Control of Copper Resistance and Inorganic Sulfur Metabolism by Paralogous Regulators in Staphylococcus aureus

Abstract

All strains of Staphylococcus aureus encode a putative copper-sensitive operon repressor (CsoR) and one other CsoR-like protein of unknown function. We show here that NWMN_1991 encodes a bona fide Cu(I)-inducible CsoR of a genetically unlinked copA-copZ copper resistance operon in S. aureus strain Newman. In contrast, an unannotated open reading frame found between NWMN_0027 and NWMN_0026 (denoted NWMN_0026.5) encodes a CsoR-like regulator that represses expression of adjacent genes by binding specifically to a pair of canonical operator sites positioned in the NWMN_0027-0026.5 intergenic region. Inspection of these regulated genes suggests a role in assimilation of inorganic sulfur from thiosulfate and vectorial sulfur transfer, and we designate NWMN_ 0026.5 as CstR (CsoR-like sulfur transferase repressor). Expression analysis demonstrates that CsoR and CstR control their respective regulons in response to distinct stimuli with no overlap in vivo. Unlike CsoR, CstR does not form a stable complex with Cu(I); operator binding is instead inhibited by oxidation of the intersubunit cysteine pair to a mixture of disulfide and trisulfide linkages by a likely metabolite of thiosulfate assimilation, sulfite. CsoR is unreactive toward sulfite under the same conditions. We conclude that CsoR and CstR are paralogs in S. aureus that function in the same cytoplasm to control distinct physiological processes.The Gram-positive opportunistic human pathogen Staphylococcus aureus is the causative agent of a wide range of hospital and community-acquired infections that are associated with significant morbidity (1). With the incidence of methicillinresistant strains increasing in previously low prevalence areas (2), new antibiotic therapies that target novel metabolic pathways are urgently needed. One approach is to target those processes that allow a pathogen to respond to environmental stresses that might change depending on the microenvironmental host niche in which the organism finds itself. Resistance to host-mediated copper killing of Escherichia coli (3), Salmonella enterica (4), and Mycobacterium tuberculosis (5, 6) and sulfur assimilation and cysteine biosynthesis in M. tuberculosis (7,8) are two such processes. S. aureus is particularly sensitive to rapid killing when exposed to copper or copper alloy surfaces, justifying this therapeutic direction (9, 10).M. tuberculosis CsoR 6 (copper-sensitive operon repressor) is a founding member of large family of regulators now known collectively to respond to Cu(I), Ni(II), and perhaps other stressors, the structural basis of which is not fully understood (11, 12). All CsoR family proteins lack a known canonical DNA binding domain and are projected to adopt the flat disc-shaped dimer of dimers homotetrameric structure characteristic of Cu(I)-sensing CsoRs, with individual dimers consisting of an antiparallel four-helix bundle flanked by a C-terminal ␣3 helix (13,14). Two cysteine residues on opposite subunits within a dimer make coordination bonds to the Cu(I) ion, with the third ...

MntABC and MntH Contribute to Systemic Staphylococcus aureus Infection by Competing with Calprotectin for Nutrient Manganese

“…Following rehydration, the tissue was analyzed using a Bruker Autoflex speed tandem time-of-flight (TOF/TOF) mass spectrometer (Bruker Daltonics, Billerica, MA) equipped with a Gaussian beam profile laser (Nd:YAG; 355 nm) in linear positive-ion mode at 75-m spatial resolu- 25 Mg, 31 P, 44 Ca, 55 Mn, 56 Fe, 63 Cu, 66 Zn Runs (no.) 10 Passes (no.)…”

“…The cultures were grown at 37°C with shaking until reaching mid-exponential phase, when the samples were harvested and preserved in acetone-ethanol at Ϫ80°C. RNA was extracted and cDNA was created, as previously described (55,56). Expression of mntA and mntH was assessed by quantitative reverse transcription-PCR (qRT-PCR) using the indicated primer sets (Table 1), and 16S RNA was used as a normalizing control.…”

Copper-responsive gene regulation in bacteria

“…In CstR, the residue corresponding to His 66 of CsoR is replaced by asparagine, and consequently CstR does not act as a copper sensor (Grossoehme et al, 2011). Unlike CsoR, CstR forms disulfide cross-linked dimers upon anaerobic incubation with sulfite.…”

“…aureus synthesizes two CsoR-like regulators designated CsoR and CstR (Grossoehme et al, 2011). In its copper-free form, CsoR represses transcription of the copper-ATPase gene copA.…”

Cu(I)-mediated Allosteric Switching in a Copper-sensing Operon Repressor (CsoR)

“…A second M. tuberculosis CsoR paralog, RicR, was shown to be involved in the regulation of several genes only found in pathogenic mycobacteria in response to copper stress (2). Several non-copper-sensing CsoR-like proteins, including the nickel-sensing repressors RcnR in Escherichia coli (21,22) and InrS in Synechocystis (23) and the sulfur transferase regulator CstR in S. aureus (24,25), have also been identified, revealing that this family of proteins has evolved to sense a range of inducers.…”