Discovery of sulfated metabolites in mycobacteria with a genetic and mass spectrometric approach

doi:10.1073/pnas.252514899

Discovery of sulfated metabolites in mycobacteria with a genetic and mass spectrometric approach

^*Howard Hughes Medical Institute, ^†Department of Molecular and Cell Biology, ^§Department of Chemistry, and^¶ School of Public Health, University of California, Berkeley, CA 94720

Edited by Chi-Huey Wong, The Scripps Research Institute, La Jolla, CA, and approved October 23, 2002 (received for review August 16, 2002)

Abstract

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. Sulfurcontaining molecules were initially identified by mass spectral analysis of cell extracts from bacteria labeled metabolically with a stable sulfur isotope (³⁴SO $\text{[math]}$ ). 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.

Footnotes

↵ ‡ J.D.M and M.D.L. contributed equally to this work.
↵ ∥ To whom correspondence may be addressed. E-mail: bertozzi{at}cchem.berkeley.edu or leary{at}socrates.berkeley.edu.
This paper was submitted directly (Track II) to the PNAS office.
Abbreviations:
1. SL-1, Sulfatide-1
2. r-sulfur, compounds containing reduced sulfur
3. APS, adenosine-5′-phosphosulfate
4. MS/MS, tandem MS
5. FT-ICRMS, Fourier transform–ion cyclotron resonance MS