Transition metals such as copper, zinc and nickel are required in many enzymatic processes that require redox changes. When transition metal concentration exceeds a certain threshold, their redox and metal binding properties make these elements extremely toxic. Bacteria regulate the cellular concentration of these important, yet toxic, elements using elaborate homeostatic systems. One such mechanism is the chemiosmotic extrusion of copper by the Cus system in the Gram-negative bacterium Escherichia coli. This work studies the regulation of the Cus system in response to copper and silver ions. Copper is an essential cofactor required in many enzymatic processes. But its redox properties can lead to toxicity. Silver is chemically similar to copper, but is not bioactive and its presence in cells can lead to extreme cytotoxicity. Transcription from cusCFBA genes is controlled by the CusR/CusS TCS in response to elevated levels of copper or silver in the periplasmic space of E. coli. Extracellular signals are transduced into the cell through phosphotransfer reactions between the prototypical histidine kinase CusS and the response regulator CusR. Copper sensing by the periplasmic domain of CusS is proposed to initiate signal transduction in the Cus system. Despite the frequency with which bacteria employ histidine kinases to sense their environment, signal recognition and incorporation by the protein is not well understood. The goal of this research is to investigate the role of CusS in regulating metal homeostasis in E. coli and characterize the periplasmic domain of the protein to determine its metal binding properties. The experiments described in this work reveal that the CusS is essential for copper and silver resistance and regulates expression from the cusCFBA promoter region. Signal recognition occurs by direct metal binding by the periplasmic domain of CusS. Metal binding causes a change in the secondary structure of the domain and its tendency to dimerize is enhanced under these conditions. The possibility of signal attenuation by interaction with the metallochaperone CusF is also discussed. These data help construct a model for signal transduction in the Cus system and help characterize, for the first time, a metal-responsive sensor histidine kinase in E. coli.

Two component systems (TCSs) are regulatory pathways in bacteria and lower eukaryotes that integrate multiple stimuli and bring about appropriate responses to promote adaptation of the bacteria to their niches. They are commonly insulated from cross-talk and form discrete regulatory systems where the sensor histidine kinase (SK) and the response regulator (RR) share high fidelity for one another. The GacS network controls the switch between acute and chronic virulence of P. aeruginosa. The network is unusual in having a 'core' SK, GacS, which is modulated directly by one other SK, RetS. Here the complex relationship between GacS and RetS is dissected to reveal three distinct mechanisms by which they interact. Two of these mechanisms involve the dephosphorylation of GacS-P by RetS and it is these mechanisms that are important in vivo for the regulation of biofilm formation, rsmY and rsmZ expression, swarming, and virulence in both Galleria mellonella and an acute model of infection in mice. This study reveals an unprecedented level of complexity in the ability of RetS to interact with GacS and suggests that RetS has a number of mechanisms by which it can downregulate the GacS network output. Furthermore, the interactions of additional SKs that have previously been linked to the GacS network were investigated. Here I demonstrate that many of these kinases can interact with one another but that RetS remained the only kinase tested that could directly interact with GacS. The interactions observed between kinases could be either stimulatory, having a synergistic impact on phosphorylation levels, or inhibitory. I also show that kinase-kinase interactions allow for the regulation of phosphorylation of downstream proteins. Finally, we searched for additional SKs that may be able to interact with the GacS network. Here I identify three new kinases, which show differing interactions with the kinases of the GacS network. The discovery of additional SKs in the GacS network indicates that the network is likely to respond to a far greater number of different signals than previously realised as it decides between acute and chronic virulence.

Phosphate is an essential compound for life. Escherichia coli employs a signal transduction pathway that controls the expression of genes that are required for the high-affinity acquisition of phosphate and the utilization of alternate sources of phosphorous. These genes are only expressed when environmental phosphate is limiting. The seven genes for this signaling pathway encode the two-component regulatory proteins PhoB and PhoR, as well as the high-affinity phosphate transporter PstSCAB and an auxiliary protein called PhoU. As the sensor kinase PhoR has no periplasmic sensory domain, the mechanism by which these cells sense environmental phosphate is not known. This paper explores the hypothesis that it is the alternating conformations of the PstSCAB transporter which are formed as part of the normal phosphate transport cycle that signal phosphate sufficiency or phosphate limitation. We tested two variants of PstB that are predicted to lock the protein in either of two conformations for their signaling output. We observed that the pstBQ160K mutant, predicted to reside in an inward facing, open conformation signaled phosphate sufficiency whereas the pstBE179Q mutant, predicted to reside in an outward facing, closed conformation signaled phosphate starvation. Neither mutant showed phosphate transport.

