Structural insights into redox signal transduction mechanisms in the control of nitrogen fixation by the NifLA system.

NifL is a conformationally dynamic flavoprotein responsible for regulating the activity of the σ54-dependent activator NifA to control the transcription of nitrogen fixation (nif) genes in response to intracellular oxygen, cellular energy, or nitrogen availability. The NifL-NifA two-component system is the master regulatory system for nitrogen fixation. NifL serves as a sensory protein, undergoing signal-dependent conformational changes that modulate its interaction with NifA, forming the NifL-NifA complex, which inhibits NifA activity in conditions unsuitable for nitrogen fixation. While NifL-NifA regulation is well understood, these conformationally flexible proteins have eluded previous attempts at structure determination. In work described here, we advance a structural model of the NifL dimer supported by a combination of scattering techniques and mass spectrometry (MS)-coupled structural analyses that report on the average structure in solution. Using a combination of small angle X-ray scattering-derived electron density maps and MS-coupled surface labeling, we investigate the conformational dynamics responsible for NifL oxygen and energy responses. Our results reveal conformational differences in the structure of NifL under reduced and oxidized conditions that provide the basis for a model for modulating NifLA complex formation in the regulation of nitrogen fixation in response to oxygen in the model diazotroph, Azotobacter vinelandii.

Phosphorylation chemistry of the Bordetella PlrSR TCS and its contribution to bacterial persistence in the lower respiratory tract.

Bordetella species cause lower respiratory tract infections in mammals. B. pertussis and B. bronchiseptica are the causative agents of whooping cough and kennel cough, respectively. The current acellular vaccine for B. pertussis protects against disease but does not prevent transmission or colonization. Cases of pertussis are on the rise even in areas of high vaccination. The PlrSR two-component system, is required for persistence in the mouse lung. A partial plrS deletion strain and a plrS H521Q strain cannot survive past 3 days in the lung, suggesting PlrSR works in a phosphorylation-dependent mechanism. We characterized the biochemistry of B. bronchiseptica PlrSR and found that both proteins function as a canonical two-component system. His521 was essential and Glu522 was critical for PlrS autophosphorylation. Asn525 was essential for phosphatase activity. The PAS domain was critical for both PlrS autophosphorylation and phosphatase activities. PlrS could both phosphotransfer to and exert phosphatase activity toward PlrR. Unexpectedly, PlrR formed a tetramer when unphosphorylated and a dimer upon phosphorylation. Finally, we demonstrated the importance of PlrS phosphatase activity for persistence within the murine lung. By characterizing PlrSR we hope to guide future in vivo investigation for development of new vaccines and therapeutics.

An overview of the two-component system GarR/GarS role on antibiotic production in Streptomyces coelicolor.

The Streptomyces genus comprises Gram-positive bacteria known to produce over two-thirds of the antibiotics used in medical practice. The biosynthesis of these secondary metabolites is highly regulated and influenced by a range of nutrients present in the growth medium. In Streptomyces coelicolor, glucose inhibits the production of actinorhodin (ACT) and undecylprodigiosin (RED) by a process known as carbon catabolite repression (CCR). However, the mechanism mediated by this carbon source still needs to be understood. It has been observed that glucose alters the transcriptomic profile of this actinobacteria, modifying different transcriptional regulators, including some of the one- and two-component systems (TCSs). Under glucose repression, the expression of one of these TCSs SCO6162/SCO6163 was negatively affected. We aimed to study the role of this TCS on secondary metabolite formation to define its influence in this general regulatory process and likely establish its relationship with other transcriptional regulators affecting antibiotic biosynthesis in the Streptomyces genus. In this work, in silico predictions suggested that this TCS can regulate the production of the secondary metabolites ACT and RED by transcriptional regulation and protein-protein interactions of the transcriptional factors (TFs) with other TCSs. These predictions were supported by experimental procedures such as deletion and complementation of the TFs and qPCR experiments. Our results suggest that in the presence of glucose, the TCS SCO6162/SCO6163, named GarR/GarS, is an important negative regulator of the ACT and RED production in S. coelicolor. KEY POINTS: • GarR/GarS is a TCS with domains for signal transduction and response regulation • GarR/GarS is an essential negative regulator of the ACT and RED production • GarR/GarS putatively interacts with and regulates activators of ACT and RED.

