The Bacillus subtilis cell envelope stress-inducible ytpAB operon modulates membrane properties and contributes to bacitracin resistance.

Antibiotics that inhibit peptidoglycan synthesis trigger the activation of both specific and general protective responses. σM responds to diverse antibiotics that inhibit cell wall synthesis. Here, we demonstrate that cell wall-inhibiting drugs, such as bacitracin and cefuroxime, induce the σM-dependent ytpAB operon. YtpA is a predicted hydrolase previously proposed to generate the putative lysophospholipid antibiotic bacilysocin (lysophosphatidylglycerol), and YtpB is the branchpoint enzyme for the synthesis of membrane-localized C35 terpenoids. Using targeted lipidomics, we reveal that YtpA is not required for the production of lysophosphatidylglycerol. Nevertheless, ytpA was critical for growth in a mutant strain defective for homeoviscous adaptation due to a lack of genes for the synthesis of branched chain fatty acids and the Des phospholipid desaturase. Consistently, overexpression of ytpA increased membrane fluidity as monitored by fluorescence anisotropy. The ytpA gene contributes to bacitracin resistance in mutants additionally lacking the bceAB or bcrC genes, which directly mediate bacitracin resistance. These epistatic interactions support a model in which σM-dependent induction of the ytpAB operon helps cells tolerate bacitracin stress, either by facilitating the flipping of the undecaprenyl phosphate carrier lipid or by impacting the assembly or function of membrane-associated complexes involved in cell wall homeostasis.IMPORTANCEPeptidoglycan synthesis inhibitors include some of our most important antibiotics. In Bacillus subtilis, peptidoglycan synthesis inhibitors induce the σM regulon, which is critical for intrinsic antibiotic resistance. The σM-dependent ytpAB operon encodes a predicted hydrolase (YtpA) and the enzyme that initiates the synthesis of C35 terpenoids (YtpB). Our results suggest that YtpA is critical in cells defective in homeoviscous adaptation. Furthermore, we find that YtpA functions cooperatively with the BceAB and BcrC proteins in conferring intrinsic resistance to bacitracin, a peptide antibiotic that binds tightly to the undecaprenyl-pyrophosphate lipid carrier that sustains peptidoglycan synthesis.

The Bacillus subtilis yqgC-sodA operon protects magnesium-dependent enzymes by supporting manganese efflux.

Microbes encounter a myriad of stresses during their life cycle. Dysregulation of metal ion homeostasis is increasingly recognized as a key factor in host-microbe interactions. Bacterial metal ion homeostasis is tightly regulated by dedicated metalloregulators that control uptake, sequestration, trafficking, and efflux. Here, we demonstrate that deletion of the Bacillus subtilis yqgC-sodA (YS) complex operon, but not deletion of the individual genes, causes hypersensitivity to manganese (Mn). YqgC is an integral membrane protein of unknown function and SodA is a Mn-dependent superoxide dismutase (MnSOD). The YS strain has reduced expression of two Mn efflux proteins, MneP and MneS, consistent with the observed Mn sensitivity. The YS strain accumulated high levels of Mn, had increased reactive radical species (RRS), and had broad metabolic alterations that can be partially explained by the inhibition of Mg-dependent enzymes. Although the YS operon deletion strain and an efflux-deficient mneP mneS double mutant both accumulate Mn and have similar metabolic perturbations they also display phenotypic differences. Several mutations that suppressed Mn intoxication of the mneP mneS efflux mutant did not benefit the YS mutant. Further, Mn intoxication in the YS mutant, but not the mneP mneS strain, was alleviated by expression of Mg-dependent, chorismate-utilizing enzymes of the menaquinone, siderophore, and tryptophan (MST) family. Therefore, despite their phenotypic similarities, the Mn sensitivity in the mneP mneS and the yqgC-sodA deletion mutants results from distinct enzymatic vulnerabilities.

TerC proteins function during protein secretion to metalate exoenzymes.

