Increased sulfate uptake by E. coli overexpressing the SLC26-related SulP protein Rv1739c from Mycobacterium tuberculosis

Abstract

Growth and virulence of mycobacteria requires sulfur uptake. The Mycobacterium tuberculosis genome contains, in addition to the ABC sulfate permease cysTWA, three SLC26-related SulP genes of unknown function. We report that induction of Rv1739c expression in E. coli increased bacterial uptake of sulfate, but not Cl − , formate, or oxalate. Uptake was time-dependent, maximal at pH 6.0, and exhibited a K 1/2 for sulfate of 4.0 μM. Na + -independent sulfate uptake was not reduced by bicarbonate, nitrate, or phosphate, but was inhibited by sulfite, selenate, thiosulfate, Nethylmaleimide and carbonyl cyanide 3-chloro-phenylhydrazone. Sulfate uptake was also increased by overexpression of the Rv1739c transmembrane domain, but not of the cytoplasmic C-terminal STAS domain. Mutation to serine of the three cysteine residues of Rv1739c did not affect magnitude, pH-dependence, or pharmacology of sulfate uptake. Expression of Rv1739c in a M. bovis BCG strain lacking the ABC sulfate permease subunit CysA could not complement sulfate auxotrophy. Moreover, inducible expression of Rv1739c in an E. coli strain lacking CysA did not increase sulfate uptake by intact cells. Our data show that facilitation of bacterial sulfate uptake by Rv1739c requires CysA and its associated sulfate permease activity, and suggest that Rv1739c may be a CysTWAdependent sulfate transporter.

Solution Structure of the Guanine Nucleotide-binding STAS Domain of SLC26-related SulP Protein Rv1739c from Mycobacterium tuberculosis

“…Protein identity was confirmed by mass spectrometry. Unlabeled and uniformly 13 C/ 15 N-or 15 N-labeled STAS-His 6 NMR samples of 0.7-1.0 mM concentration in 50 mM sodium phosphate, 275 mM NaCl, pH 7.2, were prepared and subjected to NMR spectroscopy (supplemental "Methods" and Ref. 30).…”

“…S7. In the lowest energy pose, the GDP-interacting amino acid residues include Gln 9 , Gly 15 Many of these residues are, or are adjacent to, those exhibiting above-threshold CSP in NMR titration studies. GDP binding is preferentially clustered to C-terminal STAS residues, with 7/9 poses supporting this preferred docked complex.…”

“…4 and Table 2). The amino acids exhibiting the largest GDP-induced CSPs are 15 N HSQC spectra of Rv1739c STAS in the absence (blue contours) and presence of 20 mM GDP (red contours). Circled resonances indicate residues detected only in the presence of nucleotide.…”

“…In yeast, plants, and worms, the sulp gene products mediate transport of SO 4 2Ϫ , whereas in mammals the related SLC26 gene products transport a wide range of monovalent anions in addition to divalent SO 4 2Ϫ and oxalate. Although sulp genes are present in genomes throughout the eubacteriae and archeae, indirect evidence of anion transport function by bacterial SulP polypeptides has to date been presented only for Mycobacterium tuberculosis Rv1739c (15), Synechococcus BicA (16,17), and Escherichia coli YchM (18).…”

“…We have overexpressed and purified the 560-aa putative SO 4 2Ϫ transporter Rv1739c from M. tuberculosis, comprising an N-terminal trans-membrane domain (aa 1-414) and a Cterminal cytoplasmic STAS domain (aa 442-560) (15). Previously, we reported the secondary structural features present in the C-terminal STAS domain using heteronuclear NMR spectroscopy (30).…”