Characterization of the extracellular domain of sensor histidine kinase NagS from Paenibacillus sp. str. FPU-7: nagS interacts with oligosaccharide binding protein NagB1 in complexes with N, N'-diacetylchitobiose.

We have previously isolated the Gram-positive chitin-degrading bacterium Paenibacillus sp. str. FPU-7. This bacterium traps chitin disaccharide (GlcNAc)2 on its cell surface using two homologous solute-binding proteins, NagB1 and NagB2. Bacteria use histidine kinase (HK) of the two-component regulatory system as an extracellular environment sensor. In this study, we found that nagS, which encodes a HK, is located next to the nagB1 gene. Biochemical experiments revealed that the NagS sensor domain (NagS30-294) interacts with the NagB1-(GlcNAc)2 complex. However, proof of NagS30-294 interacting with NagB1 without (GlcNAc)2 is currently unavailable. In contrast to NagB1, no complex formation was observed between NagS30-294 and NagB2, even in the presence of (GlcNAc)2. The NagS30-294 crystal structure at 1.8 Å resolution suggested that the canonical tandem-Per-Arnt-Sim fold recognizes the NagB1-(GlcNAc)2 complex. This study provides insight into the recognition of chitin oligosaccharides by bacteria.

Regulation of Bacterial Two-Component Systems by Cardiolipin.

The regulation of membrane protein activity for cellular functions is critically dependent on the composition of phospholipid membranes. Cardiolipin, a unique phospholipid found in bacterial membranes and mitochondrial membranes of eukaryotes, plays a crucial role in stabilizing membrane proteins and maintaining their function. In the human pathogen Staphylococcus aureus, the SaeRS two-component system (TCS) controls the expression of key virulence factors essential for the bacterium's virulence. The SaeS sensor kinase activates the SaeR response regulator via phosphoryl transfer to bind its gene target promoters. In this study, we report that cardiolipin is critical for sustaining the full activity of SaeRS and other TCSs in S. aureus. The sensor kinase protein SaeS binds directly to cardiolipin and phosphatidylglycerol, enabling SaeS activity. Elimination of cardiolipin from the membrane reduces SaeS kinase activity, indicating that bacterial cardiolipin is necessary for modulating the kinase activities of SaeS and other sensor kinases during infection. Moreover, the deletion of cardiolipin synthase genes cls1 and cls2 leads to reduced cytotoxicity to human neutrophils and lower virulence in a mouse model of infection. These findings suggest a model where cardiolipin modulates the kinase activity of SaeS and other sensor kinases after infection to adapt to the hostile environment of the host and expand our knowledge of how phospholipids contribute to membrane protein function.

The sensor histidine kinase (SLN1) and acetyl-CoA carboxylase (ACC1) coordinately regulate the response of Neurospora crassa to the springtail Sinella curviseta (Collembola: Entomobryidae) attack.

IMPORTANCE: Understanding the regulatory pathways by which fungi respond to environmental signals through interlinked genes provides insights into the interactions between fungi and insects. The coordinated optimization of the regulatory networks is necessary for fungi to adapt to their habitats. We demonstrated that the synergistic regulation of sensor histidine kinase (SLN1) and acetyl-CoA carboxylase (ACC1) plays a critical role in regulating the fungal response to Sinella curviseta stress. Furthermore, we found that the enhanced production of trehalose, carotenoids, and 5-MTHF plays crucial role in the resistance to the fungivore. Our results provide insights into the understanding of the adaptation of N. crassa to environmental stimuli.

Trends in the two-component system's role in the synthesis of antibiotics by Streptomyces.

