Bacterial Two-component Signal Transduction Systems Research Articles

Molecular insights into Escherichia coli Cpx envelope stress response activation by the sensor lipoprotein NlpE.

Bacterial two-component signal transduction systems provide sensory inputs for appropriately adapting gene expression. These systems rely on a histidine kinase that phosphorylates a response regulator which alters gene expression. Several two-component systems include additional sensory components that can activate the histidine kinase. In Escherichia coli, the lipoprotein NlpE was identified as a sensor for the Cpx cell envelope stress response. It has remained unclear how NlpE signals to Cpx in the periplasm. In this study, we used a combination of genetics, biochemistry, and AlphaFold2 complex modeling to uncover the molecular details of how NlpE triggers the Cpx response through an interaction with the CpxA histidine kinase. Remarkably, only a short loop of NlpE is required to activate the Cpx response. A single substitution in this loop inactivates NlpE signaling to Cpx and abolishes an in vivo biochemical NlpE:CpxA interaction. An independent AlphaFold multimer prediction supported a role for the loop and predicted an interaction interface at CpxA. Mutations in this CpxA region specifically blind the histidine kinase to NlpE activation but preserve the ability to respond to other cell envelope stressors. Hence, our work additionally reveals a previously unrecognized complexity in signal integration by the CpxA periplasmic sensor domain.

QseBC regulates in vitro and in vivo virulence of Aeromonas hydrophila in response to norepinephrine.

The inter-kingdom communication between host and pathogenic bacteria mediated by the host hormones epinephrine (Epi)/norepinephrine (NE)/autoinducer-3 (AI-3) and transduced by the bacterial two-component signal transduction system QseBC has been well demonstrated in mammalian pathogens. Aeromonas hydrophila, a common opportunistic pathogen in freshwater aquaculture, responds to NE by increased bacterial growth and enhanced virulence. However, the underlying mechanisms remain poorly understood. Our study demonstrated that deletion of qseB and qseC significantly inhibited NE-promoted growth, biofilm formation, and hemolytic activity of A. hydrophila. The adhesion ability of ΔqseB and ΔqseC to J774a.1cells was significantly decreased compared with the wild-type strain in the presence and absence of NE, whereas NE still enhanced the adhesion ability of the mutant and wild-type strains with a similar effect, suggesting that NE-enhanced cell adhesion was independent of QseBC. Moreover, QseBC did not affect the swimming and swarming motility of A. hydrophila with or without NE. Quantitative real-time PCR analyses revealed the down-regulated expression of some virulence-related genes (hly, ast, act, aerA) in each mutant compared with the wild-type strain in the presence of NE. Tilapia infection experiments indicated that deletion of qseB or qseC weakened NE-promoted virulence of A. hydrophila. In conclusion, our study suggests that NE stimulates the growth, biofilm formation, and hemolytic activity of A. hydrophila and enhances the virulence of the pathogen in fish via the QseBC system.

TorR/TorS Two-Component system resists extreme acid environment by regulating the key response factor RpoS in Escherichia coli

Response to acid stress is critical for Escherichia coli to successfully complete its life-cycle. Acid resistance is an indispensable mechanism that allows neutralophilic bacteria, such as E. coli, to survive in the gastrointestinal tract. Escherichia coli acid tolerance has been extensively studied over the past decades, and most studies have focused on mechanisms of gene regulation. Bacterial two-component signal transduction systems sense and respond to external environmental changes through regulating genes expression. However, there has been little research on the mechanism of the TorR/TorS system in acid resistance, and how TorR/TorS regulate the expression ofacid-resistantgenes is still unclear. We found that TorR/TorS deletion in E. coli cells led to a growth defect in extreme acid conditions,andthis defectmightdepend on the nutritional conditionsand growth phase.TorS/TorR sensed an extremely acidic environment, and this TorR phosphorylation process might not be entirely dependent on TorS.RNA-seqand RT-qPCR results suggested that TorR regulated expressions of gadB, gadC, hdeA, gadE, mdtE, mdtF, gadX, and slp acid-resistant genes. Compared with wild-type cells, the stress response factor RpoSlevels and itsexpressions were significantly decreased in Δ torR cellsstimulated by extreme acid. And under these circumstances, the expression of iraM was significantly reduced to 0.6-fold inΔ torR cells. Electrophoreticmobility shift assay showed that TorR-His6 could interact with the rpoS promoter sequence in vitro. β-galactosidase activity assayresultsapprovedthat TorR might bind the rpoS promoter region in vivo. After the mutation of the TorR-box in the rpoS promoter region, these interactions were no longer observed. Taken together, we propose thatTorS and potential Hanks model Ser/Thr kinase received an external acid stress signal and then phosphorylated TorR, which guided the expressions of a variety of acid resistance genes. Moreover,TorRcoped with extreme acid environmentsthroughRpoS, levels of which might be maintained byIraM. Finally,TorR may confer E. coli with the abilityto resist gastric acid, allowing the bacterium to reach the surface of the terminal ileum and large intestine mucosal epithelial cells through the gastric acid barrier, andestablishcolonization and pathogenicity.