Cytosolic metalloenzymes acquire metals from buffered intracellular pools. How exported metalloenzymes are appropriately metalated is less clear. We provide evidence that TerC family proteins function in metalation of enzymes during export through the general secretion (Sec-dependent) pathway. Bacillus subtilis strains lacking MeeF(YceF) and MeeY(YkoY) have a reduced capacity for protein export and a greatly reduced level of manganese (Mn) in the secreted proteome. MeeF and MeeY copurify with proteins of the general secretory pathway, and in their absence the FtsH membrane protease is essential for viability. MeeF and MeeY are also required for efficient function of the Mn2+-dependent lipoteichoic acid synthase (LtaS), a membrane-localized enzyme with an extracytoplasmic active site. Thus, MeeF and MeeY, representative of the widely conserved TerC family of membrane transporters, function in the co-translocational metalation of Mn2+-dependent membrane and extracellular enzymes.

TerC Proteins Function During Protein Secretion to Metalate Exoenzymes.

Cytosolic metalloenzymes acquire metals from buffered intracellular pools. How exported metalloenzymes are appropriately metalated is less clear. We provide evidence that TerC family proteins function in metalation of enzymes during export through the general secretion (Sec-dependent) pathway. Bacillus subtilis strains lacking MeeF(YceF) and MeeY(YkoY) have a reduced capacity for protein export and a greatly reduced level of manganese (Mn) in the secreted proteome. MeeF and MeeY copurify with proteins of the general secretory pathway, and in their absence the FtsH membrane protease is essential for viability. MeeF and MeeY are also required for efficient function of the Mn2+-dependent lipoteichoic acid synthase (LtaS), a membrane-localized enzyme with an extracytoplasmic active site. Thus, MeeF and MeeY, representative of the widely conserved TerC family of membrane transporters, function in the co-translocational metalation of Mn2+-dependent membrane and extracellular enzymes.

A Decrease in Fatty Acid Synthesis Rescues Cells with Limited Peptidoglycan Synthesis Capacity.

Proper synthesis and maintenance of a multilayered cell envelope are critical for bacterial fitness. However, whether mechanisms exist to coordinate synthesis of the membrane and peptidoglycan layers is unclear. In Bacillus subtilis, synthesis of peptidoglycan (PG) during cell elongation is mediated by an elongasome complex acting in concert with class A penicillin-binding proteins (aPBPs). We previously described mutant strains limited in their capacity for PG synthesis due to a loss of aPBPs and an inability to compensate by upregulation of elongasome function. Growth of these PG-limited cells can be restored by suppressor mutations predicted to decrease membrane synthesis. One suppressor mutation leads to an altered function repressor, FapR*, that functions as a super-repressor and leads to decreased transcription of fatty acid synthesis (FAS) genes. Consistent with fatty acid limitation mitigating cell wall synthesis defects, inhibition of FAS by cerulenin also restored growth of PG-limited cells. Moreover, cerulenin can counteract the inhibitory effect of ß-lactams in some strains. These results imply that limiting PG synthesis results in impaired growth, in part, due to an imbalance of PG and cell membrane synthesis and that B. subtilis lacks a robust physiological mechanism to reduce membrane synthesis when PG synthesis is impaired. IMPORTANCE Understanding how a bacterium coordinates cell envelope synthesis is essential to fully appreciate how bacteria grow, divide, and resist cell envelope stresses, such as ß-lactam antibiotics. Balanced synthesis of the peptidoglycan cell wall and the cell membrane is critical for cells to maintain shape and turgor pressure and to resist external cell envelope threats. Using Bacillus subtilis, we show that cells deficient in peptidoglycan synthesis can be rescued by compensatory mutations that decrease the synthesis of fatty acids. Further, we show that inhibiting fatty acid synthesis with cerulenin is sufficient to restore growth of cells deficient in peptidoglycan synthesis. Understanding the coordination of cell wall and membrane synthesis may provide insights relevant to antimicrobial treatment.

A model industrial workhorse: Bacillus subtilis strain 168 and its genome after a quarter of a century.

