Engineering a cell-free biosensor signal amplification circuit with polymerase strand recycling.

Cell-free systems are powerful synthetic biology technologies because of their ability to recapitulate sensing and gene expression without the complications of living cells. Cell-free systems can perform even more advanced functions when genetic circuits are incorporated as information processing components. Here we expand cell-free biosensing by engineering a highly specific isothermal signal amplification circuit called polymerase strand recycling (PSR) that leverages T7 RNA polymerase off-target transcription to recycle nucleic acid inputs within DNA strand displacement circuits. We develop design rules for PSR circuit components and use these rules to construct modular biosensors that can directly sense different RNA targets with limits of detection in the nM range and high specificity. We then use PSR for signal amplification within allosteric transcription factor-based biosensors for small molecule detection. We use a double equilibrium model of transcription factor:DNA and transcription factor:ligand binding interactions to predict biosensor sensitivity enhancement by PSR, and then demonstrate this approach experimentally by achieving 3.6-4.6-fold decreases in biosensor EC50 to sub micromolar ranges. We believe this work expands the current capabilities of cell-free circuits by incorporating PSR, which we anticipate will have a wide range of uses within biotechnology.

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.

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.

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).

Bacterial Transcriptional Regulators: A Road Map for Functional, Structural, and Biophysical Characterization.

The different niches through which bacteria move during their life cycle require a fast response to the many environmental queues they encounter. The sensing of these stimuli and their correct response is driven primarily by transcriptional regulators. This kind of protein is involved in sensing a wide array of chemical species, a process that ultimately leads to the regulation of gene transcription. The allosteric-coupling mechanism of sensing and regulation is a central aspect of biological systems and has become an important field of research during the last decades. In this review, we summarize the state-of-the-art techniques applied to unravel these complex mechanisms. We introduce a roadmap that may serve for experimental design, depending on the answers we seek and the initial information we have about the system of study. We also provide information on databases containing available structural information on each family of transcriptional regulators. Finally, we discuss the recent results of research about the allosteric mechanisms of sensing and regulation involving many transcriptional regulators of interest, highlighting multipronged strategies and novel experimental techniques. The aim of the experiments discussed here was to provide a better understanding at a molecular level of how bacteria adapt to the different environmental threats they face.

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.

Molecular Evolution of Transition Metal Bioavailability at the Host-Pathogen Interface.

The molecular evolution of the adaptive response at the host-pathogen interface has been frequently referred to as an 'arms race' between the host and bacterial pathogens. The innate immune system employs multiple strategies to starve microbes of metals. Pathogens, in turn, develop successful strategies to maintain access to bioavailable metal ions under conditions of extreme restriction of transition metals, or nutritional immunity. However, the processes by which evolution repurposes or re-engineers host and pathogen proteins to perform or refine new functions have been explored only recently. Here we review the molecular evolution of several human metalloproteins charged with restricting bacterial access to transition metals. These include the transition metal-chelating S100 proteins, natural resistance-associated macrophage protein-1 (NRAMP-1), transferrin, lactoferrin, and heme-binding proteins. We examine their coevolution with bacterial transition metal acquisition systems, involving siderophores and membrane-spanning metal importers, and the biological specificity of allosteric transcriptional regulatory proteins tasked with maintaining bacterial metallostasis. We also discuss the evolution of metallo-ß-lactamases; this illustrates how rapid antibiotic-mediated evolution of a zinc metalloenzyme obligatorily occurs in the context of host-imposed nutritional immunity.

Structural basis for persulfide-sensing specificity in a transcriptional regulator.

