Identification of Rv3852 as a nucleoid-associated protein in Mycobacterium tuberculosis

Tuberculosis remains the major cause of mortality due to a bacterial pathogen, Mycobacterium tuberculosis. The molecular mechanisms of infection and persistence have not been completely elucidated for this pathogen. Studies involving nucleoid-associated proteins (NAPs), which have been related to the control and influence of virulence genes in pathogenic bacteria, can help unveil the virulence process of M. tuberculosis. Here, we describe the initial characterization of an ORF for an M. tuberculosis putative NAP. The Rv3852 gene was cloned and expressed, and its product purified to homogeneity. A qualitative protein-DNA binding assay was carried out by gel-retardation and the protein affinity for specific DNA sequences was assessed quantitatively by surface plasmon resonance (SPR). A stoichiometry of 10 molecules of monomeric protein per molecule of DNA was determined. The monophasic apparent dissociation rate constant values increased to a saturable level as a function of protein concentration, yielding two limiting values for the molecular recognition of proU2 DNA. A protein-DNA binding mechanism is proposed. In addition, functional complementation studies with an Escherichia coli has mutant reinforce the likelihood that the Rv3852 protein represents a novel NAP in M. tuberculosis.