Despite the advances in understanding the regulatory networks for secondary metabolite production in Streptomyces, the participation of the two-component systems (TCS) in this process still requires better characterization. These sensing systems and their responses to environmental stimuli have been described by evaluating mutant strains with techniques that allow in-depth regulatory responses. However, defining the stimulus that triggers their activation is still a task. The transmembrane nature of the sensor kinases and the high content of GC in the streptomycetes represent significant challenges in their study. In some examples, adding elements to the assay medium has determined the respective ligand. However, a complete TCS description and characterization requires specific amounts of the involved proteins that are most difficult to obtain. The availability of enough sensor histidine kinase concentrations could facilitate the identification of the ligand-protein interaction, and besides would allow the establishment of its phosphorylation mechanisms and determine their tridimensional structure. Similarly, the advances in the development of bioinformatics tools and novel experimental techniques also promise to accelerate the TCSs description and provide knowledge on their participation in the regulation processes of secondary metabolite formation. This review aims to summarize the recent advances in the study of TCSs involved in antibiotic biosynthesis and to discuss alternatives to continue their characterization. KEY POINTS: • TCSs are the environmental signal transducers more abundant in nature. • The Streptomyces have some of the highest number of TCSs found in bacteria. • The study of signal transduction between SHKs and RRs domains is a big challenge.

The SrrAB two-component system regulates Staphylococcus aureus pathogenicity through redox sensitive cysteines.

Staphylococcus aureus infections can lead to diseases that range from localized skin abscess to life-threatening toxic shock syndrome. The SrrAB two-component system (TCS) is a global regulator of S. aureus virulence and critical for survival under environmental conditions such as hypoxic, oxidative, and nitrosative stress found at sites of infection. Despite the critical role of SrrAB in S. aureus pathogenicity, the mechanism by which the SrrAB TCS senses and responds to these environmental signals remains unknown. Bioinformatics analysis showed that the SrrB histidine kinase contains several domains, including an extracellular Cache domain and a cytoplasmic HAMP-PAS-DHp-CA region. Here, we show that the PAS domain regulates both kinase and phosphatase enzyme activity of SrrB and present the structure of the DHp-CA catalytic core. Importantly, this structure shows a unique intramolecular cysteine disulfide bond in the ATP-binding domain that significantly affects autophosphorylation kinetics. In vitro data show that the redox state of the disulfide bond affects S. aureus biofilm formation and toxic shock syndrome toxin-1 production. Moreover, with the use of the rabbit infective endocarditis model, we demonstrate that the disulfide bond is a critical regulatory element of SrrB function during S. aureus infection. Our data support a model whereby the disulfide bond and PAS domain of SrrB sense and respond to the cellular redox environment to regulate S. aureus survival and pathogenesis.

Insights into histidine kinase activation mechanisms from the monomeric blue light sensor EL346.

Translation of environmental cues into cellular behavior is a necessary process in all forms of life. In bacteria, this process frequently involves two-component systems in which a sensor histidine kinase (HK) autophosphorylates in response to a stimulus before subsequently transferring the phosphoryl group to a response regulator that controls downstream effectors. Many details of the molecular mechanisms of HK activation are still unclear due to complications associated with the multiple signaling states of these large, multidomain proteins. To address these challenges, we combined complementary solution biophysical approaches to examine the conformational changes upon activation of a minimal, blue-light-sensing histidine kinase from Erythrobacter litoralis HTCC2594, EL346. Our data show that multiple conformations coexist in the dark state of EL346 in solution, which may explain the enzyme's residual dark-state activity. We also observe that activation involves destabilization of the helices in the dimerization and histidine phosphotransfer-like domain, where the phosphoacceptor histidine resides, and their interactions with the catalytic domain. Similar light-induced changes occur to some extent even in constitutively active or inactive mutants, showing that light sensing can be decoupled from activation of kinase activity. These structural changes mirror those inferred by comparing X-ray crystal structures of inactive and active HK fragments, suggesting that they are at the core of conformational changes leading to HK activation. More broadly, our findings uncover surprising complexity in this simple system and allow us to outline a mechanism of the multiple steps of HK activation.

