Analysis of genetic requirements and nutrient availability for Staphylococcus aureus growth in cystic fibrosis sputum.

Staphylococcus aureus is one of the most common pathogens isolated from the lungs of people with cystic fibrosis (CF), but little is known about its ability to colonize this niche. We performed a Tn-seq screen to identify genes necessary for S. aureus growth in media prepared from ex vivo CF sputum. We identified 19 genes that were required for growth in all sputum media tested and dozens more that were required for growth in at least one sputum medium. Depleted mutants of interest included insertions in many genes important for surviving metal starvation as well as the primary regulator of cysteine metabolism cymR . To investigate the mechanisms by which these genes contribute to S. aureus growth in sputum, we quantified low-molecular-weight thiols, nutrient transition metals, and the host metal-sequestration protein calprotectin in sputum from 11 individuals with CF. In all samples, the abundance of calprotectin exceeded nutrient metal concentration, explaining the S. aureus requirement for metal-starvation genes. Further, all samples contain potentially toxic quantities of cysteine and sufficient glutathione to satisfy the organic sulfur requirements of S. aureus . Deletion of the cysteine importer genes tcyA and tcyP in the Δ cymR background restored growth to wild-type levels in CF sputum, suggesting that the mechanism by which cymR is required for growth in sputum is to prevent uncontrolled import of cysteine or cystine from this environment. Overall, this work demonstrates that calprotectin and cysteine limit S. aureus growth in CF sputum. IMPORTANCE: Staphylococcus aureus is a major cause of lung infections in people with cystic fibrosis (CF). This work identifies genes required for S. aureus growth in this niche, which represent potential targets for anti-Staphylococcal treatments. We show that genes involved in surviving metal starvation are required for growth in CF sputum. We also found that the primary regulator of cysteine metabolism, CymR, plays a critical role in preventing cysteine intoxication during growth in CF sputum. To support these models, we analyzed sputum from 11 individuals with CF to determine concentrations of calprotectin, nutrient metals, and low-molecular-weight thiols, which have not previously been quantified together in the same samples.

The five homologous CiaR-controlled Ccn sRNAs of Streptococcus pneumoniae modulate Zn-resistance.

Zinc is a vital transition metal for all bacteria; however, elevated intracellular free Zn levels can result in mis-metalation of Mn-dependent enzymes. For Mn-centric bacteria such as Streptococcus pneumoniae that primarily use Mn instead of Fe as an enzyme cofactor, Zn is particularly toxic at high concentrations. Here, we report our identification and characterization of the function of the five homologous, CiaRH-regulated Ccn sRNAs in controlling S. pneumoniae virulence and metal homeostasis. We show that deletion of all five ccn genes (ccnA, ccnB, ccnC, ccnD, and ccnE) from S. pneumoniae strains D39 (serotype 2) and TIGR4 (serotype 4) causes Zn hypersensitivity and an attenuation of virulence in a murine invasive pneumonia model. We provide evidence that bioavailable Zn disproportionately increases in S. pneumoniae strains lacking the five ccn genes. Consistent with a response to Zn intoxication or relatively high intracellular free Zn levels, expression of genes encoding the CzcD Zn exporter and the Mn-independent ribonucleotide reductase, NrdD-NrdG, were increased in the ΔccnABCDE mutant relative to its isogenic ccn+ parent strain. The growth inhibition by Zn that occurs as the result of loss of the ccn genes is rescued by supplementation with Mn or Oxyrase, a reagent that removes dissolved oxygen. Lastly, we found that the Zn-dependent growth inhibition of the ΔccnABCDE strain was not altered by deletion of sodA, whereas the ccn+ ΔsodA strain phenocopied the ΔccnABCDE strain. Overall, our results indicate that the Ccn sRNAs have a crucial role in preventing Zn intoxication in S. pneumoniae.

Coupling of zinc and GTP binding drives G-domain folding in Acinetobacter baumannii ZigA.