Long-Term Intrahost Evolution of Staphylococcus aureus Among Diabetic Patients With Foot Infections.

Staphylococcus aureus is one of the main pathogens isolated from diabetic foot infections (DFI). The purpose of this study was to evaluate the importance of the persistence of S. aureus in this environment and the possible modifications of the bacterial genome content over time. Molecular typing of S. aureus isolates cultured from patients with the same DFI over a 7-year study revealed a 25% rate of persistence of this species in 48 patients, with a short median persistence time of 12weeks (range: 4–52weeks). Non-specific clonal complexes were linked to this persistence. During the follow-up, bla genes were acquired in three cases, whereas some virulence markers were lost in all cases after a long period of colonization (21.5weeks). Only one patient (2%) had a long-term persistence of 48weeks. The genome sequencing of a clonal pair of early/late strains isolated in this patient showed mutations in genes encoding bacterial defence and two-component signal transduction systems. Although, this study suggests that the long-term persistence of S. aureus in DFI is a rare event, genomic evolution is observed, highlighting the low adaptive ability of S. aureus to the specific environment and stressful conditions of diabetic foot ulcers. These results provide the basis for better understanding of S. aureus dynamics during persistent colonization in chronic wounds.

Polymyxins, the last-resort antibiotics: Mode of action, resistance emergence, and potential solutions

Infections caused by multi-drug resistant (MDR) bacterial pathogens are a leading cause of mortality and morbidity across the world. Indiscriminate use of broad-spectrum antibiotics has seriously affected this situation. With the diminishing discovery of novel antibiotics, new treatment methods are urgently required to combat MDR pathogens. Polymyxins, the cationic lipopeptide antibiotics, discovered more than half a century ago, are considered to be the last-line of antibiotics available at the moment. This antibiotic shows a great bactericidal effect against Gram-negative bacteria. Polymyxins primarily target the bacterial membrane and disrupt them, causing lethality. Because of their membrane interacting mode of action, polymyxins cause nephrotoxicity and neurotoxicity in humans, limiting their usability. However, recent modifications in their chemical structure have been able to reduce the toxic effects. The development of better dosing regimens has also helped in getting better clinical outcomes in the infections caused by MDR pathogens. Since the mid-1990s the use of polymyxins has increased manifold in clinical settings, resulting in the emergence of polymyxin-resistant strains. The risk posed by the polymyxin-resistant nosocomial pathogens such as the Enterobacteriaceae group, Pseudomonas aeruginosa, and Acinetobacter baumannii, etc. is very serious considering these pathogens are resistant to almost all available antibacterial drugs. In this review article, the mode of action of the polymyxins and the genetic regulatory mechanism responsible for the emergence of resistance are discussed. Specifically, this review aims to update our current understanding in the field and suggest possible solutions that can be pursued for future antibiotic development. As polymyxins primarily target the bacterial membranes, resistance to polymyxins arises primarily by the modification of the lipopolysaccharides (LPS) in the outer membrane (OM). The LPS modification pathways are largely regulated by the bacterial two-component signal transduction (TCS) systems. Therefore, targeting or modulating the TCS signalling mechanisms can be pursued as an alternative to treat the infections caused by polymyxin-resistant MDR pathogens. In this review article, this aspect is also highlighted.

First-generation structure-activity relationship studies of 2,3,4,9-tetrahydro-1H-carbazol-1-amines as CpxA phosphatase inhibitors.

