Direct Interaction of Polar Scaffolding Protein Wag31 with Nucleoid-Associated Protein Rv3852 Regulates Its Polar Localization.

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.

NapA (Rv0430), a Novel Nucleoid-Associated Protein that Regulates a Virulence Operon in Mycobacterium tuberculosis in a Supercoiling-Dependent Manner.

Comparison of Mycobacterium tuberculosis with Escherichia coli reveals a reduction in the diversity of DNA-managing proteins, such as DNA topoisomerases, although genome sizes are similar for the two species. The same is true for nucleoid-associated proteins (NAPs), important factors in bacterial chromosome compaction, chromosome remodeling, and regulation of gene expression. In a search for still uncharacterized NAPs, we found that M. tuberculosis protein Rv0430 has NAP-like features: it binds to DNA in a length- and supercoil-dependent fashion, prefers A/T-rich DNA sequences, protects DNA from damaging agents, and modulates DNA supercoiling. At a ratio of 1 dimer/40 bps of DNA, Rv0430 bridges distant DNA segments; at 1 dimer/20 bps, it coats DNA, forming inflexible rods. Rv0430 also stimulates the DNA relaxation activity of topoisomerase I. Remarkably, Rv0430 stimulates its own promoter in a supercoil-dependent manner. It is the first gene of an operon harboring two regulators of M. tuberculosis virulence (virR and sodC), and controls the expression of these downstream virulence regulators and therefore itself is a virulence regulator. The sensitivity of rv0430 expression to supercoiling is consistent with supercoiling being important for infection by M. tuberculosis. Thus, Rv0430 is a novel NAP, doubling up as a topology modulator of M. tuberculosis.

Physical and functional interaction between nucleoid-associated proteins HU and Lsr2 of Mycobacterium tuberculosis: altered DNA binding and gene regulation.

Nucleoid-associated proteins (NAPs) in bacteria contribute to key activities such as DNA compaction, chromosome organization and regulation of gene expression. HU and Lsr2 are two principal NAPs in Mycobacterium tuberculosis (Mtb). HU is essential for Mtb survival and is one of the most abundant NAPs. It differs from other eubacterial HU proteins in having a long, flexible lysine- and arginine-rich carboxy-terminal domain. Lsr2 of Mtb is the functional analogue of the bacterial NAP commonly called H-NS. Lsr2 binds to and regulates expression of A/T-rich portions of the otherwise G/C-rich mycobacterial chromosome. Here, we demonstrate that HU and Lsr2 interact to form a complex. The interaction occurs primarily through the flexible carboxy-terminal domain of HU and the acidic amino-terminal domain of Lsr2. The resulting complex, upon binding to DNA, forms thick nucleoprotein rods, in contrast to the DNA bridging seen with Lsr2 and the DNA compaction seen with HU. Furthermore, transcription assays indicate that the HU-Lsr2 complex is a regulator of gene expression. This physical and functional interaction between two NAPs, which has not been reported previously, is likely to be important for DNA organization and gene expression in Mtb and perhaps other bacterial species.