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 muM. Na(+)-independent sulfate uptake was not reduced by bicarbonate, nitrate, or phosphate, but was inhibited by sulfite, selenate, thiosulfate, N-ethylmaleimide 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 CysTWA-dependent sulfate transporter.

Figure 1. Overexpression of Rv1739c increases sulfate uptake

A. Schematic of Rv1739c polypeptide from M. tb. The hypothetical disposition of the transmembrane spans is based on hydropathy analysis described by (Felce et al. 2004). B. Left: Anti-6His immunoblot of clarified SDS lysate of uninduced (lane 1) and IPTG-induced Tuner(DE3)pLacI cells (lane 2). Right: Coomassie Blue R250-stained gel of Rv1739c eluted from Ni-NTA column by 250 mM imidazole. C. ³⁵S-sulfate uptake in uninduced Tuner cells or cells induced to express either M. Tb. Rv1739c (left bars) or E. coli ychM (right bars). Number of measurements is indicated within parentheses. *, p < 0.05. D. Anti-6His immunoblot of clarified SDS lysate of uninduced Tuner cells (lane 1) or Tuner cells induced to express ychM polypeptide (lane 2).

Figure 2

A. Growth phase-dependence of ³⁵S-sulfate uptake by native Tuner cells untreated with IPTG. B. Time-dependence of ³⁵S-sulfate uptake by uninduced Tuner cells (diamonds) and by cells induced to express Rv1739c (squares). C. ³⁵S-sulfate uptake into uninduced cells and cells induced to express Rv1739c. D. Sulfate uptake into cells harboring (Rv1739c) or lacking the Rv1739c expression plasmid (Tuner), after treatment without (uninduced) or with IPTG (induced). Number of measurements is indicated within parentheses. *, p < 0.05.

Figure 3

A. pH-dependence of ³⁵S-sulfate uptake into cells induced to express Rv1739c. Number of measurements is indicated within parentheses. *, p < 0.05. B. Sulfate concentration dependence of ³⁵S-sulfate uptake into cells expressing Rv1739c (n=6), fit with a hyperbolic equation (SigmaPlot).

Figure 4

Tests of inhibition of ³⁵S-sulfate uptake into cells expressing Rv1739c by (A) 1 mM NEM, (B) 50 μM CCCP, (C) 5 mM Na sulfite, (D) 5 mM Na selenate, (E) 5 mM Na molybdate or Na thiosulfate, (F) 1 mM DIDS or 100 μM niflumate. ³⁵S-sulfate concentration was 100 μM in the presence of sulfite, molybdate, or thiosulfate, and 50 μM in the presence of the other antagonists. Number of measurements is within parentheses.

Figure 5

A. ³⁵S-sulfate uptake is increased by induction of expression of the Rv1739c transmembrane domain (aa 1–436). B. ³⁵S-sulfate uptake is not increased by induction of expression of the C-terminal cytoplasmic domain of Rv1739c (aa 437–560) including its STAS domain. Number of measurements is within parentheses. *, p < 0.05.

Figure 6

A. Schematic of the Cys-less Rv1739c polypeptide with its three cysteine-to-serine substitutions indicated. B. Induction of ³⁵S-sulfate uptake in cells expressing Cys-less Rv1739c. C. Inhibition by NEM (1 mM) and by CCCP (50 μM) of ³⁵S-sulfate uptake into cells induced to express Cys-less Rv1739c. Number of measurements is within parentheses. *, p < 0.05.

Figure 7

A. Schematic of single cysteine substitutions introduced into the Cys-less Rv1739c polypeptide. B. Increased ³⁵S-sulfate uptake into cells induced to express Cys-less Rv1739c polypeptide containing the indicated single reintroduced cysteine residues. C. Effects of MTSET and MTSES on sulfate uptake induced in Cys-less Rv1739 and its P54C variant. Number of measurements is indicated within parentheses.

Figure 8

A. Cell density-dependent induction of ³⁵S-sulfate transport in E. coli strain BW25113, but not in the cysA deletion strain JD21556. B. Arabinose treatment does not increase ³⁵S-sulfate uptake into JD21556 bacteria expressing Rv1739c in plasmid pBAD33. Number of measurements is indicated within parentheses. *, p < 0.05.