Genetic activation of the bacterial two-component signal transduction system, CpxRA, abolishes the virulence of a number of pathogens in human and murine infection models. Recently, 2,3,4,9-tetrahydro-1H-carbazol-1-amines were shown to activate the CpxRA system by inhibiting the phosphatase activity of CpxA. Herein we report the initial structure-activity relationships of this scaffold by focusing on three approaches 1) A-ring substitution, 2) B-ring deconstruction to provide N-arylated amino acid derivatives, and 3) C-ring elimination to give 2-ethylamino substituted indoles. These studies demonstrate that the A-ring is amenable to functionalization and provides a promising avenue for continued optimization of this chemotype. Further investigations revealed that the C-ring is not necessary for activity, although it likely provides conformational constraint that is beneficial to potency, and that the (R) stereochemistry is required at the primary amine. Simplification of the scaffold through deconstruction of the B-ring led to inactive compounds, highlighting the importance of the indole core. A new lead compound 26 was identified, which manifests a ∼30-fold improvement in CpxA phosphatase inhibition over the initial hit. Comparison of amino and des-amino derivatives in bacterial strains differing in membrane permeability and efflux capabilities demonstrate that the amine is required not only for target engagement but also for permeation and accumulation in Escherichia coli.

Structural Studies on the Extracellular Domain of Sensor Histidine Kinase YycG from Staphylococcus aureus and Its Functional Implications

Bacterial two-component signal transduction systems are used to adapt to fluctuations in the environment. YycG, a key two-component histidine kinase in Staphylococcus aureus, plays an essential role in cell viability and regulates cell wall metabolism, biofilm formation, virulence, and antibiotic resistance. For these reasons, YycG is considered a compelling target for the development of novel antibiotics. However, to date, the signaling mechanism of YycG and its stimulus are poorly understood mainly because of a lack of structural information on YycG. To address this deficiency, we determined the crystal structure of the extracellular domain of S. aureus YycG (YycGex) at 2.0-Å resolution. The crystal structure indicated two subunits with an extracellular Per-Arnt-Sim (PAS) topology packed into a dimer with interloop interactions. Disulfide scanning using cysteine-substituted mutants revealed that YycGex possessed dimeric interfaces not only in the loop but also in the helix α1. Cross-linking studies using intact YycG demonstrated that it was capable of forming high molecular weight oligomers on the cell membrane. Furthermore, we also observed that two auxiliary proteins of YycG, YycH and YycI, cooperatively interfered with the multimerization of YycG. From these results, we propose that signaling through YycG is regulated by multimerization and binding of YycH and YycI. These structural studies, combined with biochemical analyses, provide a better understanding of the signaling mechanism of YycG, which is necessary for developing novel antibacterial drugs targeting S. aureus.

An external sensing system in Plasmodium falciparum-infected erythrocytes.

BackgroundA number of experiments have previously indicated that Plasmodium falciparum-infected erythrocytes (pRBC) were able to sense host environment. The basis of this ability to detect external cues is not known but in screening signalling molecules from pRBC using commercial antibodies, a 34 kDa phosphorylated molecule that possesses such ability was identified.MethodsThe pRBC were exposed to different culture conditions and proteins were extracted for 1D or 2D gel electrophoresis followed by Western blot. The localization of 34 kDa protein was examined by biochemical fractionation followed by Western blot. High-resolution mass spectrometric analysis of immune precipitants was used to identify this protein and real-time quantitative reverse transcriptase polymerase chain reaction was used for detecting mRNA expression level.ResultsThe 34 kDa protein was called PfAB4 has immediate responses (dephosphorylation and rapid turnover) to host environmental stimuli such as serum depletion, osmolality change and cytokine addition. PfAB4 is expressed constitutively throughout the erythrocytic lifecycle with dominant expression in trophozoites 30 h post-infection. Tumour necrosis factor (TNF) treatment induced a transient detectable dephosphorylation of PfAB4 in the ItG strain (2 min after addition) and the level of expression and phosphorylation returned to normal within 1–2 h. PfAB4 localized dominantly in pRBC cytoplasm, with a transient shift to the nucleus under TNF stimulation as shown by biochemical fractionation. High-resolution mass spectrometric analysis of immune precipitants of AB4 antibodies revealed a 34 kDa PfAB4 component as a mixture of proliferating cellular nuclear antigen-1 (PCNA1) and exported protein-2 (EXP2), along with a small number of other inconsistently identified peptides. Different parasite strains have different PfAB4 expression levels, but no significant association between mRNA and PfAB4 levels was seen, indicating that the differences may be at the post-transcriptional, presumably phosphorylation, level. A triple serine phosphorylated PCNA1 peptide was identified from the PfAB4 high expression strain only, providing further evidence that the identity of PfAB4 is PCNA1 in P.falciparum.ConclusionA protein element in the human malaria parasite that responds to external cues, including the pro-inflammatory cytokine TNF have been discovered. Treatment results in a transient change in phosphorylation status of the response element, which also migrates from the parasite cytoplasm to the nucleus. The response element has been identified as PfPCNA1. This sensing response could be regulated by a parasite checkpoint system and be analogous to bacterial two-component signal transduction systems.Electronic supplementary materialThe online version of this article (doi:10.1186/s12936-016-1144-6) contains supplementary material, which is available to authorized users.

