Lactobacillus casei es una bacteria del ácido láctico de interés aplicado por su uso como cultivo iniciador en la industria alimentaria y por el carácter probiótico de algunas cepas. Como probiótico, L. casei debe sobrevivir a las condiciones de producción industrial y a su paso por el tracto gastrointestinal manteniendo sus propiedades. Para ello, L. casei posee rutas de reconocimiento de señales ambientales específicas y convierten esta información en una respuesta fisiológica adecuada. Un mecanismo comúnmente encontrado en bacterias para la transducción de señal son los sistemas de dos componentes (Two Components Systems, TCS). Los TCS están formados por una proteína sensora con actividad histidina quinasa (HK) y un regulador de respuesta (RR). La detección de un estímulo específico por la proteína sensora induce su autofosforilación y la transferencia del fosfato al regulador de respuesta, produciéndose la activación del mismo. Los sistemas de dos componentes median la respuesta adaptativa a una amplia gama de estímulos ambientales en bacterias. En el laboratorio de Bacterias Lácticas del Instituto de Agroquímica y Tecnología de Alimentos se ha iniciado el estudio de los TCS codificados por L. casei BL23 dentro del cual se incluye el presente proyecto de tesis doctoral. / Revilla Guarinos, A. (2014). Characterization of Two Component Systems of Lactobacillus casei BL23 and their involvement in stress response [Tesis doctoral no publicada]. Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/43589 / TESIS

Streptococci resident in the oral cavity have been linked to infective endocarditis (IE). While viridans streptococci are commonly studied and associated with IE, less research has been focused on Streptococcus pneumoniae. Two-component systems (TCSs), consisting of a histidine kinase (HK) protein and response regulator (RR) protein, are bacterial signaling systems that may mediate S. pneumoniae TIGR4 strain virulence in IE. To test this hypothesis, TCS RR mutants of TIGR4 were examined in vivo through use of rabbit models. There were 14 RR proteins identified and 13 RR mutants synthesized because SP_1227 was found to be essential. The requirement of the 13 RRs for S. pneumoniae growth in IE models was assessed by quantifying mutants after overnight inoculation in IE infected rabbits through use of real time PCR (qPCR), colony enumeration on antibiotic selection plates, and competitive index assays. Real time PCR pinpointed several candidate virulence factors. Candidate RR SP_0798 was selected to be further examined. In the in vivo model, mutant SP_0798 grew significantly less than our control mutant SP_1678, which encodes a hypothetical protein and grew at a comparable rate to wild-type TIGR4 strains. Literature and databases identified SP_0798 as the ciaR gene, which has roles in regulating many diverse cellular functions. Our data suggests that RR SP_0798 is a virulence factor of S. pneumoniae TIGR4 strain in IE. This research may place more emphasis on virulence factors and lead to novel methods to combat pneumococcal endocarditis.

Two-component systems (TCS) are the predominant signal transduction pathways in prokaryotes, being present also in eukaryotic organisms, such as algae, fungi and yeast, and higher plants. TCSs play an important role in environmental signal perception and response, essentially implementing adaptation to the surrounding environment. Histidine Kinase 2 (Hik2) in cyanobacteria is a typical sensor histidine kinase, one component of a TCS, and has been identified to be a homologue protein of Arabidopsis Chloroplast Sensor Kinase (CSK). Previous research has elucidated Hik2 to regulate photosynthetic gene transcription with two response regulators, Rre1 and RppA via phosphorylation. A typical histidine kinase contains a variable sensor domain and a conserved kinase domain. It usually functions as a homodimer. This thesis describes the structural characterisation of Hik2, probing particularly its discovered oligomeric states. Results obtained from size exclusion chromatography, native-PAGE, chemical cross-linking analyses and mass spectrometry, amongst others, have shown a variety of Hik2 structural populations exist, further validated by negative stain transmission electron microscopy coupled to single particle analysis. Hik2 protein exists predominantly as a hexamer in low salt conditions, and adding NaCl dissociates hexamers into tetramers, critical for the autophosphorylation activity of Hik2. Thus, a model is proposed for the constitution change of Hik2 oligomers when salt concentration differs. In addition, the sensor domain is typically responsible for detecting environmental input, however, it is not yet clear how Hik2 and CSK sense signals. In this thesis, the structures of Hik2 and CSK sensor domains were analysed and discussed, to aid our understanding of their mechanism of signal perception and transduction.

