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

Chemotaxis is the ability of an organism to sense its environment and move towards attractants and away from repellents. The two-component system controlling chemotaxis in bacteria contains a histidine kinase CheA, which is autophosphorylated in response to a signal from a ligand-bound transmembrane methyl-accepting chemotaxis protein. CheA transfers the phosphate group to its cognate response regulator which modulates flagellar rotation. Signal termination by dephosphorylation of the response regulator is necessary for the organism to react rapidly to changes in the environment. The phosphorylated response regulator CheY in <i>Escherichia coli</i> is dephosphorylated by CheZ, a phosphatase; certain organisms, such as <i>Sinorhizobium meliloti</i>, that lack a CheZ homolog have developed alternate methods of signal termination. The signaling chain of S. meliloti contains two response regulators, CheY1 and CheY2, in which CheY2 modulates flagellar rotation and CheY1 causes signal termination by acting as a phosphate sink. In addition to known chemotaxis components, the second gene in the chemotaxis operon of <i>S. meliloti</i> codes a 97 amino acid protein, called CheS. The phenotype of a cheS deletion strain is similar to that of a cheY1 deletion strain. Therefore, the possibility that CheS causes signal termination was explored in this work. The derived amino acid sequence of CheS showed similarities with its orthologs from other °-proteobacteria. Sequence conservation was highest at the centrally located °4 and °5 helices. Earlier observations that CheS localizes at the polar chemotaxis cluster in a CheA-dependent manner were confirmed, and the co-localization of CheS with CheA was demonstrated by fluorescence microscopy. The stable expression of CheS in the presence of CheA was confirmed by immunoblot. The same approach was used to establish the stable expression of CheS only in the presence of the P2 domain of CheA, but not with the P1 or P345 domains. Limited proteolysis followed by mass spectrometry defined CheA_163-256 as the CheS binding domain, and this domain overlapped the previously defined CheY2-binding domain, CheA_174-316. The role of CheS in the phosphate flux in S. meliloti chemotaxis was analyzed by assays using radio-labeled [?-?°P]ATP. CheS does not play a role in the autophosphorylation of CheA. However, CheS accelerated the rate of CheY1~P dephosphorylation by almost two-fold, but did not affect the rate of CheY2~P dephosphorylation. CheS also does not seem to affect phosphate flow in the retrophosphorylation from CheY2~P to CheA using acetyl [?°P]phosphate as phosphodonor. Since CheS increases the rate of CheY1 dephosphorylation, it can be envisioned that it either increases the association of CheY1 to CheA, increasing the flow of phosphate from CheA to CheY1, or directly accelerates the dephosphorylation of CheY1~P. The presence of a STAS domain and a conserved serine residue in CheS also raises the possibility that CheS may be phosphorylated by a yet unknown kinase, in a mechanism similar to the phosphorylation of <i>Bacillus subtilis</i> SpoIIAA by its cognate kinase SpoIIAB. Phosphorylated CheS may then switch CheA between a kinase or phosphotransferase ON/OFF state or activated CheS may directly interact with CheY1. Further studies are needed to determine the association of CheY1 with CheS to elucidate the mechanism of CheY1 dephosphorylation. This work has confirmed the <i>in vitro</i> association of CheS with CheA, determined the CheS binding domain on CheA, and indicated that CheS accelerates the dephosphorylation of CheY1~P. This has advanced our understanding of the role of CheS in the chemotaxis signaling chain of <i>S. meliloti</i>. / Master of Science

