Signaling by a bacterial phytochrome histidine kinase involves a conformational cascade reorganizing the dimeric photoreceptor.

Phytochromes (Phys) are a divergent cohort of bili-proteins that detect light through reversible interconversion between dark-adapted Pr and photoactivated Pfr states. While our understandings of downstream events are emerging, it remains unclear how Phys translate light into an interpretable conformational signal. Here, we present models of both states for a dimeric Phy with histidine kinase (HK) activity from the proteobacterium Pseudomonas syringae, which were built from high-resolution cryo-EM maps (2.8-3.4-Å) of the photosensory module (PSM) and its following signaling (S) helix together with lower resolution maps for the downstream output region augmented by RoseTTAFold and AlphaFold structural predictions. The head-to-head models reveal the PSM and its photointerconversion mechanism with strong clarity, while the HK region is interpretable but relatively mobile. Pr/Pfr comparisons show that bilin phototransformation alters PSM architecture culminating in a scissoring motion of the paired S-helices linking the PSMs to the HK bidomains that ends in reorientation of the paired catalytic ATPase modules relative to the phosphoacceptor histidines. This action apparently primes autophosphorylation enroute to phosphotransfer to the cognate DNA-binding response regulator AlgB which drives quorum-sensing behavior through transient association with the photoreceptor. Collectively, these models illustrate how light absorption conformationally translates into accelerated signaling by Phy-type kinases.

Photoreception and signaling in bacterial phytochrome revealed by single-particle cryo-EM.

Phytochromes are red-light photoreceptors discovered in plants with homologs in bacteria and fungi that regulate a variety of physiological responses. They display a reversible photocycle between two distinct states: a red-light-absorbing Pr state and a far-red light-absorbing Pfr state. The photoconversion regulates the activity of an enzymatic domain, usually a histidine kinase (HK). The molecular mechanism that explains how light controls the HK activity is not understood because structures of unmodified bacterial phytochromes with HK activity are missing. Here, we report three cryo-electron microscopy structures of a wild-type bacterial phytochrome with HK activity determined as Pr and Pfr homodimers and as a Pr/Pfr heterodimer with individual subunits in distinct states. We propose that the Pr/Pfr heterodimer is a physiologically relevant signal transduction intermediate. Our results offer insight into the molecular mechanism that controls the enzymatic activity of the HK as part of a bacterial two-component system that perceives and transduces light signals.

Structural and functional insights underlying recognition of histidine phosphotransfer protein in fungal phosphorelay systems.

In human pathogenic fungi, receiver domains from hybrid histidine kinases (hHK) have to recognize one HPt. To understand the recognition mechanism, we have assessed phosphorelay from receiver domains of five hHKs of group III, IV, V, VI, and XI to HPt from Chaetomium thermophilum and obtained the structures of Ct_HPt alone and in complex with the receiver domain of hHK group VI. Our data indicate that receiver domains phosphotransfer to Ct_HPt, show a low affinity for complex formation, and prevent a Leu-Thr switch to stabilize phosphoryl groups, also derived from the structures of the receiver domains of hHK group III and Candida albicans Sln1. Moreover, we have elucidated the envelope structure of C. albicans Ypd1 using small-angle X-ray scattering which reveals an extended flexible conformation of the long loop αD-αE which is not involved in phosphotransfer. Finally, we have analyzed the role of salt bridges in the structure of Ct_HPt alone.

Atomic insights into the signaling landscape of E. coli PhoQ histidine kinase from molecular dynamics simulations.

Bacteria rely on two-component systems to sense environmental cues and regulate gene expression for adaptation. The PhoQ/PhoP system exemplifies this crucial role, playing a key part in sensing magnesium (Mg2+) levels, antimicrobial peptides, mild acidic pH, osmotic upshift, and long-chain unsaturated fatty acids, promoting virulence in certain bacterial species. However, the precise details of PhoQ activation remain elusive. To elucidate PhoQ's signaling mechanism at atomic resolution, we combined AlphaFold2 predictions with molecular modeling and carried out extensive Molecular Dynamics (MD) simulations. Our MD simulations revealed three distinct PhoQ conformations that were validated by experimental data. Notably, one conformation was characterized by Mg2+ bridging the acidic patch in the sensor domain to the membrane, potentially representing a repressed state. Furthermore, the high hydration observed in a putative intermediate state lends support to the hypothesis of water-mediated conformational changes during PhoQ signaling. Our findings not only revealed specific conformations within the PhoQ signaling pathway, but also hold significant promise for understanding the broader histidine kinase family due to their shared structural features. Our approach paves the way for a more comprehensive understanding of histidine kinase signaling mechanisms across various bacterial species and opens the door for developing novel therapeutics that target PhoQ modulation.

