Contribution of the Sensor Histidine Kinases PhcS and VsrA to the Quorum Sensing of Ralstonia pseudosolanacearum Strain OE1-1.

The soilborne Gram-negative phytopathogenic beta-proteobacterium Ralstonia pseudosolanacearum strain OE1-1 produces methyl 3-hydroxymyristate (3-OH MAME) as the quorum sensing (QS) signal by the methyltransferase PhcB and senses the chemical, activating the LysR family transcriptional regulator PhcA, which regulates the QS-dependent genes responsible for QS-dependent phenotypes including virulence. The sensor histidine kinases PhcS and VsrA are reportedly involved in the regulation of QS-dependent genes. To elucidate the function of PhcS and VsrA in the active QS, we generated the phcS-deletion and vsrA-deletion mutants, which exhibited weak changes to their QS-dependent phenotypes including virulence. The phcS and vsrA-deletion mutant (ΔphcS/vsrA) had significant changes in its QS-dependent phenotypes and was nonvirulent, similar to the phcA-deletion mutant. The mutant (PhcS-H230Q) with a substitution of histidine to glutamine at amino acid position 230 in PhcS but not the mutant (VsrA-H256Q) with a substitution of histidine to glutamine at amino acid position 256 in VsrA exhibited significant changes in QS-dependent phenotypes and lost virulence. The transcriptome analysis with RNA-sequencing revealed significant alterations to the expression of QS-dependent genes in the ΔphcS/vsrA and PhcS-H230Q but not VsrA-H256Q, similar to the phcA-deletion mutant. The exogenous 3-OH MAME application led to a significantly enhanced QS-inducible major exopolysaccharide EPS I production of the strain OE1-1 and phcB-deletion mutant but not ΔphcS/vsrA and PhcS-H230Q. Collectively, results of the present genetic study suggested that PhcS contributes to QS along with VsrA and that histidine at amino acid position 230 of PhcS is required for 3-OH MAME sensing, thereby influencing QS-dependent phenotypes including virulence of the strain OE1-1. [Formula: see text] The author(s) have dedicated the work to the public domain under the Creative Commons CC0 "No Rights Reserved" license by waiving all of his or her rights to the work worldwide under copyright law, including all related and neighboring rights, to the extent allowed by law, 2024.

Structure and distribution of sensor histidine kinases in the fungal kingdom.

Two-component systems (TCSs) are diverse cell signaling pathways that play a significant role in coping with a wide range of environmental cues in both prokaryotic and eukaryotic organisms. These transduction circuitries are primarily governed by histidine kinases (HKs), which act as sensing proteins of a broad variety of stressors. To date, nineteen HK groups have been previously described in the fungal kingdom. However, the structure and distribution of these prominent sensing proteins were hitherto investigated in a limited number of fungal species. In this study, we took advantage of recent genomic resources in fungi to refine the fungal HK classification by deciphering the structural diversity and phylogenetic distribution of HKs across a large number of fungal clades. To this end, we browsed the genome of 91 species representative of different fungal clades, which yielded 726 predicted HK sequences. A domain organization analysis, coupled with a robust phylogenomic approach, led to an improved categorization of fungal HKs. While most of the compiled sequences were categorized into previously described fungal HK groups, some new groups were also defined. Overall, this study provides an improved overview of the structure, distribution, and evolution of HKs in the fungal kingdom.

Adaptative responses of Neurospora crassa by histidine kinases upon the attack of the arthropod Sinella curviseta.

Histidine kinases (HKs) are important sensor proteins in fungi and play an essential role in environmental adaptation. However, the mechanisms by which fungi sense and respond to fungivores attack via HKs are not fully understood. In this study, we utilized Neurospora crassa to investigate the involvement of HKs in responding to fungivores attack. We found that the 11 HKs in N. crassa not only affected the growth and development, but also led to fluctuations in antioxidant production. Ten mutants in the genes encoding HKs (except ∆phy1) showed increased production of reactive oxygen species (ROS), especially upon Sinella curviseta attack. The ROS burst triggered changes in conidia and perithecial beaks formation, as well as accumulation of ß-glucan, ergothioneine, ergosterol, and carotenoids. ß-glucan was increased in ∆hk9, ∆os1, ∆hcp1, ∆nik2, ∆sln1, ∆phy1 and ∆phy2 mutants compared to the wild-type strain. In parallel, ergothioneine accumulation was improved in ∆phy1 and ∆hk16 mutants and further increased upon attack, except in ∆os1 and ∆hk16 mutants. Additionally, fungivores attack stimulated ergosterol and dehydroergosterol production in ∆hk9 and ∆os1 mutants. Furthermore, deletion of these genes altered carotenoid accumulation, with wild-type strain, ∆hk9, ∆os1, ∆hcp1, ∆sln1, ∆phy2, and ∆dcc1mutants showing an increase in carotenoids upon attack. Taken together, HKs are involved in regulating the production of conidia and antioxidants. Thus, HKs may act as sensors of fungivores attack and effectively improve the adaptive capacity of fungi to environmental stimuli.

