Abstract
Two genes, pafB and pafC, are organized in an operon with the Pup-ligase gene pafA, which is part of the Pup-proteasome system (PPS) present in mycobacteria and other actinobacteria. The PPS is crucial for Mycobacterium tuberculosis resistance towards reactive nitrogen intermediates (RNI). However, pafB and pafC apparently play only a minor role in RNI resistance. To characterize their function, we generated a pafBC deletion in Mycobacterium smegmatis (Msm). Proteome analysis of the mutant strain revealed decreased cellular levels of various proteins involved in DNA damage repair, including recombinase A (RecA). In agreement with this finding, Msm ΔpafBC displayed increased sensitivity to DNA damaging agents. In mycobacteria two pathways regulate DNA repair genes: the LexA/RecA-dependent SOS response and a predominant pathway that controls gene expression via a LexA/RecA-independent promoter, termed P1. PafB and PafC feature winged helix-turn-helix DNA binding motifs and we demonstrate that together they form a stable heterodimer in vitro, implying a function as a heterodimeric transcriptional regulator. Indeed, P1-driven transcription of recA was decreased in Msm ΔpafBC under standard conditions and induction of recA expression upon DNA damage was strongly impaired. Taken together, our data indicate an important regulatory function of PafBC in the mycobacterial DNA damage response.
Highlights
A hallmark of activated macrophages is the production of reactive oxygen species and nitric oxide, which damage bacterial proteins, lipids and nucleic acids[3,4]
Like in its pathogenic relative Mtb[26], these three genes are co-transcribed in M. smegmatis as evidenced by the presence of a single mRNA encoding both PafC and PafB in the Mycobacterium smegmatis (Msm) wild type strain (Fig. 1A)
The winged helixturn-helix (wHTH) motif is typically comprised of three helices forming a compact helical bundle, which is capped by an adjacent hairpin (“wing”) (Supplementray Fig. S1)[30]
Summary
A hallmark of activated macrophages is the production of reactive oxygen species and nitric oxide, which damage bacterial proteins, lipids and nucleic acids[3,4]. In an Mtb recA deletion strain, induction of many DNA repair genes upon exposure to mitomycin C is either the same as in wild type or is only partly decreased. Deletion of clgR in Msm results in impaired induction of various genes related to DNA repair mechanisms under DNA damaging conditions. This indicates a regulatory function of ClgR in the LexA/RecA-independent DNA damage repair pathway in mycobacteria. Our findings establish pafBC as a new regulatory element in the LexA/RecA-independent DNA damage response and provide new insights into RecA regulation as well as the control of DNA repair mechanisms in mycobacteria
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