Post translational modification and DNA binding studies with two nucleoid associated proteins of Mycobacterium tuberculosis

Abstract

Genome in prokaryotes is found in a small confinement called nucleoid. The nucleoid compaction is brought about by the combined action of topoisomerases and Nucleoid associated proteins (NAPs). Topoisomerases maintain topological homeostasis of bacterial chromosomes by catalysing changes in DNA linking number by supercoiling, relaxation and decatenation. NAPs are small basic proteins that bind to the DNA and bring about coating, wrapping, and bridging of the genome. Unlike many well studied prokaryotes like Escherichia coli, Mycobacterium tuberculosis (M. tuberculosis) has underrepresentation of NAPs. Earlier work carried out in the laboratory has shown that NAPs collaborate with other NAPs, other proteins such as Topoisomerase I, as well as show different post translational modifications that alter their DNA binding properties, in order to cope up with the situation. The work shows the effect of acylation on the properties of mycobacterial NAP MtHU. It also covers the occupancy of Rv3852, a unique NAP, on the mycobacterial genome and its effect.HU is one of the abundant NAPs in mycobacteria. It is a histone like protein that undergoes post translational modification, as previously reported by the laboratory. Study was carried out to elucidate the effect of post translational modification of HU, specifically acylation, upon its DNA binding properties. Two modifiers, belonging to GNAT superfamily of proteins, namely Rv2416c (Eis) and Rv0802c, were found to acetylate as well as succinylate MtHU. Eis could acetylate as well as succinylate HU very effectively. However, Rv0802c was found to show better succinyltransferase activity than acetyltransferase activity. Both these modifications modulated the DNA binding properties of HU. Ectopic expression of Eis in Mycobacterium smegmatis (M. smegmatis) caused decompaction of genome, unlike ectopic expression of Rv0802c. Rv1151c is the only characterized Sirtuin like protein in M. tuberculosis. The gene was cloned, expressed and protein was purified. Rv1151c catalysed deacetylation as well as desuccinylation of HU in the presence of NAD. Its activity was however inhibited in the presence of nicotinamide, a known inhibitor of NAD-dependent deacetylases. Deacetylation restored the DNA binding properties of HU. However, its ectopic expression in M. smegmatis didn’t have any effect on the compaction status of the genome. Rv3852 is a unique NAP found only in slow growing mycobacteria. Although thought to be the mycobacterial counter part of H-NS, the studies from the laboratory showed that Rv3852 is unique in the sense that it binds to the genome as well as tethers to the membrane through its carboxyl terminal region. Genome-wide binding study of Rv3852 on M. tuberculosis was carried out by chromatin immunoprecipitation followed by sequencing. It was found to bind to DNA in a sequence non-specific manner. By binding to genome, it seems to regulate the expression of genes associated with iron homeostasis, polyketide synthases and genes associated with the synthesis of pthiocerol dimycocerosates.