Sulfide Homeostasis and Nitroxyl Intersect via Formation of Reactive Sulfur Species in .

is a commensal human pathogen and a major cause of nosocomial infections. As gaseous signaling molecules, endogenous hydrogen sulfide (HS) and nitric oxide (NO·) protect from antibiotic stress synergistically, which we propose involves the intermediacy of nitroxyl (HNO). Here, we examine the effect of exogenous sulfide and HNO on the transcriptome and the formation of low-molecular-weight (LMW) thiol persulfides of bacillithiol, cysteine, and coenzyme A as representative of reactive sulfur species (RSS) in wild-type and Δ strains of .

Resolution of Stepwise Cooperativities of Copper Binding by the Homotetrameric Copper-Sensitive Operon Repressor (CsoR): Impact on Structure and Stability.

The cooperativity of ligand binding is central to biological regulation and new approaches are needed to quantify these allosteric relationships. Herein, we exploit a suite of mass spectrometry (MS) experiments to provide novel insights into homotropic Cu-binding cooperativity, gas-phase stabilities and conformational ensembles of the D2 -symmetric, homotetrameric copper-sensitive operon repressor (CsoR) as a function of Cu(I) ligation state. Cu(I) binding is overall positively cooperative, but is characterized by distinct ligation state-specific cooperativities.

Electrostatic occlusion and quaternary structural ion pairing are key determinants of Cu(I)-mediated allostery in the copper-sensing operon repressor (CsoR).

The copper-sensing operon repressor (CsoR) is an all-α-helical disc-shaped D2-symmetric homotetramer that forms a 2:1 tetramer/DNA operator complex and represses the expression of copper-resistance genes in a number of bacteria. A previous bioinformatics analysis of CsoR-family repressors distributes Cu(I)-sensing CsoRs in four of seven distinct clades on the basis of global sequence similarity. In this work, we define energetically important determinants of DNA binding in the apo-state (ΔΔGbind), and for allosteric negative coupling of Cu(I) binding to DNA binding (ΔΔGc) in a model clade IV CsoR from Geobacillus thermodenitrificans (Gt) of known structure, by selectively targeting for mutagenesis those charged residues uniquely conserved in clade IV CsoRs.

Conformational analysis and chemical reactivity of the multidomain sulfurtransferase, Staphylococcus aureus CstA.

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.

Copper transport and trafficking at the host-bacterial pathogen interface.

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.

Cu(I)-mediated allosteric switching in a copper-sensing operon repressor (CsoR).

The copper-sensing operon repressor (CsoR) is representative of a major Cu(I)-sensing family of bacterial metalloregulatory proteins that has evolved to prevent cytoplasmic copper toxicity. It is unknown how Cu(I) binding to tetrameric CsoRs mediates transcriptional derepression of copper resistance genes. 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.

Solution structure of Mycobacterium tuberculosis NmtR in the apo state: insights into Ni(II)-mediated allostery.

Mycobacterium tuberculosis is an obligate human respiratory pathogen that encodes approximately 10 arsenic repressor (ArsR) family regulatory proteins that allow the organism to respond to a wide range of changes in its immediate microenvironment. How individual ArsR repressors have evolved to respond to selective stimuli is of intrinsic interest. The Ni(II)/Co(II)-specific repressor NmtR and related actinomycete nickel sensors harbor a conserved N-terminal α-NH(2)-Gly2-His3-Gly4 sequence.

Ratiometric pulse-chase amidination mass spectrometry as a probe of biomolecular complex formation.

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 Bacillus subtilis as a model system. CsoR in various allosteric states was reacted with S-methyl thioacetimidate (SMTA) for pulse time, t, and chased with excess of S-methyl thiopropionimidate (SMTP) (Δ = 14 amu), quenched and digested with chymotrypsin or Glu-C protease, and peptides were quantified by high-resolution matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) mass spectrometry and/or liquid chromatography electrospray ionization tandem mass spectrometry (LC-ESI-MS/MS).