Letteratura scientifica selezionata sul tema "Phosphorelais EnvZ"

ABSTRACT In a test of the hypothesis that DevR is a response regulator protein that functions in a phosphorelay signal transduction system involved in heterocyst development in Nostoc punctiformeATCC 29133, purified affinity-tagged DevR was shown to be phosphorylated in vitro by the noncognate sensor kinase EnvZ. Site-directed mutagenesis was used to generate N. punctiforme mutants with single amino acid substitutions at the putative phosphorylation site of DevR. These mutants exhibited a Fox− phenotype like the original devRinsertion mutant UCD 311, consistent with a phosphotransferase role for DevR.

ABSTRACT Transcription of the K+ transport operonkdp in Escherichia coli is induced during K+-limited growth by the action of a dual-component phosphorelay regulatory system comprised of a sensor kinase (integral membrane protein), KdpD, and a DNA-binding response regulator (cytoplasmic protein), KdpE. In this study, we screened for newdke (named dke for decreased kdpexpression) mutations (in loci other than kdpDE) that led to substantially decreased kdp expression. Onedke mutation was shown to be in hns, encoding the nucleoid protein H-NS. Another dke mutation was mapped to trxB (encoding thioredoxin reductase), and an equivalent reduction in kdp expression was demonstrated also fortrxA mutants that are deficient in thioredoxin 1. Exogenously provided dithiothreitol rescued the kdpexpression defect in trxB but not trxA mutants. Neither trxB nor trxA affected gene regulation mediated by another dual-component system tested, EnvZ-OmpR. Mutations in genes dsbC and dsbD did not affectkdp expression, suggesting that the trx effects on kdp are not mediated by alterations in protein disulfide bond status in the periplasm. Reduced kdp expression was observed even in a trxB strain that harbored a variant KdpD polypeptide bearing no Cys residues. A trxB hns double mutant was even more severely affected for kdp expression than either single mutant. The dke mutations themselves had no effect on strength of the signal controlling kdpexpression, and constitutive mutations in kdpDE were epistatic to hns and trxB. These results indicate that perturbations in cytoplasmic thiol oxidation status and in levels of the H-NS protein exert additive effects, direct or indirect, at a step(s) upstream of KdpD in the signal transduction pathway, which significantly influence the magnitude of KdpD kinase activity obtained for a given strength of the inducing signal forkdp transcription.

ABSTRACTAdaptation to osmotic stress is crucial for bacterial growth and survival in changing environments. Although a large number of osmotic stress response genes have been identified in various bacterial species, how osmotic changes affect bacterial motility, biofilm formation, and colonization of host niches remains largely unknown. In this study, we report that the LrhA regulator is an osmoregulated transcription factor that directly binds to the promoters of theflhDC,eps, andopgGHoperons and differentially regulates their expression, thus inhibiting motility and promoting exopolysaccharide (EPS) production, synthesis of osmoregulated periplasmic glucans (OPGs), biofilm formation, and root colonization of the plant growth-promoting bacteriumPantoea alhagiLTYR-11Z. Further, we observed that the LrhA-regulated OPGs control RcsCD-RcsB activation in a concentration-dependent manner, and a high concentration of OPGs induced by increased medium osmolarity is maintained to achieve the high level of activation of the Rcs phosphorelay, which results in enhanced EPS synthesis and decreased motility inP. alhagi. Moreover, we showed that the osmosensing regulator OmpR directly binds to the promoter oflrhAand promotes its expression, whilelrhAexpression is feedback inhibited by the activated Rcs phosphorelay system. Overall, our data support a model wherebyP. alhagisenses environmental osmolarity changes through the EnvZ-OmpR two-component system and LrhA to regulate the synthesis of OPGs, EPS production, and flagellum-dependent motility, thereby employing a hierarchical signaling cascade to control the transition between a motile lifestyle and a biofilm lifestyle.IMPORTANCEMany motile bacterial populations form surface-attached biofilms in response to specific environmental cues, including osmotic stress in a range of natural and host-related systems. However, cross talk between bacterial osmosensing, swimming, and biofilm formation regulatory networks is not fully understood. Here, we report that the pleiotropic regulator LrhA inPantoea alhagiis involved in the regulation of flagellar motility, biofilm formation, and host colonization and responds to osmotic upshift. We further show that this sensing relies on the EnvZ-OmpR two-component system that was known to detect changes in external osmotic stress. The EnvZ-OmpR-LrhA osmosensing signal transduction cascade is proposed to increase bacterial fitness under hyperosmotic conditions inside the host. Our work proposes a novel regulatory mechanism that links osmosensing and motile-sessile lifestyle transitions, which may provide new approaches to prevent or promote the formation of biofilms and host colonization inP. alhagiand other bacteria possessing a similar osmoregulatory mechanism.