[Truncated abstract] Protein phosphorylation, one of the most important forms of post-translational modification, has been demonstrated to play crucial roles in regulation of cell function. Phosphorylation of protein serine, threonine and tyrosine residues has been the most thoroughly investigated, taking advantage of the acid-stable character of these phosphohydroxyamino acids. Whereas, the cellular occurrence of acid-labile phosphoamino acids, such as phosphohistidine, phosphoarginine and phospholysine was often underestimated due to the acid treatments employed by most of the traditional phosphoamino acid analysis methods. The biological roles of histidine kinases (HKs) in prokaryotes are well understood in contrast to those of HKs in eukaryotes, especially in mammalian cells. However, the evidence has shown that phosphohistidine comprised 6% of phosphoamino acids of the basic nuclear proteins in eukaryotes (Matthews, 1995) and there was more phosphohistidine than phosphoserine in rat liver mitochondria (Bieber and Boyer, 1966). More significantly, phosphohistidine was revealed to be the major phosphoamino acid in phosphorylated histone H4 in regenerating liver in vivo (Chen et al., 1974) and the Walker-256 carcinosarcoma cells in vitro (Smith et al., 1974). Recently, the histone H4 histidine kinase (HHK) activity of human hepatocellular carcinoma (HCC) tumour tissue was measured to be 400 times higher than the normal liver tissue surrounding the tumour. HepG2 cells (HCC cell line) and PIL-2 cells (a p53 knockout mouse tumorigenic liver progenitor cell line) also displayed high HHK activity (Tan et al., 2004). The above observations suggested that HKs and HHKs are playing important roles in both prokaryotes and eukaryotes, including mammals. One major obstacle in the study of HHK study has been the lack of knowledge of the amino acid sequence of an HHK. Attempts at purifying and identifying the HHK from yeast led to the partial purification of a yeast HHK protein(s) at 32kDa (Huang et al., 1991). However, the amino acid sequence of the HHK has not yet been established. ... The success of the separation was demonstrated by the MALDI-TOF-MS and/or ESI-MS spectra of the RP-HPLC fractions. These achievements suggested that it is possible to detect phosphohistidyl histone H4 in vivo using MS under experimental conditions where phosphohistidine is relatively stable. The study in this thesis represents the progression of HHK research in various aspects, including the yeast HHK purification and identification, mammalian HHK partial purification and the methodological developments in detecting histone H4 histidine phosphorylation using MS. Furthermore, new information regarding the physical characteristics of yeast HHKs and its potential role in cellular biology have been documented. It is anticipated that knowledge generated in these studies will contribute to the insight and the understanding of the biological significance of HHK in yeast and mammalian cells.

EN This Ph.D. thesis focuses on the heme containing gas sensor proteins. These proteins are predominantly present in bacteria, in which play an important role in processes like, sporulation, antibiotic resistance and so on. Heme containing sensor proteins composed of two domains. First one is a globin domain, which contains the heme molecule. Interaction of heme with gas molecule acts as a signal for the activation/inactivation of the second functional domain. Part of this thesis is formed by a review, which summarized the current knowledge about heme containing sensor proteins. In the next part of this thesis we focused on three representatives from the group of oxygen sensor proteins - histidine kinase AfGcHK, diguanylate cyclase YddV and phosphodiesterase EcDOS. The main aim of this thesis was to solve the mechanism of interdomain/intraprotein signal transduction in two oxygen sensor proteins with globin fold of their sensor domain (AfGcHK, YddV). For this purpose, we used the kinetic analysis of their functional domain activity and the methods of structural biology. We also studied the mechanism of interprotein signal transduction in AfGcHK and its cognate partner RR protein. It was also tested, how the presence of sodium disulfide affects the functional properties of oxygen sensor proteins...