A Homolog of the Histidine Kinase RetS Controls the Synthesis of Alginates, PHB, Alkylresorcinols, and Motility in Azotobacter vinelandii.

The two-component system GacS/A and the posttranscriptional control system Rsm constitute a genetic regulation pathway in Gammaproteobacteria; in some species of Pseudomonas, this pathway is part of a multikinase network (MKN) that regulates the activity of the Rsm system. In this network, the activity of GacS is controlled by other kinases. One of the most studied MKNs is the MKN-GacS of Pseudomonas aeruginosa, where GacS is controlled by the kinases RetS and LadS; RetS decreases the kinase activity of GacS, whereas LadS stimulates the activity of the central kinase GacS. Outside of the Pseudomonas genus, the network has been studied only in Azotobacter vinelandii. In this work, we report the study of the RetS kinase of A. vinelandii; as expected, the phenotypes affected in gacS mutants, such as production of alginates, polyhydroxybutyrate, and alkylresorcinols and swimming motility, were also affected in retS mutants. Interestingly, our data indicated that RetS in A. vinelandii acts as a positive regulator of GacA activity. Consistent with this finding, mutation in retS also negatively affected the expression of small regulatory RNAs belonging to the Rsm family. We also confirmed the interaction of RetS with GacS, as well as with the phosphotransfer protein HptB.

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.

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.

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.

Histidine Kinase QseC in Glaesserella parasuis Enhances the Secretion of Proinflammatory Cytokines by Macrophages via the p38 and NF-κB Signaling Pathways.

The qseC gene is a two-component system that encodes a histidine protein kinase and is highly conserved among different Glaesserella parasuis strains. In this study, we used qRT-PCR and enzyme-linked immunosorbent assay to confirm that Toll-like receptor 4 (TLR4) plays a role in the expression of proinflammatory cytokines interleukin (IL)-1ß and IL-6 by stimulating RAW 264.7 macrophages with QseC. Furthermore, we revealed that blocking the p38 and NF-κB pathways that regulate signaling can significantly reduce the production of proinflammatory cytokines induced by QseC. In summary, our data suggest that QseC is a novel proinflammatory mediator that induces TLR4-dependent proinflammatory activity in RAW 264.7 macrophages through the p38 and NF-κB pathways.

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.

Evolution of orphan and atypical histidine kinases and response regulators for microbial signaling diversity.

Two-component signaling systems (TCS) are the predominant means of microbes for sensing and responding to environmental stimuli. Typically, TCS is comprised of a sensor histidine kinase (HK) and a cognate response regulator (RR), which might have coevolved together. They usually involve the phosphoryl transfer signaling mechanism. However, there are also some orphan and atypical HK and RR homologs, and their evolutionary origins are still not very clear. They are not associated with cognate pairs or lack the conserved residues for phosphoryl transfer, but they could receive or respond to signals from other regulators. The objective of this study is to reveal the evolutionary history of these orphan and atypical HK and RR homologs. Structural, domain, sequence, and phylogenetic analyses indicated that their evolution process might undergo gene duplication, divergence, and domain shuffling. Meanwhile, lateral gene transfer might also be involved for their gene distribution. Evolution of orphan and atypical HK and RR homologs have increased their signaling diversity, which could be helpful for microbial adaption in complex environments.

Engineering two-component systems for advanced biosensing: From architecture to applications in biotechnology.

