Role of the DinB homologs Rv1537 and Rv3056 in Mycobacterium tuberculosis

Abstract

The environment encountered by Mycobacterium tuberculosis during infection is genotoxic. Most bacteria tolerate DNA damage by engaging specialized DNA polymerases that catalyze translesion synthesis (TLS) across sites of damage. M. tuberculosis possesses two putative members of the DinB class of Y-family DNA polymerases, DinB1 (Rv1537) and DinB2 (Rv3056); however, their role in damage tolerance, mutagenesis, and survival is unknown. Here, both dinB1 and dinB2 are shown to be expressed in vitro in a growth phase-dependent manner, with dinB2 levels 12- to 40-fold higher than those of dinB1. Yeast two-hybrid analyses revealed that DinB1, but not DinB2, interacts with the beta-clamp, consistent with its canonical C-terminal beta-binding motif. However, knockout of dinB1, dinB2, or both had no effect on the susceptibility of M. tuberculosis to compounds that form N(2)-dG adducts and alkylating agents. Similarly, deletion of these genes individually or in combination did not affect the rate of spontaneous mutation to rifampin resistance or the spectrum of resistance-conferring rpoB mutations and had no impact on growth or survival in human or mouse macrophages or in mice. Moreover, neither gene conferred a mutator phenotype when expressed ectopically in Mycobacterium smegmatis. The lack of the effect of altering the complements or expression levels of dinB1 and/or dinB2 under conditions predicted to be phenotypically revealing suggests that the DinB homologs from M. tuberculosis do not behave like their counterparts from other organisms.

FIG. 1.

Y2H analysis of polymerase interactions with the β-clamp. (A) Growth on selective media. Potential β-interacting partners, cloned as GAL4 BD fusions, were tested for interaction with AD-DnaN by cotransformation and scoring for growth on selection (LT), medium stringency (LTH), and high stringency (LTHA) media. Individual colonies of each strain were resuspended in sterile water, and the cell density was adjusted to an OD₆₀₀ of 1. Tenfold serial dilutions were then plated on media of differing stringencies. LT, Leu-Trp; LTH, Leu-Trp-His; LTHA, Leu-Trp-His-Ade dropout-supplemented media. Screening for autoactivation of the HISD reporter was carried out by plating cotransformants on dropout-supplemented media containing 3-amino-1,2,4-triazole (3-AT) at 0.5 to 25 mM. A 3-AT concentration of 1 mM was found to inhibit all background growth, and all further interactions were assessed at this concentration. (B) LacZ activity. Positive interactions were confirmed by cotransformation of the corresponding vectors into strain Y187, and LacZ assays were carried out according to the manufacturer's instructions. *, P < 0.01; **, P < 0.005. + and − denote growth or lack of growth of AH109 recombinants on LTH dropout-supplemented media.

FIG. 2.

Effect of DinB1 and/or DinB2 deficiency on susceptibility of M. tuberculosis to genotoxic agents and novobiocin. Ten or five microliters of neat culture (∼10⁶ CFU) and 10-fold (A) or 3-fold (B) serial dilutions thereof were spotted on plates containing compounds at the indicated concentrations. Plates were incubated for 2 or 3 weeks before scoring growth.

FIG. 3.

Growth and survival of DinB1 and DinB2 mutants of M. tuberculosis H37Rv in infected mice. Growth of the dinB1::hyg (A) and ΔdinB2 (B) mutants with the parental wild type was monitored over a period of 350 and 315 days postinfection, respectively. The inset in panel B shows growth and survival of the ΔdinB2 mutant, its complemented derivative, and the parental wild type over a period of 77 days postinfection. Three mice were sacrificed at each time point for each strain. ⋄, wild type; □, dinB1::hyg mutant; ▵, ΔdinB2 mutant; ○, ΔdinB2 attB::dinB2 mutant.