Design of a water-soluble transmembrane receptor kinase with intact molecular function by QTY code.

Membrane proteins are critical to biological processes and central to life sciences and modern medicine. However, membrane proteins are notoriously challenging to study, mainly owing to difficulties dictated by their highly hydrophobic nature. Previously, we reported QTY code, which is a simple method for designing water-soluble membrane proteins. Here, we apply QTY code to a transmembrane receptor, histidine kinase CpxA, to render it completely water-soluble. The designed CpxAQTY exhibits expected biophysical properties and highly preserved native molecular function, including the activities of (i) autokinase, (ii) phosphotransferase, (iii) phosphatase, and (iv) signaling receptor, involving a water-solubilized transmembrane domain. We probe the principles underlying the balance of structural stability and activity in the water-solubilized transmembrane domain. Computational approaches suggest that an extensive and dynamic hydrogen-bond network introduced by QTY code and its flexibility may play an important role. Our successful functional preservation further substantiates the robustness and comprehensiveness of QTY code.

The N-terminus of the Aspergillus fumigatus group III hybrid histidine kinase TcsC is essential for its physiological activity and targets the protein to the nucleus.

Group III hybrid histidine kinases are fungal-specific proteins and part of the multistep phosphorelay, representing the initial part of the high osmolarity glycerol (HOG) pathway. TcsC, the corresponding kinase in Aspergillus fumigatus, was expected to be a cytosolic protein but is targeted to the nucleus. Activation of TcsC by the antifungal fludioxonil has lethal consequences for the fungus. The agent triggers a fast and TcsC-dependent activation of SakA and later on a redistribution of TcsC to the cytoplasm. High osmolarity also activates TcsC, which then exits the nucleus or concentrates in spot-like, intra-nuclear structures. The sequence corresponding to the N-terminal 208 amino acids of TcsC lacks detectable domains. Its loss renders TcsC cytosolic and non-responsive to hyperosmotic stress, but it has no impact on the antifungal activity of fludioxonil. A point mutation in one of the three putative nuclear localization sequences, which are present in the N-terminus, prevents the nuclear localization of TcsC, but not its ability to respond to hyperosmotic stress. Hence, this striking intracellular localization is no prerequisite for the role of TcsC in the adaptive response to hyperosmotic stress, instead, TcsC proteins that are present in the nuclei seem to modulate the cell wall composition of hyphae, which takes place in the absence of stress. The results of the present study underline that the spatiotemporal dynamics of the individual components of the multistep phosphorelay is a crucial feature of this unique signaling hub. IMPORTANCE: Signaling pathways enable pathogens, such as Aspergillus fumigatus, to respond to a changing environment. The TcsC protein is the major sensor of the high osmolarity glycerol (HOG) pathway of A. fumigatus and it is also the target of certain antifungals. Insights in its function are therefore relevant for the pathogenicity and new therapeutic treatment options. TcsC was expected to be cytoplasmic, but we detected it in the nucleus and showed that it translocates to the cytoplasm upon activation. We have identified the motif that guides TcsC to the nucleus. An exchange of a single amino acid in this motif prevents a nuclear localization, but this nuclear targeting is no prerequisite for the TcsC-mediated stress response. Loss of the N-terminal 208 amino acids prevents the nuclear localization and renders TcsC unable to respond to hyperosmotic stress demonstrating that this part of the protein is of crucial importance.

A conserved inhibitory interdomain interaction regulates DNA-binding activities of hybrid two-component systems in Bacteroides.