Bacteria respond to physical forces perceived as mechanical stress as part of their comprehensive environmental sensing strategy. Histidine kinases can then funnel diverse environmental stimuli into changes in gene expression through a series of phosphorelay reactions. Because histidine kinases are most often embedded in the inner membrane, they can be sensitive to changes in membrane tension that occurs, for example, in response to osmotic stress, or when deformation of the cell body occurs upon encountering a surface before forming biofilms, or inside the host in response to shear stress in the kidney, intestine, lungs, or blood stream. A summary of our recent work that links the histidine kinase EnvZ to mechanical changes in the inner membrane is provided and placed in a context of other bacterial systems that respond to mechanical stress.

ABSTRACT A thorough understanding of the mechanisms of Rcs and EnvZ/OmpR phosphorelay systems that allow Yersinia enterocolitica to thrive in various environments is crucial to prevent and control Y. enterocolitica infections. In this study, we showed that RcsB and OmpR have the ability to function differently in modulating a diverse array of physiological processes in Y. enterocolitica. The rcsB mutant stimulated flagella biosynthesis and increased motility, biofilm formation and c-di-GMP production by upregulating flhDC, hmsHFRS and hmsT. However, mutation in ompR exhibited a non-motile phenotype due to the lack of flagella. Biofilm formation was reduced and less c-di-GMP was produced through the downregulation of flhDC, hmsHFRS and hmsT expression when Y. enterocolitica was exposed to low osmolarity conditions. Furthermore, OmpR was identified to be important for Y. enterocolitica to grow in extreme temperature conditions. Importantly, ompR mutations in Y. enterocolitica were more sensitive to polymyxin B and sodium dodecyl sulfate than rcsB mutations. Since motility, biofilm formation and environmental tolerance are critical for bacterial colonization of the host, these findings indicated that OmpR is more critical than RcsB in shaping the pathogenic phenotype of Y. enterocolitica.

