materials and Methods using the 20-mer/100-mer DNA template, a schematic of which is shown at the top of panel A. Specific conditions for each assay are indicated at the bottom of the figure. (B) Assays were as described for panel A but used either the 20-mer primer alone (lanes 1 and 2) or the 20-mer primer annealed to M13mp18 ssDNA (NEB) (see schematic at the top of panel B). Conditions for each assay are indicated. See the text for further details.

FIG. 2.

Steady-state levels of DinB_Pa⁺, DinB(D8A)_Pa, DinB(R49A)_Pa, and DinB(D103A)_Pa. Whole-cell extracts of E. coli strain FC1237 [relevant genotype, Δ(dinB-yafN)::kan (54)], bearing the indicated plasmids, were prepared and separated by SDS-PAGE, and blotting was performed using anti-DinB_Pa antibodies as described in Materials and Methods. Lanes 1 and 2 contain 1.0 and 0.1 μg, respectively, of the purified hexahistidine-tagged DinB_Pa protein.

FIG. 3.

Expression of dinB is DNA damage regulated. (A) Real-time RT-PCR analysis of dinB mRNA from P. aeruginosa strains PAO1 (lexA⁺) and WFPA340 (ΔlexA::aacC1). PAO1+MMC represents strain PAO1 treated for 2 h with 1 μg/ml MMC before RNA harvest. WFPA340 is the mean of five replicate experiments (range, 3.05 to 11.52), and PAO1+MMC is the mean of nine replicate experiments (range, 2.35 to 10.36). All values are normalized against the levels of rpoD (see Materials and Methods). (B) Western blot showing DinB_Pa levels in P. aeruginosa strains PAO1 (lexA⁺), WFPA340 (ΔlexA::aacC1), and WFPA334 (ΔdinB::aacC1). Cell extracts were prepared and separated by SDS-PAGE, and blotting was performed using anti-DinB_Pa antibodies as described in Materials and Methods. Upper panel, DinB; lower panel, nonspecific cross-reactive band used for a normalizing control. Lane 1, purified His-tagged DinB_Pa; lane 2, PAO1 extract; lane 3, WFPA340 extract; lane 4, PAO1+MMC extract; lane 5, WFPA334 extract.

FIG. 4.

LexA_Pa and LexA_Ec DNA binding studies. (A) A radiolabeled dinB_Pa promoter DNA fragment (pdinB_Pa) was left untreated (lane 1) or incubated with increasing amounts of purified LexA_Ec (2.5 nM, 10 nM, 50 nM, 100 nM; lanes 2 to 5, respectively). (B) Radiolabeled umuDC_Ec promoter DNA (pumuDC_Ec) was incubated with the following extracts or purified LexA_Ec samples: lane 1, no protein; lane 2, 50 nM; lane 3, 100 nM; lane 4, 10 μg extract; lane 5, 10 μg extract. (C) A radiolabeled pdinB_Pa fragment was incubated with the following: lane 1, no protein; lane 2 to 4, 5.0 μg extract; lane 5 to 7, 10 μg extract; lane 8, no protein; lane 9 to 14, increasing amounts of LexA_Pa (100 ng, 500 ng, 750 ng, 1,000 ng, 1,500 ng, and 2,000 ng, respectively).

FIG. 5.

Role of DinB_Pa in tolerating NFZ- and 4-NQO-induced DNA damage. Relative sensitivities to NFZ (A) or 4-NQO (C) of dinB⁺ and ΔdinB E. coli and P. aeruginosa strains. Cultures of E. coli strains RW118 (dinB⁺) and RM137 [Δ(dinB-yafN)::kan], and of P. aeruginosa strains PAO1 (dinB⁺) and WFPA334 (ΔdinB::accC1) were serially diluted in 0.8% NaCl and spotted on LB plates containing the indicated concentration of NFZ or 4-NQO. Plates were incubated overnight at 37°C prior to photographing. NFZ- and 4-NQO-induced mutation frequencies were measured by inoculating 5 ml of LB containing either N,N-dimethylformamide (DMF), 160 μM NFZ, or 320 μM NFZ (B) or either 160 μM or 320 μM 4-NQO (D) (each made freshly in DMF [NFZ] or ethanol [4-NQO]) with 0.1 ml of P. aeruginosa strains PAO1 or WFPA334 grown to an OD₅₉₅ of ∼0.5. Following growth for ∼16 h at 37°C, cells were washed twice with 0.8% NaCl and appropriate dilutions were spread onto LB or LB-Rif agar plates. Mutation frequencies were calculated by dividing the number of Rif^r CFU by the total number of viable cells for each strain. Control experiments indicated that the presence of DMF alone had no effect on the frequency of Rif^r for either PAO1 or WFPA334. Mutation frequencies are expressed relative to those observed for strain PAO1 grown in the presence of DMF alone (2.9 ± 1.9 Rif^r CFU per 10⁸ CFU for the NFZ experiment), or in LB (4.9 ± 2.2 Rif^r CFU per 10⁸ CFU for the 4-NQO experiment), which were each set equal to 1.0. Results shown represent the averages of at least three independent experiments, each performed in duplicate. Error bars represent standard deviations. Differences in the frequencies of Rif^r between strains PAO1 and WFPA334 grown in LB or in the presence of DMF alone (P value ≥ 0.100), relative to that grown in the presence of 4-NQO or NFZ (P values ≥ 0.220, respectively, relative to the DMF-alone or LB-only controls), were not statistically significant at the 95% confidence level based on the Student t test. Ec dinB⁺, dinB_Ec⁺; Pa dinB⁺, dinB_Pa⁺.

FIG. 6.

UV-induced mutagenesis in P. aeruginosa. UV-induced mutagenesis was performed as described previously (36, 44) using the indicated isogenic P. aeruginosa strains expressing all five known P. aeruginosa DNA polymerases (MPAO1), or bearing a transposon insertion in the gene encoding either the DNA polymerase I (polA; MPA50455), DNA polymerase II (polB; MPA17464) or the second DNA polymerase III enzyme (polC; MPA34086). Results shown represent the averages of at least three independent experiments, each performed in duplicate. Error bars represent standard deviations. Based on the Student t test, differences between strains MPAO1 and MPA17464 are not statistically significant at the 95% confidence level (P value of 0.79), while those between strains MPAO1 and MPA50455 (P value of 0.03) as well as MPAO1 and MPA34086 (P value of 0.002) are statistically significant.