COG0523 proteins, also known as nucleotide-dependent metallochaperones, are a poorly understood class of small P-loop G3E GTPases. Multiple family members play critical roles in bacterial pathogen survival during an infection as part of the adaptive response to host-mediated "nutritional immunity." Our understanding of the structure, dynamics, and molecular-level function of COG0523 proteins, apart from the eukaryotic homolog, Zng1, remains in its infancy. Here, we use X-ray absorption spectroscopy to establish that Acinetobacter baumannii (Ab) ZigA coordinates ZnII using all three cysteines derived from the invariant CXCC motif to form an S3(N/O) coordination complex, a feature inconsistent with the ZnII-bound crystal structure of a distantly related COG0523 protein of unknown function from Escherichia coli, EcYjiA. The binding of ZnII and guanine nucleotides is thermodynamically linked in AbZigA, and this linkage is more favorable for the substrate GTP relative to the product GDP. Part of this coupling originates with nucleotide-induced stabilization of the G-domain tertiary structure as revealed by global thermodynamics measurements and hydrogen-deuterium exchange mass spectrometry (HDX-MS). HDX-MS also reveals that the HDX behavior of the G2 (switch 1) loop is highly sensitive to nucleotide status and becomes more exchange labile in the GDP (product)-bound state. Significant long-range perturbation of local stability in both the G-domain and the C-terminal domain define a candidate binding pocket for a client protein that appears sensitive to nucleotide status (GDP versus GTP). We place these new insights into the structure, dynamics, and energetics of intermolecular metal transfer into the context of a model for AbZigA metallochaperone function.

Characterization of the Zinc Uptake Repressor (Zur) from Acinetobacter baumannii.

Bacterial cells tightly regulate the intracellular concentrations of essential transition metal ions by deploying a panel of metal-regulated transcriptional repressors and activators that bind to operator-promoter regions upstream of regulated genes. Like other zinc uptake regulator (Zur) proteins, Acinetobacter baumannii Zur represses transcription of its regulon when ZnII is replete and binds more weakly to DNA when ZnII is limiting. Previous studies established that Zur proteins are homodimeric and harbor at least two metal sites per protomer or four per dimer. CdII X-ray absorption spectroscopy (XAS) of the Cd2Zn2 AbZur metalloderivative with CdII bound to the allosteric sites reveals a S(N/O)3 first coordination shell. Site-directed mutagenesis suggests that H89 and C100 from the N-terminal DNA binding domain and H107 and E122 from the C-terminal dimerization domain comprise the regulatory metal site. KZn for this allosteric site is 6.0 (±2.2) × 1012 M-1 with a functional "division of labor" among the four metal ligands. N-terminal domain ligands H89 and C100 contribute far more to KZn than H107 and E122, while C100S AbZur uniquely fails to bind to DNA tightly as measured by an in vitro transcription assay. The heterotropic allosteric coupling free energy, ΔGc, is negative, consistent with a higher KZn for the AbZur-DNA complex and defining a bioavailable ZnII set-point of ≈6 × 10-14 M. Small-angle X-ray scattering (SAXS) experiments reveal that only the wild-type Zn homodimer undergoes allosteric switching, while the C100S AbZur fails to switch. These data collectively suggest that switching to a high affinity DNA-binding conformation involves a rotation/translation of one protomer relative to the other in a way that is dependent on the integrity of C100. We place these findings in the context of other Zur proteins and Fur family repressors more broadly.

The five homologous CiaR-controlled Ccn sRNAs of Streptococcus pneumoniae modulate Zn-resistance.

Zinc is a vital transition metal for Streptococcus pneumoniae, but is deadly at high concentrations. In S. pneumoniae, elevated intracellular free Zn levels result in mis-metallation of key Mn-dependent metabolic and superoxide detoxifying enzymes resulting in Zn intoxication. Here, we report our identification and characterization of the function of the five homologous, CiaRH-regulated Ccn sRNAs in controlling S. pneumoniae virulence and metal homeostasis. We show that deletion of all five ccn genes (ccnA, ccnB, ccnC, ccnD, and ccnE) from S. pneumoniae strains D39 (serotype 2) and TIGR4 (serotype 4) causes Zn hypersensitivity and an attenuation of virulence in a murine invasive pneumonia model. We provide evidence that bioavailable Zn disproportionately increases in S. pneumoniae strains lacking the five ccn genes. Consistent with a response to Zn intoxication or relatively high intracellular free Zn levels, expression of genes encoding the CzcD Zn exporter and the Mn-independent ribonucleotide reductase, NrdD-NrdG, were increased in the ΔccnABCDE mutant relative to its isogenic ccn+ parent strain. The growth inhibition by Zn that occurs as the result of loss of the ccn genes is rescued by supplementation with Mn or Oxyrase™, a reagent that removes dissolved oxygen. Lastly, we found that the Zn-dependent growth inhibition of the ΔccnABCDE strain was not altered by deletion of sodA, whereas the ccn+ ΔsodA strain phenocopied the ΔccnABCDE strain. Overall, our results indicate that the Ccn sRNAs have a crucial role in preventing Zn intoxication in S. pneumoniae.

