The CbrA-CbrB two-component regulatory system controls the utilization of multiple carbon and nitrogen sources in Pseudomonas aeruginosa.

A novel two-component system, CbrA-CbrB, was discovered in Pseudomonas aeruginosa; cbrA and cbrB mutants of strain PAO were found to be unable to use several amino acids (such as arginine, histidine and proline), polyamines and agmatine as sole carbon and nitrogen sources. These mutants were also unable to use, or used poorly, many other carbon sources, including mannitol, glucose, pyruvate and citrate. A 7 kb EcoRI fragment carrying the cbrA and cbrB genes was cloned and sequenced. The cbrA and cbrB genes encode a sensor/histidine kinase (Mr 108 379, 983 residues) and a cognate response regulator (Mr 52 254, 478 residues) respectively. The amino-terminal half (490 residues) of CbrA appears to be a sensor membrane domain, as predicted by 12 possible transmembrane helices, whereas the carboxy-terminal part shares homology with the histidine kinases of the NtrB family. The CbrB response regulator shows similarity to the NtrC family members. Complementation and primer extension experiments indicated that cbrA and cbrB are transcribed from separate promoters. In cbrA or cbrB mutants, as well as in the allelic argR9901 and argR9902 mutants, the aot-argR operon was not induced by arginine, indicating an essential role for this two-component system in the expression of the ArgR-dependent catabolic pathways, including the aruCFGDB operon specifying the major aerobic arginine catabolic pathway. The histidine catabolic enzyme histidase was not expressed in cbrAB mutants, even in the presence of histidine. In contrast, proline dehydrogenase, responsible for proline utilization (Pru), was expressed in a cbrB mutant at a level comparable with that of the wild-type strain. When succinate or other C4-dicarboxylates were added to proline medium at 1 mM, the cbrB mutant was restored to a Pru+ phenotype. Such a succinate-dependent Pru+ property was almost abolished by 20 mM ammonia. In conclusion, the CbrA-CbrB system controls the expression of several catabolic pathways and, perhaps together with the NtrB-NtrC system, appears to ensure the intracellular carbon: nitrogen balance in P. aeruginosa.

DNA interaction and phosphotransfer of the C-4-dicarboxylate-responsive DcuS-DcuR two-component regulatory system from Escherichia coli

The DcuS-DcuR system of Escherichia coli is a two-component sensor-regulator that controls gene expression in response to external C-4-dicarboxylates and citrate. The DcuS protein is particularly interesting since it contains two PAS domains, namely a periplasmic C-4-dicarboxylate-sensing PAS domain (PASp) and a cytosolic PAS domain (PASc) of uncertain function. For a study of the role of the PASc domain, three different fragments of DcuS were overproduced and examined: they were PASc-kinase, PASc, and kinase. The two kinase-domain-containing fragments were autophosphorylated by [gamma-P-32]ATP. The rate was not affected by fumarate or succinate, supporting the role of the PASp domain in C-4-dicarboxylate sensing. Both of the phosphorylated DcuS constructs were able to rapidly pass their phosphoryl groups to DcuR, and after phosphorylation, DcuR dephosphorylated rapidly. No prosthetic group or significant quantity of metal was found associated with either of the PASc-containing proteins. The DNA-binding specificity of DcuR was studied by use of the pure protein. It was found to be converted from a monomer to a dimer upon acetylphosphate treatment, and native polyacrylamide gel electrophoresis suggested that it can oligomerize. DcuR specifically bound to the promoters of the three known DcuSR-regulated genes (dctA, dcuB, and frdA), with apparent K(D)s of 6 to 32 muM for untreated DcuR and less than or equal to1 to 2 muM for the acetylphosphate-treated form. The binding sites were located by DNase I footprinting, allowing a putative DcuR-binding motif [tandemly repeated (T/A)(A/T)(T/C)(A/T)AA sequences] to be identified. The DcuR-binding sites of the dcuB, dctA, and frdA genes were located 27, 94, and 86 bp, respectively, upstream of the corresponding +1 sites, and a new promoter was identified for dcuB that responds to DcuR.