The vast majority of genomic sequences are automatically annotated using various software programs. The accuracy of these annotations depends heavily on the very few manual annotation efforts that combine verified experimental data with genomic sequences from model organisms. Here, we summarize the updated functional annotation of Bacillus subtilis strain 168, a quarter century after its genome sequence was first made public. Since the last such effort 5 years ago, 1168 genetic functions have been updated, allowing the construction of a new metabolic model of this organism of environmental and industrial interest. The emphasis in this review is on new metabolic insights, the role of metals in metabolism and macromolecule biosynthesis, functions involved in biofilm formation, features controlling cell growth, and finally, protein agents that allow class discrimination, thus allowing maintenance management, and accuracy of all cell processes. New 'genomic objects' and an extensive updated literature review have been included for the sequence, now available at the International Nucleotide Sequence Database Collaboration (INSDC: AccNum AL009126.4).

Meddling with Metal Sensors: Fur-Family Proteins as Signaling Hubs.

The ferric uptake regulator (Fur) protein is the founding member of the FUR superfamily of metalloregulatory proteins that control metal homeostasis in bacteria. FUR proteins regulate metal homeostasis in response to the binding of iron (Fur), zinc (Zur), manganese (Mur), or nickel (Nur). FUR family proteins are generally dimers in solution, but the DNA-bound complex can involve a single dimer, a dimer-of-dimers, or an extended array of bound protein. Elevated FUR levels due to changes in cell physiology increase DNA occupancy and may also kinetically facilitate protein dissociation. Interactions between FUR proteins and other regulators are commonplace, often including cooperative and competitive DNA-binding interactions within the regulatory region. Further, there are many emerging examples of allosteric regulators that interact directly with FUR family proteins. Here, we focus on newly uncovered examples of allosteric regulation by diverse Fur antagonists (Escherichia coli YdiV/SlyD, Salmonella enterica EIIANtr, Vibrio parahaemolyticus FcrX, Acinetobacter baumannii BlsA, Bacillus subtilis YlaN, and Pseudomonas aeruginosa PacT) as well as one Zur antagonist (Mycobacterium bovis CmtR). Small molecules and metal complexes may also serve as regulatory ligands, with examples including heme binding to Bradyrhizobium japonicum Irr and 2-oxoglutarate binding to Anabaena FurA. How these protein-protein and protein-ligand interactions act in conjunction with regulatory metal ions to facilitate signal integration is an active area of investigation.

TerC Proteins Function During Protein Secretion to Metalate Exoenzymes.

Cytosolic metalloenzymes acquire metals from buffered intracellular pools. How exported metalloenzymes are appropriately metalated is less clear. We provide evidence that TerC family proteins function in metalation of enzymes during export through the general secretion (Sec-dependent) pathway. Bacillus subtilis strains lacking MeeF(YceF) and MeeY(YkoY) have a reduced capacity for protein export and a greatly reduced level of manganese (Mn) in the secreted proteome. MeeF and MeeY copurify with proteins of the general secretory pathway, and in their absence the FtsH membrane protease is essential for viability. MeeF and MeeY are also required for efficient function of the Mn 2+ -dependent lipoteichoic acid synthase (LtaS), a membrane-localized enzyme with an extracytoplasmic active site. Thus, MeeF and MeeY, representative of the widely conserved TerC family of membrane transporters, function in the co-translocational metalation of Mn 2+ -dependent membrane and extracellular enzymes.

The elements of life: A biocentric tour of the periodic table.

Living systems are built from a small subset of the atomic elements, including the bulk macronutrients (C,H,N,O,P,S) and ions (Mg,K,Na,Ca) together with a small but variable set of trace elements (micronutrients). Here, we provide a global survey of how chemical elements contribute to life. We define five classes of elements: those that are (i) essential for all life, (ii) essential for many organisms in all three domains of life, (iii) essential or beneficial for many organisms in at least one domain, (iv) beneficial to at least some species, and (v) of no known beneficial use. The ability of cells to sustain life when individual elements are absent or limiting relies on complex physiological and evolutionary mechanisms (elemental economy). This survey of elemental use across the tree of life is encapsulated in a web-based, interactive periodic table that summarizes the roles chemical elements in biology and highlights corresponding mechanisms of elemental economy.