The structure of plastocyanin tunes the midpoint potential by restricting axial ligation of the reduced copper ion.

Blue copper proteins are models for illustrating how proteins tune metal properties. Nevertheless, the mechanisms by which the protein controls the metal site remain to be fully elucidated. A hindrance is that the closed shell Cu(I) site is inaccessible to most spectroscopic analyses. Carbon deuterium (C-D) bonds used as vibrational probes afford nonperturbative, selective characterization of the key cysteine and methionine copper ligands in both redox states. The structural integrity of Nostoc plastocyanin was perturbed by disrupting potential hydrogen bonds between loops of the cupredoxin fold via mutagenesis (S9A, N33A, N34A), variably raising the midpoint potential. The C-D vibrations show little change to suggest substantial alteration to the Cu(II) coordination in the oxidized state or in the Cu(I) interaction with the cysteine ligand. They rather indicate, along with visible and NMR spectroscopy, that the methionine ligand distinctly interacts more strongly with the Cu(I) ion, in line with the increases in midpoint potential. Here we show that the protein structure determines the redox properties by restricting the interaction between the methionine ligand and Cu(I) in the reduced state.

Increased intracellular persulfide levels attenuate HlyU-mediated hemolysin transcriptional activation in Vibrio cholerae.

The vertebrate host's immune system and resident commensal bacteria deploy a range of highly reactive small molecules that provide a barrier against infections by microbial pathogens. Gut pathogens, such as Vibrio cholerae, sense and respond to these stressors by modulating the expression of exotoxins that are crucial for colonization. Here, we employ mass spectrometry-based profiling, metabolomics, expression assays, and biophysical approaches to show that transcriptional activation of the hemolysin gene hlyA in V. cholerae is regulated by intracellular forms of sulfur with sulfur-sulfur bonds, termed reactive sulfur species (RSS). We first present a comprehensive sequence similarity network analysis of the arsenic repressor superfamily of transcriptional regulators, where RSS and hydrogen peroxide sensors segregate into distinct clusters of sequences. We show that HlyU, transcriptional activator of hlyA in V. cholerae, belongs to the RSS-sensing cluster and readily reacts with organic persulfides, showing no reactivity or DNA dissociation following treatment with glutathione disulfide or hydrogen peroxide. Surprisingly, in V. cholerae cell cultures, both sulfide and peroxide treatment downregulate HlyU-dependent transcriptional activation of hlyA. However, RSS metabolite profiling shows that both sulfide and peroxide treatment raise the endogenous inorganic sulfide and disulfide levels to a similar extent, accounting for this crosstalk, and confirming that V. cholerae attenuates HlyU-mediated activation of hlyA in a specific response to intracellular RSS. These findings provide new evidence that gut pathogens may harness RSS-sensing as an evolutionary adaptation that allows them to overcome the gut inflammatory response by modulating the expression of exotoxins.

Sensing and regulation of reactive sulfur species (RSS) in bacteria.

The infected host deploys generalized oxidative stress caused by small inorganic reactive molecules as antibacterial weapons. An emerging consensus is that hydrogen sulfide (H2S) and forms of sulfur with sulfur-sulfur bonds termed reactive sulfur species (RSS) provide protection against oxidative stressors and antibiotics, as antioxidants. Here, we review our current understanding of RSS chemistry and its impact on bacterial physiology. We start by describing the basic chemistry of these reactive species and the experimental approaches developed to detect them in cells. We highlight the role of thiol persulfides in H2S-signaling and discuss three structural classes of ubiquitous RSS sensors that tightly regulate cellular H2S/RSS levels in bacteria, with a specific focus on the chemical specificity of these sensors.

Polysulfide metabolizing enzymes influence SqrR-mediated sulfide-induced transcription by impacting intracellular polysulfide dynamics.

