Molecular Dissection of Mycobacterium tuberculosis Integration Host Factor Reveals Novel Insights into the Mode of DNA Binding and Nucleoid Compaction

Abstract

The annotated whole-genome sequence of Mycobacterium tuberculosis indicated that Rv1388 (Mtihf) likely encodes a putative 20 kDa integration host factor (mIHF). However, very little is known about the functional properties of mIHF or organization of mycobacterial nucleoid. Molecular modeling of the mIHF three-dimensional structure, based on the cocrystal structure of Streptomyces coelicolor IHF-duplex DNA, a bona fide relative of mIHF, revealed the presence of Arg170, Arg171, and Arg173, which might be involved in DNA binding, and a conserved proline (P150) in the tight turn. The phenotypic sensitivity of Escherichia coli ΔihfA and ΔihfB strains to UV and methylmethanesulfonate could be complemented with the wild-type Mtihf, but not its alleles bearing mutations in the DNA-binding residues. Protein-DNA interaction assays revealed that wild-type mIHF, but not its DNA-binding variants, bind with high affinity to fragments containing attB and attP sites and curved DNA. Strikingly, the functionally important amino acid residues of mIHF and the mechanism(s) underlying its binding to DNA, DNA bending, and site-specific recombination are fundamentally different from that of E. coli IHFαβ. Furthermore, we reveal novel insights into IHF-mediated DNA compaction depending on the placement of its preferred binding sites; mIHF promotes compaction of DNA into nucleoid-like or higher-order filamentous structures. We hence propose that mIHF is a distinct member of a subfamily of proteins that serve as essential cofactors in site-specific recombination and nucleoid organization and that these findings represent a significant advance in our understanding of the role(s) of nucleoid-associated proteins.

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

“…Consistent with previous studies (23), it was found that the E. coli ⌬ihfA or ΔihfB cells were highly sensitive to MMS compared to the wild-type cells (Fig. 1B).…”

“…To understand the functional importance of the extra 86 amino acid residues at the N terminus of mIHF, we prepared two derivatives of mIHF, a truncated derivative containing the N-terminal 86 amino acid residues (pMtihfntd) and a C-terminal derivative lacking the 86 amino acids from the N terminus (pMtihfctd). These vectors encoding either the N-terminal (mIHF-NTD) or C-terminal (mIHF-CTD) regions of mIHF were tested for their ability to suppress the sensitivity of E. coli ⌬ihfA or ΔihfB cells using a previously established complementation assay (23). Wild-type (WT) E. coli ihfA or ihfB strains treated with methyl methanesulfonate (MMS) and UV served as positive controls.…”

“…Homologous NAPs are widely distributed across bacterial species, including Mycobacteria, Streptomyces, Rhodococcus, and Corynebacteria (10)(11)(12)(13)(14)(15)(16)(17)(18)(19)(20)(21)(22)(23)(24); however, they are far less well characterized. The focus of the present study is mainly on the characterization of the IHF of M. tuberculosis; consequently, the existing literature relevant to IHF is discussed here.…”

“…The whole-genome sequence and annotation of Mycobacterium tuberculosis H37Rv revealed the presence of a putative ihf gene (Rv1388) capable of encoding a single polypeptide chain of 20 kDa, designated mIHF (for M. tuberculosis integration host factor) (45,46). However, the purified mIHF showed an apparent mass of 25 kDa because of its anomalous mobility on SDS-polyacrylamide gels (23). Moreover, our work revealed that the mechanisms underlying binding of mIHF to DNA and integrative recombination are different from those of the E. coli IHF␣␤ heterodimer (23).…”

“…However, the purified mIHF showed an apparent mass of 25 kDa because of its anomalous mobility on SDS-polyacrylamide gels (23). Moreover, our work revealed that the mechanisms underlying binding of mIHF to DNA and integrative recombination are different from those of the E. coli IHF␣␤ heterodimer (23). Bioinformatics analysis suggested that the IHF of M. tuberculosis contains a region of 86 additional amino acid residues at the N terminus, which is absent from both ␣-and ␤-subunits of E. coli IHF.…”