Mutations in rpoB That Confer Rifampicin Resistance Can Alter Levels of Peptidoglycan Precursors and Affect ß-Lactam Susceptibility.

Bacteria can adapt to stressful conditions through mutations affecting the RNA polymerase core subunits that lead to beneficial changes in transcription. In response to selection with rifampicin (RIF), mutations arise in the RIF resistance-determining region (RRDR) of rpoB that reduce antibiotic binding. These changes can also alter transcription and thereby have pleiotropic effects on bacterial fitness. Here, we studied the evolution of resistance in Bacillus subtilis to the synergistic combination of RIF and the ß-lactam cefuroxime (CEF). Two independent evolution experiments led to the recovery of a single rpoB allele (S487L) that was able to confer resistance to RIF and CEF through a single mutation. Two other common RRDR mutations made the cells 32 times more sensitive to CEF (H482Y) or led to only modest CEF resistance (Q469R). The diverse effects of these three mutations on CEF resistance are correlated with differences in the expression of peptidoglycan (PG) synthesis genes and in the levels of two metabolites crucial in regulating PG synthesis, glucosamine-6-phosphate (GlcN-6-P) and UDP-N-acetylglucosamine (UDP-GlcNAc). We conclude that RRDR mutations can have widely varying effects on pathways important for cell wall biosynthesis, and this may restrict the spectrum of mutations that arise during combination therapy. IMPORTANCE Rifampicin (RIF) is one of the most valued drugs in the treatment of tuberculosis. TB treatment relies on a combination therapy and for multidrug-resistant strains may include ß-lactams. Mutations in rpoB present a common route for emergence of resistance to RIF. In this study, using B. subtilis as a model, we evaluate the emergence of resistance for the synergistic combination of RIF and the ß-lactam cefuroxime (CEF). One clinically relevant rpoB mutation conferred resistance to both RIF and CEF, whereas one other increased CEF sensitivity. We were able to link these CEF sensitivity phenotypes to accumulation of UDP-N-acetylglucosamine (UDP-GlcNAc), which feedback regulates GlmS activity and thereby peptidoglycan synthesis. Further, we found that higher CEF concentrations precluded the emergence of high RIF resistance. Collectively, these results suggest that multidrug treatment regimens may limit the available pathways for the evolution of antibiotic resistance.

Iron homeostasis in Bacillus subtilis relies on three differentially expressed efflux systems.

In Bacillus subtilis, iron homeostasis is maintained by the ferric uptake regulator (Fur) and manganese homeostasis relies on the manganese transport regulator (MntR). Both Fur and MntR function as bi-functional metalloregulators that repress import and activate metal ion efflux systems. The ferrous iron efflux ATPase, PfeT, is derepressed by hydrogen peroxide (H2O2) as sensed by PerR and induced by iron as sensed by Fur. Mutants lacking PfeT are sensitive to iron intoxication. Here, we show that mntR mutants are also iron-sensitive, largely due to decreased expression of the MntR-activated MneP and MneS cation diffusion facilitator (CDF) proteins previously defined for their role in Mn2+ export. The ability of MneP and MneS to export iron is apparent even when their expression is not induced by Mn2+. Our results demonstrate that PfeT, MneP and MneS each contribute to iron homeostasis, and a triple mutant lacking all three is more iron-sensitive than any single mutant. We further show that sensitivity to H2O2 does not correlate with iron sensitivity. For example, an mntR mutant is H2O2-sensitive due to elevated Mn(II) that increases PerR-mediated repression of peroxide resistance genes, and this repression is antagonized by elevated Fe2+ in an mntR pfeT mutant. Thus, H2O2-sensitivity reflects the relative levels of Mn2+ and Fe2+ as sensed by the PerR regulatory protein. These results underscore the complex interplay between manganese, iron and oxidative stress in B. subtilis.

A Simplified Method for CRISPR-Cas9 Engineering of Bacillus subtilis.