Two-component systems (TCSs) are prevalent signaling pathways in bacteria. These systems mediate phosphotransfer between histidine kinase and a response regulator, facilitating responses to diverse physical, chemical, and biological stimuli. Advancements in synthetic and structural biology have repurposed TCSs for applications in monitoring heavy metals, disease-associated biomarkers, and the production of bioproducts. However, the utility of many TCS biosensors is hindered by undesired performance due to the lack of effective engineering methods. Here, we briefly discuss the architectures and regulatory mechanisms of TCSs. We also summarize the recent advancements in TCS engineering by experimental or computational-based methods to fine-tune the biosensor functional parameters, such as response curve and specificity. Engineered TCSs have great potential in the medical, environmental, and biorefinery fields, demonstrating a crucial role in a wide area of biotechnology.

Analysis of CenKR essentiality in Sinorhizobium meliloti and its activity at a target gene promoter in vivo.

The two-component regulatory system CenK-CenR has recently emerged as a regulator of cell envelope and cell division processes in the alpha-proteobacteria. In Sinorhizobium meliloti, CenK-CenR regulates the expression of SrlA, a thioredoxin-domain protein of unknown function. Deletion of srlA causes sensitivity to salt and oxidizing agents on solid growth medium. In this work, we report that the response regulator CenR, but not the histidine kinase CenK, is essential for cell viability in S. meliloti. We also demonstrate that phosphorylation of the target residue D55 is not required for viability, suggesting that the unphosphorylated transcription factor sufficiently regulates expression of one or more essential genes in the genome. Using transcription assays and phenotype testing we examine CenK-CenR-dependent activation of the srlA promoter and demonstrate its absolute dependence on phosphoryl-CenR for activity and that the CenR substitution D55E acts as a phosphomimetic that partially restores activity at the srlA promoter in the absence of phosphorylation by CenK. Finally, we report a mutational analysis of the CenR binding site in the srlA promoter required for transcriptional activation.

Histidine Phosphorylation: Protein Kinases and Phosphatases.

Phosphohistidine (pHis) is a reversible protein post-translational modification (PTM) that is currently poorly understood. The P-N bond in pHis is heat and acid-sensitive, making it more challenging to study than the canonical phosphoamino acids pSer, pThr, and pTyr. As advancements in the development of tools to study pHis have been made, the roles of pHis in cells are slowly being revealed. To date, a handful of enzymes responsible for controlling this modification have been identified, including the histidine kinases NME1 and NME2, as well as the phosphohistidine phosphatases PHPT1, LHPP, and PGAM5. These tools have also identified the substrates of these enzymes, granting new insights into previously unknown regulatory mechanisms. Here, we discuss the cellular function of pHis and how it is regulated on known pHis-containing proteins, as well as cellular mechanisms that regulate the activity of the pHis kinases and phosphatases themselves. We further discuss the role of the pHis kinases and phosphatases as potential tumor promoters or suppressors. Finally, we give an overview of various tools and methods currently used to study pHis biology. Given their breadth of functions, unraveling the role of pHis in mammalian systems promises radical new insights into existing and unexplored areas of cell biology.

Leveraging the histidine kinase-phosphatase duality to sculpt two-component signaling.

Bacteria must constantly probe their environment for rapid adaptation, a crucial need most frequently served by two-component systems (TCS). As one component, sensor histidine kinases (SHK) control the phosphorylation of the second component, the response regulator (RR). Downstream responses hinge on RR phosphorylation and can be highly stringent, acute, and sensitive because SHKs commonly exert both kinase and phosphatase activity. With a bacteriophytochrome TCS as a paradigm, we here interrogate how this catalytic duality underlies signal responses. Derivative systems exhibit tenfold higher red-light sensitivity, owing to an altered kinase-phosphatase balance. Modifications of the linker intervening the SHK sensor and catalytic entities likewise tilt this balance and provide TCSs with inverted output that increases under red light. These TCSs expand synthetic biology and showcase how deliberate perturbations of the kinase-phosphatase duality unlock altered signal-response regimes. Arguably, these aspects equally pertain to the engineering and the natural evolution of TCSs.

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.

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.

Synthesis and biochemical characterization of naphthoquinone derivatives targeting bacterial histidine kinases.

