Copper is an essential transition metal for living organisms but it is detrimental in excess. The metalloregulatory protein copper‐sensing operon repressor (CsoR) in bacteria has evolved to prevent cytoplasmic copper toxicity. Cu(I)‐binding to tetrameric CsoRs mediates transcriptional derepression of copper resistance genes but the mechanism is unknown. A phylogenetic analysis of 227 DUF156 protein members including biochemically or structurally characterized CsoR/RcnR repressors reveals that Geobacillus thermodenitrificans (Gt) CsoR characterized here is representative of CsoRs from pathogenic bacilli Listeria monocytogenes and Bacillus anthracis. The 2.56 Å structure of Cu(I)‐bound Gt CsoR reveals that Cu(I) binding induces a kink in the α2‐helix between two conserved copper‐ligating residues and folds an N‐terminal tail (residues 12‐19) over the Cu(I) binding site. NMR studies of Gt CsoR reveal that this tail is flexible in the apo‐state with these dynamics quenched upon Cu(I) binding. Small angle X‐ray scattering (SAXS) experiments on an N‐terminally truncated Gt CsoR (∆2‐10) reveal that the Cu(I)‐bound tetramer is hydrodynamically more compact than is the apo‐state. A mutational analysis of residues critical to N‐terminal tail folding reveals that these residues function to stabilize the apoprotein‐DNA complex and/or control the extent of allosteric negative regulation of DNA binding by Cu(I), but to varying degrees. The mechanism of Cu(I)‐mediated allosteric switching in CsoRs is discussed. Grant Funding Source: Supported by NIH grant GM042569

CONSPECTUS The human innate immune system has evolved the means to reduce the bioavailability of first-row late d-block transition metal ions to invading microbial pathogens in a process termed “nutritional immunity”. Transition metals from Mn(II) to Zn(II) function as metalloenzyme cofactors in all living cells, and the successful pathogen is capable of mounting an adaptive response to mitigate the effects of host control of transition metal bioavailability. Emerging evidence suggests that Mn, Fe, and Zn are withheld from the pathogen in classically defined nutritional immunity, while Cu is used to kill invading microorganisms. This Account summarizes new molecular-level insights into copper trafficking across cell membranes from studies of a number of important bacterial I pathogens and model organisms, including Escherichia coli, Salmonella species, Mycobacterium tuberculosis, and Streptococcus pneumoniae, to illustrate general principles of cellular copper resistance. Recent highlights of copper chemistry at the host—microbial pathogen interface include the first high resolution structures and functional characterization of a Cu(I)-effluxing P1B-ATPase, a new class of bacterial copper chaperone, a fungal Cu-only superoxide dismutase SOD5, and the discovery of a small molecule Cu-bound SOD mimetic. Successful harnessing by the pathogen of host-derived bactericidal Cu to reduce the bacterial load of reactive oxygen species (ROS) is an emerging theme; in addition, recent studies continue to emphasize the importance of short lifetime protein–protein interactions that orchestrate the channeling of Cu(I) from donor to target without dissociation into bulk solution; this, in turn, mitigates the off-pathway effects of Cu(I) toxicity in both the periplasm in Gram negative organisms and in the bacterial cytoplasm. It is unclear as yet, outside of the photosynthetic bacteria, whether Cu(I) is trafficked to other cellular destinations, for example, to cuproenzymes or other intracellular storage sites, or the general degree to which copper chaperones vs copper efflux transporters are essential for bacterial pathogenesis in the vertebrate host. Future studies will be directed toward the identification and structural characterization of other cellular targets of Cu(I) trafficking and resistance, the physical and mechanistic characterization of Cu(I)-transfer intermediates, and elucidation of the mutual dependence of Cu(I) trafficking and cellular redox status on thiol chemistry in the cytoplasm. Crippling bacterial control of Cu(I) sensing, trafficking, and efflux may represent a viable strategy for the development of new antibiotics.

Hydrogen sulfide (H2S) is a toxic molecule and a recently described gasotransmitter in vertebrates whose function in bacteria is not well understood. In this work, we describe the transcriptomic response of the major human pathogen Staphylococcus aureus to quantified changes in levels of cellular organic reactive sulfur species, which are effector molecules involved in H2S signaling. We show that nitroxyl (HNO), a recently described signaling intermediate proposed to originate from the interplay of H2S and nitric oxide, also induces changes in cellular sulfur speciation and transition metal homeostasis, thus linking sulfide homeostasis to an adaptive response to antimicrobial reactive nitrogen species.

The cst operon of the major human pathogen Staphylococcus aureus (S. aureus) is under the transcriptional control of CsoR-like sulfurtransferase repressor (CstR). Expression of this operon is induced by hydrogen sulfide, and two components of the cst operon, cstA and cstB, protect S. aureus from sulfide toxicity. CstA is a three-domain protein, and each domain harbors a single cysteine that is proposed to function in vectorial persulfide shuttling. We show here that single cysteine substitution mutants of CstA fail to protect S. aureus against sulfide toxicity in vivo. The N-terminal domain of CstA exhibits thiosulfate sulfurtransferase (TST; rhodanese) activity, and a Cys66 (34)S-persulfide is formed as a catalytic intermediate in both the presence and absence of the adjacent TusA-like domain using (34)S-SO3(2-) as a substrate. Cysteine persulfides can be trapped on both C66 in CstA(Rhod) and on C66 and C128 in CstA(Rhod-TusA) when incubated with thiosulfate, sodium tetrasulfide (Na2S4), and in situ persulfurated SufS. C66A substitution in CstA(Rhod-TusA) abolishes C128 S-sulfhydration, consistent with directional persulfide shuttling in CstA. Fully reduced CstA(Rhod-TusA) is predominately monomeric, and high resolution tandem mass spectrometry reveals that Cys66 and Cys128 can form a C66-C128 disulfide bond using a number of oxidants, which leads to a significant change in conformation. A competing intermolecular C128-C128' disulfide bond is also formed. Small-angle X-ray scattering measurements and gel filtration chromatography of reduced CstA(Rhod-TusA) reveal an elongated molecule (Rg ≈ 30 Å, 21.6 kDa) where the two domains pack "side-by-side" that likely places Cys66 and Cys128 far apart. These studies are consistent with the low yield of C66-C128 cross-link as a mimic of a persulfide transfer intermediate in CstA, and small, but measurable persulfide transfer from Cys66 to Cys128 within the CstA(Rhod-TusA) with inorganic sulfur donors.

Selective chemical modification of protein side chains coupled with mass spectrometry is often most informative when used to compare residue-specific reactivities in a number of functional states or macromolecular complexes. Herein, we develop ratiometric pulse-chase amidination mass spectrometry (rPAm-MS) as a site-specific probe of lysine reactivities at equilibrium using the Cu(I)-sensing repressor CsoR from B. subtilis as a model system. CsoR in various allosteric states was reacted with S-methylthioacetimidate (SMTA) for pulse time, t, and chased with excess of S-methylthiopropionimidate (SMTP) (Δ=14 amu), quenched and digested with chymotrypsin or Glu-C protease and peptides quantified by high resolution MALDI-TOF mass spectrometry and/or LC-ESI tandem mass spectrometry. We show that the reactivities of individual lysines from peptides containing up to three Lys residues are readily quantified using this method. New insights into operator DNA binding and the Cu(I)-mediated structural transition in the tetrameric copper sensor CsoR are also obtained.