The biology and pathogenicity of Clostridium perfringens type F: a common human enteropathogen with a new(ish) name.

SUMMARYIn the 2018-revised Clostridium perfringens typing classification system, isolates carrying the enterotoxin (cpe) and alpha toxin genes but no other typing toxin genes are now designated as type F. Type F isolates cause food poisoning and nonfoodborne human gastrointestinal (GI) diseases, which most commonly involve type F isolates carrying, respectivefooly, a chromosomal or plasmid-borne cpe gene. Compared to spores of other C. perfringens isolates, spores of type F chromosomal cpe isolates often exhibit greater resistance to food environment stresses, likely facilitating their survival in improperly prepared or stored foods. Multiple factors contribute to this spore resistance phenotype, including the production of a variant small acid-soluble protein-4. The pathogenicity of type F isolates involves sporulation-dependent C. perfringens enterotoxin (CPE) production. C. perfringens sporulation is initiated by orphan histidine kinases and sporulation-associated sigma factors that drive cpe transcription. CPE-induced cytotoxicity starts when CPE binds to claudin receptors to form a small complex (which also includes nonreceptor claudins). Approximately six small complexes oligomerize on the host cell plasma membrane surface to form a prepore. CPE molecules in that prepore apparently extend ß-hairpin loops to form a ß-barrel pore, allowing a Ca2+ influx that activates calpain. With low-dose CPE treatment, caspase-3-dependent apoptosis develops, while high-CPE dose treatment induces necroptosis. Those effects cause histologic damage along with fluid and electrolyte losses from the colon and small intestine. Sialidases likely contribute to type F disease by enhancing CPE action and, for NanI-producing nonfoodborne human GI disease isolates, increasing intestinal growth and colonization.

The impact of orphan histidine kinases and phosphotransfer proteins on the regulation of clostridial sporulation initiation.

Sporulation is an important feature of the clostridial life cycle, facilitating survival of these bacteria in harsh environments, contributing to disease transmission for pathogenic species, and sharing common early steps that are also involved in regulating industrially important solvent production by some non-pathogenic species. Initial genomics studies suggested that Clostridia lack the classical phosphorelay that phosphorylates Spo0A and initiates sporulation in Bacillus, leading to the hypothesis that sporulation in Clostridia universally begins when Spo0A is phosphorylated by orphan histidine kinases (OHKs). However, components of the classical Bacillus phosphorelay were recently identified in some Clostridia. Similar Bacillus phosphorelay components have not yet been found in the pathogenic Clostridia or the solventogenic Clostridia of industrial importance. For some of those Clostridia lacking a classical phosphorelay, the involvement of OHKs in sporulation initiation has received support from genetic studies demonstrating the involvement of several apparent OHKs in their sporulation. In addition, several clostridial OHKs directly phosphorylate Spo0A in vitro. Interestingly, there is considerable protein domain diversity among the sporulation-associated OHKs in Clostridia. Further adding to the emergent complexity of sporulation initiation in Clostridia, several candidate OHK phosphotransfer proteins that were OHK candidates were shown to function as phosphatases that reduce sporulation in some Clostridia. The mounting evidence indicates that no single pathway explains sporulation initiation in all Clostridia and supports the need for further study to fully understand the unexpected and biologically fascinating mechanistic diversity of this important process among these medically and industrially important bacteria.

The Sensory Histidine Kinase CusS of Escherichia coli Senses Periplasmic Copper Ions.

