Abstract
Methionine can be used as the sole sulfur source by the Mycobacterium tuberculosis complex although it is not obvious from examination of the genome annotation how these bacteria utilize methionine. Given that genome annotation is a largely predictive process, key challenges are to validate these predictions and to fill in gaps for known functions for which genes have not been annotated. We have addressed these issues by functional analysis of methionine metabolism. Transport, followed by metabolism of (35)S methionine into the cysteine adduct mycothiol, demonstrated the conversion of exogenous methionine to cysteine. Mutational analysis and cloning of the Rv1079 gene showed it to encode the key enzyme required for this conversion, cystathionine gamma-lyase (CGL). Rv1079, annotated metB, was predicted to encode cystathionine gamma-synthase (CGS), but demonstration of a gamma-elimination reaction with cystathionine as well as the gamma-replacement reaction yielding cystathionine showed it encodes a bifunctional CGL/CGS enzyme. Consistent with this, a Rv1079 mutant could not incorporate sulfur from methionine into cysteine, while a cysA mutant lacking sulfate transport and a methionine auxotroph was hypersensitive to the CGL inhibitor propargylglycine. Thus, reverse transsulfuration alone, without any sulfur recycling reactions, allows M. tuberculosis to use methionine as the sole sulfur source. Intracellular cysteine was undetectable so only the CGL reaction occurs in intact mycobacteria. Cysteine desulfhydrase, an activity we showed to be separable from CGL/CGS, may have a role in removing excess cysteine and could explain the ability of M. tuberculosis to recycle sulfur from cysteine, but not methionine.
Highlights
Methionine can be used as the sole sulfur source by the Mycobacterium tuberculosis complex it is not obvious from examination of the genome annotation how these bacteria utilize methionine
Cystathionine ␥-lyase (CGL)1 is the key enzyme required for the operation of the reverse transsulfuration pathway (Fig. 1, unique reactions shown by blue arrows inside yellow box) from methionine to cysteine most likely to be needed for methionine auxotrophy
Growth Experiments with M. bovis BCG cysA::Tn5367 That Can Be Used to Interpret Biochemical Pathways—If a methionine auxotroph is more susceptible to propargylglycine than its parent strain, this provides a line of evidence that it is dependent upon the propargylglycine-sensitive steps shown in Fig. 1, and that alternative pathways are not operating
Summary
Vol 280, No 9, Issue of March 4, pp. 8069 –8078, 2005 Printed in U.S.A. Functional Demonstration of Reverse Transsulfuration in the Mycobacterium tuberculosis Complex Reveals That Methionine Is the Preferred Sulfur Source for Pathogenic Mycobacteria*□S. Key sulfur-containing metabolites derived from methionine include N-formyl methionine for the initiation of peptide chain biosynthesis and S-adenosylmethionine for many one-carbon transfer reactions Compounds, such as glutathione or its replacement in the actinomyctes, mycothiol, are synthesized from cysteine for redox maintenance. Our interest in the pathways for the biosynthesis and utilization of cysteine and methionine arose from the observation that methionine, but never cysteine, auxotrophs of the M. tuberculosis complex could be isolated [3, 4] These observations, together with the lack of further attenuation of a methionine auxotroph of Mycobacterium bovis BCG [4], suggested methionine might be an important source of sulfur in the host. Given that members of this protein family are enzymes with broad substrate specificity [5, 6, 8, 9], we decided to investigate the activities Rv1079 encodes by both mutational analysis and cloning the gene
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