Identification and characterization of the two-component NtrY/NtrX regulatory system in Azospirillum brasilense

Two Azospirillum brasilense open reading frames (ORFs) exhibited homology with the two-component NtrY/NtrX regulatory system from Azorhizobium caulinodans. These A. brasilense ORFs, located downstream to the nifR3ntrBC operon, were isolated, sequenced and characterized. The present study suggests that ORF1 and ORF2 correspond to the A. brasilense ntrY and ntrX genes, respectively. The amino acid sequences of A. brasilense NtrY and NtrX proteins showed high similarity to sensor/kinase and regulatory proteins, respectively. Analysis of lacZ transcriptional fusions by the ß-galactosidase assay in Escherichia coli ntrC mutants showed that the NtrY/NtrX proteins failed to activate transcription of the nifA promoter of A. brasilense. The ntrYX operon complemented a nifR3ntrBC deletion mutant of A. brasilense for nitrate-dependent growth, suggesting a possible cross-talk between the NtrY/X and NtrB/C sensor/regulator pairs. Our data support the existence of another two-component regulatory system in A. brasilense, the NtrY/NtrX system, probably involved in the regulation of nitrate assimilation.

Understanding the two-component sensing System of virulent bacteria

Report for the scientific sojourn carried out at the l’ Institute for Computational Molecular Science of the Temple University, United States, from 2010 to 2012. Two-component systems (TCS) are used by pathogenic bacteria to sense the environment within a host and activate mechanisms related to virulence and antimicrobial resistance. A prototypical example is the PhoQ/PhoP system, which is the major regulator of virulence in Salmonella. Hence, PhoQ is an attractive target for the design of new antibiotics against foodborne diseases. Inhibition of the PhoQ-mediated bacterial virulence does not result in growth inhibition, presenting less selective pressure for the generation of antibiotic resistance. Moreover, PhoQ is a histidine kinase (HK) and it is absent in animals. Nevertheless, the design of satisfactory HK inhibitors has been proven to be a challenge. To compete with the intracellular ATP concentrations, the affinity of a HK inhibidor must be in the micromolar-nanomolar range, whereas the current lead compounds have at best millimolar affinities. Moreover, the drug selectivity depends on the conformation of a highly variable loop, referred to as the “ATP-lid, which is difficult to study by X-Ray crystallography due to its flexibility. I have investigated the binding of different HK inhibitors to PhoQ. In particular, all-atom molecular dynamics simulations have been combined with enhanced sampling techniques in order to provide structural and dynamic information of the conformation of the ATP-lid. Transient interactions between these drugs and the ATP-lid have been identified and the free energy of the different binding modes has been estimated. The results obtained pinpoint the importance of protein flexibility in the HK-inhibitor binding, and constitute a first step in developing more potent and selective drugs. The computational resources of the hosting institution as well as the experience of the members of the group in drug binding and free energy methods have been crucial to carry out this work.

The ethylene hormone response in Arabidopsis: a eukaryotic two-component signaling system.

The simple gas ethylene affects numerous physiological processes in the growth and development of higher plants. With the use of molecular genetic approaches, we are beginning to learn how plants perceive ethylene and how this signal is transduced. Components of ethylene signal transduction are defined by ethylene response mutants in Arabidopsis thaliana. The genes corresponding to two of these mutants, etr1 and etr1, have been cloned. The ETR1 gene encodes a homolog of two-component regulators that are known almost exclusively in prokaryotes. The two-component regulators in prokaryotes are involved in the perception and transduction of a wide range of environmental signals leading to adaptive responses. The CTR1 gene encodes a homolog of the Raf family of serine/threonine protein kinases. Raf is part of a mitogen-activated protein kinase cascade known to regulate cell growth and development in mammals, worms, and flies. The ethylene response pathway may, therefore, exemplify a conserved protein kinase cascade regulated by a two-component system. The dominance of all known mutant alleles of ETR1 may be due to either constitutive activation of the ETR1 protein or dominant interference of wild-type activity. The discovery of Arabidopsis genes encoding proteins related to ETR1 suggests that the failure to recover recessive etr1 mutant alleles may be due to the presence of redundant genes.