The clustered regularly interspaced short palindromic repeat (CRISPR)-Cas9 system from Streptococcus pyogenes has been widely deployed as a tool for bacterial strain construction. Conventional CRISPR-Cas9 editing strategies require design and molecular cloning of an appropriate guide RNA (gRNA) to target genome cleavage and a repair template for introduction of the desired site-specific genome modification. Here, we present a streamlined method that leverages the existing collection of nearly 4,000 Bacillus subtilis strains (the BKE collection) with individual genes replaced by an integrated erythromycin (erm) resistance cassette. A single plasmid (pAJS23) with a gRNA targeted to erm allows cleavage of the genome at any nonessential gene and at sites nearby to many essential genes. This plasmid can be engineered to include a repair template, or the repair template can be cotransformed with the plasmid as either a PCR product or genomic DNA. We demonstrate the utility of this system for generating gene replacements, site-specific mutations, modification of intergenic regions, and introduction of gene-reporter fusions. In sum, this strategy bypasses the need for gRNA design and allows the facile transfer of mutations and genetic constructions with no requirement for intermediate cloning steps. IMPORTANCE Bacillus subtilis is a well-characterized Gram-positive model organism and a popular platform for biotechnology. Although many different CRISPR-based genome editing strategies have been developed for B. subtilis, they generally involve the design and cloning of a specific guide RNA (gRNA) and repair template for each application. By targeting the erm resistance cassette with an anti-erm gRNA, genome editing can be directed to any of nearly 4,000 gene disruptants within the existing BKE collection of strains. Repair templates can be engineered as PCR products, or specific alleles and constructions can be transformed as chromosomal DNA, thereby bypassing the need for plasmid construction. The described method is rapid and facilitates a wide range of genome manipulations.

Manganese impairs the QoxABCD terminal oxidase leading to respiration-associated toxicity.

Cell physiology relies on metalloenzymes and can be easily disrupted by imbalances in metal ion pools. Bacillus subtilis requires manganese for growth and has highly regulated mechanisms for import and efflux that help maintain homeostasis. Cells defective for manganese (Mn) efflux are highly sensitive to intoxication, but the processes impaired by Mn excess are often unknown. Here, we employed a forward genetics approach to identify pathways affected by manganese intoxication. Our results highlight a central role for the membrane-localized electron transport chain in metal intoxication during aerobic growth. In the presence of elevated manganese, there is an increased generation of reactive radical species associated with dysfunction of the major terminal oxidase, the cytochrome aa3 heme-copper menaquinol oxidase (QoxABCD). Intoxication is suppressed by diversion of menaquinol to alternative oxidases or by a mutation affecting heme A synthesis that is known to convert QoxABCD from an aa3 to a bo3 -type oxidase. Manganese sensitivity is also reduced by derepression of the MhqR regulon, which protects cells against reactive quinones. These results suggest that dysfunction of the cytochrome aa3 -type quinol oxidase contributes to metal-induced intoxication.

Mini Review: Bacterial Membrane Composition and Its Modulation in Response to Stress.

Antibiotics and other agents that perturb the synthesis or integrity of the bacterial cell envelope trigger compensatory stress responses. Focusing on Bacillus subtilis as a model system, this mini-review summarizes current views of membrane structure and insights into how cell envelope stress responses remodel and protect the membrane. Altering the composition and properties of the membrane and its associated proteome can protect cells against detergents, antimicrobial peptides, and pore-forming compounds while also, indirectly, contributing to resistance against compounds that affect cell wall synthesis. Many of these regulatory responses are broadly conserved, even where the details of regulation may differ, and can be important in the emergence of antibiotic resistance in clinical settings.

The Bacillus subtilis monothiol bacilliredoxin BrxC (YtxJ) and the Bdr (YpdA) disulfide reductase reduce S-bacillithiolated proteins.