Waldiomycin is an inhibitor of histidine kinases (HKs). Although most HK inhibitors target the ATP-binding region, waldiomycin binds to the intracellular dimerization domain (DHp domain) with its naphthoquinone moiety presumed to interact with the conserved H-box region. To further develop inhibitors targeting the H-box, various 2-aminonaphthoquinones with cyclic, aliphatic, or aromatic amino groups and naphtho [2,3-d] isoxazole-4,9-diones were synthesized. These compounds were tested for their inhibitory activity (IC50) against WalK, an essential HK for Bacillus subtilis growth, and their minimum inhibitory concentrations (MIC) against B. subtilis. As a result, 11 novel HK inhibitors were obtained as naphthoquinone derivatives (IC50: 12.6-305 µM, MIC: 0.5-128 µg ml-1). The effect of representative compounds on the expression of WalK/WalR regulated genes in B. subtilis was investigated. Four naphthoquinone derivatives induced the expression of iseA (formerly yoeB), whose expression is negatively regulated by the WalK/WalR system. This suggests that these compounds inhibit WalK in B. subtilis cells, resulting in antibacterial activity. Affinity selection/mass spectrometry analysis was performed to identify whether these naphthoquinone derivatives interact with WalK in a manner similar to waldiomycin. Three compounds were found to competitively inhibit the binding of waldiomycin to WalK, suggesting that they bind to the H-box region conserved in HKs and inhibit HK activity.

Proteolytic control of FixT by the Lon protease impacts FixLJ signaling in Caulobacter crescentus.

Responding to changes in oxygen levels is critical for aerobic microbes. In Caulobacter crescentus, low oxygen is sensed by the FixL-FixJ two-component system which induces multiple genes, including those involved in heme biosynthesis, to accommodate microaerobic conditions. The FixLJ inhibitor FixT is also induced under low oxygen conditions and is degraded by the Lon protease when the oxygen levels are sufficient, which together provides negative feedback proposed to adjust FixLJ signaling thresholds during changing conditions. Here, we address whether degradation of FixT by the Lon protease contributes to phenotypic defects associated with loss of Lon. We find that ∆lon strains are deficient in FixLJ-dependent heme biosynthesis, consistent with elevated FixT levels as deletion of fixT suppresses this defect. Transcriptomics validate this result as, along with heme biosynthesis, there is diminished expression of many FixL-activated genes in ∆lon. However, stabilization of FixT in ∆lon strains does not contribute to restoring any known Lon-related fitness defect, such as cell morphology defects or stress sensitivity. In fact, cells lacking both FixT and Lon are compromised in viability during growth in standard aerobic conditions. Our work highlights the complexity of protease-dependent regulation of transcription factors and explains the molecular basis of defective heme accumulation in Lon-deficient Caulobacter. IMPORTANCE: The Lon protease shapes protein quality control, signaling pathways, and stress responses in many bacteria species. Loss of Lon often results in multiple phenotypic consequences. In this work, we found a connection between the Lon protease and deficiencies in heme accumulation that then led to our finding of a global change in gene expression due in part to degradation of a regulator of the hypoxic response. However, loss of degradation of this regulator did not explain other phenotypes associated with Lon deficiencies demonstrating the complex and multiple pathways that this highly conserved protease can impact.

New insights into the signal transduction mechanism of O2-sensing FixL and other biological heme-based sensor proteins.

Recent structural and biophysical studies of O2-sensing FixL, NO-sensing soluble guanylate cyclase, and other biological heme-based sensing proteins have begun to reveal the details of their molecular mechanisms and shed light on how nature regulates important biological processes such as nitrogen fixation, blood pressure, neurotransmission, photosynthesis and circadian rhythm. The O2-sensing FixL protein from S. meliloti, the eukaryotic NO-sensing protein sGC, and the CO-sensing CooA protein from R. rubrum transmit their biological signals through gas-binding to the heme domain of these proteins, which inhibits or activates the regulatory, enzymatic domain. These proteins appear to propagate their signal by specific structural changes in the heme sensor domain initiated by the appropriate gas binding to the heme, which is then propagated through a coiled-coil linker or other domain to the regulatory, enzymatic domain that sends out the biological signal. The current understanding of the signal transduction mechanisms of O2-sensing FixL, NO-sensing sGC, CO-sensing CooA and other biological heme-based gas sensing proteins and their mechanistic themes are discussed, with recommendations for future work to further understand this rapidly growing area of biological heme-based gas sensors.

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.