A direct comparison of one- and two-component dendritic self-assembled materials: elucidating molecular recognition pathways

This paper compares and contrasts, for the first time, one- and two-component gelation systems that are direct structural analogues and draws conclusions about the molecular recognition pathways that underpin fibrillar self-assembly. The new one-component systems comprise L-lysine-based dendritic headgroups covalently connected to an aliphatic diamine spacer chain via an amide bond, One-component gelators with different generations of headgroup (from first to third generation) and different length spacer chains are reported. The self-assembly of these dendrimers in toluene was elucidated using thermal measurements, circular dichroism (CD) and NMR spectroscopies, scanning electron microscopy (SEM), and small-angle X-ray scattering (SAXS). The observations are compared with previous results for the analogous two-component gelation system in which the dendritic headgroups are bound to the aliphatic spacer chain noncovalently via acid-amine interactions. The one-component system is inherently a more effective gelator, partly as a consequence of the additional covalent amide groups that provide a new hydrogen bonding molecular recognition pathway, whereas the two-component analogue relies solely on intermolecular hydrogen bond interactions between the chiral dendritic headgroups. Furthermore, because these amide groups are important in the assembly process for the one-component system, the chiral information preset in the dendritic headgroups is not always transcribed into the nanoscale assembly, whereas for the two-component system, fiber formation is always accompanied by chiral ordering because the molecular recognition pathway is completely dependent on hydrogen bond interactions between well-organized chiral dendritic headgroups.

Sistema de secreción de tipo III en Aeromonas mesòfilas

Aeromonas hydrophila és un bacil gram-negatiu, patogen oportunista d’animal i humans. La patogènesi d’A. Hydrophila és multifactorial. A fi d'identificar gens implicats en la virulència de la soca PPD134/91 d’A. hydrophila, vam realitzar experiments de substracció gènica, que van dur a la detecció de 22 fragments d’ADN que codificaven 19 potencials factors de virulencia, incloent un gen que codificava una proteïna de sistema de secreció de tipus III (T3SS). La importància creixent del T3SS en la patogènesi de diversos bacteris, ens va dur a identificar i analitzar l'agrupació gènica del T3SS de les soques AH-1 i AH-3 d’A. hydrophila. La inactivació dels gens de T3SS aopB i aopD d’A. hydrophila AH-1, i ascV d’A. hydrophila AH-3, comporta una disminució de la citotoxicitat, un increment de la fagocitosi, i una reducció de la virulència en diferents models animals. Aquests resultats demostren que el T3SS és necessari per a la patogenicitat. També vam clonar i seqüenciar una ADP-ribosiltransferasa (AexT) a la soca AH-3 d’A. hydrophila, i vam demostrar que aquesta toxina és translocada via el T3SS, sistema que al seu torn sembla ser induïble in vitro en condicions de depleció de calci. El mutant en el gen aexT de la soca AH-3 d’A. hydrophila va mostrar una lleugera reducció de la virulència, assajada amb diferents mètodes. Mitjançant l'ús de diferents sondes d’ADN, vam determinar la presència del T3SS en soques tant clíniques com ambientals de diferents espècies del gènere Aeromonas: A. hydrophila, A. veronii, i A. caviae, i la codistribució d'aquesta agrupació gènica i el gen aexT. Finalment, amb la finalitat d'estudiar la regulació transcripcional de l'agrupació gènica de T3SS i de l’efector AexT A. hydrophila AH-3, vam aïllar els promotors predits per l’operó aopN-aopD i el gen aexT, i els vam fusionar amb el gen reporter gfp (Green Fluorescence Protein). A més, vam demostrar que l'expressió d'ambdós promotors depèn de diferents components bacterians, com per exemple el sistema de dos components PhoP/PhoQ, el sistema de quorum sensing AhyI/AhyR, o el complex piruvat deshidrogenasa.

Analysis of teichoic acid biosynthesis regulation reveals that the extracytoplasmic function sigma factor sigmaM is induced by phosphate depletion in Bacillus subtilis W23.

The expression of the Bacillus subtilis W23 tar genes specifying the biosynthesis of the major wall teichoic acid, the poly(ribitol phosphate), was studied under phosphate limitation using lacZ reporter fusions. Three different regulation patterns can be deduced from these beta-galactosidase activity data: (i) tarD and tarL gene expression is downregulated under phosphate starvation; (ii) tarA and, to a minor extent, tarB expression after an initial decrease unexpectedly increases; and (iii) tarO is not influenced by phosphate concentration. To dissect the tarA regulatory pattern, its two promoters were analysed under phosphate limitation: The P(tarA)-ext promoter is repressed under phosphate starvation by the PhoPR two-component system, whereas, under the same conditions, the P(tarA)-int promoter is upregulated by the action of an extracytoplasmic function (ECF) sigma factor, sigma(M). In contrast to strain 168, sigma(M) is activated in strain W23 in phosphate-depleted conditions, a phenomenon indirectly dependent on PhoPR, the two-component regulatory system responsible for the adaptation to phosphate starvation. These results provide further evidence for the role of sigma(M) in cell-wall stress response, and suggest that impairment of cell-wall structure is the signal activating this ECF sigma factor.

