Mass Spectrometric Analysis of Mycothiol levels in Wild-Type and Mycothiol Disulfide Reductase Mutant Mycobacterium smegmatis.

Mycothiol (MSH), the primary low-molecular weight thiol produced in mycobacteria, acts to protect the cell from oxidative stress and to maintain redox homeostasis, notably in the pathogenic Mycobacterium tuberculosis in the course of human infection. The mycothiol disulfide reductase (Mtr) enzyme reduces the oxidized form of mycothiol, mycothione (MSSM), back to MSH, however its role in bacterial viability is not clear. In this study, we sought to determine the MSH levels of wild-type (WT) and Mtr mutant mycobacteria during oxidative stress. We describe a rapid method for the relative quantification of MSH using high-sensitivity mass spectrometry (MS) with selected ion monitoring (SIM). This method uses only minimal sample cleanup, and does not require advanced chromatographic equipment or fluorescent compounds. MSH levels decreased in the Mtr mutant only upon treatment with peroxide, and the results were consistent between our method and previously-described thiol quantification methods. Our results indicate that our MS-based method is a useful, high-throughput alternative tool for the quantification of MSH from mycobacteria.

Drug targets in mycobacterial sulfur metabolism.

The identification of new antibacterial targets is urgently needed to address multidrug resistant and latent tuberculosis infection. Sulfur metabolic pathways are essential for survival and the expression of virulence in many pathogenic bacteria, including Mycobacterium tuberculosis. In addition, microbial sulfur metabolic pathways are largely absent in humans and therefore, represent unique targets for therapeutic intervention. In this review, we summarize our current understanding of the enzymes associated with the production of sulfated and reduced sulfur-containing metabolites in Mycobacteria. Small molecule inhibitors of these catalysts represent valuable chemical tools that can be used to investigate the role of sulfur metabolism throughout the Mycobacterial lifecycle and may also represent new leads for drug development. In this light, we also summarize recent progress in the development of inhibitors of sulfur metabolism enzymes.

Nitrogen regulation of the codBA (cytosine deaminase) operon from Escherichia coli by the nitrogen assimilation control protein, NAC.

Transcription of the cytosine deaminase (codBA) operon of Escherichia coli is regulated by nitrogen, with about three times more codBA expression in cells grown in nitrogen-limiting medium than in nitrogen-excess medium. Beta-galactosidase expression from codBp-lacZ operon fusions showed that the nitrogen assimilation control protein NAC was necessary for this regulation. In vitro transcription from the codBA promoter with purified RNA polymerase was stimulated by the addition of purified NAC, confirming that no other factors are required. Gel mobility shifts and DNase I footprints showed that NAC binds to a site centered at position -59 relative to the start site of transcription and that mutants that cannot bind NAC there cannot activate transcription. When a longer promoter region (positions -120 to +67) was used, a double footprint was seen with a second 26-bp footprint separated from the first by a hypersensitive site. When a shorter fragment was used (positions -83 to +67), only the primary footprint was seen. Nevertheless, both the shorter and longer fragments showed NAC-mediated regulation in vivo. Cytosine deaminase expression in Klebsiella pneumoniae was also regulated by nitrogen in a NAC-dependent manner. K. pneumoniae differs from E. coli in having two cytosine deaminase genes, an intervening open reading frame between the codB and codA orthologs, and a different response to hypoxanthine which increased cod expression in K. pneumoniae but decreased it in E. coli.