Hybrid two-component systems (HTCSs) comprise a major class of transcription regulators of polysaccharide utilization genes in Bacteroides. Distinct from classical two-component systems in which signal transduction is carried out by intermolecular phosphotransfer between a histidine kinase (HK) and a cognate response regulator (RR), HTCSs contain the membrane sensor HK and the RR transcriptional regulator within a single polypeptide chain. Tethering the DNA-binding domain (DBD) of the RR with the dimeric HK domain in an HTCS could potentially promote dimerization of the DBDs and would thus require a mechanism to suppress DNA-binding activity in the absence of stimulus. Analysis of phosphorylation and DNA-binding activities of several HTCSs from Bacteroides thetaiotaomicron revealed a DBD suppression mechanism in which an inhibitory interaction between the DBD and the phosphoryl group-accepting receiver domain (REC) decreases autophosphorylation rates of HTCS-RECs and represses DNA-binding activities in the absence of phosphorylation. Sequence analyses and structure predictions identified a highly conserved sequence motif correlated with a conserved inhibitory domain arrangement of REC and DBD. The presence of the motif, as in most HTCSs, or its absence, in a small subset of HTCSs, is likely predictive of two distinct regulatory mechanisms evolved for different glycans. Substitutions within the conserved motif relieve the inhibitory interaction and result in elevated DNA-binding activities in the absence of phosphorylation. Our data suggest a fundamental regulatory mechanism shared by most HTCSs to suppress DBD activities using a conserved inhibitory interdomain arrangement to overcome the challenge of the fused HK and RR components. IMPORTANCE: Different dietary and host-derived complex carbohydrates shape the gut microbial community and impact human health. In Bacteroides, the prevalent gut bacteria genus, utilization of these diverse carbohydrates relies on different gene clusters that are under sophisticated control by various signaling systems, including the hybrid two-component systems (HTCSs). We have uncovered a highly conserved regulatory mechanism in which the output DNA-binding activity of HTCSs is suppressed by interdomain interactions in the absence of stimulating phosphorylation. A consensus amino acid motif is found to correlate with the inhibitory interaction surface while deviations from the consensus can lead to constitutive activation. Understanding of such conserved HTCS features will be important to make regulatory predictions for individual systems as well as to engineer novel systems with substitutions in the consensus to explore the glycan regulation landscape in Bacteroides.

Repurposing Hsp90 inhibitors as antimicrobials targeting two-component systems identifies compounds leading to loss of bacterial membrane integrity.

The discovery of antimicrobials with novel mechanisms of action is crucial to tackle the foreseen global health crisis due to antimicrobial resistance. Bacterial two-component signaling systems (TCSs) are attractive targets for the discovery of novel antibacterial agents. TCS-encoding genes are found in all bacterial genomes and typically consist of a sensor histidine kinase (HK) and a response regulator. Due to the conserved Bergerat fold in the ATP-binding domain of the TCS HK and the human chaperone Hsp90, there has been much interest in repurposing inhibitors of Hsp90 as antibacterial compounds. In this study, we explore the chemical space of the known Hsp90 inhibitor scaffold 3,4-diphenylpyrazole (DPP), building on previous literature to further understand their potential for HK inhibition. Six DPP analogs inhibited HK autophosphorylation in vitro and had good antimicrobial activity against Gram-positive bacteria. However, mechanistic studies showed that their antimicrobial activity was related to damage of bacterial membranes. In addition, DPP analogs were cytotoxic to human embryonic kidney cell lines and induced the cell arrest phenotype shown for other Hsp90 inhibitors. We conclude that these DPP structures can be further optimized as specific disruptors of bacterial membranes providing binding to Hsp90 and cytotoxicity are lowered. Moreover, the X-ray crystal structure of resorcinol, a substructure of the DPP derivatives, bound to the HK CheA represents a promising starting point for the fragment-based design of novel HK inhibitors. IMPORTANCE: The discovery of novel antimicrobials is of paramount importance in tackling the imminent global health crisis of antimicrobial resistance. The discovery of novel antimicrobials with novel mechanisms of actions, e.g., targeting bacterial two-component signaling systems, is crucial to bypass existing resistance mechanisms and stimulate pharmaceutical innovations. Here, we explore the possible repurposing of compounds developed in cancer research as inhibitors of two-component systems and investigate their off-target effects such as bacterial membrane disruption and toxicity. These results highlight compounds that are promising for further development of novel bacterial membrane disruptors and two-component system inhibitors.

The S-component fold: a link between bacterial transporters and receptors.