Cysteine thiol-based transcriptional regulators orchestrate the coordinated regulation of redox homeostasis and other cellular processes by 'sensing' or detecting a specific redox-active molecule, which in turn activates the transcription of a specific detoxification pathway. The extent to which these sensors are truly specific in cells for a singular class of reactive small-molecule stressors, for example, reactive oxygen or sulfur species, is largely unknown. Here, we report structural and mechanistic insights into the thiol-based transcriptional repressor SqrR, which reacts exclusively with oxidized sulfur species such as persulfides, to yield a tetrasulfide bridge that inhibits DNA operator-promoter binding. Evaluation of crystallographic structures of SqrR in various derivatized states, coupled with the results of a mass spectrometry-based kinetic profiling strategy, suggest that persulfide selectivity is determined by structural frustration of the disulfide form. These findings led to the identification of an uncharacterized repressor from the bacterial pathogen Acinetobacter baumannii as a persulfide sensor.

Cell-free biosensors for rapid detection of water contaminants.

Lack of access to safe drinking water is a global problem, and methods to reliably and easily detect contaminants could be transformative. We report the development of a cell-free in vitro transcription system that uses RNA Output Sensors Activated by Ligand Induction (ROSALIND) to detect contaminants in water. A combination of highly processive RNA polymerases, allosteric protein transcription factors and synthetic DNA transcription templates regulates the synthesis of a fluorescence-activating RNA aptamer. The presence of a target contaminant induces the transcription of the aptamer, and a fluorescent signal is produced. We apply ROSALIND to detect a range of water contaminants, including antibiotics, small molecules and metals. We also show that adding RNA circuitry can invert responses, reduce crosstalk and improve sensitivity without protein engineering. The ROSALIND system can be freeze-dried for easy storage and distribution, and we apply it in the field to test municipal water supplies, demonstrating its potential use for monitoring water quality.

Multi-metal nutrient restriction and crosstalk in metallostasis systems in microbial pathogens.

Transition metals from manganese to zinc function as catalytic and structural cofactors for an amazing diversity of proteins and enzymes, and thus are essential for all forms of life. During infection, inflammatory host proteins limit the accessibility of multiple transition metals to invading pathogens in a process termed nutritional immunity. In order to respond to host-mediated metal starvation, bacteria employ both protein and RNA-based mechanisms to sense prevailing transition metal concentrations that collectively regulate systems-level strategies to maintain cellular metallostasis. In this review, we discuss a number of recent advances in our understanding of how bacteria orchestrate the adaptive response to host-mediated multi-metal restriction, highlighting crosstalk among these regulatory systems.

Mechanistic Insights into the Metal-Dependent Activation of ZnII-Dependent Metallochaperones.

Members of the COG0523 subfamily of candidate GTPase metallochaperones function in bacterial transition-metal homeostasis, but the nature of the cognate metal, mechanism of metal transfer, and identification of target protein(s) for metal delivery remain open questions. Here, we explore the multifunctionality of members of the subfamily linked to delivering ZnII to apoprotein targets under conditions of host-imposed transition-metal depletion. We examine two zinc-uptake repressor (Zur)-regulated COG0523 family members, each from a major human pathogen, Acinetobacter baumannii (AbZigA) and Staphylococcus aureus (SaZigA), in an effort to develop a model for ZnII metallochaperone activity. ZnII chelator competition experiments reveal one high-affinity (KZn1 ≈ 1010-1011 M-1) metal-binding site in each GTPase, while AbZigA and SaZigA are characterized by an additional one and two (lower-affinity) metal-binding sites, respectively. CoII titrations reveal that both metallochaperones have similar electronic absorption characteristics that indicate the presence of two tetrahedral metal coordination sites. High-affinity metal binding at the CXCC motif activates the GTPase activity of both enzymes, with ZnII more effective than CoII. Both GTPases bind the product, GDP, more tightly in the apoprotein than the ZnII-bound state and exhibit what is best described as a "locked" conformation around the GTP substrate. Negative thermodynamic linkage is observed between nucleotide binding and metal binding, leading to a new mechanistic model for COG0523-catalyzed metal delivery.

A Mn-sensing riboswitch activates expression of a Mn2+/Ca2+ ATPase transporter in Streptococcus.