Global GacA-steered control of cyanide and exoprotease production in Pseudomonas fluorescens involves specific ribosome binding sites.

The conserved two-component regulatory system GacS/GacA determines the expression of extracellular products and virulence factors in a variety of Gram-negative bacteria. In the biocontrol strain CHA0 of Pseudomonas fluorescens, the response regulator GacA is essential for the synthesis of extracellular protease (AprA) and secondary metabolites including hydrogen cyanide. GacA was found to exert its control on the hydrogen cyanide biosynthetic genes (hcnABC) and on the aprA gene indirectly via a posttranscriptional mechanism. Expression of a translational hcnA'-'lacZ fusion was GacA-dependent whereas a transcriptional hcnA-lacZ fusion was not. A distinct recognition site overlapping with the ribosome binding site appears to be primordial for GacA-steered regulation. GacA-dependence could be conferred to the Escherichia coli lacZ mRNA by a 3-bp substitution in the ribosome binding site. The gene coding for the global translational repressor RsmA of P. fluorescens was cloned. RsmA overexpression mimicked partial loss of GacA function and involved the same recognition site, suggesting that RsmA is a downstream regulatory element of the GacA control cascade. Mutational inactivation of the chromosomal rsmA gene partially suppressed a gacS defect. Thus, a central, GacA-dependent switch from primary to secondary metabolism may operate at the level of translation.

Molecular basis and regulation of insect pathogenicity in plant-beneficial pseudomonads