Two-component regulatory systems composed of a membrane-bound sensor/sensory histidine kinase (HK) and a cytoplasmic, DNA-binding response regulator (RR) are often associated with transenvelope efflux systems, which export transition metal cations from the periplasm directly out of the cell. Although much work has been done in this field, more evidence is needed for the hypothesis that the respective two-component regulatory systems are indeed sensing periplasmic ions. If so, a regulatory circuit between the concentration of periplasmic metal cations, sensing of these metals, and control of expression of the genes for transenvelope efflux systems that remove periplasmic cations can be assumed. Escherichia coli possesses only one transenvelope efflux system for metal cations, the Cus system for export of Cu(I) and Ag(I). It is composed of the transenvelope efflux system CusCBA, the periplasmic copper chaperone CusF, and the two-component regulatory system CusS (HK) and CusR (RR). Using phoA- and lacZ-reporter gene fusions, it was verified that an assumed periplasmic part of CusS is located in the periplasm. CusS was more important for copper resistance in E. coli under anaerobic conditions than under aerobic conditions and in complex medium more than in mineral salts medium. Predicted copper-binding sites in the periplasmic part of CusS were identified that, individually, were not essential for copper resistance but were in combination. In summary, evidence was obtained that the two-component regulatory system CusSR that controls expression of cusF and cusCBA does indeed sense periplasmic copper ions. IMPORTANCE Homeostasis of essential-but-toxic transition metal cations such as Zn(II) and Cu(II)/Cu(I) is an important contributor to the fitness of environmental bacteria and pathogenic bacteria during their confrontation with an infected host. Highly efficient removal of threatening concentrations of these metals can be achieved by the combined actions of an inner membrane with a transenvelope efflux system, which removes periplasmic ions after their export from the cytoplasm to this compartment. To understand the resulting metal cation homeostasis in the periplasm, it is important to know if a regulatory circuit exists between periplasmic metal cations, their sensing, and the subsequent control of the expression of the transenvelope efflux system. This publication adds evidence to the hypothesis that two-component regulatory systems in control of the expression of genes for transenvelope efflux systems do indeed sense metal cations in the periplasm.

The Cellular Abundance of Chemoreceptors, Chemosensory Signaling Proteins, Sensor Histidine Kinases, and Solute Binding Proteins of Pseudomonas aeruginosa Provides Insight into Sensory Preferences and Signaling Mechanisms.

Chemosensory pathways and two-component systems are important bacterial signal transduction systems. In the human pathogen Pseudomonas aeruginosa, these systems control many virulence traits. Previous studies showed that inorganic phosphate (Pi) deficiency induces virulence. We report here the abundance of chemosensory and two-component signaling proteins of P. aeruginosa grown in Pi deficient and sufficient media. The cellular abundance of chemoreceptors differed greatly, since a 2400-fold difference between the most and least abundant receptors was observed. For many chemoreceptors, their amount varied with the growth condition. The amount of chemoreceptors did not correlate with the magnitude of chemotaxis to their cognate chemoeffectors. Of the four chemosensory pathways, proteins of the Che chemotaxis pathway were most abundant and showed little variation in different growth conditions. The abundance of chemoreceptors and solute binding proteins indicates a sensing preference for amino acids and polyamines. There was an excess of response regulators over sensor histidine kinases in two-component systems. In contrast, ratios of the response regulators CheY and CheB to the histidine kinase CheA of the Che pathway were all below 1, indicative of different signaling mechanisms. This study will serve as a reference for exploring sensing preferences and signaling mechanisms of other bacteria.

Recent Advances in Histidine Kinase-Targeted Antimicrobial Agents.

The prevalence of antimicrobial-resistant pathogens significantly limited the number of effective antibiotics available clinically, which urgently requires new drug targets to screen, design, and develop novel antibacterial drugs. Two-component system (TCS), which is comprised of a histidine kinase (HK) and a response regulator (RR), is a common mechanism whereby bacteria can sense a range of stimuli and make an appropriate adaptive response. HKs as the sensor part of the bacterial TCS can regulate various processes such as growth, vitality, antibiotic resistance, and virulence, and have been considered as a promising target for antibacterial drugs. In the current review, we highlighted the structural basis and functional importance of bacterial TCS especially HKs as a target in the discovery of new antimicrobials, and summarize the latest research progress of small-molecule HK-inhibitors as potential novel antimicrobial drugs reported in the past decade.

Activity-based ATP analog probes for bacterial histidine kinases.