Maintaining manganese (Mn) homeostasis is important for the virulence of numerous bacteria. In the human respiratory pathogen Streptococcus pneumoniae, the Mn-specific importer PsaBCA, exporter MntE, and transcriptional regulator PsaR establish Mn homeostasis. In other bacteria, Mn homeostasis is controlled by yybP-ykoY family riboswitches. Here, we characterize a yybP-ykoY family riboswitch upstream of the mgtA gene encoding a PII-type ATPase in S. pneumoniae, suggested previously to function in Ca2+ efflux. We show that the mgtA riboswitch aptamer domain adopts a canonical yybP-ykoY structure containing a three-way junction that is compacted in the presence of Ca2+ or Mn2+ at a physiological Mg2+ concentration. Although Ca2+ binds to the RNA aptamer with higher affinity than Mn2+, in vitro activation of transcription read-through of mgtA by Mn2+ is much greater than by Ca2+. Consistent with this result, mgtA mRNA and protein levels increase ≈5-fold during cellular Mn stress, but only in genetic backgrounds of S. pneumoniae and Bacillus subtilis that exhibit Mn2+ sensitivity, revealing that this riboswitch functions as a failsafe 'on' signal to prevent Mn2+ toxicity in the presence of high cellular Mn2+. In addition, our results suggest that the S. pneumoniae yybP-ykoY riboswitch functions to regulate Ca2+ efflux under these conditions.

Tuning site-specific dynamics to drive allosteric activation in a pneumococcal zinc uptake regulator.

MarR (multiple antibiotic resistance repressor) family proteins are bacterial repressors that regulate transcription in response to a wide range of chemical signals. Although specific features of MarR family function have been described, the role of atomic motions in MarRs remains unexplored thus limiting insights into the evolution of allostery in this ubiquitous family of repressors. Here, we provide the first experimental evidence that internal dynamics play a crucial functional role in MarR proteins. Streptococcus pneumoniae AdcR (adhesin-competence repressor) regulates ZnII homeostasis and ZnII functions as an allosteric activator of DNA binding. ZnII coordination triggers a transition from somewhat independent domains to a more compact structure. We identify residues that impact allosteric activation on the basis of ZnII-induced perturbations of atomic motions over a wide range of timescales. These findings appear to reconcile the distinct allosteric mechanisms proposed for other MarRs and highlight the importance of conformational dynamics in biological regulation.

Functional Role of Solvent Entropy and Conformational Entropy of Metal Binding in a Dynamically Driven Allosteric System.

Allostery is a regulatory phenomenon whereby ligand binding to one site influences the binding of the same or a different ligand to another site on a macromolecule. The physical origins of allosteric regulation remain under intense investigation. In general terms, ligand-induced structural changes, perturbations of residue-specific dynamics, and surrounding solvent molecules all potentially contribute to the global energetics of allostery. While the role of solvent is generally well understood in regulatory events associated with major protein structural rearrangements, the degree to which protein dynamics impact solvent degrees of freedom is unclear, particularly in cases of dynamically driven allostery. With the aid of new crystal structures, extensive calorimetric and residue-specific dynamics studies over a range of time scales and temperatures, we dissect for the first time the relative degree to which changes in solvent entropy and residue-specific dynamics impact dynamically driven, allosteric inhibition of DNA binding by Zn in the zinc efflux repressor, CzrA (chromosomal zinc-regulated repressor). We show that non-native residue-specific dynamics in allosterically impaired CzrA mutants are accompanied by significant perturbations in solvent entropy that cannot be predicted from crystal structures. We conclude that functional dynamics are not necessarily restricted to protein residues but involve surface water molecules that may be responding to ligand (Zn)-mediated perturbations in protein internal motions that define the conformational ensemble, rather than major structural rearrangements.

Metal-dependent allosteric activation and inhibition on the same molecular scaffold: the copper sensor CopY from Streptococcus pneumoniae.