Investigating the effect of an identified mutation within a critical site of PAS domain of WalK protein in a vancomycin-intermediate resistant Staphylococcus aureus by computational approaches.

BACKGROUND: Vancomycin-intermediate resistant Staphylococcus aureus (VISA) is becoming a common cause of nosocomial infections worldwide. VISA isolates are developed by unclear molecular mechanisms via mutations in several genes, including walKR. Although studies have verified some of these mutations, there are a few studies that pay attention to the importance of molecular modelling of mutations. METHOD: For genomic and transcriptomic comparisons in a laboratory-derived VISA strain and its parental strain, Sanger sequencing and reverse transcriptase quantitative PCR (RT-qPCR) methods were used, respectively. After structural protein mapping of the detected mutation, mutation effects were analyzed using molecular computational approaches and crystal structures of related proteins. RESULTS: A mutation WalK-H364R was occurred in a functional zinc ion coordinating residue within the PAS domain in the VISA strain. WalK-H364R was predicted to destabilize protein and decrease WalK interactions with proteins and nucleic acids. The RT-qPCR method showed downregulation of walKR, WalKR-regulated autolysins, and agr locus. CONCLUSION: Overall, WalK-H364R mutation within a critical metal-coordinating site was presumably related to the VISA development. We assume that the WalK-H364R mutation resulted in deleterious effects on protein, which was verified by walKR gene expression changes.. Therefore, molecular modelling provides detailed insight into the molecular mechanism of VISA development, in particular, where allelic replacement experiments are not readily available.

MifS, a DctB family histidine kinase, is a specific regulator of α-ketoglutarate response in Pseudomonas aeruginosa PAO1.

The C5-dicarboxylate α-ketoglutarate (α-KG) is a preferred nutrient source for the opportunistic pathogen Pseudomonas aeruginosa. However, very little is known about how P. aeruginosa detects and responds to α-KG in the environment. Our laboratory has previously shown that the MifS/MifR two-component signal transduction system regulates α-KG assimilation in P. aeruginosa PAO1. In an effort to better understand how this bacterium detects α-KG, we characterized the MifS sensor histidine kinase. In this study we show that although MifS is a homologue of the C4-dicarboxylate sensor DctB, it specifically responds to the C5-dicarboxylate α-KG. MifS activity increased >10-fold in the presence of α-KG, while the related C5-dicarboxylate glutarate caused only a 2-fold increase in activity. All other dicarboxylates tested did not show any significant effect on MifS activity. Homology modelling of the MifS sensor domain revealed a substrate binding pocket for α-KG. Using protein modelling and mutational analysis, we identified nine residues that are important for α-KG response, including one residue that determines the substrate specificity of MifS. Further, we found that MifS has a novel cytoplasmic linker domain that is required for α-KG response and is probably involved in signal transduction from the sensor domain to the cytoplasmic transmitter domain. Until this study, DctB family histidine kinases were known to only respond to C4-dicarboxylates. Our work shows that MifS is a novel member of the DctB family histidine kinase that specifically responds to α-KG.

Modulation of CrbS-Dependent Activation of the Acetate Switch in Vibrio cholerae.

