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.

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Among the nucleoid-associated proteins (NAPs), HU is the most conserved in eubacteria, engaged in overall chromosome organization and regulation of gene expression. Unlike other bacteria, HU from Mycobacterium tuberculosis (MtHU), has a long carboxyl terminal domain enriched in basic amino acids, resembling eukaryotic histone N-terminal tails. As with histones, MtHU undergoes post-translational modifications and we have previously identified interacting kinases, methyltransferases, an acetyltransferase and a deacetylase. Here we show that Rv0802c interacts and succinylates MtHU. Although categorized as a succinyltransferase, we show that this GNAT superfamily member can catalyse both succinylation and acetylation of MtHU with comparable kinetic parameters. Like acetylation of MtHU, succinylation of MtHU caused reduced interaction of the NAP with DNA, determined by electrophoretic mobility shift assay and surface plasmon resonance. However, in vivo expression of Rv0802c did not significantly alter the nucleoid architecture. Although such succinylation of NAPs is rare, these modifications of the archetypal NAP may provide avenues to the organism to compensate for the underrepresentation of NAPs in its genome to control the dynamics of nucleoid architecture and cellular functions.

In the present work, detailed laser-based diagnostic experiments were conducted to characterise the spray from low pressure 2-hole and 4-hole Port Fuel Injection (PFI) injectors. The main objective of the work included obtaining quantitative information of the spatio-temporal spray structure of such low-pressure gasoline sprays. A novel approach involving a combination of techniques such as Mie scattering, Granulometry, and Laser Sheet Dropsizing (LSD) was used to study the spray structure. The droplet sizes, distributions with time, Sauter Mean Diameters (SMD), droplet velocities, cone angles and spray tip penetrations of the sprays from the injectors were determined. The spray from these injectors is found to be 'pencil like' and not dispersed as in high pressure sprays. The application of the above mentioned techniques provides two-dimensional SMD contours of the entire spray at different instants of time, with reasonable accuracy.

Rv3852 is a unique nucleoid-associated protein (NAP) found exclusively in Mycobacterium tuberculosis (Mtb) and closely related species. Although annotated as H-NS, we showed previously that it is very different from H-NS in its properties and is distinct from other NAPs, anchoring to cell membrane by virtue of possessing a C-terminal transmembrane helix. Here, we investigated the role of Rv3852 in Mtb in organizing architecture or synthesis machinery of cell wall by protein–protein interaction approach. We demonstrated a direct physical interaction of Rv3852 with Wag31, an important cell shape and cell wall integrity determinant essential in Mtb. Wag31 localizes to the cell poles and possibly acts as a scaffold for cell wall synthesis proteins, resulting in polar cell growth in Mtb. Ectopic expression of Rv3852 in M. smegmatis resulted in its interaction with Wag31 orthologue DivIVAMsm. Binding of the NAP to Wag31 appears to be necessary for fine-tuning Wag31 localization to the cell poles, enabling complex cell wall synthesis in Mtb. In Rv3852 knockout background, Wag31 is mislocalized resulting in disturbed nascent peptidoglycan synthesis, suggesting that the NAP acts as a driver for localization of Wag31 to the cell poles. While this novel association between these two proteins presents one of the mechanisms to structure the elaborate multi-layered cell envelope of Mtb, it also exemplifies a new function for a NAP in mycobacteria.