Discovery of Sulfated Metabolites in Mycobacteria with a Genetic and Mass Spectrometric Approach

• Published in: Proceedings of the National Academy of Sciences - PNAS Vol. 99; no. 26; pp. 17037 - 17042
• Main Authors: Mougous, Joseph D., Leavell, Michael D., Senaratne, Ryan H., Leigh, Clifton D., Williams, Spencer J., Riley, Lee W., Leary, Julie A., Bertozzi, Carolyn R.
• Format: Journal Article
• Language: English
• Published: United States National Academy of Sciences 24.12.2002; National Acad Sciences
• Subjects: Average linear density; Bacteria; Biological Sciences; Cellular metabolism; Cysteine; Disaccharides - metabolism; Genetic Engineering; Genetics; Glycopeptides; Inositol; Ions; Mass Spectrometry; Metabolism; Metabolites; Microbiology; Molecules; Mycobacterium smegmatis - metabolism; Mycobacterium tuberculosis; Mycobacterium tuberculosis - metabolism; Pyrazoles - metabolism; Scientific imaging; Sulfates; Sulfates - metabolism; Sulfation; Sulfhydryl Compounds - metabolism; Sulfoglycosphingolipids - metabolism; Sulfur; Sulfur Isotopes; Tuberculosis
• ISSN: 0027-8424; 1091-6490
• DOI: 10.1073/pnas.252514899

The study of the metabolome presents numerous challenges, first among them being the cataloging of its constituents. A step in this direction will be the development of tools to identify metabolites that share common structural features. The importance of sulfated molecules in cell-cell communication motivated us to develop a rapid two-step method for identifying these metabolites in microorganisms, particularly in pathogenic mycobacteria. Sulfur-containing molecules were initially identified by mass spectral analysis of cell extracts from bacteria labeled metabolically with a stable sulfur isotope (34SO4 2-). To differentiate sulfated from reduced-sulfur-containing molecules, we employed a mutant lacking the reductive branch of the sulfate assimilation pathway. In these sulfur auxotrophs, heavy sulfate is channeled exclusively into sulfated metabolites. The method was applied to the discovery of several new sulfated molecules in Mycobacterium tuberculosis and Mycobacterium smegmatis. Because a sulfur auxotrophic strain is the only requirement of the approach, many microorganisms can be studied in this manner. Such genetic engineering in combination with stable isotopic labeling can be applied to various metabolic pathways and their products.