Sulfide plays essential roles in controlling various physiological activities in almost all organisms. Although recent evidence has demonstrated that sulfide is endogenously generated and metabolized into polysulfides inside the cells, the relationship between polysulfide metabolism and polysulfide-sensing mechanisms is not well understood. To better define this interplay between polysulfide metabolism and sensing in cells, we investigated the role of polysulfide-metabolizing enzymes such as sulfide:quinone oxidoreductase (SQR) on the temporal dynamics of cellular polysulfide speciation and on the transcriptional regulation by the persulfide-responsive transcription factor SqrR in Rhodobacter capsulatus. We show that disruption of the sqr gene resulted in the loss of SqrR repression by exogenous sulfide at longer culture times, which impacts the speciation of intracellular polysulfides of Δsqr vs. wild-type strains. Both the attenuated response of SqrR and the change in polysulfide dynamics of the Δsqr strain is fully reversed by the addition to cells of cystine-derived polysulfides, but not by glutathione disulfide (GSSG)-derived polysulfides. Furthermore, cysteine persulfide (CysSSH) yields a higher rate of oxidation of SqrR relative to glutathione persulfide (GSSH), which leads to DNA dissociation in vitro. The oxidation of SqrR was confirmed by a mass spectrometry-based kinetic profiling strategy that showed distinct polysulfide-crosslinked products obtained with CysSSH vs. GSSH. Taken together, these results establish a novel association between the metabolism of polysulfides and the mechanisms for polysulfide sensing inside the cells.

Increased intracellular persulfide levels attenuate HlyU-mediated hemolysin transcriptional activation in Vibrio cholerae.

The vertebrate host’s immune system and resident commensal bacteria deploy a range of highly reactive small molecules that provide a barrier against infections by microbial pathogens. Gut pathogens, such as Vibrio cholerae , sense and respond to these stressors by modulating the expression of exotoxins that are crucial for colonization. Here, we employ mass-spectrometry-based profiling, metabolomics, expression assays and biophysical approaches to show that transcriptional activation of the hemolysin gene hlyA in V. cholerae is regulated by intracellular reactive sulfur species (RSS), specifically sulfane sulfur. We first present a comprehensive sequence similarity network analysis of the arsenic repressor (ArsR) superfamily of transcriptional regulators where RSS and reactive oxygen species (ROS) sensors segregate into distinct clusters. We show that HlyU, transcriptional activator of hlyA in V. cholerae , belongs to the RSS-sensing cluster and readily reacts with organic persulfides, showing no reactivity and remaining DNA-bound following treatment with various ROS in vitro, including H 2 O 2 . Surprisingly, in V. cholerae cell cultures, both sulfide and peroxide treatment downregulate HlyU-dependent transcriptional activation of hlyA . However, RSS metabolite profiling shows that both sulfide and peroxide treatment raise the endogenous inorganic sulfide and disulfide levels to a similar extent, accounting for this crosstalk, and confirming that V. cholerae attenuates HlyU-mediated activation of hlyA in a specific response to intracellular RSS. These findings provide new evidence that gut pathogens may harness RSS-sensing as an evolutionary adaptation that allows them to overcome the gut inflammatory response by modulating the expression of exotoxins.

Discovery and structure of a widespread bacterial ABC transporter specific for ergothioneine.

L-Ergothioneine (ET), the 2-thioimidazole derivative of trimethylhistidine, is biosynthesized by select fungi and bacteria, notably Mycobacterium tuberculosis, and functions as a scavenger of reactive oxygen species. The extent to which ET broadly functions in bacterial cells unable to synthesize it is unknown. Here we show that spd_1642-1643 in Streptococcus pneumoniae, a Gram-positive respiratory pathogen, encodes an ET uptake ATP-binding cassette (ABC) transporter, designated EgtU. The solute binding domain (SBD) of EgtU, EgtUC, binds ET with high affinity and exquisite specificity in a cleft between the two subdomains, with cation-π interactions engaging the betaine moiety and a network of water molecules that surround the thioimidazole ring. EgtU is highly conserved among known quaternary amine compound-specific transporters and widely distributed in Firmicutes, including the human pathogens Listeria monocytogenes, as BilEB, Enterococcus faecalis and Staphylococcus aureus. ET increases the chemical diversity of the low molecular weight thiol pool in Gram-positive human pathogens and may contribute to antioxidant defenses in the infected host.

