The Mycobacterium tuberculosis cysD and cysNC genes form a stress-induced operon that encodes a tri-functional sulfate-activating complex

1 Mycobacterial Research Group, Centenary Institute of Cancer Medicine and Cell Biology, Locked Bag no. 6, Newtown, NSW 2042, Australia 2 Department of Biochemistry, Albert Einstein College of Medicine, 1300 Morris Park Avenue, Bronx, NY 10461-1926, USA 3 Department of Medicine, University of Sydney, NSW 2006, Australia Correspondence James A. Triccas J.Triccas{at}centenary.usyd.edu.au Sulfur metabolism has been implicated in the virulence, antibiotic resistance and anti-oxidant defence of Mycobacterium tuberculosis . Despite its human disease relevance, sulfur metabolism in mycobacteria has not yet been fully characterized. ATP sulfurylase catalyses the synthesis of activated sulfate (adenosine 5'-phosphosulfate, APS), the first step in the reductive assimilation of sulfate. Expression of the M. tuberculosis cysD gene, predicted to encode the adenylyl-transferase subunit of ATP sulfurylase, is upregulated by the bacilli inside its preferred host, the macrophage. This study demonstrates that cysD and cysNC orthologues exist in M. tuberculosis and constitute an operon whose expression is induced by sulfur limitation and repressed by the presence of cysteine, a major end-product of sulfur assimilation. The cysDNC genes are also induced upon exposure to oxidative stress, suggesting regulation of sulfur assimilation by M. tuberculosis in response to toxic oxidants. To ensure that the cysDNC operon encoded the activities predicted by its primary sequence, and to begin to characterize the products of the operon, they were expressed in Escherichia coli , purified to homogeneity, and tested for their catalytic activities. The CysD and CysNC proteins were shown to form a multifunctional enzyme complex that exhibits the three linked catalytic activities that constitute the sulfate activation pathway. Abbreviations: APS, adenosine 5'-phosphosulfate; PAPS, 3'-phosphoadenosine 5'-phosphosulfate; PB, Proskauer and Beck