Development and validation of a high-throughput cell-based screen to identify activators of a bacterial two-component signal transduction system.

CpxRA is a two-component signal transduction system (2CSTS) found in many drug-resistant Gram-negative bacteria. In response to periplasmic stress, CpxA autophosphorylates and donates a phosphoryl group to its cognate response regulator, CpxR. Phosphorylated CpxR (CpxR-P) upregulates genes involved in membrane repair and downregulates multiple genes that encode virulence factors, which are trafficked across the cell membrane. Mutants that constitutively activate CpxRA in Salmonella enterica serovar Typhimurium and Haemophilus ducreyi are avirulent in mice and humans, respectively. Thus, the activation of CpxRA has high potential as a novel antimicrobial/antivirulence strategy. Using a series of Escherichia coli strains containing a CpxR-P-responsive lacZ reporter and deletions in genes encoding CpxRA system components, we developed and validated a novel cell-based high-throughput screen (HTS) for CpxRA activators. A screen of 36,000 compounds yielded one hit compound that increased reporter activity in wild-type cells. This is the first report of a compound that activates, rather than inhibits, a 2CSTS. The activity profile of the compound against CpxRA pathway mutants in the presence of glucose suggested that the compound inhibits CpxA phosphatase activity. We confirmed that the compound induced the accumulation of CpxR-P in treated cells. Although the hit compound contained a nitro group, a derivative lacking this group retained activity in serum and had lower cytotoxicity than that of the initial hit. This HTS is amenable for the screening of larger libraries to find compounds that activate CpxRA by other mechanisms, and it could be adapted to find activators of other two-component systems.

An intimate link: two-component signal transduction systems and metal transport systems in bacteria.

Bacteria have evolved various strategies to contend with high concentrations of environmental heavy metal ions for rapid, adaptive responses to maintain cell viability. Evidence gathered in the past two decades suggests that bacterial two-component signal transduction systems (TCSTSs) are intimately involved in monitoring cation accumulation, and can regulate the expression of related metabolic and virulence genes to elicit adaptive responses to changes in the concentration of these ions. Using examples garnered from recent studies, we summarize the cross-regulatory relationships between metal ions and TCSTSs. We present evidence of how bacterial TCSTSs modulate metal ion homeostasis and also how metal ions, in turn, function to control the activities of these signaling systems linked with bacterial survival and virulence.

Response of S. thermophilus LMD-9 to bacitracin: Involvement of a BceRS/AB-like module and of the rhamnose–glucose polysaccharide synthesis pathway