Vibrio cholerae controls the pathogenicity of interactions with arthropod hosts via the activity of the CrbS/R two-component system. This signaling pathway regulates the consumption of acetate, which in turn alters the relative virulence of interactions with arthropods, including Drosophila melanogaster CrbS is a histidine kinase that links a transporter-like domain to its signaling apparatus via putative STAC and PAS domains. CrbS and its cognate response regulator are required for the expression of acetyl coenzyme A (acetyl-CoA) synthetase (product of acs), which converts acetate to acetyl-CoA. We demonstrate that the STAC domain of CrbS is required for signaling in culture; without it, acs transcription is reduced in LB medium, and V. cholerae cannot grow on acetate minimal media. However, the strain remains virulent toward Drosophila and expresses acs similarly to the wild type during infection. This suggests that there is a unique signal or environmental variable that modulates CrbS in the gastrointestinal tract of Drosophila Second, we present evidence in support of CrbR, the response regulator that interacts with CrbS, binding directly to the acs promoter, and we identify a region of the promoter that CrbR may target. We further demonstrate that nutrient signals, together with the cAMP receptor protein (CRP)-cAMP system, control acs transcription, but regulation may occur indirectly, as CRP-cAMP activates the expression of the crbS and crbR genes. Finally, we define the role of the Pta-AckA system in V. cholerae and identify redundancy built into acetate excretion pathways in this pathogen.IMPORTANCE CrbS is a member of a unique family of sensor histidine kinases, as its structure suggests that it may link signaling to the transport of a molecule. However, mechanisms through which CrbS senses and communicates information about the outside world are unknown. In the Vibrionaceae, orthologs of CrbS regulate acetate metabolism, which can, in turn, affect interactions with host organisms. Here, we situate CrbS within a larger regulatory framework, demonstrating that crbS is regulated by nutrient-sensing systems. Furthermore, CrbS domains may play various roles in signaling during infection and growth in culture, suggesting a unique mechanism of host recognition. Finally, we define the roles of additional pathways in acetate flux, as a foundation for further studies of this metabolic nexus point.

Probing the nucleotide-binding activity of a redox sensor: two-component regulatory control in chloroplasts.

Two-component signal transduction systems mediate adaptation to environmental changes in bacteria, plants, fungi, and protists. Each two-component system consists of a sensor histidine kinase and a response regulator. Chloroplast sensor kinase (CSK) is a modified sensor histidine kinase found in chloroplasts-photosynthetic organelles of plants and algae. CSK regulates the transcription of chloroplast genes in response to changes in photosynthetic electron transport. In this study, the full-length and truncated forms of Arabidopsis CSK proteins were overexpressed and purified in order to characterise their kinase and redox sensing activities. Our results show that CSK contains a modified kinase catalytic domain that binds ATP with high affinity and forms a quinone adduct that may confer redox sensing activity.

Exploring solute binding proteins in Pseudomonas aeruginosa that bind to γ-aminobutyrate and 5-aminovalerate and their role in activating sensor kinases.

The standard method of receptor activation involves the binding of signals or signal-loaded solute binding proteins (SBPs) to sensor domains. Many sensor histidine kinases (SHKs), which are activated by SBP binding, are encoded adjacent to their corresponding sbp gene. We examined three SBPs of Pseudomonas aeruginosa PAO1, encoded near the genes for the AgtS (PA0600) and AruS (PA4982) SHKs, to determine how common this arrangement is. Ligand screening and microcalorimetric studies revealed that the SBPs PA0602 and PA4985 preferentially bind to GABA (KD = 2.3 and 0.58 µM, respectively), followed by 5-aminovalerate (KD = 30 and 1.6 µM, respectively) and ethanoldiamine (KD = 2.3 and 0.58 µM, respectively). In contrast, AgtB (PA0604) exclusively recognizes 5-aminovaleric acid (KD = 2.9 µM). However, microcalorimetric titrations did not show any binding between the AgtS sensor domain and AgtB or PA0602, regardless of the presence of ligands. Similarly, bacterial two-hybrid assays did not demonstrate an interaction between PA4985 and the AruS sensor domain. Therefore, sbp and shk genes located nearby are not always functionally linked. We previously identified PA0222 as a GABA-specific SBP. The presence of three SBPs for GABA may be linked to GABA's role as a trigger for P. aeruginosa virulence.

PAS domain of flagellar histidine kinase FlrB has a unique architecture and binds heme as a sensory ligand in an unconventional fashion.