Histidine kinases (HKs) are sensor proteins found ubiquitously in prokaryotes. They are the first protein in two-component systems (TCSs), signaling pathways that respond to a myriad of environmental stimuli. TCSs are typically comprised of a HK and its cognate response regulator (RR) which often acts as a transcription factor. RRs will bind DNA and ultimately lead to a cellular response. These cellular outputs vary widely, but HKs are particularly interesting as they are tied to antibiotic resistance and virulence pathways in pathogenic bacteria, making them promising drug targets. We anticipate that HK inhibitors could serve as either standalone antibiotics or antivirulence therapies. Additionally, while the cellular response mediated by the HKs is often well-characterized, very little is known about which stimuli trigger the sensor kinase to begin the phosphorylation cascade. Studying HK activity and enrichment of active HKs through activity-based protein profiling will enable these stimuli to be elucidated, filling this fundamental gap in knowledge. Here, we describe methods to evaluate the potency of putative HK inhibitors in addition to methods to calculate kinetic parameters of various activity-based probes designed for the HKs.

Histidine Kinases: Diverse Functions in Plant Development and Responses to Environmental Conditions.

The two-component system (TCS), which is one of the most evolutionarily conserved signaling pathway systems, has been known to regulate multiple biological activities and environmental responses in plants. Significant progress has been made in characterizing the biological functions of the TCS components, including signal receptor histidine kinase (HK) proteins, signal transducer histidine-containing phosphotransfer proteins, and effector response regulator proteins. In this review, our scope is focused on the diverse structure, subcellular localization, and interactions of the HK proteins, as well as their signaling functions during development and environmental responses across different plant species. Based on data collected from scientific studies, knowledge about acting mechanisms and regulatory roles of HK proteins is presented. This comprehensive summary ofthe HK-related network provides a panorama of sophisticated modulating activities of HK members and gaps in understanding these activities, as well as the basis for developing biotechnological strategies to enhance the quality of crop plants.

Nitrate- and Nitrite-Sensing Histidine Kinases: Function, Structure, and Natural Diversity.

Under anaerobic conditions, bacteria may utilize nitrates and nitrites as electron acceptors. Sensitivity to nitrous compounds is achieved via several mechanisms, some of which rely on sensor histidine kinases (HKs). The best studied nitrate- and nitrite-sensing HKs (NSHKs) are NarQ and NarX from Escherichia coli. Here, we review the function of NSHKs, analyze their natural diversity, and describe the available structural information. In particular, we show that around 6000 different NSHK sequences forming several distinct clusters may now be found in genomic databases, comprising mostly the genes from Beta- and Gammaproteobacteria as well as from Bacteroidetes and Chloroflexi, including those from anaerobic ammonia oxidation (annamox) communities. We show that the architecture of NSHKs is mostly conserved, although proteins from Bacteroidetes lack the HAMP and GAF-like domains yet sometimes have PAS. We reconcile the variation of NSHK sequences with atomistic models and pinpoint the structural elements important for signal transduction from the sensor domain to the catalytic module over the transmembrane and cytoplasmic regions spanning more than 200 Å.

Evidence of a New MoYpd1p Phosphotransferase Isoform in the Multistep Phosphorelay System of Magnaporthe oryzae.

Different external stimuli are perceived by multiple sensor histidine kinases and transmitted by phosphorylation via the phosphotransfer protein Ypd1p in the multistep phosphorelay system of the high osmolarity glycerol signaling pathway of filamentous fungi. How the signal propagation takes place is still not known in detail since multiple sensor histidine kinase genes in most filamentous fungi are coded in the genome, whereas only one gene for Ypd1p exists. That raises the hypothesis that various Ypd1p isoforms are produced from a single gene sequence, perhaps by alternative splicing, facilitating a higher variability in signal transduction. We found that the mRNA of MoYPD1 in the rice blast fungus Magnaporthe oryzae is subjected to an increased structural variation and amplified putative isoforms on a cDNA level. We then generated mutant strains overexpressing these isoforms, purified the products, and present here one previously unknown MoYpd1p isoform on a proteome level. Alternative splicing was found to be a valid molecular mechanism to increase the signal diversity in eukaryotic multistep phosphorelay systems.

Two-component signaling system in plants: interaction network and specificity in response to stress and hormones.