Streptococcus thermophilus is a lactic acid bacterium of major importance to the dairy industry as it is found in numerous cheeses and is one of the two bacterial species involved in the fermentation of yogurt. Bacterial two-component signal transduction systems (TCSs) play important roles in the process of bacterial environmental adaptation. S. thermophilus LMD-9 possesses eight such TCS systems; however, their functions have thus far been only poorly investigated. Here, we focused on two of the TCSs in LMD-9, TCS06 and TCS07, whose encoding genes are located close to each other on the chromosome, and are associated with those of ABC transporters. TCS06 homologs are frequently found in Lactobacillales, but their function has not yet been determined, while TCS07 and its upstream potential ABC transporter are homologous to the BceRS/AB system, which is involved in bacitracin resistance in Bacillus and Streptococcus species. To investigate the function(s) of TCS06 and TCS07, we constructed and characterized deletion mutants and performed transcriptional analysis in the presence and absence of bacitracin. We show here that both TCS06 and TCS07 regulate the genes in their close vicinity, in particular those encoding ABC transporters. We propose that the response of S. thermophilus to bacitracin includes i) a bacitracin export system, regulated by TCS07 and constituting a BceRS/AB-like detoxification module, and ii) the modification of cell-envelope properties via modulation of rhamnose–glucose polysaccharide synthesis, at least partially regulated by TCS06.

Role of the CpxAR Two-Component Signal Transduction System in Control of Fosfomycin Resistance and Carbon Substrate Uptake

Although fosfomycin is an old antibiotic, it has resurfaced with particular interest. The antibiotic is still effective against many pathogens that are resistant to other commonly used antibiotics. We have found that fosfomycin resistance of enterohemorrhagic Escherichia coli (EHEC) O157:H7 is controlled by the bacterial two-component signal transduction system CpxAR. A cpxA mutant lacking its phosphatase activity results in constitutive activation of its cognate response regulator, CpxR, and fosfomycin resistance. We have shown that fosfomycin resistance requires CpxR because deletion of the cpxR gene in the cpxA mutant restores fosfomycin sensitivity. We have also shown that CpxR directly represses the expression of two genes, glpT and uhpT, which encode transporters that cotransport fosfomycin with their native substrates glycerol-3-phosphate and glucose-6-phosphate, and repression of these genes leads to a decrease in fosfomycin transport into the cpxA mutant. However, the cpxA mutant had an impaired growth phenotype when cultured with glycerol-3-phosphate or glucose-6-phosphate as a sole carbon substrate and was outcompeted by the parent strain, even in nutrient-rich medium. This suggests a trade-off between fosfomycin resistance and the biological fitness associated with carbon substrate uptake. We propose a role for the CpxAR system in the reversible control of fosfomycin resistance. This may be a beneficial strategy for bacteria to relieve the fitness burden that results from fosfomycin resistance in the absence of fosfomycin.

Interactions between an M. tuberculosis strain overexpressing mtrA and mononuclear phagocytes

It was previously shown that the bacterial two-component regulatory signal transduction (2CR) system MtrAB may be associated with the ability of M. tuberculosis (Mtb) to survive in macrophages. In the present work Mtb mutants: Rv-78 with overexpression of mtrA and Rv-129 with elevated level of phosphorylation-defective MtrA were used for further investigation of the potential influence of the MtrAB system on Mtb interaction with human monocytes. Flow cytometry was used to determine the expression of MHC class II molecules. The expression of genes for inducible nitric oxide synthase (iNOS) and cathepsin G was quantified by RT-PCR. The association of Mtb strains with Rab5 and Rab7 positive vacuoles was investigated applying confocal microscopy. IL-10 and IL-12 secretion by monocytes as well as the Mtb susceptibility to cathepsin G were investigated. Mutation-carried and wild type Mtb strains inhibited MHC class II expression on monocytes to a similar extent. Monocyte stimulation with mycobacteria led to the increased production of IL-10 but no detectable amounts of IL-12 or NO were observed. Expression of the gene for iNOS was not detected while that for cathepsin G was shown, however its intensity was not associated with MtrA mutation. Mtb mutant strains were more effectively enclosed in phagosomes containing the late endosome marker Rab7 as compared to the control. The results may confirm the importance of the MtrAB system in mycobacterial capacity for successful survival in phagocytes, especially in the context of high degree of colocalization of Mtb Rv-78 to mature phagosomes.

