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

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 α- and ß-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 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 ΔihfA 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 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 α- and ß-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.

Ubiquitous Micro-Modular Homologies among Genomes from Viruses to Bacteria to Human Mitochondrial DNA: Platforms for Recombination during Evolution?

The emerging Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2) and its variants have raised tantalizing questions about evolutionary mechanisms that continue to shape biology today. We have compared the nucleotide sequence of SARS-CoV-2 RNA to that of genomes of many different viruses, of endosymbiotic proteobacterial and bacterial DNAs, and of human mitochondrial DNA. The entire 4,641,652 nt DNA sequence of Escherichia coli K12 has been computer-matched to SARS-CoV-2 RNA. Numerous, very similar micro-modular clusters of 3 to 13 nucleotides lengths were detected with sequence identities of 40 to >50% in specific genome segments between SARS-CoV-2 and the investigated genomes. These clusters were part of patch-type homologies. Control sequence comparisons between 1000 randomly computer-composed sequences of 29.9 kb and with the A, C, G, T base composition of SARS-CoV-2 genome versus the reference Wuhan SARS-CoV-2 sequence showed similar patterns of sequence homologies. The universal A, C, G, T genetic coding mode might have succeeded in evolution due in part to its built-in capacity to select for a substantial reservoir of micro-modular domains and employ them as platforms for integrative recombination. Their role in SARS-CoV-2 interspecies transition and the generation of variants appears likely, but their actual involvement will require detailed investigations.