Defining the Functionally Important Domain and Amino Acid Residues in Mycobacterium tuberculosis Integration Host Factor for Genome Stability, DNA Binding, and Integrative Recombination

Abstract

The integration host factor of Mycobacterium tuberculosis (mIHF) consists of a single polypeptide chain, the product of the ihf gene. We previously revealed that mIHF is a novel member of a new class of nucleoid-associated proteins that have important roles in DNA damage response, nucleoid compaction, and integrative recombination. The mIHF contains a region of 86 amino acids at its N terminus, absent from both ␣-and ␤-subunits of Escherichia coli IHF. However, the functional significance of an extra 86-amino-acid region in the full-length protein remains unknown. Here, we report the structure/function relationship of the DNA-binding and integrative recombinationstimulating activity of mIHF. Deletion mutagenesis showed that an extra 86-amino-acid region at the N terminus is dispensable; the C-terminal region possesses the sequences essential for its known biological functions, including the ability to suppress the sensitivity of E. coli ΔihfA and ΔihfB cells to DNA-damaging agents, DNA binding, DNA multimerization-circularization, and stimulation of phage L5 integrase-catalyzed integrative recombination. Single and double alanine substitutions at positions Arg170 and Arg171, located at the mIHF DNA-binding site, abrogated its capacity to suppress the sensitivity of E. coli ⌬ihfA and ΔihfB cells to DNA-damaging agents. The variants encoded by these mutant alleles failed to bind DNA and stimulate integrative recombination. Interestingly, the DNA-binding activity of the mIHF-R173A variant remained largely unaffected; however, it was unable to stimulate integrative recombination, thus revealing a separationof-function allele of mIHF. The functional and structural characterization of this separationof-function allele of mIHF could reveal previously unknown functions of IHF. IMPORTANCEThe integration host factor of Mycobacterium tuberculosis is a novel nucleoid-associated protein. mIHF plays a vital role in DNA damage response, nucleoid compaction, and integrative recombination. Intriguingly, mIHF contains an extra 86-amino-acid region at its N terminus, absent from both ␣-and ␤-subunits of Escherichia coli IHF, whose functional significance is unknown. Furthermore, a triad of arginine residues located at the mIHF-DNA interface have been implicated in a range of its functions. Here, we reveal the roles of N-and C-terminal regions of mIHF and the individual residues in the Arg triad for their ability to provide protection in vivo against DNA damage, bind DNA, and stimulate integrase-catalyzed site-specific recombination.KEYWORDS nucleoid-associated proteins, integration host factor, DNA damage response, integrase, integrative recombination, separation of function T he nucleoid-associated proteins (NAPs) are a set of small, abundant, and generally basic proteins that not only compact the bacterial chromosome and modulate DNA supercoiling but also regulate its structure and influence various cellular processes,

Mycobacterial IHF is a highly dynamic nucleoid-associated protein that assists HupB in organizing chromatin

“…S1C,E). Some reports suggested that (Sharadamma et al, 2015(Sharadamma et al, , 2017, in contrast to M. smegmatis IHF (msIHF), M. tuberculosis (mtIHF) includes an additional N-terminal domain (85 amino acids); recently, however, this hypothesis was rejected based on the finding that the longer mihf gene sequence arose from an incorrect annotation of the transcription start (Mishra et al, 2013;Odermatt et al, 2018). Moreover, we confirmed expression of the shorter version of mtIHF by Western blotting comparing lysates from M. smegmatis strains producing either mtIHF-EGFP or msIHF-EGFP fusion proteins (expressed under native promoters; Supplementary Figure S1D).…”