Bacillus cereus sensu stricto (ss) est un pathogène alimentaire majeur représentant la 2e cause de toxiinfectionalimentaire en France en 2012. Cette espèce fait partie du groupe Bacillus cereus sensu lato (sl)constitué d’espèces ubiquitaires génétiquement très proches et incluant d’autres pathogènes comme B.anthracis, B. thuringensis et B. cytotoxicus. Les souches de B. cereus sl sont d’autre part réparties en septgroupes phylogénétiques présentant des gammes de température de croissance variées et caractérisés partrois thermotypes principaux: thermotolérants, mésophiles, psychrotolérants. L’adaptation au froid dessouches B. cereus ss est un mécanisme clé car il conditionne sa capacité à se développer dans les alimentsréfrigéré pour atteindre des doses qui peuvent être dangereuse pour les consommateurs. Le but de cetteétude a été d’étudier les mécanismes moléculaires impliqués dans l’adaptation au froid de la diversité desouches représentant B. cereus sl.Nous avons mis en évidence que les gènes codant pour le système à deux composants CasK/R sontsurexprimés à basse température. CasK/R s’est révélé être un système générique d’adaptation de B. cereussl au froid, car son rôle a été mis en évidence lors de l’étude de quatre souches de thermotypes différents etleurs mutants isogéniques ΔcasK/R respectifs. Une étude transcriptomique réalisée sur une souche ATCC14579 et son mutant ΔcasK/R a révélé que seize des gènes différentiellement exprimés en début de phaseexponentielle et en phase stationnaire, à basse température, codent pour des protéines impliquées dans lemétabolisme des acides gras. Nous avons mis en évidence le rôle de CasK /R dans la modification de lacomposition en acides gras membranaires via une augmentation de la proportion en acides gras insaturéslors de la croissance de B. cereus au froid. Par ailleurs, le gène codant pour la désaturase DesA,principalement responsable des insaturations des acides gras à basse température est régulée positivementpar CasK/R au froid.Nous avons également démontré que les gènes casK/R sont organisés en opéron avec un gène codant pourun régulateur RpiR-like. De manière originale, cet opéron est négativement régulé par CasK/R à bassetempérature en phase stationnaire. Le promoteur individuel du rpiR est réprimé à basse température maisaussi à température optimale de croissance, ce qui suggère un rôle de CasK/R, même à températureoptimale / Bacillus cereus sensu stricto (ss) is a major foodborne pathogen representing the second cause of foodpoisoning in France in 2012. This species belongs to Bacillus cereus sensu lato (sl) consisting of ubiquitousspecies genetically close-related and including other pathogens such as B. anthracis, B. thuringiensis and B.cytotoxicus. The strains of B. cereus sl are divided into seven phylogenetic groups with various growthtemperature ranges and characterized by three main thermotypes: thermotolerant, mesophilic,psychrotolerant. The B. cereus ss cold adaptation is a key mechanism because it determines B. cereusability to grow in refrigerated foods and achieve doses that can be dangerous to consumers. The aim of thisstudy was to study the molecular mechanisms involved in the cold adaptation of strains representing B.cereus sl diversity.We demonstrated that the genes encoding the two component system CasK/R are overexpressed at lowtemperature. CasK/R was found to be a generic mechanism for B. cereus sl cold adaptation as its role washighlighted in the study of four strains with different thermotypes and their respective isogenic mutantsΔcasK/R. A transcriptomic study on a B. cereus ATCC 14579 strain and its ΔcasK/R mutant strain revealedthat sixteen of the genes differentially expressed in both early log phase and stationary phase at lowtemperature encode proteins involved in the fatty acids metabolism. We showed the role of CasK/R in themodification of the membrane fatty acid composition via an increase of the proportion of unsaturated fattyacids during growth of B. cereus at low temperature. Furthermore, the gene encoding the desaturase DesA,mainly responsible of the fatty acids unsaturation at a low temperature is upregulated by CasK/R at lowtemperature.We also demonstrated that casK/R genes were organized in operon with a gene encoding a RpiR-likeregulator. Interstingly,, this operon is negatively regulated by CasK/R at low temperature in the stationaryphase. The individual rpiR promoter is repressed by CasK/R at low temperature but also optimal growthtemperature, suggesting also a role for CasK/R at optimal temperature

