Lysine acetylation of the Mycobacterium tuberculosis HU protein modulates its DNA binding and genome organization

Abstract

Nucleoid-associated protein HU, a conserved protein across eubacteria is necessary for maintaining the nucleoid organization and global regulation of gene expression. Mycobacterium tuberculosis HU (MtHU) is distinct from the other orthologues having 114 amino acid long carboxyl terminal extensions with a high degree of sequence similarity to eukaryotic histones. In this study, we demonstrate that the DNA binding property of MtHU is regulated by posttranslational modifications akin to eukaryotic histones. MtHU purified from M. tuberculosis cells is found to be acetylated on multiple lysine residues unlike the E. coli expressed recombinant protein. Using coimmunoprecipitation assay, we identified Eis as one of the acetyl transferases that interacts with MtHU and modifies it. Although Eis is known to acetylate aminoglycosides, the kinetics of acetylation showed that its protein acetylation activity on MtHU is robust. In vitro Eis modified MtHU at various lysine residues, primarily those located at the carboxyl terminal domain. Acetylation of MtHU caused reduced DNA interaction and alteration in DNA compaction ability of the NAP. Over-expression of the Eis leads to hyperacetylation of HU and decompaction of genome. These results provide first insights into the modulation of the nucleoid structure by lysine acetylation in bacteria.

Regulation, Function, and Detection of Protein Acetylation in Bacteria

“…Acetylation is measured by the incorporation or removal of radioactive acetyl derivatives or by mass spectrometry to determine site specificity. One problem is that acetylases are often promiscuous in vitro and can acetylate nonendogenous sites, making potential downstream biochemical assays difficult to interpret (61). Gln substitution mutants purified from E. coli can be used for such in vitro studies (93,101), but the same caveats apply as discussed above for the in vivo interpretation of results using mutations to mimic acetylated proteins.…”

“…If we find acetylation occupancy to be high at a particular site, the likelihood of an inherently slow nonenzymatic mechanism may be diminished and may therefore suggest enzymatic acetylation. With a large number of acetylated proteins, it seems probable that more enzymes exist, and new ones continue to be discovered in both bacteria (61,67) and mitochondria (140). Answering these questions is important in moving toward an understanding of the biological significance of protein acetylation in bacteria.…”

“…2). In M. tuberculosis, the histone-like nucleoid protein MtHU is acetylated at eight sites by the KAT Eis (61). Acetylation leads to a change in DNAbinding capacity and altered DNA structure in vitro.…”

“…These approaches were used either in a targeted manner, focused on a given protein of interest, or in global studies, which aimed to characterize cellular acetylomes under different conditions and in various species. Targeted analyses can involve the purification of the protein of interest by immunoaffinity purification, followed by digestion with a protease and mass spectrometry analysis of the resulting peptides to identify the acetylated sites (56,61,111,112). Global acetylome studies begin with protein extraction from cells grown under particular conditions, followed by enzymatic digestion of the entire proteome.…”

Nε- and O-Acetylation in Mycobacterium tuberculosis Lineage 7 and Lineage 4 Strains: Proteins Involved in Bioenergetics, Virulence, and Antimicrobial Resistance Are Acetylated

“…13–15 Acetylation of Mtb histone-like nucleoid protein MtHU modulates DNA binding and genome organization. 16 It has also been shown that reversible lysine acetylation regulates fatty acid metabolism in Mtb and acetate and propionate metabolism in M. smegmatis . 6,8 Furthermore, Liu et al have demonstrated the regulatory role of lysine acetylation in the immunogenicity of the secreted protein HspX in Mtb.…”