Plants are exposed to various environmental challenges that can hamper their growth, development, and productivity. Being sedentary, plants cannot escape from these unfavorable environmental conditions and have evolved various signaling cascades to endure them. The two-component signaling (TCS) system is one such essential signaling circuitry present in plants regulating responses against multiple abiotic and biotic stresses. It is among the most ancient and evolutionary conserved signaling pathways in plants, which include membrane-bound histidine kinases (HKs), cytoplasmic histidine phosphotransfer proteins (Hpts), and nuclear or cytoplasmic response regulators (RRs). At the same time, TCS also involved in many signaling circuitries operative in plants in response to diverse hormones. These plant growth hormones play a significant role in diverse physiological and developmental processes, and their contribution to plant stress responses is coming up in a big way. Therefore, it is intriguing to know how TCS and various plant growth regulators, along with the key transcription factors, directly or indirectly control the responses of plants towards diverse stresses. The present review attempts to explore this relationship, hoping that this knowledge will contribute towards developing crop plants with enhanced climate resilience.

Electrochemical biosensor for cytokinins based on the CHASE domain of Arabidopsis histidine kinases 4.

In this study, An AHK4 CHASE domain was used to construct an electrochemical cytokinin biosensor using ferrocene as the electrochemical mediator. Upon addition of cytokinin, the binding of cytokinin and AHK4 led to dimerization, which blocked electron transfer between ferrocene and the electrode, and the redox peak current of ferrocene was gradually reduced. Cytokinin was detected by recording the change of the ferrocene redox peak current. The biosensor shows a linear range of 50-400 nM with a linear regression equation of ip = 0.0086c + 0.732 (R2 = 0.993) with ip in µA and c in nM and a detection limit (LOD) of 1.5 nM (S/N = 3). The biosensor exhibits excellent performance that avoids interference of other types of plant hormones and was successfully applied to the detection of cytokinins in bean sprouts.

The Journey from Two-Step to Multi-Step Phosphorelay Signaling Systems.

BACKGROUND: The two-component signaling (TCS) system is an important signal transduction machinery in prokaryotes and eukaryotes, excluding animals, that uses a protein phosphorylation mechanism for signal transmission. CONCLUSION: Prokaryotes have a primitive type of TCS machinery, which mainly comprises a membrane-bound sensory histidine kinase (HK) and its cognate cytoplasmic response regulator (RR). Hence, it is sometimes referred to as two-step phosphorelay (TSP). Eukaryotes have more sophisticated signaling machinery, with an extra component - a histidine-containing phosphotransfer (HPT) protein that shuttles between HK and RR to communicate signal baggage. As a result, the TSP has evolved from a two-step phosphorelay (His-Asp) in simple prokaryotes to a multi-step phosphorelay (MSP) cascade (His-Asp-His-Asp) in complex eukaryotic organisms, such as plants, to mediate the signaling network. This molecular evolution is also reflected in the form of considerable structural modifications in the domain architecture of the individual components of the TCS system. In this review, we present TCS system's evolutionary journey from the primitive TSP to advanced MSP type across the genera. This information will be highly useful in designing the future strategies of crop improvement based on the individual members of the TCS machinery.

Bacterial Protein Kinases.

Bacteria are able to inhabit and survive vastly diverse environments. This enormous adaptive capacity depend on their ability to perceive cues from the micro-environment and process this information accordingly to mount appropriate metabolic responses and ultimately sustain homeostasis. From systems perspective, microbial cells conceal significant degree of organismal complexity, which may only be managed by continuous bulk cellular information flow and processing, inside the cell, between other cells and the environment. In this respect, reversible covalent modification of proteins is one of the universal mode of information flow mechanism used to regulate metabolism in all organisms. More than 30 types of post translational modifications have been identified, where phosphorylation constitutes nearly half of them. Bacterial cells possess several modes of phosphoprotein mediated information flow mechanisms. Histidine kinases and two component systems, bacterial tyrosine kinases, Hanks type serine/threonine kinases, atypical serine kinases and arginine kinases have been identified in many species.

Phos-Tag Fluorescent Gel Staining for the Quantitative Detection of His- and Asp-Phosphorylated Proteins.

