Essential roles for imuA'- and imuB-encoded accessory factors in DnaE2-dependent mutagenesis in Mycobacterium tuberculosis

Abstract

In Mycobacterium tuberculosis (Mtb), damage-induced mutagenesis is dependent on the C-family DNA polymerase, DnaE2. Included with dnaE2 in the Mtb SOS regulon is a putative operon comprising Rv3395c, which encodes a protein of unknown function restricted primarily to actinomycetes, and Rv3394c, which is predicted to encode a Y-family DNA polymerase. These genes were previously identified as components of an imuA-imuB-dnaE2-type mutagenic cassette widespread among bacterial genomes. Here, we confirm that Rv3395c (designated imuA') and Rv3394c (imuB) are individually essential for induced mutagenesis and damage tolerance. Yeast two-hybrid analyses indicate that ImuB interacts with both ImuA' and DnaE2, as well as with the beta-clamp. Moreover, disruption of the ImuB-beta clamp interaction significantly reduces induced mutagenesis and damage tolerance, phenocopying imuA', imuB, and dnaE2 gene deletion mutants. Despite retaining structural features characteristic of Y-family members, ImuB homologs lack conserved active-site amino acids required for polymerase activity. In contrast, replacement of DnaE2 catalytic residues reproduces the dnaE2 gene deletion phenotype, strongly implying a direct role for the alpha-subunit in mutagenic lesion bypass. These data implicate differential protein interactions in specialist polymerase function and identify the split imuA'-imuB/dnaE2 cassette as a compelling target for compounds designed to limit mutagenesis in a pathogen increasingly associated with drug resistance.

Fig. 1.

ImuA′ and ImuB are essential for induced mutagenesis. UV-induced mutation frequencies to rifampicin resistance (Rif^R) in (A) Mtb at 24 and 48 h and (B) Msm at 4.5 h post UV treatment. Data represent single experiments performed in triplicate.

Fig. 2.

ImuB is a homolog of Y-family polymerases but lacks active-site acidic residues. (A) Comparison of the Mtb ImuB homology model with the X-ray structure of Sulfolobus solfataricus Dpo4 complexed with DNA and incoming nucleotide (PDB id: 1jx4) (21). ImuB is colored from N terminus (blue) to C terminus (red). (Center) The superimposed structures. (B) Close-up of the active site of Dpo4 and corresponding residues in ImuB. Only the palm domain is shown in both structures. The incoming nucleotide in the Dpo4–DNA complex is colored pink, and the magnesium ion is shown as a cyan sphere.

Fig. 3.

DnaE2 catalytic activity is required for induced mutagenesis and damage tolerance. (A) UV-induced mutation frequencies to rifampicin resistance (Rif^R). (B) Sensitivity to MMC treatment. Log-fold dilutions were plated on antibiotic-free medium or on medium containing MMC at 0.02 and 0.04 μg·ml⁻¹. Data are from a representative experiment performed in triplicate.

Fig. 4.

Interactions of cassette components. Summary of Mtb protein interactions detected (●) or absent (×) on highest stringency growth medium as identified in this study and previously (25). ND, not determined. An open circle (o) indicates interactions maintained where bait or prey vector contains full-length ImuB; ImuB self-association is eliminated when both vectors carry the ImuB^C168 allele. A blue bar indicates the position of the β-clamp–binding motif (³⁵⁴QLPLWG³⁵⁹) deleted in ImuB^CB and mutated (XXX) in ImuB^ALPLWG. The ⁴³⁹AIA⁴⁴¹ mutation in Mtb DnaE2 is indicated (XXX). Lines are to scale.

Fig. 5.

ImuA′ is required for damage tolerance. (A) Comparison of the Mtb ImuA′ homology model and the X-ray structure of E. coli RecA (PDB id: 1u94) (57). ImuA′ is colored according to truncation variants: blue and cyan indicate N-terminal truncations, and red is the C-terminal truncation. (B) Deletion of 44 C-terminal amino acids (imuA′^C) renders Msm sensitive to MMC treatment. Log-fold dilutions were plated on antibiotic-free medium (Left and Right, Rows 1 and 4), 0.02 μg·ml⁻¹ (Left and Right, Rows 2 and 5), and 0.04 μg·mL⁻¹ (Left and Right, Rows 3 and 6) MMC. Data are from a representative experiment performed in duplicate.

Fig. 6.

The ImuB–β-clamp interaction is required for induced mutagenesis and damage tolerance. (A) UV-induced mutation frequencies to rifampicin resistance (Rif^R). (B) Sensitivity to MMC treatment. Log-fold dilutions of Msm ΔimuA′ ΔimuB complemented with full-length imuA′-imuB containing wild-type (WT) and mutant imuB alleles were plated on 0.02 μg·mL⁻¹ (Rows 1, 3, 5, 7, and 9) and 0.04 μg·ml⁻¹ (Rows 2, 4, 6, 8, and 10) MMC; at right is the untreated control. Data are from a representative experiment performed in duplicate.