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1.
Molecules ; 26(16)2021 Aug 23.
Artigo em Inglês | MEDLINE | ID: mdl-34443697

RESUMO

There is an urgent need to find new antibacterial agents to combat bacterial infections, including agents that inhibit novel, hitherto unexploited targets in bacterial cells. Amongst novel targets are two-component signal transduction systems (TCSs) which are the main mechanism by which bacteria sense and respond to environmental changes. TCSs typically comprise a membrane-embedded sensory protein (the sensor histidine kinase, SHK) and a partner response regulator protein. Amongst promising targets within SHKs are those involved in environmental signal detection (useful for targeting specific SHKs) and the common themes of signal transmission across the membrane and propagation to catalytic domains (for targeting multiple SHKs). However, the nature of environmental signals for the vast majority of SHKs is still lacking, and there is a paucity of structural information based on full-length membrane-bound SHKs with and without ligand. Reasons for this lack of knowledge lie in the technical challenges associated with investigations of these relatively hydrophobic membrane proteins and the inherent flexibility of these multidomain proteins that reduces the chances of successful crystallisation for structural determination by X-ray crystallography. However, in recent years there has been an explosion of information published on (a) methodology for producing active forms of full-length detergent-, liposome- and nanodisc-solubilised membrane SHKs and their use in structural studies and identification of signalling ligands and inhibitors; and (b) mechanisms of signal sensing and transduction across the membrane obtained using sensory and transmembrane domains in isolation, which reveal some commonalities as well as unique features. Here we review the most recent advances in these areas and highlight those of potential use in future strategies for antibiotic discovery. This Review is part of a Special Issue entitled "Interactions of Bacterial Molecules with Their Ligands and Other Chemical Agents" edited by Mary K. Phillips-Jones.


Assuntos
Antibacterianos/farmacologia , Membrana Celular/enzimologia , Descoberta de Drogas , Histidina Quinase/química , Histidina Quinase/metabolismo , Transdução de Sinais , Ligantes , Domínios Proteicos , Transdução de Sinais/efeitos dos fármacos
2.
Biomolecules ; 11(7)2021 06 25.
Artigo em Inglês | MEDLINE | ID: mdl-34201916

RESUMO

DesK is a Histidine Kinase that allows Bacillus subtilis to maintain lipid homeostasis in response to changes in the environment. It is located in the membrane, and has five transmembrane helices and a cytoplasmic catalytic domain. The transmembrane region triggers the phosphorylation of the catalytic domain as soon as the membrane lipids rigidify. In this research, we study how transmembrane inter-helical interactions contribute to signal transmission; we designed a co-expression system that allows studying in vivo interactions between transmembrane helices. By Alanine-replacements, we identified a group of polar uncharged residues, whose side chains contain hydrogen-bond donors or acceptors, which are required for the interaction with other DesK transmembrane helices; a particular array of H-bond- residues plays a key role in signaling, transmitting information detected at the membrane level into the cell to finally trigger an adaptive response.


Assuntos
Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Histidina Quinase/genética , Histidina Quinase/metabolismo , Transporte Proteico/fisiologia , Sequência de Aminoácidos , Bacillus subtilis/genética , Bacillus subtilis/metabolismo , Proteínas de Bactérias/química , Histidina Quinase/química , Ligação de Hidrogênio
3.
Nat Commun ; 12(1): 4394, 2021 07 20.
Artigo em Inglês | MEDLINE | ID: mdl-34285211

RESUMO

Bacterial phytochrome photoreceptors usually belong to two-component signaling systems which transmit environmental stimuli to a response regulator through a histidine kinase domain. Phytochromes switch between red light-absorbing and far-red light-absorbing states. Despite exhibiting extensive structural responses during this transition, the model bacteriophytochrome from Deinococcus radiodurans (DrBphP) lacks detectable kinase activity. Here, we resolve this long-standing conundrum by comparatively analyzing the interactions and output activities of DrBphP and a bacteriophytochrome from Agrobacterium fabrum (Agp1). Whereas Agp1 acts as a conventional histidine kinase, we identify DrBphP as a light-sensitive phosphatase. While Agp1 binds its cognate response regulator only transiently, DrBphP does so strongly, which is rationalized at the structural level. Our data pinpoint two key residues affecting the balance between kinase and phosphatase activities, which immediately bears on photoreception and two-component signaling. The opposing output activities in two highly similar bacteriophytochromes suggest the use of light-controllable histidine kinases and phosphatases for optogenetics.