Metal retention and replacement in QueD2 protect queuosine-tRNA biosynthesis in metal-starved Acinetobacter baumannii.

In response to bacterial infection, the vertebrate host employs the metal-sequestering protein calprotectin (CP) to withhold essential transition metals, notably Zn(II), to inhibit bacterial growth. Previous studies of the impact of CP-imposed transition-metal starvation in A. baumannii identified two enzymes in the de novo biosynthesis pathway of queuosine-transfer ribonucleic acid (Q-tRNA) that become cellularly abundant, one of which is QueD2, a 6-carboxy-5,6,7,8-tetrahydropterin (6-CPH4) synthase that catalyzes the initial, committed step of the pathway. Here, we show that CP strongly disrupts Q incorporation into tRNA. As such, we compare the AbQueD2 "low-zinc" paralog with a housekeeping, obligatory Zn(II)-dependent enzyme QueD. The crystallographic structure of Zn(II)-bound AbQueD2 reveals a distinct catalytic site coordination sphere and assembly state relative to QueD and possesses a dynamic loop, immediately adjacent to the catalytic site that coordinates a second Zn(II) in the structure. One of these loop-coordinating residues is an invariant Cys18, that protects QueD2 from dissociation of the catalytic Zn(II) while maintaining flux through the Q-tRNA biosynthesis pathway in cells. We propose a "metal retention" model where Cys18 introduces coordinative plasticity into the catalytic site which slows metal release, while also enhancing the metal promiscuity such that Fe(II) becomes an active cofactor. These studies reveal a complex, multipronged evolutionary adaptation to cellular Zn(II) limitation in a key Zn(II) metalloenzyme in an important human pathogen.

Metabolic and Structural Insights into Hydrogen Sulfide Mis-Regulation in Enterococcus faecalis.

Hydrogen sulfide (H2S) is implicated as a cytoprotective agent that bacteria employ in response to host-induced stressors, such as oxidative stress and antibiotics. The physiological benefits often attributed to H2S, however, are likely a result of downstream, more oxidized forms of sulfur, collectively termed reactive sulfur species (RSS) and including the organic persulfide (RSSH). Here, we investigated the metabolic response of the commensal gut microorganism Enterococcus faecalis to exogenous Na2S as a proxy for H2S/RSS toxicity. We found that exogenous sulfide increases protein abundance for enzymes responsible for the biosynthesis of coenzyme A (CoA). Proteome S-sulfuration (persulfidation), a posttranslational modification implicated in H2S signal transduction, is also widespread in this organism and is significantly elevated by exogenous sulfide in CstR, the RSS sensor, coenzyme A persulfide (CoASSH) reductase (CoAPR) and enzymes associated with de novo fatty acid biosynthesis and acetyl-CoA synthesis. Exogenous sulfide significantly impacts the speciation of fatty acids as well as cellular concentrations of acetyl-CoA, suggesting that protein persulfidation may impact flux through these pathways. Indeed, CoASSH is an inhibitor of E. faecalis phosphotransacetylase (Pta), suggesting that an important metabolic consequence of increased levels of H2S/RSS may be over-persulfidation of this key metabolite, which, in turn, inhibits CoA and acyl-CoA-utilizing enzymes. Our 2.05 Å crystallographic structure of CoA-bound CoAPR provides new structural insights into CoASSH clearance in E. faecalis.

Protocol for using organic persulfides to measure the chemical reactivity of persulfide sensors.

Hydrogen sulfide (H2S) and downstream reactive sulfur species (RSS), including organic persulfides, protect bacterial cells against diverse oxidative stressors. Specialized dithiol-based transcriptional repressors sense persulfides directly to control cellular H2S/RSS and avoid toxicity. Here, we present a protocol to quantify the kinetics of chemical reactivity of cysteines in two bacterial persulfide sensors toward cysteine persulfide and glutathione persulfide, with a LC-ESI-MS analysis that results in a kinetic model. This protocol has potential applications to other cysteine-containing proteins and oxidants. For complete details on the use and execution of this protocol, please refer to Fakhoury et al. (2021) and Capdevila et al. (2021).

Zn-regulated GTPase metalloprotein activator 1 modulates vertebrate zinc homeostasis.