コネクターがつなぐ細菌情報伝達ネットワーク

細菌細胞において，動物細胞と同様に，情報伝達ネットワークの実態が明らかになりつつある．細菌の主要な情報伝達系は，対となるセンサーとレスポンスレギュレータータンパク質からなる二成分制御系 (TCS) である．最近，TCS間をつなぐコネクタータンパク質が発見された．このようなコネクターがTCS間をつなぐことで情報ネットワークが組織され，環境の変化に対応して細菌がより迅速に適応することが可能になっている．本稿では，大腸菌およびサルモネラ菌における新規コネクターを介する情報伝達ネットワークの分子機構を紹介するとともに，TCSのセンサーやレスポンスレギュレーターに直接結合して作用する修飾タンパク質についても解説する．

Functional Assessment of EnvZ/OmpR Two-Component System in Shewanella oneidensis

EnvZ and OmpR constitute the bacterial two-component signal transduction system known to mediate osmotic stress response in a number of Gram-negative bacteria. In an effort to understand the mechanism through which Shewanella oneidensis senses and responds to environmental osmolarity changes, structure of the ompR-envZ operon was determined with Northern blotting assay and roles of the EnvZ/OmpR two-component system in response to various stresses were investigated with mutational analysis, quantitative reverse transcriptase PCR (qRT-PCR), and phenotype microarrays. Results from the mutational analysis and qRT-PCR suggested that the EnvZ/OmpR system contributed to osmotic stress response of S. oneidensis and very likely engaged a similar strategy employed by E. coli, which involved reciprocal regulation of two major porin coding genes. Additionally, the ompR-envZ system was also found related to cell motility. We further showed that the ompR-envZ dependent regulation of porin genes and motility resided almost completely on ompR and only partially on envZ, indicating additional mechanisms for OmpR phosphorylation. In contrast to E. coli lacking ompR-envZ, however, growth of S. oneidensis did not show a significant dependence on ompR-envZ even under osmotic stress. Further analysis with phenotype microarrays revealed that the S. oneidensis strains lacking a complete ompR-envZ system displayed hypersensitivities to a number of agents, especially in alkaline environment. Taken together, our results suggest that the function of the ompR-envZ system in S. oneidensis, although still connected with osmoregulation, has diverged considerably from that of E. coli. Additional mechanism must exist to support growth of S. oneidensis under osmotic stress.

X-Ray Crystal Structure of the DNA-Binding Domain of Response Regulator WalR Essential to the Cell Viability of Staphylococcus aureus and Interaction with Target DNA

A bacterial two-component signal transduction system, WalK/WalR, is essential to the cell viability of Gram-positive bacteria and is therefore a potential target for the development of a new class of antibiotics. We have solved the X-ray crystal structure of the DNA-binding domain of the response regulator WalR (WalRc) from a Gram-positive pathogen Staphylococcus aureus, currently causing serious problems in public health through the acquisition of multi-drug resistance. The structure contains a winged helix-turn-helix motif and closely resembles those of WalRs of Bacillus subtilis and Enterococcus faecalis, and also that of PhoB of Escherichia coli. Gel mobility shift assays with mutant WalRs revealed specific interactions of WalR with the target DNA, as elaborated by in silico modeling of the WalRc-DNA complex.

The role of the TetR-family transcriptional regulator Aur1R in negative regulation of the auricin gene cluster in Streptomyces aureofaciens CCM 3239

Two regulatory genes, aur1P and aur1R, have been previously identified upstream of the aur1 polyketide gene cluster involved in biosynthesis of the angucycline-like antibiotic auricin in Streptomyces aureofaciens CCM 3239. The aur1P gene encodes a protein similar to the response regulators of bacterial two-component signal transduction systems and has been shown to specifically activate expression of the auricin biosynthetic genes. The aur1R gene encodes a protein homologous to transcriptional repressors of the TetR family. Here we describe the characterization of the aur1R gene. Expression of the gene is directed by a single promoter, aur1Rp, which is induced just before stationary phase. Disruption of aur1R in S. aureofaciens CCM 3239 had no effect on growth and differentiation. However, the disrupted strain produced more auricin than its parental wild-type S. aureofaciens CCM 3239 strain. Transcription from the aur1Ap and aur1Pp promoters, directing expression of the first biosynthetic gene in the auricin gene cluster and the pathway-specific transcriptional activator, respectively, was increased in the S. aureofaciens CCM 3239 aur1R mutant strain. However, Aur1R was shown to bind specifically only to the aur1Pp promoter in vitro. This binding was abolished by the addition of auricin and/or its intermediates. The results indicate that the Aur1R regulator specifically represses expression of the aur1P gene, which encodes a pathway-specific activator of the auricin biosynthetic gene cluster in S. aureofaciens CCM 3239, and that this repression is relieved by auricin or its intermediates.