Assuntos
Proteínas de Bactérias/metabolismo , Histidina Quinase/metabolismo , Monoéster Fosfórico Hidrolases/metabolismo , Fotorreceptores Microbianos/metabolismo , Transdução de Sinais/efeitos da radiação , Agrobacterium/enzimologia , Proteínas de Bactérias/ultraestrutura , Deinococcus/enzimologia , Histidina Quinase/ultraestrutura , Luz , Simulação de Dinâmica Molecular , Monoéster Fosfórico Hidrolases/ultraestrutura , Fotorreceptores Microbianos/ultraestrutura , Domínios Proteicos
4.
Mol Genet Genomics ; 296(5): 1135-1145, 2021 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-34196769

RESUMO

Nik1 orthologs or group III hybrid histidine kinases (HHK3) represent a unique cytoplasmic osmosensor that act upstream of HOG/p38 MAPK pathway in fungi. It is an important molecular target for developing new antifungal agents against human pathogens. HHK3 orthologs contain a linear array of alternative HAMP and HAMP-like linker domains (poly-HAMP) in the N-terminal region. HAMP domains are quite common in prokaryotic histidine kinases where it mostly functions as signal transducer mediating conformational changes in the kinase domains. In contrast, poly-HAMP in HHK3 acts as a sensor and signal transducer to regulate histidine kinase activity. However, the mechanistic detail of this is poorly understood. Interestingly, recent studies indicate that the poly-HAMP-mediated regulation of the kinase activity varies among the orthologs. Hik1 is an important HHK3 ortholog from fungus Magnaporthe oryzae. In this paper, we aimed to decipher the role HAMP and HAMP-like linker domains in regulating the activity of Hik1p. We show that Hik1p acts as a bona fide osmosensor and negatively regulates the downstream HOG/p38 MAPK pathway in Saccharomyces cerevisiae. Our data suggest a differential role of the HAMP domains in the functionality of Hik1p. Most interestingly, the deletion of individual domains in poly-HAMP resulted in distinct active forms of Hik1p and thereby indicating that the poly-HAMP domain, instead of acting as on-off switch, regulates the histidine kinase activity by transition through multiple conformational states.


Assuntos
Proteínas Fúngicas/metabolismo , Histidina Quinase/química , Histidina Quinase/metabolismo , Magnaporthe/enzimologia , Dioxóis/farmacologia , Proteínas Fúngicas/química , Proteínas Fúngicas/genética , Teste de Complementação Genética , Histidina Quinase/genética , Peptídeos e Proteínas de Sinalização Intracelular/genética , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Microrganismos Geneticamente Modificados , Mutação , Domínios Proteicos , Proteínas Quinases/genética , Proteínas Quinases/metabolismo , Pirróis/farmacologia , Saccharomyces cerevisiae/efeitos dos fármacos , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo
5.
Commun Biol ; 4(1): 841, 2021 07 06.
Artigo em Inglês | MEDLINE | ID: mdl-34230602

RESUMO

Characterizing protein-protein interactions (PPIs) is an effective method to help explore protein function. Here, through integrating a newly identified split human Rhinovirus 3 C (HRV 3 C) protease, super-folder GFP (sfGFP), and ClpXP-SsrA protein degradation machinery, we developed a fluorescence-assisted single-cell methodology (split protease-E. coli ClpXP (SPEC)) to explore protein-protein interactions for both eukaryotic and prokaryotic species in E. coli cells. We firstly identified a highly efficient split HRV 3 C protease with high re-assembly ability and then incorporated it into the SPEC method. The SPEC method could convert the cellular protein-protein interaction to quantitative fluorescence signals through a split HRV 3 C protease-mediated proteolytic reaction with high efficiency and broad temperature adaptability. Using SPEC method, we explored the interactions among effectors of representative type I-E and I-F CRISPR/Cas complexes, which combining with subsequent studies of Cas3 mutations conferred further understanding of the functions and structures of CRISPR/Cas complexes.


Assuntos
Endopeptidase Clp/metabolismo , Proteínas de Escherichia coli/metabolismo , Escherichia coli/metabolismo , Mapas de Interação de Proteínas , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Sistemas CRISPR-Cas , Endopeptidase Clp/genética , Enterovirus/enzimologia , Enterovirus/genética , Escherichia coli/genética , Proteínas de Escherichia coli/genética , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Histidina Quinase/genética , Histidina Quinase/metabolismo , Humanos , Modelos Moleculares , Ligação Proteica , Conformação Proteica , Proteólise , Serina Endopeptidases/química , Serina Endopeptidases/genética , Serina Endopeptidases/metabolismo , Proteínas Virais/genética , Proteínas Virais/metabolismo
6.
Appl Microbiol Biotechnol ; 105(14-15): 5905-5914, 2021 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-34287659