Les bactéries du genre Pseudomonas ont la capacité étonnante de s'adapter à différents habitats et d'y survivre, ce qui leur a permis de conquérir un large éventail de niches écologiques et d'interagir avec différents organismes hôte. Les espèces du groupe Pseudomonas fluorescens peuvent être facilement isolées de la rhizosphère et sont communément connues comme des Pseudomonas bénéfiques pour les plantes. Elles sont capables d'induire la résistance systémique des plantes, d'induire leur croissance et de contrer des phytopathogènes du sol. Un sous-groupe de ces Pseudomonas a de plus développé la capacité d'infecter et de tuer certaines espèces d'insectes. Approfondir les connaissances sur l'interaction de ces bactéries avec les insectes pourraient conduire au développement de nouveaux biopesticides pour la protection des cultures. Le but de cette thèse est donc de mieux comprendre la base moléculaire, l'évolution et la régulation de la pathogénicité des Pseudomonas plante-bénéfiques envers les insectes. Plus spécifiquement, ce travail a été orienté sur l'étude de la production de la toxine insecticide appelée Fit et sur l'indentification d'autres facteurs de virulence participant à la toxicité de la bactérie envers les insectes. Dans la première partie de ce travail, la régulation de la production de la toxine Fit a été évaluée par microscopie à épifluorescence en utilisant des souches rapportrices de Pseudomonas protegens CHA0 qui expriment la toxine insecticide fusionnée à une protéine fluorescente rouge, au site natif du gène de la toxine. Celle-ci a été détectée uniquement dans l'hémolymphe des insectes et pas sur les racines des plantes, ni dans les milieux de laboratoire standards, indiquant une production dépendante de l'hôte. L'activation de la production de la toxine est contrôlée par trois protéines régulatrices dont l'histidine kinase FitF, essentielle pour un contrôle précis de l'expression et possédant un domaine "senseur" similaire à celui de la kinase DctB qui régule l'absorption de carbone chez les Protéobactéries. Il est donc probable que, durant l'évolution de FitF, un réarrangement de ce domaine "senseur" largement répandu ait contribué à une production hôte-spécifique de la toxine. Les résultats de cette étude suggèrent aussi que l'expression de la toxine Fit est plutôt réprimée en présence de composés dérivés des plantes qu'induite par la perception d'un signal d'insecte spécifique. Dans la deuxième partie de ce travail, des souches mutantes ciblant des facteurs de virulence importants identifiés dans des pathogènes connus ont été générées, dans le but d'identifier ceux avec une virulence envers les insectes atténuée. Les résultats ont suggéré que l'antigène O du lipopolysaccharide (LPS) et le système régulateur à deux composantes PhoP/PhoQ contribuent significativement à la virulence de P. protegens CHA0. La base génétique de la biosynthèse de l'antigène O dans les Pseudomonas plante-bénéfiques et avec une activité insecticide a été élucidée et a révélé des différences considérables entre les lignées suite à des pertes de gènes ou des acquisitions de gènes par transfert horizontal durant l'évolution de certaines souches. Les chaînes latérales du LPS ont été montrées comme vitales pour une infection des insectes réussie par la souche CHA0, après ingestion ou injection. Les Pseudomonas plante-bénéfiques, avec une activité insecticide sont naturellement résistants à la polymyxine B, un peptide antimicrobien modèle. La protection contre ce composé antimicrobien particulier dépend de la présence de l'antigène O et de la modification du lipide A, une partie du LPS, avec du 4-aminoarabinose. Comme les peptides antimicrobiens cationiques jouent un rôle important dans le système immunitaire des insectes, l'antigène O pourrait être important chez les Pseudomonas insecticides pour surmonter les mécanismes de défense de l'hôte. Le système PhoP/PhoQ, connu pour contrôler les modifications du lipide A chez plusieurs bactéries pathogènes, a été identifié chez Pseudomonas chlororaphis PCL1391 et P. protegens CHA0. Pour l'instant, il n'y a pas d'évidence que des modifications du lipide A contribuent à la pathogénicité de cette bactérie envers les insectes. Cependant, le senseur-kinase PhoQ est requis pour une virulence optimale de la souche CHA0, ce qui suggère qu'il régule aussi l'expression des facteurs de virulence de cette bactérie. Les découvertes de cette thèse démontrent que certains Pseudomonas associés aux plantes sont de véritables pathogènes d'insectes et donnent quelques indices sur l'évolution de ces microbes pour survivre dans l'insecte-hôte et éventuellement le tuer. Les résultats suggèrent également qu'une recherche plus approfondie est nécessaire pour comprendre comment ces bactéries sont capables de contourner ou surmonter la réponse immunitaire de l'hôte et de briser les barrières physiques pour envahir l'insecte lors d'une infection orale. Pour cela, les futures études ne devraient pas uniquement se concentrer sur le côté bactérien de l'interaction hôte-microbe, mais aussi étudier l'infection du point de vue de l'hôte. Les connaissances gagnées sur la pathogénicité envers les insectes des Pseudomonas plante-bénéfiques donnent un espoir pour une future application en agriculture, pour protéger les plantes, non seulement contre les maladies, mais aussi contre les insectes ravageurs. -- Pseudomonas bacteria have the astonishing ability to survive within and adapt to different habitats, which has allowed them to conquer a wide range of ecological niches and to interact with different host organisms. Species of the Pseudomonas fluorescens group can readily be isolated from plant roots and are commonly known as plant-beneficial pseudomonads. They are capable of promoting plant growth, inducing systemic resistance in the plant host and antagonizing soil-borne phytopathogens. A defined subgroup of these pseudomonads evolved in addition the ability to infect and kill certain insect species. Profound knowledge about the interaction of these particular bacteria with insects could lead to the development of novel biopesticides for crop protection. This thesis thus aimed at a better understanding of the molecular basis, evolution and regulation of insect pathogenicity in plant-beneficial pseudomonads. More specifically, it was outlined to investigate the production of an insecticidal toxin termed Fit and to identify additional factors contributing to the entomopathogenicity of the bacteria. In the first part of this work, the regulation of Fit toxin production was probed by epifluorescence microscopy using reporter strains of Pseudomonas protegens CHAO that express a fusion between the insecticidal toxin and a red fluorescent protein in place of the native toxin gene. The bacterium was found to express its insecticidal toxin only in insect hemolymph but not on plant roots or in common laboratory media. The host-dependent activation of Fit toxin production is controlled by three local regulatory proteins. The histidine kinase of this regulatory system, FitF, is essential for the tight control of toxin expression and shares a sensing domain with DctB, a sensor kinase regulating carbon uptake in Proteobacteria. It is therefore likely that shuffling of a ubiquitous sensor domain during the evolution of FitF contributed to host- specific production of the Fit toxin. Findings of this study additionally suggest that host-specific expression of the Fit toxin is mainly achieved by repression in the presence of plant-derived compounds rather than by induction upon perceiving an insect-specific signal molecule. In the second part of this thesis, mutant strains were generated that lack factors previously shown to be important for virulence in prominent pathogens. A screening for attenuation in insect virulence suggested that lipopolysaccharide (LPS) O-antigen and the PhoP-PhoQ two-component regulatory system significantly contribute to virulence of P. protegens CHAO. The genetic basis of O-antigen biosynthesis in plant-beneficial pseudomonads displaying insect pathogenicity was elucidated and revealed extensive differences between lineages due to reduction and horizontal acquisition of gene clusters during the evolution of several strains. Specific 0 side chains of LPS were found to be vital for strain CHAO to successfully infect insects by ingestion or upon injection. Insecticidal pseudomonads with plant-beneficial properties were observed to be naturally resistant to polymyxin B, a model antimicrobial peptide. Protection against this particular antimicrobial compound was dependent on the presence of O-antigen and modification of the lipid A portion of LPS with 4-aminoarabinose. Since cationic antimicrobial peptides play a major role in the immune system of insects, O-antigenic polysaccharides could be important for insecticidal pseudomonads to overcome host defense mechanisms. The PhoP-PhoQ system, which is well-known to control lipid A modifications in several pathogenic bacteria, was identified in Pseudomonas chlororaphis PCL1391 and P. protegens CHAO. No evidence was found so far that lipid A modifications contribute to insect pathogenicity in this bacterium. However, the sensor kinase PhoQ was required for full virulence of strain CHAO suggesting that it additionally regulates the expression of virulence factors in this bacterium. The findings of this thesis demonstrate that certain plant-associated pseudomonads are true insect pathogens and give some insights into how these microbes evolved to survive within and eventually kill the insect host. Results however also point out that more in-depth research is needed to know how exactly these fascinating bacteria manage to bypass or overcome host immune responses and to breach physical barriers to invade insects upon oral infection. To achieve this, future studies should not only focus on the bacterial side of the microbe-host interactions but also investigate the infection from a host-oriented view. The knowledge gained about the entomopathogenicity of plant-beneficial pseudomonads gives hope for their future application in agriculture to protect plants not only against plant diseases but also against insect pests.