Signal transduction networks enable organisms to respond to environmental stimuli. Bacteria utilize two-component systems (TCSs) and phosphorelays as their primary means of signal transduction. Histidine kinase (HK) and response regulator (RR) proteins comprise these TCSs and phosphorelays. Previously, signal transduction within TCSs and phosphorelays was thought to only occur through a linear series of phosphotransfers between HKs and RRs. Recently multikinase networks have been shown to be involved in TCS and phosphorelay signal transmission. A multikinase network that includes the HKs RetS and GacS controls the switch between the motile invasive lifestyle and the sessile biofilm lifestyle of the opportunistic human pathogen Pseudomonas aeruginosa. GacS promotes the sessile biofilm lifestyle, while RetS promotes the motile invasive lifestyle via the inhibition of GacS. This inhibition occurs through three distinct mechanisms. Two of the mechanisms are dephosphorylating mechanisms and the third mechanism is a direct interaction between RetS and GacS which results in the inhibition of GacS autophosphorylation. This study examines the direct binding interaction between RetS and GacS using structural biology. We observed a heterodimeric RetS-GacS complex in which the canonical homodimerization interface was replaced with a heterodimeric interface. Heterodimerization between bacterial HKs is currently a novel observation, but it is likely that other HKs heterodimerize. The RetS-GacS direct interaction can serve as a model for HK-HK binding in multikinase networks. / Doctor of Philosophy / The way in which bacteria assess and respond to their environment is of great interest to microbiologists. Bacteria transmit environmental signals via protein interactions. Some of these interactions involve the transfer of phosphate groups, and some involve a direct binding interaction between proteins. We are investigating a direct binding interaction between two proteins, RetS and GacS. These proteins control whether Pseudomonas aeruginosa, an opportunistic pathogen of humans, causes an acute infection, which is characterized by motility and invasiveness, or a chronic infection, which is characterized by a sessile biofilm lifestyle, in a human host. Through the use of structural biology techniques we have visualized the three-dimensional structure of the complex between RetS and GacS. This complex has provided insight into the role of the RetS-GacS interaction in controlling the infection state of P. aeruginosa.