RESUMO

In the model actinomycete strain, Streptomyces coelicolor, an orphan histidine kinase (HK) named OhkA (encoded by SCO1596), which belongs to bacterial two-component regulatory systems (TCSs), has been identified as being involved in the regulation of both antibiotic biosynthesis and morphological development. However, its cognate response regulator (RR) remains unknown due to its isolated genetic location on the genome, which impedes the elucidation of the mechanism underlying OhkA-mediated regulation. Here, we identified the orphan RR OrrA (encoded by SCO3008) as the cognate RR of OhkA according to mutant phenotypic changes, transcriptomics analysis, and bacterial two-hybrid experiment. Considering that the partner RR of the orphan HK is also orphan, a library of mutants with in-frame individual deletion of these functionally unknown orphan RR-encoding genes were generated. Through phenotypic analysis, it was found that the ∆orrA mutant exhibited similar phenotypic changes as that of the ∆ohkA mutant, showing increased production of actinorhodin (ACT) and undecylprodigiosin (RED), and pink colony surface. Further transcriptomics analysis showed these two mutants exhibited highly similar transcriptomics profiles. Finally, the direct interaction between OhkA and OrrA was revealed by bacterial two-hybrid system. The identification of the partner RR of OhkA lays a good foundation for an in-depth elucidation of the molecular mechanism underlying OhkA-mediated regulation of development and antibiotic biosynthesis in Streptomyces. KEY POINTS: • OrrA was identified as the partner RR of the orphan histidine kinase OhkA. • The ∆orrA and ∆ohkA mutants showed similar phenotype and transcriptomic profiling. • Specific interaction of OrrA and OhkA was revealed by bacterial two-hybrid system.


Assuntos
Streptomyces coelicolor , Streptomyces , Antibacterianos , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Perfilação da Expressão Gênica , Regulação Bacteriana da Expressão Gênica , Histidina Quinase/genética , Histidina Quinase/metabolismo , Metabolismo Secundário/genética , Streptomyces/genética , Streptomyces coelicolor/genética , Streptomyces coelicolor/metabolismo
7.
Int J Mol Sci ; 22(11)2021 May 31.
Artigo em Inglês | MEDLINE | ID: mdl-34072989

RESUMO

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


Assuntos
Bactérias/enzimologia , Proteínas de Bactérias , Histidina Quinase , Proteínas de Membrana , Nitratos/metabolismo , Nitritos/metabolismo , Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Histidina Quinase/química , Histidina Quinase/classificação , Histidina Quinase/metabolismo , Proteínas de Membrana/química , Proteínas de Membrana/metabolismo , Domínios Proteicos
8.
J Biol Chem ; 296: 100771, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33989637

RESUMO

The KdpDE two-component system regulates potassium homeostasis and virulence in various bacterial species. The KdpD histidine kinases (HK) of this system contain a universal stress protein (USP) domain which binds to the second messenger cyclic-di-adenosine monophosphate (c-di-AMP) for regulating transcriptional output from this two-component system in Firmicutes such as Staphylococcus aureus. However, the structural basis of c-di-AMP specificity within the KdpD-USP domain is not well understood. Here, we resolved a 2.3 Å crystal structure of the S. aureus KdpD-USP domain (USPSa) complexed with c-di-AMP. Binding affinity analyses of USPSa mutants targeting the observed USPSa:c-di-AMP structural interface enabled the identification of the sequence residues that are required for c-di-AMP specificity. Based on the conservation of these residues in other Firmicutes, we identified the binding motif, (A/G/C)XSXSX2N(Y/F), which allowed us to predict c-di-AMP binding in other KdpD HKs. Furthermore, we found that the USPSa domain contains structural features distinct from the canonical standalone USPs that bind ATP as a preferred ligand. These features include inward-facing conformations of its ß1-α1 and ß4-α4 loops, a short α2 helix, the absence of a triphosphate-binding Walker A motif, and a unique dual phospho-ligand binding mode. It is therefore likely that USPSa-like domains in KdpD HKs represent a novel subfamily of the USPs.


Assuntos
Proteínas de Bactérias/metabolismo , AMP Cíclico/metabolismo , Histidina Quinase/metabolismo , Proteínas Quinases/metabolismo , Staphylococcus aureus/metabolismo , Proteínas de Bactérias/química , Cristalografia por Raios X , Histidina Quinase/química , Humanos , Modelos Moleculares , Conformação Proteica , Domínios Proteicos , Proteínas Quinases/química , Sistemas do Segundo Mensageiro , Infecções Estafilocócicas/microbiologia , Staphylococcus aureus/química
9.
Appl Environ Microbiol ; 87(13): e0072021, 2021 06 11.
Artigo em Inglês | MEDLINE | ID: mdl-33893120

RESUMO

Bacteriocins are useful for controlling the composition of microorganisms in fermented food. Bacteriocin synthesis is regulated by quorum sensing mediated by autoinducing peptides. In addition, short-chain fatty acids, especially acetic acid, reportedly regulate bacteriocin synthesis. Five histidine kinases that regulated the synthesis of bacteriocins were selected to verify their interactions with acetate. Acetate activated the kinase activity of PlnB, SppK, and HpK3 in vitro and increased the yield of their cognate bacteriocins plantaricin EF, sakacin A, and rhamnosin B in vivo. The antimicrobial activity against Staphylococcus aureus of the fermentation supernatants of Lactobacillus plantarum, Lactobacillus sakei, and Lactobacillus rhamnosus with addition of acetate increased to 298%, 198%, and 289%, respectively, compared with that in the absence of acetate. Our study elucidated the activation activity of acetate in bacteriocin synthesis, and it might provide a potential strategy to increase the production of bacteriocin produced by Lactobacillus. IMPORTANCE Bacteriocins produced by lactic acid bacteria (LAB) are particularly useful in food preservation and food safety. Bacteriocins might increase bacterial competitive advantage against the indigenous microbiota of the intestines; at the same time, bacteriocins could limit the growth of undesired microorganisms in yogurt and other dairy products. This study confirmed that three kinds of histidine kinases were activated by acetate and upregulated bacteriocin synthesis both in vitro and in vivo. The increasing yield of bacteriocins reduced the number of pathogens and increased the number of probiotics in milk. Bacteriocin synthesis activation by acetate may have a broad application in the preservation of dairy products and forage silage.