Bacteria use Two Component Systems (TCS) to sense and respond to changes in their external environment. TCS are used to navigate to nutrients or away from toxins (chemotaxis) and to adapt to changes in osmolarity (osomosensing). TCS are composed of a histidine protein kinase (HPK) which trans-autophosphorylates in response to environmental change, transferring the phosphoryl group to a cognate response regulator (RR). Phosphorylated RRs modulate an output response such as protein-protein interaction for chemotaxis, and transcription for osmosensing. RRs are composed of a conserved amino terminal REC domain, and where present a variable effector domain. CheY, the chemotaxis RR, contains only a REC domain, whilst OmpR, the osmosensing RR, also contains a DNA binding effector domain. Recently, TCS have been used in synthetic biology applications due to their modularity and conserved signalling mechanism. This thesis aimed to investigate whether it was possible to design a synthetic TCS composed of fused chemotaxis and osmosensing components. Synthetic RRs were designed, fusing the highly conserved REC domains of CheY and OmpR upstream of the OmpR effector domain. REC domains were fused across the α₄-β₅-α₅ region, a region which transmits REC domain phosphorylation into effector domain activation. Synthetic RRs were designed to undergo phosphotransfer to their fused REC domains from the chemotaxis HPK, CheA, activate the attached OmpR effector domain and bind promoter DNA. Four chimeric RRs were created, although only three were structurally viable; F2, F3 and F4. Each fusion bound CheA, and F3 and F4 bound CheA with a significantly higher affinity than CheY. The chimeric RRs could all be phosphorylated byCheA-P; F4 and F3 were phosphorylated to wild-type levels. DNA binding affinitywas investigated with fluorescence anisotropy, hosphorylated and unphosphorylated F3 could not bind promoter DNA. F2 bound promoter DNA regardless of phosphorylation state. These data indicate that phosphorylation of the F2 REC domain does not lead to activation of the effector domain. F2 is likely to be constitutively active suggesting a previously unknown role for OmpR α₅ as a mediator of effector domain activation. Furthermore, using a simple fusion approach to design RRs is not a viable method to create a synthetic TCS with a controllable output.