The bacterial cytosol is generally a reducing environment with protein cysteine residues maintained in their thiol form. The low molecular weight thiol bacillithiol (BSH) serves as a general thiol reductant, analogous to glutathione, in a wide range of bacterial species. Proteins modified by disulfide bond formation with BSH (S-bacillithiolation) are reduced by the action of bacilliredoxins, BrxA and BrxB. Here, the YtxJ protein is identified as a monothiol bacilliredoxin, renamed BrxC, and is implicated in BSH removal from oxidized cytosolic proteins, including the glyceraldehyde 3-phosphate dehydrogenases GapA and GapB. BrxC can also debacillithiolate the mixed disulfide form of the bacilliredoxin BrxB. Bdr is a thioredoxin reductase-like flavoprotein with bacillithiol-disulfide (BSSB) reductase activity. Here, Bdr is shown to additionally function as a bacilliredoxin reductase. Bdr and BrxB function cooperatively to debacillithiolate OhrR, a transcription factor regulated by S-bacillithiolation on its sole cysteine residue. Collectively, these results expand our understanding of the BSH redox network comprised of three bacilliredoxins and a BSSB reductase that serve to counter the widespread protein S-bacillithiolation that results from conditions of disulfide stress.

Resource sharing between central metabolism and cell envelope synthesis.

Synthesis of the bacterial cell envelope requires a regulated partitioning of resources from central metabolism. Here, we consider the key metabolic junctions that provide the precursors needed to assemble the cell envelope. Peptidoglycan synthesis requires redirection of a glycolytic intermediate, fructose-6-phosphate, into aminosugar biosynthesis by the highly regulated branchpoint enzyme GlmS. MurA directs the downstream product, UDP-GlcNAc, specifically into peptidoglycan synthesis. Other shared resources required for cell envelope synthesis include the isoprenoid carrier lipid undecaprenyl phosphate and amino acids required for peptidoglycan cross-bridges. Assembly of the envelope requires a sharing of limited resources between competing cellular pathways and may additionally benefit from scavenging of metabolites released from neighboring cells or the formation of symbiotic relationships with a host.

A Central Role for Magnesium Homeostasis during Adaptation to Osmotic Stress.

Osmotic stress is a significant physical challenge for free-living cells. Cells from all three domains of life maintain viability during osmotic stress by tightly regulating the major cellular osmolyte potassium (K+) and by import or synthesis of compatible solutes. It has been widely established that in response to high salt stress, many bacteria transiently accumulate high levels of K+, leading to bacteriostasis, with growth resuming only when compatible solutes accumulate and K+ levels are restored to biocompatible levels. Using Bacillus subtilis as a model system, we provide evidence that K+ fluxes perturb Mg2+ homeostasis: import of K+ upon osmotic upshift is correlated with Mg2+ efflux, and Mg2+ reimport is critical for adaptation. The transient growth inhibition resulting from hyperosmotic stress is coincident with loss of Mg2+ and a decrease in protein translation. Conversely, the reimport of Mg2+ is a limiting factor during resumption of growth. Furthermore, we show the essential signaling dinucleotide cyclic di-AMP fluctuates dynamically in coordination with Mg2+ and K+ levels, consistent with the proposal that cyclic di-AMP orchestrates the cellular response to osmotic stress. IMPORTANCE Environments with high concentrations of salt or other solutes impose an osmotic stress on cells, ultimately limiting viability by dehydration of the cytosol. A very common cellular response to high osmolarity is to immediately import high levels of potassium ion (K+), which helps prevent dehydration and allows time for the import or synthesis of biocompatible solutes that allow a resumption of growth. Here, using Bacillus subtilis as a model, we demonstrate that concomitant with K+ import there is a large reduction in intracellular magnesium (Mg2+) mediated by specific efflux pumps. Further, it is the reimport of Mg2+ that is rate-limiting for the resumption of growth. These coordinated fluxes of K+ and Mg2+ are orchestrated by cyclic-di-AMP, an essential second messenger in Firmicutes. These findings amend the conventional model for osmoadaptation and reveal that Mg2+ limitation is the proximal cause of the bacteriostasis that precedes resumption of growth.

A regulatory pathway that selectively up-regulates elongasome function in the absence of class A PBPs.