Assuntos
Acetatos/farmacologia , Antibacterianos/biossíntese , Bacteriocinas/biossíntese , Lactobacillus/efeitos dos fármacos , Percepção de Quorum/efeitos dos fármacos , Fermentação , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Histidina Quinase/genética , Histidina Quinase/metabolismo , Lactobacillus/metabolismo , Lactobacillus/fisiologia , Staphylococcus aureus/crescimento & desenvolvimento
10.
mBio ; 12(2)2021 04 20.
Artigo em Inglês | MEDLINE | ID: mdl-33879593

RESUMO

The ability to sense and respond to environmental cues is essential for adaptation and survival in living organisms. In bacteria, this process is accomplished by multidomain sensor histidine kinases that undergo autophosphorylation in response to specific stimuli, thereby triggering downstream signaling cascades. However, the molecular mechanism of allosteric activation is not fully understood in these important sensor proteins. Here, we report the full-length crystal structure of a blue light photoreceptor LOV histidine kinase (LOV-HK) involved in light-dependent virulence modulation in the pathogenic bacterium Brucella abortus Joint analyses of dark and light structures determined in different signaling states have shown that LOV-HK transitions from a symmetric dark structure to a highly asymmetric light state. The initial local and subtle structural signal originated in the chromophore-binding LOV domain alters the dimer asymmetry via a coiled-coil rotary switch and helical bending in the helical spine. These amplified structural changes result in enhanced conformational flexibility and large-scale rearrangements that facilitate the phosphoryl transfer reaction in the HK domain.IMPORTANCE Bacteria employ two-component systems (TCSs) to sense and respond to changes in their surroundings. At the core of the TCS signaling pathway is the multidomain sensor histidine kinase, where the enzymatic activity of its output domain is allosterically controlled by the input signal perceived by the sensor domain. Here, we examine the structures and dynamics of a naturally occurring light-sensitive histidine kinase from the pathogen Brucella abortus in both its full-length and its truncated constructs. Direct comparisons between the structures captured in different signaling states have revealed concerted protein motions in an asymmetric dimer framework in response to light. Findings of this work provide mechanistic insights into modular sensory proteins that share a similar modular architecture.


Assuntos
Proteínas de Bactérias/metabolismo , Brucella abortus/enzimologia , Brucella abortus/metabolismo , Cor , Histidina Quinase/química , Histidina Quinase/metabolismo , Luz , Proteínas de Bactérias/genética , Brucella abortus/genética , Brucella abortus/patogenicidade , Histidina Quinase/genética , Modelos Moleculares , Domínios Proteicos , Transdução de Sinais
11.
Int J Mol Sci ; 22(6)2021 Mar 22.
Artigo em Inglês | MEDLINE | ID: mdl-33810099

RESUMO

The histidine phosphocarrier protein (HPr) kinase/phosphorylase (HPrK/P) modulates the phosphorylation state of the HPr protein, and it is involved in the use of carbon sources by Gram-positive bacteria. Its X-ray structure, as concluded from crystals of proteins from several species, is a hexamer; however, there are no studies about its conformational stability, and how its structure is modified by the pH. We have embarked on the conformational characterization of HPrK/P of Bacillus subtilis (bsHPrK/P) in solution by using several spectroscopic (namely, fluorescence and circular dichroism (CD)) and biophysical techniques (namely, small-angle X-ray-scattering (SAXS) and dynamic light-scattering (DLS)). bsHPrK/P was mainly a hexamer in solution at pH 7.0, in the presence of phosphate. The protein had a high conformational stability, with an apparent thermal denaturation midpoint of ~70 °C, at pH 7.0, as monitored by fluorescence and CD. The protein was very pH-sensitive, precipitated between pH 3.5 and 6.5; below pH 3.5, it had a molten-globule-like conformation; and it acquired a native-like structure in a narrow pH range (between pH 7.0 and 8.0). Guanidinium hydrochloride (GdmCl) denaturation occurred through an oligomeric intermediate. On the other hand, urea denaturation occurred as a single transition, in the range of concentrations between 1.8 and 18 µM, as detected by far-UV CD and fluorescence.