Étude du rôle spécifique du système à deux composantes PhoBR et du système Pst (phosphate specific transport) dans la virulence d’une souche pathogène aviaire de Escherichia coli (APEC)

Les souches d’Escherichia coli pathogènes aviaires (APEC) sont responsables d’infections respiratoires et de septicémies chez la volaille. Le régulon Pho est contrôlé conjointement par le système à deux composantes PhoBR et par le système de transport spécifique du phosphate (Pst). Afin de déterminer l’implication de PhoBR et du système Pst dans la pathogenèse de la souche APEC O78 χ7122, différentes souche mutantes phoBR et pst ont été testées pour divers traits de virulence in vivo et in vitro. Les mutations menant à l’activation constitutive du régulon Pho rendaient les souches plus sensibles au peroxyde d’hydrogène et au sérum de lapin comparativement à la souche sauvage. De plus, l’expression des fimbriae de type 1 était affectée chez ces souches. L’ensemble des mutants Pho-constitutifs étaient aussi significativement moins virulents que la souche sauvage dans un modèle de coinfection de poulet, incluant les souches avec un système Pst fonctionnel. De plus, l’inactivation du régulateur PhoB chez un mutant Pst restaure la virulence. Par ailleurs, l’inactivation de PhoB n’affecte pas la virulence de la souche χ7122 dans notre modèle. De manière intéressante, le degré d’atténuation des souches mutantes corrèle directement avec le niveau d’activation du régulon Pho. Globalement, les résultats indiquent que l’activation du régulon Pho plutôt que le transport du phosphate via le système Pst joue un rôle majeur dans l’atténuation des APEC.

