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 recombination-stimulating 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 ?ihfA and ?ihfB 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 ?ihfB 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 separation-of-function allele of mIHF. The functional and structural characterization of this separation-of-function allele of mIHF could reveal previously unknown functions of IHF.IMPORTANCE The 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.