Assuntos
Bacillus subtilis/enzimologia , Histidina Quinase/química , Multimerização Proteica , Algoritmos , Estabilidade Enzimática , Histidina Quinase/metabolismo , Concentração de Íons de Hidrogênio , Modelos Químicos , Modelos Moleculares , Fosforilação , Conformação Proteica , Desnaturação Proteica , Análise Espectral , Relação Estrutura-Atividade , Temperatura
12.
FASEB J ; 35(4): e21475, 2021 04.
Artigo em Inglês | MEDLINE | ID: mdl-33772870

RESUMO

Cell signaling relies on second messengers to transduce signals from the sensory apparatus to downstream signaling pathway components. In bacteria, one of the most important and ubiquitous second messenger is the small molecule cyclic diguanosine monophosphate (c-di-GMP). While the biosynthesis, degradation, and regulatory pathways controlled by c-di-GMP are well characterized, the mechanisms through which c-di-GMP controls these processes are not entirely understood. Herein we present the report of a c-di-GMP sensing sensor histidine kinase PdtaS (Rv3220c), which binds to c-di-GMP at submicromolar concentrations, subsequently perturbing signaling of the PdtaS-PdtaR (Rv1626) two-component system. Aided by biochemical analysis, genetics, molecular docking, FRET microscopy, and structural modelling, we have characterized the binding of c-di-GMP in the GAF domain of PdtaS. We show that a pdtaS knockout in Mycobacterium smegmatis is severely compromised in growth on amino acid deficient media and exhibits global transcriptional dysregulation. The perturbation of the c-di-GMP-PdtaS-PdtaR axis results in a cascade of cellular changes recorded by a multiparametric systems' approach of transcriptomics, unbiased metabolomics, and lipid analyses.


Assuntos
Carbono/metabolismo , Regulação Bacteriana da Expressão Gênica/fisiologia , Histidina Quinase/metabolismo , Bactérias , Proteínas de Bactérias/metabolismo , Simulação de Acoplamento Molecular/métodos , Mycobacterium/metabolismo , Mycobacterium smegmatis/crescimento & desenvolvimento , Mycobacterium smegmatis/metabolismo , Sistemas do Segundo Mensageiro/fisiologia , Transdução de Sinais/fisiologia
13.
Appl Environ Microbiol ; 87(11)2021 05 11.
Artigo em Inglês | MEDLINE | ID: mdl-33771779

RESUMO

Pseudomonas aeruginosa is an opportunistic pathogen that can cause problematic infections at different sites throughout the human body. P. aeruginosa encodes a large suite of over 60 two-component signaling systems that enable cells to rapidly sense and respond to external signals. Previous work has shown that some of these sensory systems contribute to P. aeruginosa pathogenesis, but the virulence-associated processes and phenotypic traits that each of these systems controls are still largely unclear. To aid investigations of these sensory systems, we have generated deletion strains for each of 64 genes encoding histidine kinases and one histidine phosphotransferase in P. aeruginosa PA14. We carried out initial phenotypic characterizations of this collection by assaying these mutants for over a dozen virulence-associated traits, and we found that each of these phenotypes is regulated by multiple sensory systems. Our work highlights the usefulness of this collection for further studies of P. aeruginosa two-component signaling systems and provides insight into how these systems may contribute to P. aeruginosa infection.IMPORTANCE Pseudomonas aeruginosa can grow and survive under a wide range of conditions, including as a human pathogen. As such, P. aeruginosa must be able to sense and respond to diverse signals and cues in its environment. This sensory capability is endowed in part by the hundreds of two-component signaling proteins encoded in the P. aeruginosa genome, but the precise roles of each remain poorly defined. To facilitate systematic study of the signaling repertoire of P. aeruginosa PA14, we generated a library of deletion strains, each lacking one of the 64 histidine kinases. By subjecting these strains to a battery of phenotypic assays, we confirmed the functions of many and unveiled roles for dozens of previously uncharacterized histidine kinases in controlling various traits, many of which are associated with P. aeruginosa virulence. Thus, this work provides new insight into the functions of two-component signaling proteins and provides a resource for future investigations.


Assuntos
Proteínas de Bactérias/genética , Genes Bacterianos , Pseudomonas aeruginosa/genética , Pseudomonas aeruginosa/patogenicidade , Proteínas de Bactérias/metabolismo , Deleção de Genes , Histidina Quinase/genética , Histidina Quinase/metabolismo , Fosfotransferases/genética , Fosfotransferases/metabolismo , Transdução de Sinais/genética , Virulência
14.
Proc Natl Acad Sci U S A ; 118(8)2021 02 23.
Artigo em Inglês | MEDLINE | ID: mdl-33608460

RESUMO

Early root growth is critical for plant establishment and survival. We have identified a molecular pathway required for helical root tip movement known as circumnutation. Here, we report a multiscale investigation of the regulation and function of this phenomenon. We identify key cell signaling events comprising interaction of the ethylene, cytokinin, and auxin hormone signaling pathways. We identify the gene Oryza sativa histidine kinase-1 (HK1) as well as the auxin influx carrier gene OsAUX1 as essential regulators of this process in rice. Robophysical modeling and growth challenge experiments indicate circumnutation is critical for seedling establishment in rocky soil, consistent with the long-standing hypothesis that root circumnutation facilitates growth past obstacles. Thus, the integration of robotics, physics, and biology has elucidated the functional importance of root circumnutation and uncovered the molecular mechanisms underlying its regulation.