Der Carrier DcuB als zweiter Sensor des Zweikomponentensystems DcuSR in Escherichia coli

Escherichia coli kann C4-Dicarboxylate und andere Carbonsäuren als Substrate für den aeroben und anaeroben Stoffwechsel nutzen. Die Anwesenheit von C4-Dicarboxylaten im Außenmedium wird über das Zweikomponentensystem DcuSR, bestehend aus der membranständigen Sensorkinase DcuS und dem cytoplasmatischen Responseregulator DcuR, erkannt. Die Bindung von C4-Dicarboxylaten an die periplasmatische Domäne von DcuS führt zu einer Induktion der Zielgene. Hierzu zählen die Gene für den anaeroben Fumarat/Succinat-Antiporter DcuB (dcuB), die anaerobe Fumarase (fumB) und die Fumaratreduktase (frdABCD). Unter aeroben Bedingungen stimuliert DcuSR die Expression des dctA Gens, das für den aeroben C4-Dicarboxylat-Carrier DctA kodiert. Für den Carrier DcuB konnte eine regulatorische Funktion bei der Expression der DcuSR-regulierten Gene gezeigt werden. Die Inaktivierung des dcuB Gens führte bereits ohne Fumarat zu einer maximalen Expression einer dcuB´-´lacZ Reportergenfusion und anderer DcuSR-abhängiger Gene. Diese Stimulierung erfolgte nur in einem dcuS-positiven Hintergrund. DcuB unterscheidet sich damit von den alternativen Carriern DcuA und DcuC, die diesen Effekt nicht zeigten. Mithilfe ungerichteter Mutagenese wurden DcuB-Punktmutanten hergestellt (Thr394Ile und Asp398Asn), die eine Geninduktion verursachten, aber eine intakte Transportfunktion besaßen. Dies zeigt, dass der regulatorische Effekt von DcuB unabhängig von dessen Transportfunktion ist. Durch gerichtete Mutagenese wurde die Funktion einer Punktmutation (Thr394) näher charakterisiert. Es werden zwei Modelle zur Membrantopologie von DcuB und der Lage der Punktmutationen im Protein vorgestellt. Da DcuB seine regulatorische Funktion über eine Interaktion mit DcuS vermitteln könnte, wurden mögliche Wechselwirkungen zwischen DcuB und DcuS als auch DcuR mithilfe von Two-Hybrid-Systemen untersucht. Für biochemische Untersuchungen von DcuB wurde außerdem die Expression des Proteins in vivo und in vitro versucht. Unter aeroben Bedingungen beeinflusst der C4-Dicarboxylat-Carrier DctA die Expression der DcuSR-abhängigen Gene. Eine Mutation des dctA Gens bewirkte eine stärkere Expression einer dctA´-´lacZ Reportergenfusion im Vergleich zum Wildtyp. Diese Expression nahm in einem dcuS-negativen Hintergrund ab, die Succinat-abhängige Induktion blieb jedoch erhalten. Unter anaeroben Bedingungen kann das dctA Gen auch durch Inaktivierung von DcuB induziert werden. Es wird ein Modell vorgestellt, das die Beteiligung beider Carrier an der DcuSR-abhängigen Regulation erklärt.

Oligomerization and protein-protein interactions of the sensory histidine kinase DcuS in Escherichia coli