La pourriture grise, causée par le champignon Botrytis cinerea, est l'une des principales maladies aériennes fongiques sur diverses cultures d’importance agronomique. La diversité génétique de B. cinerea est très forte et la capacité rapide d’adaptation de ce champignon à une pression sélective est également avérée. Ce champignon est ainsi capable de développer des résistances à une grande variété de composés fongicides de synthèse ou d'origine naturelle. Des méthodes alternatives de lutte ont de ce fait été développées ces dernières années : divers agents de lutte biologique (ALB) présentant différents modes d’actions ont été identifiés et pour certains d’entre eux commercialisés pour contrôler B. cinerea. Cependant la durabilité de la lutte biologique est un domaine encore très peu étudié. La perte d'efficacité d'un ALB pourrait résulter de la préexistence d’isolats moins sensibles de pathogènes dans les populations naturelles et/ou de la capacité de l’agent pathogène à produire, sous une pression de sélection continue exercée par l’ALB, des mutants ayant une sensibilité réduite. L'objectif global de la présente étude est d'évaluer le risque potentiel de perte d'efficacité de la lutte biologique vis-à-vis de B. cinerea. Dans cette étude, les efforts ont été concentrés sur la pyrrolnitrine, un antibiotique produit par divers ALBs, dont certains sont efficaces contre B. cinerea. Les objectifs spécifiques de l'étude étaient (i) d’évaluer la diversité de la sensibilité à la pyrrolnitrine au sein de la population naturelle de B. cinerea, (ii) d'estimer le risque de perte d'efficacité des ALBs produisant la pyrrolnitrine due à la pression de sélection exercée par la pyrrolnitrine et (iii) d'étudier le mécanisme de résistance à la pyrrolnitrine chez B. cinerea. Parmi 204 isolats de B. cinerea, une gamme importante de sensibilité à la pyrrolnitrine a été observée, avec un facteur de résistance de 8,4 entre l’isolat le plus sensible et l'isolat le moins sensible. La production de 20 générations successives pour 4 isolats de B. cinerea, sur des doses croissantes de pyrrolnitrine, a abouti au développement de mutants avec des niveaux élevés de résistance à l'antibiotique, et à une réduction in vitro de la sensibilité à la bactérie productrice de pyrrolnitrine Pseudomonas chlororaphis PhZ24. La comparaison entre les mutants résistants à la pyrrolnitrine et leurs parents sensibles pour la croissance mycélienne, la sporulation et l'agressivité sur plantes a révélé que la résistance à la pyrrolnitrine est associée à un fort coût adaptatif. Des observations cytohistologiques sur tomates ont confirmé que l’isolat sensible à la pyrrolnitrine attaque le pétiole rapidement et envahit la tige, alors que le mutant résistant à la pyrrolnitrine ne s'étend pas au-delà du pétiole. De plus, ce dernier mutant forme un mycélium anormal et des cellules ressemblant à des chlamydospores. Les résultats ont d'autre part révélé que les mutants de B. cinerea résistants à la pyrrolnitrine sont résistants au fongicide iprodione, suggérant ainsi qu'une pression exercée par la pyrrolnitrine sur le champignon conduit à une résistance au fongicide. Réciproquement, la production de générations successives sur iprodione conduit à une résistance à l'antibiotique. Afin d'étudier les déterminants moléculaires de la résistance de B. cinerea à la pyrrolnitrine, le gène histidine kinase Bos1, impliqué entre autres dans la résistance aux fongicides chez B. cinerea a été séquencé chez les souches sensibles et les mutants résistants. La comparaison des séquences a mis en évidence des mutations ponctuelles différentes chez les mutants de B. cinerea obtenus sur la pyrrolnitrine et ceux obtenus sur l'iprodione. De plus, les résistances à la pyrrolnitrine et à l'iprodione ne sont pas systématiquement associées à une mutation ponctuelle dans le gène Bos1. Enfin, aucune modification n'a été détectée dans la taille des allèles de neuf locus microsatellites quelle que soit la pression sélective exercée et quelle que soit le phénotype du mutant produit. Cette étude montre qu'un champignon pathogène des plantes est capable de développer progressivement une moindre sensibilité à un agent de lutte biologique mais que cette moindre sensibilité est associée à une forte perte de fitness / Gray mould, caused by Botrytis cinerea, is a severe disease on a wide range of crops. Disease control generally relies on chemicals, although biological control strategies have been intensively studied over the last decades. This pathogen can withstand a wide variety of fungitoxic compounds including fungicides and natural molecules. This capacity to adapt to different stress might, potentially, compromise the durability of biological control methods. The global purpose of that work was to estimate the potential of B. cinerea to overcome the efficacy of biological control agents. Knowledge on the potential development of resistance to biological control agents can help to devise or improve resistance management strategies. In this work, efforts have been focused on the antibiotic pyrrolnitrin produced by various bacteria described as potential biological control agents against B. cinerea. The specific objectives of the study were (i) to evaluate the diversity in susceptibility to pyrrolnitrin among natural population of B. cinerea, (ii) to estimate the risk of loss of efficacy of pyrrolnitrinproducing biological control agent due to selection pressure exerted by pyrrolnitrin and (iii) to study the mechanism of resistance to pyrrolnitrin in B. cinerea. An important range of sensitivity to pyrrolnitrin with an 8.4-fold difference in EC50 values between the most sensitive and the least sensitive isolates was observed within the 204 isolates tested. The production of 20 generations, for 4 isolates of B. cinerea, on increasing doses of pyrrolnitrin, resulted in the development of mutants of B. cinerea with high levels of resistance to the antibiotic and a reduced sensitivity in vitro to the pyrrolnitrin-producing Pseudomonas chlororaphis PhZ24. Comparison of the pyrrolnitrin-resistant mutants and their sensitive parent isolates for mycelial growth, sporulation and aggressiveness on plant tissues revealed that the high level of resistance to pyrrolnitrin has resulted in a high fitness cost. Additional cytohistological investigations revealed that while the sensitive isolate spread throughout the petiole and rapidly invaded the stem via the abscission zone, the pyrrolnitrinresistant mutant failed to extend beyond petiole to invade the stem. Moreover, the pyrrolnitrin-resistant mutant formed abnormal mycelium and chlamydospore-like cells. The comparison of resistance to pyrrolnitrin and to the iprodione fungicide in B. cinerea revealed that fungicide pressure exerted on the fungus is able to build-up resistance to pyrrolnitrin. Comparison of sequences of the osmosensing class III histidine kinase encoding gene bos1 revealed different mutations in pyrrolnitrin- and iprodione-resistant mutants. However, resistance to pyrrolnitrin and to iprodione was not systematically associated with a point mutation in the Bos1 gene. Finally, no changes were observed in the allele size at nine microsatellite loci whatever the four selective pressure endured by the fungus despite their phenotypic changes. This study provides evidence that a fungal plant pathogen is able to gradually build-up resistance to an antibiotic produced by a biocontrol agent