The processes of nutrient uptake and signal sensing are crucial for microbial survival and adaptation. Membrane-embedded proteins involved in these functions (transporters and receptors) are commonly regarded as unrelated in terms of sequence, structure, mechanism of action and evolutionary history. Here, we analyze the protein structural universe using recently developed artificial intelligence-based structure prediction tools, and find an unexpected link between prominent groups of microbial transporters and receptors. The so-called S-components of Energy-Coupling Factor (ECF) transporters, and the membrane domains of sensor histidine kinases of the 5TMR cluster share a structural fold. The discovery of their relatedness manifests a widespread case of prokaryotic "transceptors" (related proteins with transport or receptor function), showcases how artificial intelligence-based structure predictions reveal unchartered evolutionary connections between proteins, and provides new avenues for engineering transport and signaling functions in bacteria.

The sensor of the bacterial histidine kinase CpxA is a novel dimer of extracytoplasmic Per-ARNT-Sim domains.

Histidine kinases are key bacterial sensors that recognize diverse environmental stimuli. While mechanisms of phosphorylation and phosphotransfer by cytoplasmic kinase domains are relatively well-characterized, the ways in which extracytoplasmic sensor domains regulate activation remain mysterious. The Cpx envelope stress response is a conserved Gram-negative two-component system which is controlled by the sensor kinase CpxA. We report the structure of the Escherichia coli CpxA sensor domain (CpxA-SD) as a globular Per-ARNT-Sim (PAS)-like fold highly similar to that of Vibrio parahaemolyticus CpxA as determined by X-ray crystallography. Because sensor kinase dimerization is important for signaling, we used AlphaFold2 to model CpxA-SD in the context of its connected transmembrane domains, which yielded a novel dimer of PAS domains possessing a distinct dimer organization compared to previously characterized sensor domains. Gain of function cpxA∗ alleles map to the dimer interface, and mutation of other residues in this region also leads to constitutive activation. CpxA activation can be suppressed by mutations that restore inter-monomer interactions, suggesting that inhibitory interactions between CpxA-SD monomers are the major point of control for CpxA activation and signaling. Searching through hundreds of structural homologs revealed the sensor domain of Pseudomonas aeruginosa sensor kinase PfeS as the only PAS structure in the same novel dimer orientation as CpxA, suggesting that our dimer orientation may be utilized by other extracytoplasmic PAS domains. Overall, our findings provide insight into the diversity of the organization of PAS sensory domains and how they regulate sensor kinase activation.

HssS activation by membrane heme defines a paradigm for two-component system signaling in Staphylococcus aureus.

Strict management of intracellular heme pools, which are both toxic and beneficial, is crucial for bacterial survival during infection. The human pathogen Staphylococcus aureus uses a two-component heme sensing system (HssRS), which counteracts environmental heme toxicity by triggering expression of the efflux transporter HrtBA. The HssS heme sensor is a HisKA-type histidine kinase, characterized as a membrane-bound homodimer containing an extracellular sensor and a cytoplasmic conserved catalytic domain. To elucidate HssS heme-sensing mechanism, a structural simulation of the HssS dimer based on Alphafold2 was docked with heme. In this model, a heme-binding site is present in the HssS dimer between the membrane and extracellular domains. Heme is embedded in the membrane bilayer with its two protruding porphyrin propionates interacting with two conserved Arg94 and Arg163 that are located extracellularly. Single substitutions of these arginines and two highly conserved phenylalanines, Phe25 and Phe128, in the predicted hydrophobic pocket limited the ability of HssS to induce HrtBA synthesis. Combination of the four substitutions abolished HssS activation. Wild-type (WT) HssS copurified with heme from Escherichia coli, whereas heme binding was strongly attenuated in the variants. This study gives evidence that exogenous heme interacts with HssS at the membrane/extracellular interface to initiate HssS activation and induce HrtBA-mediated heme extrusion from the membrane. This "gatekeeper" mechanism could limit intracellular diffusion of exogenous heme in S. aureus and may serve as a paradigm for how efflux transporters control detoxification of exogenous hydrophobic stressors.IMPORTANCEIn the host blood, pathogenic bacteria are exposed to the red pigment heme that concentrates in their lipid membranes, generating cytotoxicity. To overcome heme toxicity, Staphylococcus aureus expresses a membrane sensor protein, HssS. Activation of HssS by heme triggers a phosphotransfer mechanism leading to the expression of a heme efflux system, HrtBA. This detoxification system prevents intracellular accumulation of heme. Our structural and functional data reveal a heme-binding hydrophobic cavity in HssS within the transmembrane domains (TM) helices at the interface with the extracellular domain. This structural pocket is important for the function of HssS as a heme sensor. Our findings provide a new basis for the elucidation of pathogen-sensing mechanisms as a prerequisite to the discovery of inhibitors.