Phosphorylation of the σ54-dependent transcription activator FlrC by the sensor histidine kinase FlrB is essential for flagellar synthesis of Vibrio cholerae. Despite that, the structure, sensory signal, and mechanistic basis of function of FlrB were elusive. Here, we report the crystal structure of the sensory PAS domain of FlrB in its functional dimeric state that exhibits a unique architecture. Series of biochemical/biophysical experiments on different constructs and mutants established that heme binds hydrophobically as sensory ligand in the shallow ligand-binding cleft of FlrB-PAS without axial coordination. Intriguingly, ATP binding to the C-terminal ATP-binding (CA) domain assists PAS domain to bind heme, vis-à-vis, heme binding to the PAS facilitates ATP binding to the CA domain. We hypothesize that synergistic binding of heme and ATP triggers conformational signaling in FlrB, leading to downstream flagellar gene transcription. Enhanced swimming motility of V. cholerae with increased heme uptake supports this proposition.

Sensor histidine kinases kdpD and aauS regulate biofilm and virulence in Pseudomonas aeruginosa PA14.

Pseudomonas aeruginosa is a multidrug-resistant opportunistic human pathogen that utilizes two-component systems (TCSs) to sense pathophysiological signals and coordinate virulence. P. aeruginosa contains 64 sensor histidine kinases (HKs) and 72 response regulators (RRs) that play important roles in metabolism, bacterial physiology, and virulence. However, the role of some TCSs in virulence remains uncharacterized. In this study, we evaluated the virulence potential of some uncharacterized sensor HK and RR knockouts in P. aeruginosa using a Galleria mellonella infection model. Furthermore, we demonstrated that KdpD and AauS HKs regulate virulence by affecting P. aeruginosa biofilm formation and motility. Both ΔkdpD and ΔaauS showed reduced biofilm and motility which were confirmed by restored phenotypes upon complementation. Moreover, ΔkdpD and ΔaauS exhibited increased survival of HeLa cells and G. mellonella during in vivo infection. Altered expression of the transcriptional regulators anR and lasR, along with the virulence genes lasA, pelA, cupA, pqsA, pqsB, pqsC, and pqsD in the mutant strains elucidated the mechanism by which ΔkdpD and ΔaauS affect virulence. These findings confirm that kdpD and aauS play important roles in P. aeruginosa pathogenesis by regulating biofilm formation and motility.

Novel RclSAR three-component system regulates expression of the intI1 gene in the stationary growth phase.

The rapid and appropriate response of Pseudomonas spp. to environmental fluctuations has been enabled by numerous signal transduction regulatory systems. Regulatory systems in Pseudomonas aeruginosa are organized in a complex network which provides quick and fine-tuned cellular response through regulation of virulence and antibiotic resistance determinants production. Mobile integrons represent genetic elements included in the rapid dissemination of multiple antibiotic resistance determinants. The key factor of integron dynamics is enzyme integrase. So far, global regulators LexA, RpoS and PsrA have been recognized as regulators of the intI1 transcription. In this study, we discovered novel activator of the intI1 transcription, sensor kinase RclS, in Pseudomonas putida WCS358. This regulation is limited to stationary growth phase and appears to be indirect, at least through regulation of the rpoS expression. Sensor kinase RclS is a part of novel three-component system Rcl (Roc-like) together with two response regulators, RclR and RclA. RclS acted as a negative regulator of the rclA transcription, while the role in the rclR transcription regulation could not be defined. The RclSAR regulatory system seems to be a part of complex intI1 regulatory network which includes major stress response (SOS and RpoS) regulons.

Comparative Metabolic Pathways Analysis and Subtractive Genomics Profiling to Prioritize Potential Drug Targets Against Streptococcus pneumoniae.