Assuntos
Regulação da Expressão Gênica de Plantas , Histidina Quinase/metabolismo , Ácidos Indolacéticos/farmacologia , Oryza/crescimento & desenvolvimento , Proteínas de Plantas/metabolismo , Raízes de Plantas/crescimento & desenvolvimento , Solo/química , Transporte Biológico , Citocininas/metabolismo , Histidina Quinase/genética , Oryza/efeitos dos fármacos , Oryza/genética , Oryza/metabolismo , Reguladores de Crescimento de Plantas/farmacologia , Proteínas de Plantas/genética , Raízes de Plantas/efeitos dos fármacos , Raízes de Plantas/genética , Raízes de Plantas/metabolismo
15.
Biochemistry ; 60(6): 465-476, 2021 02 16.
Artigo em Inglês | MEDLINE | ID: mdl-33538578

RESUMO

The anaerobic bacterium Chrysiogenes arsenatis respires using the oxyanion arsenate (AsO43-) as the terminal electron acceptor, where it is reduced to arsenite (AsO33-) while concomitantly oxidizing various organic (e.g., acetate) electron donors. This respiratory activity is catalyzed in the periplasm of the bacterium by the enzyme arsenate reductase (Arr), with expression of the enzyme controlled by a sensor histidine kinase (ArrS) and a periplasmic-binding protein (PBP), ArrX. Here, we report for the first time, the molecular structure of ArrX in the absence and presence of bound ligand arsenate. Comparison of the ligand-bound structure of ArrX with other PBPs shows a high level of conservation of critical residues for ligand binding by these proteins; however, this suite of PBPs shows different structural alterations upon ligand binding. For ArrX and its homologue AioX (from Rhizobium sp. str. NT-26), which specifically binds arsenite, the structures of the substrate-binding sites in the vicinity of a conserved and critical cysteine residue contribute to the discrimination of binding for these chemically similar ligands.


Assuntos
Arseniato Redutases/química , Bactérias/metabolismo , Sequência de Aminoácidos/genética , Arseniato Redutases/metabolismo , Arseniatos/química , Arseniatos/metabolismo , Bactérias/química , Composição de Bases/genética , Sítios de Ligação , Catálise , Cristalografia por Raios X/métodos , Histidina Quinase/metabolismo , Oxirredutases/metabolismo , Periplasma/metabolismo , Proteínas Periplásmicas de Ligação/química , Proteínas Periplásmicas de Ligação/metabolismo , Filogenia , RNA Ribossômico 16S/genética , Análise de Sequência de DNA/métodos
16.
PLoS Genet ; 17(2): e1009099, 2021 02.
Artigo em Inglês | MEDLINE | ID: mdl-33539353

RESUMO

Regulation by oxygen (O2) in rhizobia is essential for their symbioses with plants and involves multiple O2 sensing proteins. Three sensors exist in the pea microsymbiont Rhizobium leguminosarum Rlv3841: hFixL, FnrN and NifA. At low O2 concentrations (1%) hFixL signals via FxkR to induce expression of the FixK transcription factor, which activates transcription of downstream genes. These include fixNOQP, encoding the high-affinity cbb3-type terminal oxidase used in symbiosis. In free-living Rlv3841, the hFixL-FxkR-FixK pathway was active at 1% O2, and confocal microscopy showed hFixL-FxkR-FixK activity in the earliest stages of Rlv3841 differentiation in nodules (zones I and II). Work on Rlv3841 inside and outside nodules showed that the hFixL-FxkR-FixK pathway also induces transcription of fnrN at 1% O2 and in the earliest stages of Rlv3841 differentiation in nodules. We confirmed past findings suggesting a role for FnrN in fixNOQP expression. However, unlike hFixL-FxkR-FixK, Rlv3841 FnrN was only active in the near-anaerobic zones III and IV of pea nodules. Quantification of fixNOQP expression in nodules showed this was driven primarily by FnrN, with minimal direct hFixL-FxkR-FixK induction. Thus, FnrN is key for full symbiotic expression of fixNOQP. Without FnrN, nitrogen fixation was reduced by 85% in Rlv3841, while eliminating hFixL only reduced fixation by 25%. The hFixL-FxkR-FixK pathway effectively primes the O2 response by increasing fnrN expression in early differentiation (zones I-II). In zone III of mature nodules, near-anaerobic conditions activate FnrN, which induces fixNOQP transcription to the level required for wild-type nitrogen fixation activity. Modelling and transcriptional analysis indicates that the different O2 sensitivities of hFixL and FnrN lead to a nuanced spatiotemporal pattern of gene regulation in different nodule zones in response to changing O2 concentration. Multi-sensor O2 regulation is prevalent in rhizobia, suggesting the fine-tuned control this enables is common and maximizes the effectiveness of the symbioses.