Recent structural advances in bacterial chemotaxis signalling.

Bacterial chemosensory arrays have served as a model system for in-situ structure determination, clearly cataloguing the improvement of cryo-electron tomography (cryoET) over the past decade. In recent years, this has culminated in an accurately fitted atomistic model for the full-length core signalling unit (CSU) and numerous insights into the function of the transmembrane receptors responsible for signal transduction. Here, we review the achievements of the latest structural advances in bacterial chemosensory arrays and the developments which have made such advances possible.

Structural features discriminating hybrid histidine kinase Rec domains from response regulator homologs.

In two-component systems, the information gathered by histidine kinases (HKs) are relayed to cognate response regulators (RRs). Thereby, the phosphoryl group of the auto-phosphorylated HK is transferred to the receiver (Rec) domain of the RR to allosterically activate its effector domain. In contrast, multi-step phosphorelays comprise at least one additional Rec (Recinter) domain that is typically part of the HK and acts as an intermediary for phosphoryl-shuttling. While RR Rec domains have been studied extensively, little is known about discriminating features of Recinter domains. Here we study the Recinter domain of the hybrid HK CckA by X-ray crystallography and NMR spectroscopy. Strikingly, all active site residues of the canonical Rec-fold are pre-arranged for phosphoryl-binding and BeF3- binding does not alter secondary or quaternary structure, indicating the absence of allosteric changes, the hallmark of RRs. Based on sequence-covariation and modeling, we analyze the intra-molecular DHp/Rec association in hybrid HKs.

Signal transduction mechanisms in heme-based globin-coupled oxygen sensors with a focus on a histidine kinase (AfGcHK) and a diguanylate cyclase (YddV or EcDosC).

Heme is a vital cofactor of proteins with roles in oxygen transport (e.g. hemoglobin), storage (e.g. myoglobin), and activation (e.g. P450) as well as electron transfer (e.g. cytochromes) and many other functions. However, its structural and functional role in oxygen sensing proteins differs markedly from that in most other enzymes, where it serves as a catalytic or functional center. This minireview discusses the mechanism of signal transduction in two heme-based oxygen sensors: the histidine kinase AfGcHK and the diguanylate cyclase YddV (EcDosC), both of which feature a heme-binding domain containing a globin fold resembling that of hemoglobin and myoglobin.

Crystal structure of the Escherichia coli CusS kinase core.

The CusS histidine kinase is a member of Escherichia coli two-component signal transduction system, engaged in a response to copper ions excess in the cell periplasm. The periplasmic sensor domain of CusS binds the free copper ions and the CusS kinase core phosphorylates the cognate CusR which regulates transcription of the efflux pomp CusCBA. A small amount of copper ions is indispensable for the aerobic cell metabolism. Nonetheless, its excess in the cytoplasm generates damaging and reactive hydroxyl radicals. For that reason, understanding the bacterial copper sensing mechanisms can contribute to reducing bacterial copper-resistance and developing bactericidal copper-based materials. The crystal structure of the CusS kinase core was solved at the resolution of 1.4 Å. The cytoplasmic catalytic core domains formed a homodimer. Based on the obtained structure, the intramolecular and intermolecular interactions crucial for the mechanism of CusS autophosphorylation were described.

Insights into the atypical autokinase activity of the Pseudomonas aeruginosa GacS histidine kinase and its interaction with RetS.

Virulence in Pseudomonas aeruginosa (PA) depends on complex regulatory networks, involving phosphorelay systems based on two-component systems (TCSs). The GacS/GacA TCS is a master regulator of biofilm formation, swarming motility, and virulence. GacS is a membrane-associated unorthodox histidine kinase (HK) whose phosphorelay signaling pathway is inhibited by the RetS hybrid HK. Here we provide structural and functional insights into the interaction of GacS with RetS. The structure of the GacS-HAMP-H1 cytoplasmic regions reveals an unusually elongated homodimer marked by a 135 Å long helical bundle formed by the HAMP, the signaling helix (S helix) and the DHp subdomain. The HAMP and S helix regions are essential for GacS signaling and contribute to the GacS/RetS binding interface. The structure of the GacS D1 domain together with the discovery of an unidentified functional ND domain, essential for GacS full autokinase activity, unveils signature motifs in GacS required for its atypical autokinase mechanism.