Zinc (Zn) is an essential micronutrient and cofactor for up to 10% of proteins in living organisms. During Zn limitation, specialized enzymes called metallochaperones are predicted to allocate Zn to specific metalloproteins. This function has been putatively assigned to G3E GTPase COG0523 proteins, yet no Zn metallochaperone has been experimentally identified in any organism. Here, we functionally characterize a family of COG0523 proteins that is conserved across vertebrates. We identify Zn metalloprotease methionine aminopeptidase 1 (METAP1) as a COG0523 client, leading to the redesignation of this group of COG0523 proteins as the Zn-regulated GTPase metalloprotein activator (ZNG1) family. Using biochemical, structural, genetic, and pharmacological approaches across evolutionarily divergent models, including zebrafish and mice, we demonstrate a critical role for ZNG1 proteins in regulating cellular Zn homeostasis. Collectively, these data reveal the existence of a family of Zn metallochaperones and assign ZNG1 an important role for intracellular Zn trafficking.

SifR is an Rrf2-family quinone sensor associated with catechol iron uptake in Streptococcus pneumoniae D39.

Streptococcus pneumoniae (pneumococcus) is a Gram-positive commensal and human respiratory pathogen. How this bacterium satisfies its nutritional iron (Fe) requirement in the context of endogenously produced hydrogen peroxide is not well understood. Here, we characterize a novel virulence-associated Rrf2-family transcriptional repressor that we term SifR (streptococcal IscR-like family transcriptional repressor) encoded by spd_1448 and conserved in Streptococci. Global transcriptomic analysis of a ΔsifR strain defines the SifR regulon as genes encoding a candidate catechol dioxygenase CatE, an uncharacterized oxidoreductase YwnB, a candidate flavin-dependent ferric reductase YhdA, a candidate heme-based ferric reductase domain-containing protein and the Piu (pneumococcus iron uptake) Fe transporter (piuBCDA). Previous work established that membrane-anchored PiuA binds FeIII-bis-catechol or monocatechol complexes with high affinity, including the human catecholamine stress hormone, norepinephrine. We demonstrate that SifR senses quinone via a single conserved cysteine that represses its regulon when in the reduced form. Upon reaction with catechol-derived quinones, we show that SifR dissociates from the DNA leading to regulon derepression, allowing the pneumococcus to access a catechol-derived source of Fe while minimizing reactive electrophile stress induced by quinones. Consistent with this model, we show that CatE is an FeII-dependent 2,3-catechol dioxygenase with broad substrate specificity, YwnB is an NAD(P)H-dependent quinone reductase capable of reducing the oxidized and cyclized norepinephrine, adrenochrome, and YhdA is capable of reducing a number of FeIII complexes, including PiuA-binding transport substrates. These findings are consistent with a model where FeIII-catechol complexes serve as significant nutritional Fe sources in the host.

Bacterial hydrophilins promote pathogen desiccation tolerance.

Acinetobacter baumannii is a leading cause of hospital-acquired infections, where outbreaks are driven by its ability to persist on surfaces in a desiccated state. Here, we show that A. baumannii causes more virulent pneumonia following desiccation and profile the genetic requirements for desiccation. We find that desiccation tolerance is enhanced upon the disruption of Lon protease, which targets unfolded and aggregated proteins for degradation. Notably, two bacterial hydrophilins, DtpA and DtpB, are transcriptionally upregulated in Δlon via the two-component regulator, BfmR. These proteins, both hydrophilic and intrinsically disordered, promote desiccation tolerance in A. baumannii. Additionally, recombinant DtpA protects purified enzymes from inactivation and improves the desiccation tolerance of a probiotic bacterium when heterologously expressed. These results demonstrate a connection between environmental persistence and pathogenicity in A. baumannii, provide insight into the mechanisms of extreme desiccation tolerance, and reveal potential applications for bacterial hydrophilins in the preservation of protein- and live bacteria-based pharmaceuticals.

Functional asymmetry and chemical reactivity of CsoR family persulfide sensors.