Assuntos
Proteínas de Bactérias/metabolismo , Histidina Quinase/metabolismo , Oxigênio/metabolismo , Rhizobium leguminosarum/metabolismo , Simbiose/genética , Fatores de Transcrição/metabolismo , Proteínas de Bactérias/genética , Fabaceae/genética , Fabaceae/metabolismo , Regulação Bacteriana da Expressão Gênica/genética , Histidina Quinase/genética , Mutação , Fixação de Nitrogênio/genética , Óperon/genética , Rhizobium leguminosarum/genética , Fatores de Transcrição/genética
17.
Plant Signal Behav ; 16(4): 1879542, 2021 04 03.
Artigo em Inglês | MEDLINE | ID: mdl-33586610

RESUMO

N,N-dimethyl-hexadecylamine (DMHDA) is a volatile organic compound (VOC) produced by some plant growth-promoting rhizobacteria (PGPR), which inhibits the growth of pathogenic fungi and induces iron uptake by roots. In this report, through the application of a wide range of concentrations, we found that DMHDA affects Arabidopsis primary root growth and lateral root formation in a dose-dependent manner where 1 and 2 µM promoted root growth and higher (4-32 µM) concentrations repressed growth. Cytokinin-inducible TCS::GFP and ARR5::uidA gene constructs showed an increased expression in columella cells and root meristem, respectively, at 2 µM DMHDA, but their expression domains strongly diminished at growth repressing treatments. To test if either primary root growth promotion or repression could involve members of the cytokinin receptor family, the growth of WT and double mutant combinations cre1-12 ahk2-2, cre1-12 ahk3-3, and ahk2-2 ahk3-3 was tested in control conditions or supplemented with 2 µM or 16 µM DMHDA. Noteworthy, the root growth promotion disappeared in cre1-12 ahk2-2 and ahk2-2 ahk3-3 combinations, whereas all double mutants had higher repression than the WT at high doses. We further show that DMHDA fails to mimic the effects of ethylene in Arabidopsis seedlings grown in darkness that include an exaggerated apical hook, stem and root shortening, and root hair elongation. Our data help unravel how Arabidopsis senses a growth-modulating bacterial volatile through changes in cytokinin responsiveness.


Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Citocininas/metabolismo , Histidina Quinase/metabolismo , Metilaminas/farmacologia , Raízes de Plantas/crescimento & desenvolvimento , Transdução de Sinais , Compostos Orgânicos Voláteis/farmacologia , Arabidopsis/efeitos dos fármacos , Arabidopsis/genética , Arabidopsis/crescimento & desenvolvimento , Proteínas de Arabidopsis/genética , Regulação da Expressão Gênica de Plantas/efeitos dos fármacos , Genes Reporter , Histidina Quinase/genética , Mutação/genética , Raízes de Plantas/efeitos dos fármacos , Plantas Geneticamente Modificadas , Transdução de Sinais/efeitos dos fármacos
18.
Microbiol Mol Biol Rev ; 85(1)2021 02 17.
Artigo em Inglês | MEDLINE | ID: mdl-33441490

RESUMO

Bacteria have evolved a variety of signal transduction mechanisms that generate different outputs in response to external stimuli. Chemosensory pathways are widespread in bacteria and are among the most complex signaling mechanisms, requiring the participation of at least six proteins. These pathways mediate flagellar chemotaxis, in addition to controlling alternative functions such as second messenger levels or twitching motility. The human pathogen Pseudomonas aeruginosa has four different chemosensory pathways that carry out different functions and are stimulated by signal binding to 26 chemoreceptors. Recent research employing a diverse range of experimental approaches has advanced enormously our knowledge on these four pathways, establishing P. aeruginosa as a primary model organism in this field. In the first part of this article, we review data on the function and physiological relevance of chemosensory pathways as well as their involvement in virulence, whereas the different transcriptional and posttranscriptional regulatory mechanisms that govern pathway function are summarized in the second part. The information presented will be of help to advance the understanding of pathway function in other organisms.


Assuntos
Quimiotaxia/fisiologia , Pseudomonas aeruginosa/metabolismo , Transdução de Sinais/fisiologia , Proteínas de Bactérias/metabolismo , Regulação Bacteriana da Expressão Gênica/genética , Histidina Quinase/metabolismo , Proteínas Quimiotáticas Aceptoras de Metil/metabolismo , Metilação , Metiltransferases/metabolismo
19.
mBio ; 12(1)2021 01 05.
Artigo em Inglês | MEDLINE | ID: mdl-33402539