Streptococcus pneumoniae is a notorious pathogen that affects ∼450 million people worldwide and causes up to four million deaths per annum. Despite availability of antibiotics (i.e., penicillin, doxycycline, or clarithromycin) and conjugate vaccines (e.g., PCVs), it is still challenging to treat because of its drug resistance ability. The rise of antibiotic resistance in S. pneumoniae is a major source of concern across the world. Computational subtractive genomics is one of the most applied techniques in which the whole proteome of the bacterial pathogen is gradually reduced to a limited number of potential therapeutic targets. Whole-genome sequencing has greatly reduced the time required and provides more opportunities for drug target identification. The goal of this work is to evaluate and analyze metabolic pathways in serotype 14 of S. pneumonia to identify potential drug targets. In the present study, 47 potent drug targets were identified against S. pneumonia by employing the computational subtractive genomics approach. Among these, two proteins are prioritized (i.e., 4-oxalocrotonate tautomerase and Sensor histidine kinase uniquely present in S. pneumonia) as novel drug targets and selected for further structure-based studies. The identified proteins may provide a platform for the discovery of a lead drug candidate that may be capable of inhibiting these proteins and, therefore, could be helpful in minimizing the associated risk related to the drug-resistant S. pneumoniae. Finally, these enzymatic proteins could be of prime interest against S. pneumoniae to design rational targeted therapy.

The putative sensor histidine kinase VadJ coordinates development and sterigmatocystin production in Aspergillus nidulans.

The VosA-VelB heterocomplex governs expression of several genes associated with fungal development and secondary metabolism. In this study, we have investigated the functions of one of the VosA-VelB-activated developmental genes vadJ in development and production of the mycotoxin sterigmatocystin in the model fungus Aspergillus nidulans. The vadJ gene is predicted to encode a 957-amino acid length protein containing a highly conserved sensor histidine kinase domain. The deletion of vosA or velB resulted in decreased mRNA levels of vadJ throughout the life cycle, suggesting that VosA and VelB are necessary for proper expression of vadJ. Nullifying vadJ led to highly restricted colony growth, lowered formation of asexual spores, and about two-fold reduction in conidial viability. Conversely, the deletion of vadJ resulted in elevated production of sexual fruiting bodies and sterigmatocystin. These suggest that VadJ is necessary for proper coordination of asexual and sexual development, and sterigmatocystin production. In accordance with this idea, the deletion of vadJ led to elevated mRNA levels of the two key sexual developmental activators esdC and nsdD. In summary, the putative sensor histidine kinase VadJ represses sexual development and sterigmatocystin production, but activates asexual development in A. nidulans.

Structural insights into the histidine-containing phospho-transfer protein and receiver domain of sensor histidine kinase suggest a complex model in the two-component regulatory system in Pseudomonas aeruginosa.

In Pseudomonas aeruginosa, an important opportunistic pathogen that causes numerous acute and chronic infections, the hybrid two-component system (TCS) regulates the swarming ability and biofilm formation with a multistep phospho-relay, and consists of hybrid-sensor histidine kinase (HK), histidine-containing phospho-transfer protein (Hpt) and response regulator (RR). In this work, two crystal structures of HptB and the receiver domain of HK PA1611 (PA1611REC) of P. aeruginosa have been determined in order to elucidate their interactions for the transfer of the phospho-ryl group. The structure of HptB folds into an elongated four-helix bundle - helices α2, α3, α4 and α5, covered by the short N-terminal helix α1. The imidazole side chain of the conserved active-site histidine residue His57, located near the middle of helix α3, protrudes from the bundle and is exposed to solvent. The structure of PA1611REC possesses a conventional (ß/α)5 topology with five-stranded parallel ß-sheets folded in the central region, surrounded by five α-helices. The divalent Mg2+ ion is located in the negatively charged active-site cleft and interacts with Asp522, Asp565 and Arg567. The HptB-PA1611REC complex is further modeled to analyze the binding surface and interactions between the two proteins. The model shows a shape complementarity between the convex surface of PA1611REC and the kidney-shaped HptB with fewer residues and a different network involved in interactions compared with other TCS complexes, such as SLN1-R1/YPD1 from Saccharomyces cerevisiae and AHK5RD/AHP1 from Arabidopsis thaliana. These structural results provide a better understanding of the TCS in P. aeruginosa and could potentially lead to the discovery of a new treatment for infection.