CstR is a persulfide-sensing member of the functionally diverse copper-sensitive operon repressor (CsoR) superfamily. While CstR regulates the bacterial response to hydrogen sulfide (H2S) and more oxidized reactive sulfur species (RSS) in Gram-positive pathogens, other dithiol-containing CsoR proteins respond to host derived Cu(I) toxicity, sometimes in the same bacterial cytoplasm, but without regulatory crosstalk in cells. It is not clear what prevents this crosstalk, nor the extent to which RSS sensors exhibit specificity over other oxidants. Here, we report a sequence similarity network (SSN) analysis of the entire CsoR superfamily, which together with the first crystallographic structure of a CstR and comprehensive mass spectrometry-based kinetic profiling experiments, reveal new insights into the molecular basis of RSS specificity in CstRs. We find that the more N-terminal cysteine is the attacking Cys in CstR and is far more nucleophilic than in a CsoR. Moreover, our CstR crystal structure is markedly asymmetric and chemical reactivity experiments reveal the functional impact of this asymmetry. Substitution of the Asn wedge between the resolving and the attacking thiol with Ala significantly decreases asymmetry in the crystal structure and markedly impacts the distribution of species, despite adopting the same global structure as the parent repressor. Companion NMR, SAXS and molecular dynamics simulations reveal that the structural and functional asymmetry can be traced to fast internal dynamics of the tetramer. Furthermore, this asymmetry is preserved in all CstRs and with all oxidants tested, giving rise to markedly distinct distributions of crosslinked products. Our exploration of the sequence, structural, and kinetic features that determine oxidant-specificity suggest that the product distribution upon RSS exposure is determined by internal flexibility.

COG0523 proteins: a functionally diverse family of transition metal-regulated G3E P-loop GTP hydrolases from bacteria to man.

Transition metal homeostasis ensures that cells and organisms obtain sufficient metal to meet cellular demand while dispensing with any excess so as to avoid toxicity. In bacteria, zinc restriction induces the expression of one or more Zur (zinc-uptake repressor)-regulated Cluster of Orthologous Groups (COG) COG0523 proteins. COG0523 proteins encompass a poorly understood sub-family of G3E P-loop small GTPases, others of which are known to function as metallochaperones in the maturation of cobalamin (CoII) and NiII cofactor-containing metalloenzymes. Here, we use genomic enzymology tools to functionally analyse over 80 000 sequences that are evolutionarily related to Acinetobacter baumannii ZigA (Zur-inducible GTPase), a COG0523 protein and candidate zinc metallochaperone. These sequences segregate into distinct sequence similarity network (SSN) clusters, exemplified by the ZnII-Zur-regulated and FeIII-nitrile hydratase activator CxCC (C, Cys; X, any amino acid)-containing COG0523 proteins (SSN cluster 1), NiII-UreG (clusters 2, 8), CoII-CobW (cluster 4), and NiII-HypB (cluster 5). A total of five large clusters that comprise ≈ 25% of all sequences, including cluster 3 which harbors the only structurally characterized COG0523 protein, Escherichia coli YjiA, and many uncharacterized eukaryotic COG0523 proteins. We also establish that mycobacterial-specific protein Y (Mpy) recruitment factor (Mrf), which promotes ribosome hibernation in actinomycetes under conditions of ZnII starvation, segregates into a fifth SSN cluster (cluster 17). Mrf is a COG0523 paralog that lacks all GTP-binding determinants as well as the ZnII-coordinating Cys found in CxCC-containing COG0523 proteins. On the basis of this analysis, we discuss new perspectives on the COG0523 proteins as cellular reporters of widespread nutrient stress induced by ZnII limitation.

Proteomics Profiling of S-sulfurated Proteins in Acinetobacter baumannii.

Hydrogen sulfide (H2S) is emerging as an important modulator in bacterial cytoprotection against the host immune response in infected animals, which may well be attributed to downstream highly oxidized sulfur species, termed reactive sulfur species (RSS), derived from H2S. One mechanism by which H2S/RSS may signal in the cell is through proteome S-sulfuration (persulfidation), which is the conversion of protein thiols (-SH) to protein persulfides (-SSH). While several analytical methods have been developed to profile sites of protein persulfidation, few have been applied to bacterial cells. The analytical workflow presented here was recently utilized to profile proteome persulfidation in the major human pathogen Acinetobacter baumannii treated with an exogenous sulfide source, Na2S. The data obtained using this protocol allow quantitation of the change in persulfidation status of each cysteine in the proteome normalized to the change in protein abundance, thus identifying sites of persulfidation that may constitute regulatory modifications. These can be validated using follow-up biochemical studies.