Evaluation of DNA primase DnaG as a potential target for antibiotics

Abstract

Mycobacteria contain genes for several DNA-dependent RNA primases, including dnaG, which encodes an essential replication enzyme that has been proposed as a target for antituberculosis compounds. An in silico analysis revealed that mycobacteria also possess archaeo-eukaryotic superfamily primases (AEPs) of unknown function. Using a homologous recombination system, we obtained direct evidence that wild-type dnaG cannot be deleted from the chromosome of Mycobacterium smegmatis without disrupting viability, even in backgrounds in which mycobacterial AEPs are overexpressed. In contrast, single-deletion AEP mutants or mutants defective for all four identified M. smegmatis AEP genes did not exhibit growth defects under standard laboratory conditions. Deletion of native dnaG in M. smegmatis was tolerated only after the integration of an extra intact copy of the M. smegmatis or Mycobacterium tuberculosis dnaG gene, under the control of chemically inducible promoters, into the attB site of the chromosome. M. tuberculosis and M. smegmatis DnaG proteins were overproduced and purified, and their primase activities were confirmed using radioactive RNA synthesis assays. The enzymes appeared to be sensitive to known inhibitors (suramin and doxorubicin) of DnaG. Notably, M. smegmatis bacilli appeared to be sensitive to doxorubicin and resistant to suramin. The growth and survival of conditional mutant mycobacterial strains in which DnaG was significantly depleted were only slightly affected under standard laboratory conditions. Thus, although DnaG is essential for mycobacterial viability, only low levels of protein are required for growth. This suggests that very efficient inhibition of enzyme activity would be required for mycobacterial DnaG to be useful as an antibiotic target.

FIG 1

The purification and activity of M. smegmatis (Ms) and M. tuberculosis (Mtb) DnaG proteins. (A) SDS-PAGE analysis. Purified proteins were resolved on a 12% polyacrylamide gel followed by Instant Blue staining. M_w, molecular weight; nt, nucleotides. (B) Western blot analysis with antibodies raised against DnaG of M. smegmatis. (C) Protein activity assays with Mg²⁺, as described in Materials and Methods.

FIG 2

(A) Synthesis of radiolabeled RNA primers by M. smegmatis DnaG and its inhibition by doxorubicin and suramin. Phosphorimager analysis of an 18% urea–polyacrylamide gel showing RNA products of the priming reaction. The priming reaction was performed with Mn²⁺ on a 24-mer ssDNA template (5′-tactctcatcgtggaatcctgaca) in the presence of a mixture of the four nucleotides containing [α-³²P]UTP. (B) Time-dependent colony formation by wild-type M. smegmatis (M. smegmatis mc²155) growing in the presence of various concentrations of doxorubicin (0, 2, 2.5, and 3 μM). Colony formation values are means ± standard errors from three independent experiments. (C) The dose-dependent colony formation by wild-type M. smegmatis (M. smegmatis mc²155) and its mutant with downregulated DnaG expression (ΔdnaGP_tetdnaG) growing in the presence of doxorubicin. The number of CFU of 24-h-old culture is shown. Colony formation values are means ± standard errors from three independent experiments.

FIG 3

Complementation of the M. smegmatis ΔdnaG SCO strain. (A) Schematic showing the restriction-digested DNA fragment (1,862 bp) and the size of the internal deletion in the mutated gene (1,180 bp). The dnaG gene is represented by gray arrows and the internal deletion by white rectangles. The aacC1 gene (gentamicin resistance cassette) was cloned within the dnaG gene to facilitate screening of DCO mutants. The dnaG is essential for the viability of M. smegmatis. SCO strains were enriched with intact dnaG from M. smegmatis or M. tuberculosis under the control of an inducible promoter (P_amidnaG_Ms/dnaG_Mt or P_tetdnaG_Ms). (B) The genotype of selected strains was confirmed by PCR and Southern hybridization analysis.

FIG 4

Phenotypic analysis of M. smegmatis and the conditionally complemented mutant ΔdnaG. (A) Growth rate analysis of wild-type M. smegmatis and a strain complemented with an intact copy of dnaG_Ms under the control of a tetracycline promoter. Growth rate analyses were performed on rich medium (7H9/oleic acid-albumin-dextrose-catalase [OADC]). OD values are means ± standard errors from three independent experiments. (B) Densitometric analysis of DnaG protein levels in M. smegmatis and ΔdnaGP_tetdnaG_Ms. (C) Western blot analysis with antibodies raised against DnaG of M. smegmatis proteins isolated from cells growing in rich medium in indicated time intervals. Lane 1, M. smegmatis, 0 h; lane 2, ΔdnaGP_tetdnaG_Ms, 0 h; lane 3, M. smegmatis, 6 h; lane 4, ΔdnaGP_tetdnaG_Ms, 6 h; lane 5, M. smegmatis, 12 h; lane 6, ΔdnaGP_tetdnaG_Ms, 12 h; lane 7, M. smegmatis, 24 h; lane 8, ΔdnaGP_tetdnaG_Ms, 24 h.