Au cours de leur vie, les bactéries pathogènes sont confrontées à de nombreuses variations environnementales souvent appelées stress, notamment au cours du processus infectieux. Pour survivre et coloniser avec succès son hôte, la bactérie doit percevoir ce nouvel et hostile environnement pour s'y adapter rapidement. C'est le rôle principal assigné aux phosphorelais. Ces systèmes sont composés d'un couple capteur/régulateur. Sous l'action d'un stimulus, le capteur s'autophosphoryle et transmet son phosphate au régulateur, qui module l’activité d’un ensemble de gènes cibles permettant l'adaptation au nouvel environnement. Notre modèle expérimental Dickeya dadantii est une bactérie phytopathogène nécrotrophe responsable de la maladie de la pourriture molle chez un large spectre de plantes hôtes. Les variations du pH et d’osmolarité sont deux des stress souvent rencontrés et combattus par les bactéries pathogènes. Les phosphorelais EnvZ/OmpR et RcsCDB sont deux systèmes majeurs répondant à ces stress. Le laboratoire avait précédemment démontré que le niveau d'activation du système RcsCDB dépendait de la concentration en glucanes périplasmiques osmorégulés (OPG). Leur concentration est d’autant plus élevée dans le périplasme que l’osmolarité du milieu est basse ce qui fait des OPG un intermédiaire essentiel dans la perception de l'osmolarité. Cela nous a poussé à éclaircir la relation entre EnvZ/OmpR et les OPG. Dans ce travail, j’ai montré que, contrairement à l'activation du système RcsCDB, l'activation du système EnvZ/OmpR ne dépend pas de la concentration des OPG, tout en nécessitant leur présence pour l’activation correcte de ce phosphorelais. Pour mieux comprendre le rôle du système EnvZ/OmpR chez D. dadantii, l'activité de ce système a été étudiée in vivo et in planta. Alors que le système EnvZ/OmpR est activé dans un milieu à pH acide et à une osmolarité élevée chez E. coli, mes travaux montrent que seule la variation du pH active ce phosphorelais. De plus, contrairement à E. coli qui possède deux porines majeures, il ne semblait exister qu’une seule porine majeure chez D. dadantii. Mes études ont cependant révélé l’existence d’une seconde porine apparaissant à pH acide in vivo et in planta. Ces deux porines de type OmpF sont régulées par le pH via OmpR. Passée une adaptation de quelques heures dans l’hôte, le profil de ces porines dans l’enveloppe ne change plus durant l’infection. Pourtant, le niveau d’activation d’EnvZ/OmpR durant cette même période fluctue. Ainsi, au moins un autre paramètre environnemental module l’activation de EnvZ/OmpR in planta. Enfin, l’absence de variation des porines dans l’enveloppe durant cette même période suggère qu’un autre régulateur, peut-être RcsCDB, permettrait le maintien de leur niveau d’expression
During their lifetime, pathogenic bacteria are confronted with numerous environmental variations often referred to stress, particularly during infection. In order to survive and successfully colonize its host, the bacterium must perceive this new and dangerous environment to adapt quickly. This is the main role assigned to phosphorelays. These systems are composed of a sensor and a cognate regulator. Under the action of a stimulus, the sensor autophosphorylates and transmits the phosphate group to its regulator, which in turn modulates the activity of a set of target genes allowing adaptation to the new environment. Our experimental model Dickeya dadantii is a necrotrophic plant pathogen bacterium responsible for soft rot disease in a wide range of plant species. The variation of pH and osmolarity are two stresses often faced and fought by pathogenic bacteria. EnvZ/OmpR and RcsCDB phosphorelays are two major systems known to respond to these stresses. The laboratory had previously demonstrated that the level of activation of the RcsCDB system was dependent on the concentration of periplasmic osmoregulated glucans (OPG). Their concentration in the periplasm increases as the medium osmolarity decreases, making OPGs a major intermediate in the perception of osmolarity. This prompted us to decipher the relationship between EnvZ/OmpR and OPGs. I showed that, unlike for the activation of the RcsCDB system, the activation of EnvZ/OmpR doesn’t depend on the concentration of OPGs, but still requires its presence for proper activation of the phosphorelay. To go deeper into the EnvZ/OmpR system, activities of this system have been studied in vivo and in planta. While the EnvZ/OmpR system is activated in a medium with an acidic pH and a high osmolarity in E. coli, my work shows that only pH variation activates this phosphorelay in D. dadantii. In addition, only one major porin (versus two in E. coli) was previously detected in D. dadantii. My studies revealed the existence of a second porin expressed at acidic pH in vivo and in planta. These two OmpF-like porins are regulated by the pH via OmpR. After adaptation for a few hours in planta, the pattern of these two porines remains the same over the rest of the infection. However, the level of OmpR activation during the same period fluctuates indicating that at least one other environmental parameter modulates the activation of EnvZ/OmpR in planta. The steady state level of the porines in the envelope during this same period suggests that another regulatory system, perhaps RcsCDB may maintain their expression level