We describe a standard protocol for phosphate-affinity fluorescent gel staining that uses a fluorophore-labeled dizinc(II) complex of a derivative of the phosphate-binding tag molecule Phos-tag to detect His- and Asp-phosphorylated proteins separated by SDS-PAGE. The procedure permits the quantitative monitoring of phosphorylated histidine kinases (His-phosphoproteins) and their cognate phosphorylated response regulators (Asp-phosphoproteins) in bacterial two-component signaling transduction systems. The total time required for each gel staining operation is about 2 h at room temperature.

Post-translational Modifications of Serine/Threonine and Histidine Kinases and Their Roles in Signal Transductions in Synechocystis Sp. PCC 6803.

Cyanobacterium Synechocystis sp. PCC 6803, a popular model organism for researches in photosynthesis and biofuel production, contains plant-like photosynthetic machineries which significantly contribute to global carbon fixation. There are 12 eukaryotic-type Ser/Thr kinases (SpkA-L) and 49 His kinases (Hik1-49) of two-component systems in the genome of Synechocystis sp. PCC 6803. They are the key regulators in sensing and transmitting stimuli including light- and glucose-mediate signal transduction. Proteomic studies were able to identify all the kinases. The majority of kinases no matter whether they have a predicted transmembrane domain were identified in the membrane fractions. Six Ser/Thr kinases (SpkA-D, F and G) and ten His kinases (Hik4, 12, 14, 21, 26-27, 29, 36, 43, and 46) were identified to have one or more of the three types of post-translational modifications: phosphorylation, acetylation, and thiol oxidation. Interestingly, SpkG has the phosphorylatable threonine residue that was aligned with the phosphorylated threonine residue in the activation loop of human CDK7, demonstrating conserved phosphorylation between cyanobacterial and human kinases. Transcriptomics and proteomics revealed differential expression of the kinases in heterotrophic and photoheterotrophic compared with photoautotrophic conditions, indicating their roles in regulating the growth modes of cyanobacteria. In summary, this review focuses on the discussions on post-transcriptional modifications, transcriptomic, and proteomic studies of Ser/Thr and His kinases. This together with our published review in 2019 present a complete story of an overview of sequences, domain architectures, and biochemical and physiological functions of cyanobacterial kinases with adequate details in the context of high throughput systems. We also emphasize the importance of discovering upstream molecules and substrates to understand the exact functions of the kinases in vivo. As an attempt, a model is proposed in which Hik31, His33, Sll1334, and IcfG are hypothesized to be critical for switching between autotrophic and heterotrophic modes based on the results from the phenotypes of the gene knockout strains combined with their post-translational modifications, and gene expression profiles.

Cytokinin and Ethylene Cell Signaling Pathways from Prokaryotes to Eukaryotes.

Cytokinins (CKs) and ethylene (ET) are among the most ancient organic chemicals on Earth. A wide range of organisms including plants, algae, fungi, amoebae, and bacteria use these substances as signaling molecules to regulate cellular processes. Because of their ancestral origin and ubiquitous occurrence, CKs and ET are also considered to be ideal molecules for inter-kingdom communication. Their signal transduction pathways were first historically deciphered in plants and are related to the two-component systems, using histidine kinases as primary sensors. Paradoxically, although CKs and ET serve as signaling molecules in different kingdoms, it has been supposed for a long time that the canonical CK and ET signaling pathways are restricted to terrestrial plants. These considerations have now been called into question following the identification over recent years of genes encoding CK and ET receptor homologs in many other lineages within the tree of life. These advances shed new light on the dissemination and evolution of these hormones as both intra- and inter-specific communication molecules in prokaryotic and eukaryotic organisms.

2-Aminobenzothiazoles Inhibit Virulence Gene Expression and Block Polymyxin Resistance in Salmonella enterica.