Protein engineering strategies to stimulate the functions of bacterial pseudokinases.

Enzymes orchestrate an array of concerted functions that often culminate in the chemical conversion of substrates into products. In the bacterial kingdom, histidine kinases autophosphorylate, then transfer that phosphate to a second protein called a response regulator. Bacterial genomes can encode large numbers of histidine kinases that provide surveillance of environmental and cytosolic stresses through signal stimulation of histidine kinase activity. Pseudokinases lack these hallmark catalytic functions but often retain binding interactions and allostery. Characterization of bacterial pseudokinases then takes a fundamentally different approach than their enzymatic counterparts. Here we discuss models for how bacterial pseudokinases can utilize protein-protein interactions and allostery to serve as crucial signaling pathway regulators. Then we describe a protein engineering strategy to interrogate these models, emphasizing how signals flow within bacterial pseudokinases. This description includes design considerations, cloning strategies, and the purification of leucine zippers fused to pseudokinases. We then describe two assays to interrogate this approach. First is a C. crescentus swarm plate assay to track motility phenotypes related to a bacterial pseudokinase. Second is an in vitro coupled-enzyme assay that can be applied to test if and how a pseudokinase regulates an active kinase. Together these approaches provide a blueprint for dissecting the mechanisms of cryptic bacterial pseudokinases.

Diversity in Sensing and Signaling of Bacterial Sensor Histidine Kinases.

Two-component signal transduction systems (TCSs) are widely conserved in bacteria to respond to and adapt to the changing environment. Since TCSs are also involved in controlling the expression of virulence, biofilm formation, quorum sensing, and antimicrobial resistance in pathogens, they serve as candidates for novel drug targets. TCSs consist of a sensor histidine kinase (HK) and its cognate response regulator (RR). Upon perception of a signal, HKs autophosphorylate their conserved histidine residues, followed by phosphotransfer to their partner RRs. The phosphorylated RRs mostly function as transcriptional regulators and control the expression of genes necessary for stress response. HKs sense their specific signals not only in their extracytoplasmic sensor domain but also in their cytoplasmic and transmembrane domains. The signals are sensed either directly or indirectly via cofactors and accessory proteins. Accumulating evidence shows that a single HK can sense and respond to multiple signals in different domains. The underlying molecular mechanisms of how HK activity is controlled by these signals have been extensively studied both biochemically and structurally. In this article, we introduce the wide diversity of signal perception in different domains of HKs, together with their recently clarified structures and molecular mechanisms.

Studying bacterial chemosensory array with CryoEM.

Bacteria direct their movement in respond to gradients of nutrients and other stimuli in the environment through the chemosensory system. The behavior is mediated by chemosensory arrays that are made up of thousands of proteins to form an organized array near the cell pole. In this review, we briefly introduce the architecture and function of the chemosensory array and its core signaling unit. We describe the in vivo and in vitro systems that have been used for structural studies of chemosensory array by cryoEM, including reconstituted lipid nanodiscs, 2D lipid monolayer arrays, lysed bacterial ghosts, bacterial minicells and native bacteria cells. Lastly, we review recent advances in structural analysis of chemosensory arrays using state-of-the-art cryoEM and cryoET methodologies, focusing on the latest developments and insights with a perspective on current challenges and future directions.

Overview of protein phosphorylation in bacteria with a main focus on unusual protein kinases in Bacillus subtilis.

Protein phosphorylation is a post-translational modification that affects protein activity through the addition of a phosphate moiety by protein kinases or phosphotransferases. It occurs in all life forms. In addition to Hanks kinases found also in eukaryotes, bacteria encode membrane histidine kinases that, with their cognate response regulator, constitute two-component systems and phosphotransferases that phosphorylate proteins involved in sugar utilization on histidine and cysteine residues. In addition, they encode BY-kinases and arginine kinases that phosphorylate protein specifically on tyrosine and arginine residues respectively. They also possess unusual bacterial protein kinases illustrated here by examples from Bacillus subtilis.