Bacteria surround themselves with peptidoglycan, an adaptable enclosure that contributes to cell shape and stability. Peptidoglycan assembly relies on penicillin-binding proteins (PBPs) acting in concert with SEDS-family transglycosylases RodA and FtsW, which support cell elongation and division respectively. In Bacillus subtilis, cells lacking all four PBPs with transglycosylase activity (aPBPs) are viable. Here, we show that the alternative sigma factor σI is essential in the absence of aPBPs. Defects in aPBP-dependent wall synthesis are compensated by σI-dependent upregulation of an MreB homolog, MreBH, which localizes the LytE autolysin to the RodA-containing elongasome complex. Suppressor analysis reveals that cells unable to activate this σI stress response acquire gain-of-function mutations in the essential histidine kinase WalK, which also elevates expression of sigI, mreBH and lytE. These results reveal compensatory mechanisms that balance the directional peptidoglycan synthesis arising from the elongasome complex with the more diffusive action of aPBPs.

Group A Streptococcus AdcR Regulon Participates in Bacterial Defense against Host-Mediated Zinc Sequestration and Contributes to Virulence.

Colonization by pathogenic bacteria depends on their ability to overcome host nutritional defenses and acquire nutrients. The human pathogen group A streptococcus (GAS) encounters the host defense factor calprotectin (CP) during infection. CP inhibits GAS growth in vitro by imposing zinc (Zn) limitation. However, GAS counterstrategies to combat CP-mediated Zn limitation and the in vivo relevance of CP-GAS interactions to bacterial pathogenesis remain unknown. Here, we report that GAS upregulates the AdcR regulon in response to CP-mediated Zn limitation. The AdcR regulon includes genes encoding Zn import (adcABC), Zn sparing (rpsN.2), and Zn scavenging systems (adcAII, phtD, and phtY). Each gene in the AdcR regulon contributes to GAS Zn acquisition and CP resistance. The ΔadcC and ΔrpsN.2 mutant strains were the most susceptible to CP, whereas the ΔadcA, ΔadcAII, and ΔphtD mutant strains displayed less CP sensitivity during growth in vitro However, the ΔphtY mutant strain did not display an increased CP sensitivity. The varied sensitivity of the mutant strains to CP-mediated Zn limitation suggests distinct roles for individual AdcR regulon genes in GAS Zn acquisition. GAS upregulates the AdcR regulon during necrotizing fasciitis infection in WT mice but not in S100a9-/- mice lacking CP. This suggests that CP induces Zn deficiency in the host. Finally, consistent with the in vitro results, several of the AdcR regulon genes are critical for GAS virulence in WT mice, whereas they are dispensable for virulence in S100a9-/- mice, indicating the direct competition for Zn between CP and proteins encoded by the GAS AdcR regulon during infection.

Biphasic unbinding of a metalloregulator from DNA for transcription (de)repression in Live Bacteria.

Microorganisms use zinc-sensing regulators to alter gene expression in response to changes in the availability of zinc, an essential micronutrient. Under zinc-replete conditions, the Fur-family metalloregulator Zur binds to DNA tightly in its metallated repressor form to Zur box operator sites, repressing the transcription of zinc uptake transporters. Derepression comes from unbinding of the regulator, which, under zinc-starvation conditions, exists in its metal-deficient non-repressor forms having no significant affinity with Zur box. While the mechanism of transcription repression by Zur is well-studied, little is known on how derepression by Zur could be facilitated. Using single-molecule/single-cell measurements, we find that in live Escherichia coli cells, Zur's unbinding rate from DNA is sensitive to Zur protein concentration in a first-of-its-kind biphasic manner, initially impeded and then facilitated with increasing Zur concentration. These results challenge conventional models of protein unbinding being unimolecular processes and independent of protein concentration. The facilitated unbinding component likely occurs via a ternary complex formation mechanism. The impeded unbinding component likely results from Zur oligomerization on chromosome involving inter-protein salt-bridges. Unexpectedly, a non-repressor form of Zur is found to bind chromosome tightly, likely at non-consensus sequence sites. These unusual behaviors could provide functional advantages in Zur's facile switching between repression and derepression.