DcuS is a membrane-integral sensory histidine kinase involved in the DcuSR two-component regulatory system in Escherichia coli by regulating the gene expression of C4-dicarboxylate metabolism in response to external stimuli. How DcuS mediates the signal transduction across the membrane remains little understood. This study focused on the oligomerization and protein-protein interactions of DcuS by using quantitative Fluorescence Resonance Energy Transfer (FRET) spectroscopy. A quantitative FRET analysis for fluorescence spectroscopy has been developed in this study, consisting of three steps: (1) flexible background subtraction to yield background-free spectra, (2) a FRET quantification method to determine FRET efficiency (E) and donor fraction (fD = [donor] / ([donor]+[acceptor])) from the spectra, and (3) a model to determine the degree of oligomerization (interaction stoichiometry) in the protein complexes based on E vs. fD. The accuracy and applicability of this analysis was validated by theoretical simulations and experimental systems. These three steps were integrated into a computer procedure as an automatic quantitative FRET analysis which is easy, fast, and allows high-throughout to quantify FRET accurately and robustly, even in living cells. This method was subsequently applied to investigate oligomerization and protein-protein interactions, in particular in living cells. Cyan (CFP) and yellow fluorescent protein (YFP), two spectral variants of green fluorescent protein, were used as a donor-acceptor pair for in vivo measurements. Based on CFP- and YFP-fusions of non-interacting membrane proteins in the cell membrane, a minor FRET signal (E = 0.06 ± 0.01) can be regarded as an estimate of direct interaction between CFP and YFP moieties of fusion proteins co-localized in the cell membrane (false-positive). To confirm if the FRET occurrence is specific to the interaction of the investigated proteins, their FRET efficiency should be clearly above E = 0.06. The oligomeric state of DcuS was examined both in vivo (CFP/YFP) and in vitro (two different donor-acceptor pairs of organic dyes) by three independent experimental systems. The consistent occurrence of FRET in vitro and in vivo provides the evidence for the homo-dimerization of DcuS as full-length protein for the first time. Moreover, novel interactions (hetero-complexes) between DcuS and its functionally related proteins, citrate-specific sensor kinase CitA and aerobic dicarboxylate transporter DctA respectively, have been identified for the first time by intermolecular FRET in vivo. This analysis can be widely applied as a robust method to determine the interaction stoichiometry of protein complexes for other proteins of interest labeled with adequate fluorophores in vitro or in vivo.

Global GacA-steered control of cyanide and exoprotease production in Pseudomonas fluorescens involves specific ribosome binding sites

The conserved two-component regulatory system GacS/GacA determines the expression of extracellular products and virulence factors in a variety of Gram-negative bacteria. In the biocontrol strain CHA0 of Pseudomonas fluorescens, the response regulator GacA is essential for the synthesis of extracellular protease (AprA) and secondary metabolites including hydrogen cyanide. GacA was found to exert its control on the hydrogen cyanide biosynthetic genes (hcnABC) and on the aprA gene indirectly via a posttranscriptional mechanism. Expression of a translational hcnA′-′lacZ fusion was GacA-dependent whereas a transcriptional hcnA-lacZ fusion was not. A distinct recognition site overlapping with the ribosome binding site appears to be primordial for GacA-steered regulation. GacA-dependence could be conferred to the Escherichia coli lacZ mRNA by a 3-bp substitution in the ribosome binding site. The gene coding for the global translational repressor RsmA of P. fluorescens was cloned. RsmA overexpression mimicked partial loss of GacA function and involved the same recognition site, suggesting that RsmA is a downstream regulatory element of the GacA control cascade. Mutational inactivation of the chromosomal rsmA gene partially suppressed a gacS defect. Thus, a central, GacA-dependent switch from primary to secondary metabolism may operate at the level of translation.

Gene repression by the ferric uptake regulator in Pseudomonas aeruginosa: cycle selection of iron-regulated genes.

The expression of at least 24 distinct genes of Pseudomonas aeruginosa PAO1 is under direct control of the "ferric uptake regulator" (Fur). Novel targets of the Fur protein were isolated in a powerful SELEX (systematic evolution of ligands by exponential enrichment)-like cycle selection consisting of in vitro DNA-Fur interaction, binding to anti-Fur antibody, purification on protein G, and PCR amplification. DNA fragments obtained after at least three exponential enrichment cycles were cloned and subjected to DNA mobility-shift assays and DNase I footprint analyses to verify the specific interaction with the Fur protein in vitro. Iron-dependent expression of the corresponding genes in vivo was monitored by RNase protection analysis. In total, 20 different DNA fragments were identified which represent actual Pseudomonas iron-regulated genes (PIGs). While four PIGs are identical to already known genes (pfeR, pvdS, tonB, and fumC, respectively), 16 PIGs represent previously unknown genes. Homology studies of the putative proteins encoded by the PIGs allowed us to speculate about their possible function. Two PIG products were highly similar to siderophore receptors from various species, and three PIG products were significantly homologous to alternative sigma factors. Furthermore, homologs of the Escherichia coli ORF1-tolQ, nuoA, stringent starvation protein Ssp, and of a two-component regulatory system similar to the Pseudomonas syringae LemA sensor kinase were identified. The putative gene products of seven additional PIGs did not show significant homologies to any known proteins. The PIGs were mapped on the P.aeruginosa chromosome. Their possible role in iron metabolism and virulence of P. aeruginosa is discussed.