RESUMO

Bacteriocins are natural antimicrobial peptides produced by bacteria to kill closely related competitors. The opportunistic pathogen Streptococcus gallolyticus subsp. gallolyticus was recently shown to outcompete commensal enterococci of the murine microbiota under tumoral conditions thanks to the production of a two-peptide bacteriocin named gallocin. Here, we identified four genes involved in the regulatory control of gallocin in S. gallolyticus subsp. gallolyticus UCN34 that encode a histidine kinase/response regulator two-component system (BlpH/BlpR), a secreted peptide (GSP [gallocin-stimulating peptide]), and a putative regulator of unknown function (BlpS). While BlpR is a typical 243-amino-acid (aa) response regulator possessing a phospho-receiver domain and a LytTR DNA-binding domain, BlpS is a 108-aa protein containing only a LytTR domain. Our results showed that the secreted peptide GSP activates the dedicated two-component system BlpH/BlpR to induce gallocin transcription. A genome-wide transcriptome analysis indicates that this regulatory system (GSP-BlpH/BlpR) is specific for bacteriocin production. Importantly, as opposed to BlpR, BlpS was shown to repress gallocin gene transcription. A conserved operator DNA sequence of 30 bp was found in all promoter regions regulated by BlpR and BlpS. Electrophoretic mobility shift assays (EMSA) and footprint assays showed direct and specific binding of BlpS and BlpR to various regulated promoter regions in a dose-dependent manner on this conserved sequence. Gallocin expression appears to be tightly controlled in S. gallolyticus subsp. gallolyticus by quorum sensing and antagonistic activity of 2 LytTR-containing proteins. Competition experiments in gut microbiota medium and 5% CO2 to mimic intestinal conditions demonstrate that gallocin is functional under these in vivo-like conditions.IMPORTANCE Streptococcus gallolyticus subsp. gallolyticus, formerly known as Streptococcus bovis biotype I, is an opportunistic pathogen causing septicemia and endocarditis in the elderly often associated with asymptomatic colonic neoplasia. Recent studies indicate that S. gallolyticus subsp. gallolyticus is both a driver and a passenger of colorectal cancer. We previously showed that S. gallolyticus subsp. gallolyticus produces a bacteriocin, termed gallocin, enabling colonization of the colon under tumoral conditions by outcompeting commensal members of the murine microbiota such as Enterococcus faecalis Here, we identified and extensively characterized a four-component system that regulates gallocin production. Gallocin gene transcription is activated by a secreted peptide pheromone (GSP) and a two-component signal transduction system composed of a transmembrane histidine kinase receptor (BlpH) and a cytosolic response regulator (BlpR). Finally, a DNA-binding protein (BlpS) was found to repress gallocin genes transcription, likely by antagonizing BlpR. Understanding gallocin regulation is crucial to prevent S. gallolyticus subsp. gallolyticus colon colonization under tumoral conditions.


Assuntos
Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Bacteriocinas/genética , Regulação Bacteriana da Expressão Gênica , Streptococcus gallolyticus/genética , Streptococcus gallolyticus/metabolismo , Proteínas de Ligação a DNA/metabolismo , Microbioma Gastrointestinal , Perfilação da Expressão Gênica , Genes Bacterianos/genética , Genoma Bacteriano , Histidina Quinase/genética , Histidina Quinase/metabolismo , Percepção de Quorum , Infecções Estreptocócicas/microbiologia , Transcriptoma
20.
Mol Genet Genomics ; 296(2): 299-312, 2021 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-33386986

RESUMO

Pseudomonas syringae pv. tabaci 6605 (Pta6605) is a causal agent of wildfire disease in host tobacco plants and is highly motile. Pta6605 has multiple clusters of chemotaxis genes including cheA, a gene encoding a histidine kinase, cheY, a gene encoding a response regulator, mcp, a gene for a methyl-accepting chemotaxis protein, as well as flagellar and pili biogenesis genes. However, only two major chemotaxis gene clusters, cluster I and cluster II, possess cheA and cheY. Deletion mutants of cheA or cheY were constructed to evaluate their possible role in Pta6605 chemotaxis and virulence. Motility tests and a chemotaxis assay to known attractant demonstrated that cheA2 and cheY2 mutants were unable to swarm and to perform chemotaxis, whereas cheA1 and cheY1 mutants retained chemotaxis ability almost equal to that of the wild-type (WT) strain. Although WT and cheY1 mutants of Pta6605 caused severe disease symptoms on host tobacco leaves, the cheA2 and cheY2 mutants did not, and symptom development with cheA1 depended on the inoculation method. These results indicate that chemotaxis genes located in cluster II are required for optimal chemotaxis and host plant infection by Pta6605 and that cluster I may partially contribute to these phenotypes.


Assuntos
Histidina Quinase/genética , Proteínas Quimiotáticas Aceptoras de Metil/genética , Pseudomonas aeruginosa/fisiologia , Pseudomonas syringae/fisiologia , Tabaco/microbiologia , Quimiotaxia , Resistência à Doença , Deleção de Genes , Histidina Quinase/metabolismo , Proteínas Quimiotáticas Aceptoras de Metil/metabolismo , Família Multigênica , Filogenia , Doenças das Plantas/microbiologia , Pseudomonas aeruginosa/patogenicidade , Pseudomonas syringae/patogenicidade , Virulência
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