Alginate overproduction by Pseudomonas aeruginosa, which causes a mucoid phenotype, is a major virulence factor associated with chronic pulmonary infections in cystic fibrosis patients. Expression of the algD operon for alginate biosynthesis requires three major regulators in association with the ECF sigma factor, σ22, in mucoid strains that are typically defective in anti-sigma factor, MucA. One such algD regulator is AlgB, a member of the NtrC family of two-component systems, which typically utilize σ54. However, neither σ54 nor the cognate sensor kinase (KinB) of AlgB are required for algD expression in such mucoid strains. I hypothesized that KinB-phosphorylated AlgB must play some role in gene regulation, and so I sought to construct a constitutively active AlgB that simulated kinase-phosphorylation. I took a predictive approach and genetically introduced substitutions in AlgB that had been shown to activate DctD, a close homologue of AlgB in Rhizobium (52). When one such substitution, AlgBE125K, was transferred to a nonmucoid P. aeruginosa PAO ΔalgB-kinB (JK159) strain, alginate overproduction was observed. Interestingly, introduction of an algT mutation to remove σ22 did not block alginate production induced by AlgBE125K; although, it did stimulate the production of alginate in the presence of AlgBwt in trans to similar levels induced by the constitutive mutant. In contrast, introduction of an rpoN mutation showed that alginate production mediated by AlgBwt and AlgBE125K was σ54 dependent. The increase in expression of alginate by AlgBwt in the presence of σ54 and the absence of σ22 suggested a competition between the sigma factors for binding to PalgD. Biochemical assays were conducted to assess the constitutive property of AlgBE125K. For the ATPase assay, an equivalent amount of ATP hydrolysis was observed between the mutant and the wild type AlgB proteins. Slight differences seen for the EMSA data suggested possible higher order complex formation for AlgBE125K compared to AlgBwt. Collectively, these results suggested that in wild-type (MucA+) P. aeruginosa, expression of the algD operon is dependent on the phosphorylation of AlgB by KinB in a typical two-component fashion that is triggered by some as yet unknown environmental stimulus.

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.

abstract: Mycobacterium tuberculosis (Mtb), the causative agent of tuberculosis, is the 10th leading cause of death, worldwide. The prevalence of drug-resistant clinical isolates and the paucity of newly-approved antituberculosis drugs impedes the successful eradication of Mtb. Bacteria commonly use two-component systems (TCS) to sense their environment and genetically modulate adaptive responses. The prrAB TCS is essential in Mtb, thus representing an auspicious drug target; however, the inability to generate an Mtb ΔprrAB mutant complicates investigating how this TCS contributes to pathogenesis. Mycobacterium smegmatis, a commonly used M. tuberculosis genetic surrogate was used here. This work shows that prrAB is not essential in M. smegmatis. During ammonium stress, the ΔprrAB mutant excessively accumulates triacylglycerol lipids, a phenotype associated with M. tuberculosis dormancy and chronic infection. Additionally, triacylglycerol biosynthetic genes were induced in the ΔprrAB mutant relative to the wild-type and complementation strains during ammonium stress. Next, RNA-seq was used to define the M. smegmatis PrrAB regulon. PrrAB regulates genes participating in respiration, metabolism, redox balance, and oxidative phosphorylation. The M. smegmatis ΔprrAB mutant is compromised for growth under hypoxia, is hypersensitive to cyanide, and fails to induce high-affinity respiratory genes during hypoxia. Furthermore, PrrAB positively regulates the hypoxia-responsive dosR TCS response regulator, potentially explaining the hypoxia-mediated growth defects in the ΔprrAB mutant. Despite inducing genes encoding the F1F0 ATP synthase, the ΔprrAB mutant accumulates significantly less ATP during aerobic, exponential growth compared to the wild-type and complementation strains. Finally, the M. smegmatis ΔprrAB mutant exhibited growth impairment in media containing gluconeogenic carbon sources. M. tuberculosis mutants unable to utilize these substrates fail to establish chronic infection, suggesting that PrrAB may regulate Mtb central carbon metabolism in response to chronic infection. In conclusion, 1) prrAB is not universally essential in mycobacteria; 2) M. smegmatis PrrAB regulates genetic responsiveness to nutrient and oxygen stress; and 3) PrrAB may provide feed-forward control of the DosRS TCS and dormancy phenotypes. The data generated in these studies provide insight into the mycobacterial PrrAB TCS transcriptional regulon, PrrAB essentiality in Mtb, and how PrrAB may mediate stresses encountered by Mtb during the transition to chronic infection. / Dissertation/Thesis / Doctoral Dissertation Microbiology 2019