Tout au long de sa vie, la bactérie doit faire face à de nombreuses variations de l'environnement. Elle doit s'y adapter rapidement et efficacement afin de survivre. Pour cela, elle dispose des phosphorelais, ou systèmes à deux composants qui sont les outils moléculaires majeurs permettant la perception et l'adaptation de l'environnement chez les bactéries. Ils sont composés d'un capteur et d'un régulateur associé. Suite à la perception d'un stimulus, le capteur s'autophosphoryle et transmet le groupement phosphate au régulateur qui va alors moduler l'expression de l'ensemble des gènes cibles, appelé régulon. Durant le processus d'infection, les bactéries pathogènes doivent faire face à de multiples stress. Ainsi est retrouvé un nombre important de ces systèmes chez de nombreuses bactéries pathogènes comme notre modèle d'étude Dickeya dadantii. Responsable de la maladie de la pourriture molle, D. dadantii est une entérobactérie phytopathogène à large spectre d'hôte. Elle dispose d'une batterie de 32 phosphorelais pour affronter les défenses de l'hôte et les stress généraux de carences nutritionnelles ou des variations physico-chimique de l'environnement.Dans un premier temps, cette étude se focalise sur l'un d'entre eux, le système EnvZ/OmpR. Mes travaux montrent dans un premier temps que le pH dans la plante reste acide durant l'infection. Cependant, malgré une activation du système par le pH acide, il n'est pas activé durant ce processus. Pour comprendre la raison de cette incohérence, le régulon du système a été étudié. Il a alors été découvert que durant l'émergence du genre Dickeya, le gène ompF, codant la porine du même nom, a été dupliqué. De façon intéressante, l'expression d'ompF est constitutive tandis que celle d'ompF2, le gène dupliqué, est soumise au niveau de phosphorylation d'OmpR. L'expression de cette seconde porine est également délétère à l'infection. Ainsi, durant l'infection, l'activation d'EnvZ/OmpR est contrecarrée par la perception de molécule de défense de l'hôte afin d'éviter l'expression d'ompF2 et permettre un bon déroulement de la virulence.Dans un second temps, a été réalisée lors de mes travaux une étude globale de l'importance de chaque phosphorelais sur la virulence de D. dadantii. Les premiers résultats montrent que seuls 6 systèmes sont impliqués dans la virulence. Le nombre et la complexité des stress rencontrés par les bactéries pathogènes ne semblent pas en accord avec ce faible nombre. La baisse de la quantité de bactéries inoculées a permis d'affiner la détection des systèmes participant à la virulence, qui se comptent désormais au nombre de 12. Enfin, l'ensemble de ces résultats indiquent l'importance d'une régulation fine de l'activation d'un phosphorelais car EnvZ/OmpR doit être activé pour l'infection mais que cette activation doit être fermement contrôlée au risque d'avoir des effets néfastes sur la virulence
Throughout their life, the bacteria must confront numerous environmental variations. They must adapt rapidly and effectively to ensure their survival. To accomplish this, they possess phosphorelays, or two-component systems, which are the major molecular tools enabling perception and adaptation to the environment in bacteria. These phosphorelays consist of a sensor and an associated regulator. Following the perception of a stimulus, the sensor autophosphorylates and transmits the phosphate group to the regulator, which then modulates the expression of the entire target gene set, known as a regulon. During the infection process, pathogenic bacteria must deal with multiple stresses. A significant number of these systems are found in various pathogenic bacteria, such as our study model Dickeya dadantii. Responsible for soft rot disease, D. dadantii is a wide-host-range phytopathogenic enterobacterium. It possesses a battery of 32 phosphorelays to deal with host defenses and the general stresses of nutritional deficiencies or physicochemical variations in the environment.First this study focuses on one of them, the EnvZ/OmpR system. My work initially shows that the pH in the plant remains acidic during infection. However, despite activation of the system by acidic pH, it is not activated during this process. To understand the reason for this inconsistency, the system's regulon was studied. It was then discovered that during the emergence of the Dickeya genus, the ompF gene, encoding the porin of the same name, was duplicated. Interestingly, the expression of ompF is constitutive, whereas that of ompF2, the duplicated gene, which is dependent on OmpR phosphorylation levels. The expression of this second porin is also detrimental to infection. Thus, during infection, the activation of EnvZ/OmpR is counteracted by the perception of host defense molecules to prevent the expression of ompF2 and enable proper virulence progression.In a second phase, a comprehensive study of the importance of each phosphorelay in D. dadantii's virulence was conducted in my work. The initial results show that only 6 systems are involved in virulence. The number and complexity of stresses encountered by pathogenic bacteria do not seem to align with this low number. Reducing the quantity of inoculated bacteria allowed for a more precise detection of the systems contributing to virulence, which now totals 12. Overall, these results indicate the significance of finely regulating the activity of a phosphorelay, as EnvZ/OmpR must be activated for infection, but this activation must be strongly controlled to avoid detrimental effects on virulence