Resistance to copper (Cu) toxicity in the respiratory pathogen Streptococcus pneumoniae is regulated by the Cu-specific metallosensor CopY. CopY is structurally related to the antibiotic-resistance regulatory proteins MecI and BlaI from Staphylococcus aureus, but is otherwise poorly characterized. Here we employ a multi-pronged experimental strategy to define the Spn CopY coordination chemistry and the unique mechanism of allosteric activation by Zn(ii) and allosteric inhibition by Cu(i) of cop promoter DNA binding. We show that Zn(ii) is coordinated by a subunit-bridging 3S 1H2O complex formed by the same residues that coordinate Cu(i), as determined by X-ray absorption spectroscopy and ratiometric pulsed alkylation-mass spectrometry (rPA-MS). Apo- and Zn-bound CopY are homodimers by small angle X-ray scattering (SAXS); however, Zn stabilizes the dimer, narrows the conformational ensemble of the apo-state as revealed by ion mobility-mass spectroscopy (IM-MS), and activates DNA binding in vitro and in cells. In contrast, Cu(i) employs the same Cys pair to form a subunit-bridging, kinetically stable, multi-metallic Cu·S cluster (KCu ≈ 1016 M-1) that induces oligomerization beyond the dimer as revealed by SAXS, rPA-MS and NMR spectroscopy, leading to inhibition of DNA binding. These studies suggest that CopY employs conformational selection to drive Zn-activation of DNA binding, and a novel Cu(i)-mediated assembly mechanism that dissociates CopY from the DNA via ligand exchange-catalyzed metal substitution, leading to expression of Cu resistance genes. Mechanistic parallels to antibiotic resistance repressors MecI and BlaI are discussed.

Metallochaperones and metalloregulation in bacteria.

Bacterial transition metal homoeostasis or simply 'metallostasis' describes the process by which cells control the intracellular availability of functionally required metal cofactors, from manganese (Mn) to zinc (Zn), avoiding both metal deprivation and toxicity. Metallostasis is an emerging aspect of the vertebrate host-pathogen interface that is defined by a 'tug-of-war' for biologically essential metals and provides the motivation for much recent work in this area. The host employs a number of strategies to starve the microbial pathogen of essential metals, while for others attempts to limit bacterial infections by leveraging highly competitive metals. Bacteria must be capable of adapting to these efforts to remodel the transition metal landscape and employ highly specialized metal sensing transcriptional regulators, termed metalloregulatory proteins,and metallochaperones, that allocate metals to specific destinations, to mediate this adaptive response. In this essay, we discuss recent progress in our understanding of the structural mechanisms and metal specificity of this adaptive response, focusing on energy-requiring metallochaperones that play roles in the metallocofactor active site assembly in metalloenzymes and metallosensors, which govern the systems-level response to metal limitation and intoxication.

Entropy redistribution controls allostery in a metalloregulatory protein.

Allosteric communication between two ligand-binding sites in a protein is a central aspect of biological regulation that remains mechanistically unclear. Here we show that perturbations in equilibrium picosecond-nanosecond motions impact zinc (Zn)-induced allosteric inhibition of DNA binding by the Zn efflux repressor CzrA (chromosomal zinc-regulated repressor). DNA binding leads to an unanticipated increase in methyl side-chain flexibility and thus stabilizes the complex entropically; Zn binding redistributes these motions, inhibiting formation of the DNA complex by restricting coupled fast motions and concerted slower motions. Allosterically impaired CzrA mutants are characterized by distinct nonnative fast internal dynamics "fingerprints" upon Zn binding, and DNA binding is weakly regulated. We demonstrate the predictive power of the wild-type dynamics fingerprint to identify key residues in dynamics-driven allostery. We propose that driving forces arising from dynamics can be harnessed by nature to evolve new allosteric ligand specificities in a compact molecular scaffold.