Evaluation of NAD(+) -dependent DNA ligase of mycobacteria as a potential target for antibiotics

Abstract

Mycobacteria contain genes for several DNA ligases, including ligA, which encodes a NAD(+)-dependent enzyme that has been postulated to be a target for novel antibacterial compounds. Using a homologous recombination system, direct evidence is presented that wild-type ligA cannot be deleted from the chromosome of Mycobacterium smegmatis. Deletions of native ligA in M. smegmatis could be obtained only after the integration of an extra copy of M. smegmatis or Mycobacterium tuberculosis ligA into the attB site of the chromosome, with expression controlled by chemically inducible promoters. The four ATP-dependent DNA ligases encoded by the M. smegmatis chromosome were unable to replace the function of LigA. Interestingly, the LigA protein from M. smegmatis could be substituted with the NAD(+)-dependent DNA ligase of Escherichia coli or the ATP-dependent ligase of bacteriophage T4. The conditional mutant strains allowed the analysis of the effect of LigA depletion on the growth of M. smegmatis. The protein level of the conditional mutants was estimated by Western blot analysis using antibodies raised against LigA of M. tuberculosis. This revealed that a strong overproduction or depletion of LigA did not affect the growth or survival of mycobacteria under standard laboratory conditions. In conclusion, although NAD(+)-dependent DNA ligase is essential for mycobacterial viability, only low levels of protein are required for growth. These findings suggest that very efficient inhibition of enzyme activity would be required if NAD(+)-dependent DNA ligase is to be useful as an antibiotic target in mycobacteria. The strains developed here will provide useful tools for the evaluation of the efficacy of any appropriate compounds in mycobacteria.

FIG. 1.

DNA ligases of mycobacteria, E. coli, and bacteriophage T4. A schematic diagram of conserved domains within the various DNA ligases used in this study is shown. The number of amino acids in each specific protein is indicated. Approximate relative positions of domains conserved within more than one of the proteins are shown by the shaded regions. Domains were identified from their reference within the Pfam (8) or SMART (15) database, as indicated. (A) NAD⁺-dependent ligases from M. smegmatis (MsLigA), M. tuberculosis (MtLigA), and E. coli (EcLigA). (B) ATP-dependent DNA ligases from M. smegmatis (MsLigB, MsLigC1, MsLigC2, and MsLigD) and bacteriophage T4 (T4Dnl).

FIG. 2.

NAD⁺-dependent ligA is essential for viability of M. smegmatis. (A) A deleted version of ligA (ΔligA) of M. smegmatis was constructed by PCR and introduced into the suicide recombination vector p2NIL. SCO mutants carrying both ligA and ΔligA were obtained by the integration of plasmid DNA (p2NILΔligA) into regions of the chromosome that flank the gene of interest. SCO mutant strains were processed directly for DCO mutant strains. The genotype of selected strains (>50) was confirmed by PCR, indicating that all DCO strains carried wt ligA exclusively. (B) SCO strains from A were enriched with intact ligA from M. smegmatis or M. tuberculosis controlled by an inducible promoter (P_ami ligA_Ms/ligA_Mt). The genotype of selected strains was confirmed by PCR and Southern hybridization analysis, indicating that DCO strains were both wt (ligA) and mutant type (ΔligA). Numbers above the lanes of each gel represent the following samples: 1, 1-kb DNA ladder; 2, M. smegmatis wt control; 3, p2NILΔligA plasmid; 4, SCO mutant strains. DCO mutant strains are indicated in the figure, with those containing ΔligA highlighted by an asterisk (*). Note that the middle PstI recognition site (A) was present within the deleted region of ligA (ΔligA), thus causing the ΔligA band detected by Southern hybridization to be larger than the wt ligA band. The Southern hybridization probe was amplified using the 3′ undeleted end of ligA.

FIG. 3.

NAD⁺-dependent ligA of M. smegmatis can be substituted with NAD⁺-dependent ligA of E. coli or ATP-dependent ligase of enterobacteriophage T4. SCO strains carrying both ligA and ΔligA (Fig. 2) were enriched with ligA_Ec (A) or ligT4 (B) controlled by a P_ami promoter. DCO mutant strains resulting from the processing of these SCO strains were both wt (ligA) and mutant type (ΔligA). The genotype of selected DCO strains was confirmed by PCR (A and B) and Southern hybridization (C) analyses. Numbers above the lanes of each gel represent the following samples: 1, 1-kb DNA ladder; 2, M. smegmatis wt control; 3, p2NILΔligA plasmid; 4, SCO mutant strains; 5, DCO mutant strains carrying wt ligA; 6, DCO mutant strains carrying ΔligA. In the Southern hybridization shown in C, 6A and 6B represent the complementation by ligA_Ec and ligT4, respectively. Note that the middle PstI recognition site (A) was present within the deleted region of ligA (ΔligA), thus causing the ΔligA band detected by Southern hybridization to be larger than the wt ligA band. The Southern hybridization probe was amplified using the 3′ undeleted end of ligA.

FIG. 4.

M. smegmatis carrying only ATP-dependent ligases is defective in growth. The M. smegmatis wt control strain (wt) and conditional mutant strains carrying ligase genes driven by P_ami promoter were grown in Middlebrook 7H9/oleic acid-albumin-dextrose catalase medium with and without the induction of P_ami. All conditional mutant strains carried a single intact copy of the named ligA substituting for a deletion in M. smegmatis ligA as follows: (A) ΔligA-P_ami ligA_Ms, ΔligA mutant complemented with M. smegmatis ligA; (B) ΔligA-P_ami ligA_Ec, ΔligA mutant complemented with E. coli ligA; (C) ΔligA-P_ami ligT4, ΔligA mutant complemented with the gene for T4 DNA ligase. The growth of each culture was monitored by optical density analysis. Growth experiments were repeated three times, with the representative result being presented in the figure. OD₆₀₀, optical density at 600 nm.

FIG. 5.

Growth of M. smegmatis is insensitive to the amount of expressed LigA. As shown in the panel on the right, optical density (OD) analysis determined the extent of growth of M. smegmatis strains carrying a natural level of LigA (lane 1, wt strain), a low level of LigA (lane 2, ΔligA-P_ami ligA_Ms, without inducer), and an overproduced level of LigA (lane 3, ΔligA-P_tet ligA_Ms, with inducer). In the left panel, the level of LigA was determined by Western blot analysis with antibodies raised against LigA of M. tuberculosis. For each lane, 12 μg of total proteins was loaded.