Staphylococcus aureus is defined by its ability to agglutinate during exposure to human blood plasma. Although agglutination has long correlated with disease severity, the function of agglutination during infection remains unclear. Increasing evidence suggests the mechanisms of agglutination are highly complex and poorly understood. The goal of this dissertation was to characterize the mechanisms required for S. aureus agglutination in vitro and determine how these factors contribute to pathogenesis. Chapter II focuses on the development of two in vitro agglutination assays, which allow the process to be measured quantitatively. Through these assays, we confirmed the major factors contributing to agglutination are human fibrinogen and the bacterial surface protein, ClfA. Productive interactions between these two factors are required for agglutination to proceed. Surprisingly, we also identified a novel regulatory system that significantly contributed to agglutination. Inactivation of the ArlRS two-component system (TCS) prevents agglutination in both of the developed assays. Studies in Chapter III focused on characterizing the mechanism by which ArlRS inhibits agglutination. To examine regulation, quantitative PCR identified the major output of the ArlRS system as the gene ebh. Surprisingly, transcript levels of known extracellular matrix (ECM) binding proteins did not change. Characterization of ebh indicated that overexpression in an arlRS mutant is the major factor responsible for preventing agglutination. Deletion of ebh restores the ability of the arlRS mutant to agglutinate in both gravity and flow-based agglutination assays. Fluorescence microscopy of clumps indicates wildtype cells bind and incorporate fluorescently labeled human fibrinogen (Fg) displaying co-localization with the clumps. Surprisingly, arlRS mutants also bound human Fg, but these interactions were not productive for clumping, suggesting successful agglutination is more complex than binding ECM proteins. These studies indicate that ArlRS regulates agglutination through a unique mechanism that depends on the surface protein Ebh. Studies in Chapter IV were performed to determine the role ArlRS played in pathogenesis. A rabbit model of infective endocarditis and sepsis was employed to assess ArlRS virulence because this model has been shown to require agglutination for disease progression. Mutants in arlRS displayed reduced virulence in the rabbit model of infective endocarditis, which correlated with the mutant's inability to form a vegetation of the heart valve. These studies provide further insight into the importance of S. aureus agglutination during infection and define a mechanism of regulation through a novel surface protein.

Biofilms are a crucial adaptation for bacterial survival against stresses from external environments. Biofilms are adherent colonies of sessile bacteria embedded within a self-produced matrix. Bacterial control over formation, maintenance, and response to external stresses are strictly regulated. However, complexities of intracellular signaling for biofilm regulation are still not fully understood. In this thesis, I report on two distinct regulatory systems important for biofilm formation in the opportunistic pathogen Pseudomonas aeruginosa. The first regulatory system I report on is the two-component system TctD-TctE. This system is involved in regulating the uptake of tricarboxylic acids such as citric acid and is involved in biofilm-specific susceptibility to aminoglycoside antibiotics. Here I describe work I performed characterizing the involvement of TctD-TctE in biofilm development when citric acid is present as a carbon source in nutrient media. In further characterizing a previously observed aminoglycoside susceptibility, I found that a strain with a deletion of TctD-TctE (ΔtctED) has a heightened accumulation of tobramycin in its biofilms when grown in the presence of citric acid. In ΔtctED, I determined that there was an inhibition of overall cell growth when citric acid was present in nutrient media. Additionally, in the presence of citric acid, ΔtctED displayed high levels of biofilm formation. This contrasted with normal biofilm development observed in the PA14 wild type strain where biofilm mass was reduced in the presence citric acid. The second project of this thesis reports on a novel regulatory small RNA, the Small RNA Regulator of Biofilms (SrbA). SrbA was found to be unique to P. aeruginosa and displayed no homology with any other sequenced bacterial species. I found that loss of SrbA resulted in a significant reduction in biofilm mass. Subsequently, loss of SrbA also leads to attenuation of P. aeruginosa pathogenicity in Caenorhabditis elegans nematodes. Bacterial biofilms possess specific regulatory programs that are still just being appreciated for their complexity. This thesis work adds to our understanding of biofilm regulation by studying roles of the two-component system TctD-TctE and the small RNA SrbA in P. aeruginosa.