Hydrogen sulfide mediates various physiological functions and along with carbon monoxide and nitric oxide it is an important gaseous signaling molecule. Cellular targets for H2S are proteins, enzymes, transcriptional factors or ion channels. In many cases, the effect of H2S on the regulatory protein is mediated by modifications of its cystein residues. In hemeproteins, the regulation of catalytic activity is induced by formation of the Fe(III)-SH complex or by reduction of the heme iron with subsequent formation of Fe(II)-O2 complex. The effect of Na2S on model sensor heme-containing proteins is presented in this thesis. Protein, isolated from bacterium Anaeromyxobacter sp. strain FW109-5, containing a globine coupled sensor domain and a histidine kinase domain is one of the studied proteins, the second one is protein isolated from bacterium Escherichie coli, containing a globine coupled sensor domain and a diguanylate cyclase domain. The effect of Na2S on both model proteins and their mutants was studied by UV-Vis spectral analysis. Spectra of YddV-HD Y43A were very unique, because thery confirmed formation of a homogenous complex Fe(III)-SH in this protein, whereas only mixtures of varous heme complexes were detected in other tested proteins. Additionally the effect of Na2S on functional domain...

M. tuberculosis (Mtb) senses and responds to changes in its environment primar-ily through two-component signalling systems (TCSs). Each TCS contains a trans-membrane histidine kinase (HK ) protein and a cytoplasmic response regulator (RR) protein. HK detects a stimulus and gets phosphorylated. It then binds and transfers the phosphoryl group to the RR of the same TCS. Activated RR then triggers gene ex-pression, including upregulation of the HK and RR involved, eliciting responses that are essential for the bacterium to adapt. Though di erent TCSs detect distinct stimuli, the binding regions of the HK s and RRs share signi cant similarity. This raises the possibil-ity of crosstalk, where HK s dissipate signals to RRs that do not belong to the same TCS. Studies have argued that such dissipation of signals impairs the fitness of the organism, as it decreases the output levels as well as triggers unwanted responses. In contrast, a recent experimental study has discovered that TCSs of Mtb share extensive crosstalk, violating the widely accepted specificity paradigm. In this study, we have attempted to unravel the evolutionary underpinnings of this extensive crosstalk observed in Mtb. We hypothesised that such crosstalk may be advantageous in programmed environments, where there are well-defined sequences of stimuli. In such situations, crosstalk can up-regulate HK s and RRs of non-cognate TCSs. This up-regulation primes the latter TCSs for upcoming signals, increasing their sensitivity. We constructed a mechanistic model of the functioning of TCSs and a fitness variable to qualitatively measure the response of a TCS to a signal, to test the hypothesis. We performed population genetics simulations of the evolution of phenotypes of different crosstalk patterns. We found that in a random environment, the phenotype without any crosstalk is selected over time, which is in agreement with prevalent arguments in favour of specificity of TCSs. But when the environment is programmed, the phenotype with a crosstalk pattern mirroring the pattern of stimuli dominates the population. Finally, we found evidence for the evolutionary preference to preserve crosstalk in gene sequences of HK s and RRs encoded in Mtb. We found that the binding domains of HK s and RRs, which were predicted to share crosstalk, are under greater pressure to be similar than those domains which do not crosstalk. Our study thus provides a plausible explanation of the unexpected presence of crosstalk in Mtb. Since these cross-interactions aid the pathogen to adapt in the host, inhibitors of such interactions are likely to have therapeutic potential.

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