HupB, a Nucleoid-Associated Protein of Mycobacterium tuberculosis, Is Modified by Serine/Threonine Protein Kinases In Vivo

Abstract

e HU, a widely conserved bacterial histone-like protein, regulates many genes, including those involved in stress response and virulence. Whereas ample data are available on HU-DNA communication, the knowledge on how HU perceives a signal and transmit it to DNA remains limited. In this study, we identify HupB, the HU homolog of the human pathogen Mycobacterium tuberculosis, as a component of serine/threonine protein kinase (STPK) signaling. HupB is extracted in its native state from the exponentially growing cells of M. tuberculosis H 37 Ra and is shown to be phosphorylated on both serine and threonine residues. The STPKs capable of modifying HupB are determined in vitro and the residues modified by the STPKs are identified for both in vivo and the in vitro proteins through mass spectrometry. Of the identified phosphosites, Thr 65 and Thr 74 in the DNA-embracing ␤-strand of the N-terminal domain of HupB (N-HupB) are shown to be crucial for its interaction with DNA. In addition, Arg 55 is also identified as an important residue for N-HupB-DNA interaction. N-HupB is shown to have a diminished interaction with DNA after phosphorylation. Furthermore, hupB is shown to be maximally expressed during the stationary phase in M. tuberculosis H 37 Ra, while HupB kinases were found to be constitutively expressed (PknE and PknF) or most abundant during the exponential phase (PknB). In conclusion, HupB, a DNA-binding protein, with an ability to modulate chromatin structure is proposed to work in a growth-phase-dependent manner through its phosphorylation carried out by the mycobacterial STPKs.

Bacterial Chromosome Organization and Segregation

“…For example, S. coelicolor and Mycobacteria encode an HU paralog called HupS/Hlp that has an extensive C-terminal extension with homology to eukaryotic histone H1 (Mukherjee et al 2009, Salerno et al 2009). In M. tuberculosis , the phosphorylation of HupS decreases its interaction with DNA highlighting the possibility of post-translational regulation of DNA compaction (Gupta et al 2014). …”

Multilayer chromosome organization through DNA bending, bridging and extrusion

“…HU proteins are known to be phosphorylated in Bacillus subtilis and Mycobacterium tuberculosis. In the latter organism phosphorylation of HupB eliminates its ability to bind to DNA [15,16]. Moreover, the expression levels of several NAPs change drastically at transitions between growth phases, likely explaining, at least in parts, the distinct appearance of nucleoids at different stages of growth [9,17].…”

Systematic Analysis of Mycobacterial Acylation Reveals First Example of Acylation-mediated Regulation of Enzyme Activity of a Bacterial Phosphatase

“…The supernatant obtained after removing the cell debris was incubated with Ni 2ϩ -NTA affinity resin (Qiagen) previously equilibrated with the equilibration buffer (50 mM Tris-Cl (pH 8.5), 300 mM NaCl, 5 mM ␤-mercaptoethanol, 1 mM PMSF, 10% glycerol, and 20 mM imidazole) for 2 h. After washing the resin with the wash buffer (50 mM Tris-Cl (pH 8.5), 1 M NaCl, 5 mM ␤-mercaptoethanol, 1 mM PMSF, 10% glycerol, and 20 mM imidazole), elution was carried out in the elution buffer (50 mM Tris-Cl (pH 8.5), 10% glycerol, 150 mM NaCl, 1 mM PMSF, and 200 mM imidazole). For glutathione S-transferase (GST)-tagged PknG and Rv0998 purification, previously published protocol was used (27). The purified proteins were resolved by SDS-PAGE and confirmed by immunoblotting.…”