Hierarchical control of virginiamycin production in Streptomyces virginiae by three pathway-specific regulators: VmsS, VmsT and VmsR

Two regulatory genes encoding a Streptomyces antibiotic regulatory protein (vmsS) and a response regulator (vmsT) of a bacterial two-component signal transduction system are present in the left-hand region of the biosynthetic gene cluster of the antibiotic virginiamycin, which is composed of virginiamycin M (VM) and virginiamycin S (VS), in Streptomyces virginiae. Disruption of vmsS abolished both VM and VS biosynthesis, with drastic alteration of the transcriptional profile for virginiamycin biosynthetic genes, whereas disruption of vmsT resulted in only a loss of VM biosynthesis, suggesting that vmsS is a pathway-specific regulator for both VM and VS biosynthesis, and that vmsT is a pathway-specific regulator for VM biosynthesis alone. Gene expression profiles determined by semiquantitative RT-PCR on the virginiamycin biosynthetic gene cluster demonstrated that vmsS controls the biosynthetic genes for VM and VS, and vmsT controls unidentified gene(s) of VM biosynthesis located outside the biosynthetic gene cluster. In addition, transcriptional analysis of a deletion mutant of vmsR located in the clustered regulatory region in the virginiamycin cluster (and which also acts as a SARP-family activator for both VM and VS biosynthesis) indicated that the expression of vmsS and vmsT is under the control of vmsR, and vmsR also contributes to the expression of VM and VS biosynthetic genes, independent of vmsS and vmsT. Therefore, coordinated virginiamycin biosynthesis is controlled by three pathway-specific regulators which hierarchically control the expression of the biosynthetic gene cluster.

Identification of direct residue contacts in protein–protein interaction by message passing

Understanding the molecular determinants of specificity in protein-protein interaction is an outstanding challenge of postgenome biology. The availability of large protein databases generated from sequences of hundreds of bacterial genomes enables various statistical approaches to this problem. In this context covariance-based methods have been used to identify correlation between amino acid positions in interacting proteins. However, these methods have an important shortcoming, in that they cannot distinguish between directly and indirectly correlated residues. We developed a method that combines covariance analysis with global inference analysis, adopted from use in statistical physics. Applied to a set of >2,500 representatives of the bacterial two-component signal transduction system, the combination of covariance with global inference successfully and robustly identified residue pairs that are proximal in space without resorting to ad hoc tuning parameters, both for heterointeractions between sensor kinase (SK) and response regulator (RR) proteins and for homointeractions between RR proteins. The spectacular success of this approach illustrates the effectiveness of the global inference approach in identifying direct interaction based on sequence information alone. We expect this method to be applicable soon to interaction surfaces between proteins present in only 1 copy per genome as the number of sequenced genomes continues to expand. Use of this method could significantly increase the potential targets for therapeutic intervention, shed light on the mechanism of protein-protein interaction, and establish the foundation for the accurate prediction of interacting protein partners.

Transferred NOE and Saturation Transfer Difference NMR Studies of Novobiocin Binding to EnvZ Suggest Binding Mode Similar to DNA Gyrase

Histidine protein kinases (HPKs) are a class of receptor proteins found in bacterial two-component signal transduction systems, which allow bacteria to respond to changes in their external environment. To date, there are few potent inhibitors of histidine kinases, despite their potential ability to weaken bacteria against antibiotic treatment. EnvZ is a histidine protein kinase with osmoregulatory function in bacteria with sequence and topological similarity to DNA Gyrase B. DNA Gyrase B has several well-characterized potent inhibitors, including novobiocin and clorobiocin which have detailed structures in complex. With fluorescence competition experiments, we have determined that novobiocin binds to EnvZ with a (novo)K(D) 120 +/- 20 microm. NMR transferred NOE (trNOE) experiments, and saturation transfer difference (STD) experiments suggest that novobiocin binds to EnvZ in a conformation and orientation similar to its binding with DNA Gyrase B. These experiments suggest some similarity in the pocket despite weaker affinity for EnvZ by novobiocin.