One promising strategy to combat antibiotic-resistant bacteria is to develop compounds that block bacterial defenses against antibacterial conditions produced by the innate immune system. Salmonella enterica, which causes food-borne gastroenteritis and typhoid fever, requires histidine kinases (HKs) to resist innate immune defenses such as cationic antimicrobial peptides (CAMPs). Herein, we report that 2-aminobenzothiazoles block histidine kinase-dependent phenotypes in Salmonella enterica serotype Typhimurium. We found that 2-aminobenzothiazoles inhibited growth under low Mg2+ , a stressful condition that requires histidine kinase-mediated responses, and decreased expression of the virulence genes pagC and pagK. Furthermore, we discovered that 2-aminobenzothiazoles weaken Salmonella's resistance to polymyxin B and polymyxin E, which are last-line antibiotics and models for host defense CAMPs. These findings raise the possibilities that 2-aminobenzothiazoles can block HK-mediated bacterial defenses and can be used in combination with polymyxins to treat infections caused by Salmonella.

The Interaction Network and Signaling Specificity of Two-Component System in Arabidopsis.

Two-component systems (TCS) in plants have evolved into a more complicated multi-step phosphorelay (MSP) pathway, which employs histidine kinases (HKs), histidine-containing phosphotransfer proteins (HPts), and response regulators (RRs) to regulate various aspects of plant growth and development. How plants perceive the external signals, then integrate and transduce the secondary signals specifically to the desired destination, is a fundamental characteristic of the MSP signaling network. The TCS elements involved in the MSP pathway and molecular mechanisms of signal transduction have been best understood in the model plant Arabidopsis thaliana. In this review, we focus on updated knowledge on TCS signal transduction in Arabidopsis. We first present a brief description of the TCS elements; then, the protein-protein interaction network is established. Finally, we discuss the possible molecular mechanisms involved in the specificity of the MSP signaling at the mRNA and protein levels.

Metabolic Engineering of Histidine Kinases in Clostridium beijerinckii for Enhanced Butanol Production.

Clostridium beijerinckii, a promising industrial microorganism for butanol production, suffers from low butanol titer and lack of high-efficiency genetical engineering toolkit. A few histidine kinases (HKs) responsible for Spo0A phosphorylation have been demonstrated as functionally important components in regulating butanol biosynthesis in solventogenic clostridia such as C. acetobutylicum, but no study about HKs has been conducted in C. beijerinckii. In this study, six annotated but uncharacterized candidate HK genes sharing partial homologies (no less than 30%) with those in C. acetobutylicum were selected based on sequence alignment. The encoding region of these HK genes were deleted with CRISPR-Cas9n-based genome editing technology. The deletion of cbei2073 and cbei4484 resulted in significant change in butanol biosynthesis, with butanol production increased by 40.8 and 17.3% (13.8 g/L and 11.5 g/L vs. 9.8 g/L), respectively, compared to the wild-type. Faster butanol production rates were observed, with butanol productivity greatly increased by 40.0 and 20.0%, respectively, indicating these two HKs are important in regulating cellular metabolism in C. beijerinckii. In addition, the sporulation frequencies of two HKs inactivated strains decreased by 96.9 and 77.4%, respectively. The other four HK-deletion (including cbei2087, cbei2435, cbei4925, and cbei1553) mutant strains showed few phenotypic changes compared with the wild-type. This study demonstrated the role of HKs on sporulation and solventogenesis in C. beijerinckii, and provided a novel engineering strategy of HKs for improving metabolite production. The hyper-butanol-producing strains generated in this study have great potentials in industrial biobutanol production.

Structural and Functional Characterization of Autophosphorylation in Bacterial Histidine Kinases.

Autophosphorylation of histidine kinases (HK) is the first step for signal transduction in bacterial two-component signalling systems. As HKs dimerize, the His residue is phosphorylated in cis or trans depending on whether the ATP molecule used in the reaction is bound to the same or the neighboring subunit, respectively. The cis or trans autophosphorylation results from an alternative directionality in the connection between helices α1 and α2 in the HK DHp domain, in such a way that α2 could be oriented almost 90° counterclockwise or clockwise with respect to α1. Sequence and length variability of this connection appears to lie behind the different directionality and is implicated in partner recognition with the response regulator (RR), highlighting its importance in signal transduction. Despite this mechanistic difference, HK autophosphorylation appears to be universal, involving conserved residues neighboring the phosphoacceptor His residue. Herein, we describe a simple protocol to determine both autophosphorylation directionality of HKs and the roles of the catalytic residues in these protein kinases.