Mycobacterium tuberculosis, the causative organism of the disease tuberculosis (TB) in humans, leads to nearly two million deaths each year. This versatile pathogen can exist in highly distinct physiological states such as asymptomatic latent TB infection where bacilli lie dormant or as active TB disease in which the bacilli replicate in macrophages. The pathogenic lifestyle requires the tubercle bacillus to sense and respond to multiple environmental cues to ensure its survival. Such stimuli include hypoxia, nutrient limitation, presence of reactive oxygen and reactive nitrogen intermediates, pH alterations, and cell wall/ membrane stress. Two component systems (TCSs) form the primary apparatus for sensing and responding to environmental cues in bacteria. A prototypical TCS is composed of a sensory protein called sensor kinase (SK) and a response generating protein called response regulator (RR). M. tuberculosis encodes 11 genetically paired TCSs, 2 orphan sensor kinases and six orphan response regulator proteins. Studies of the TB bacilli using transcriptional profiling and gene knockouts have revealed that TCSs play an important role in facilitating successful adaptation to diverse environmental conditions encountered within the host. The mtrAB and prrAB genes encoding corresponding TCSs have been shown to be essential for survival, mprAB for persistence and devRS for hypoxic adaptation. Further, inactivation of the TCSs regX3-senX3, tcrXY, trcRS, phoPR or kdpDE was shown to affect the growth and/or virulence of M. tuberculosis in animal infection models. The SK and RR proteins of TCSs are modular and contain variable input and output domains coupled to conserved ‘transmitter’ and ‘receiver’ domains. Despite the modular nature and extensive homology of SK and RR proteins across TCSs, which may allow non-cognate interactions, it is believed that crosstalk across different TCSs is not favored and that individual pathways are generally well insulated. The existing profiling studies have been performed on the TCSs of bacterial species containing a relatively large number of TCSs. In those studies, specificity and insulation have been the norm and thus become the prevalent paradigm of TCS signaling. In vitro genome wide phosphotransfer profiling has revealed only a few cross- communication nodes in the TCSs of Escherichia coli (~3%), while none in Caulobacter crescentus (in 352 interactions tested, in short time duration) and Myxococcus xanthus (in 250 interactions tested). Yet, many instances of cross talk have been reported in literature. For example, E. coli TCSs PmrAB and EnvZ-OmpR show cross-communication with QseBC and ArcBA, and many more. In M. tuberculosis, indirect evidence of the existence of such cross regulation has originated from studies where mutations in phoPR have been shown to affect the expression of the TCS devRS and its regulon. It is thus interesting to examine the extent of crosstalk in the TCSs of M. tuberculosis, which has an exceptionally small number of TCS proteins compared to E. coli. As mentioned earlier, M. tuberculosis H37Rv has 11 cognate pairs of TCSs, 2 orphan sensor kinases and 6 orphan response regulators. To study the entire landscape, we aimed to study all 221 connections between SK and RR proteins including 12 cognate interactions. While 10 of the cognate TCS interactions were established in the literature, two putative systems KdpDE and NarSL and 5 orphan response regulators were still uncharacterized, therefore we initiated our work with the characterization of these TCSs. At the biochemical level, the KdpDE two component system of M. tuberculosis is not well studied, though one report showed interaction of the C-terminal domain of KdpD SK and KdpE RR using yeast two hybrid assay and another reported the interaction of the SK with LRP protein. Besides these associations, there is no evidence for the functionality of KdpDE system. Similarly, NarSL system also has not been characterized and it not known whether these putative two component proteins are functional. The initial part of the study includes the characterization of these two TCSs, NarS-NarL and KdpD-KdpE, at biochemical and physiological levels. In our studies we demonstrated that KdpDE system is a bonafide two component system of M. tuberculosis, and KdpD SK undergoes autophosphorylation at His642 residue in presence of Mg+2 ions and then it transfers phosphoryl group to a conserved Asp52 residue on the KdpE RR protein. The acid-base stability analysis revealed the nature of chemical bonds present in the KdpD and KdpE proteins, and further confirmed that KdpD and KdpE are typical SK and RR respectively. SPR analysis demonstrated that KdpD and KdpE proteins interact under basal non-phosphorylated conditions and the interaction affinity reduced when SK was phosphorylated. The reduction in the interaction affinity indicated towards a possible dissociation of SK and RR protein during phosphotransfer, which allows RRs to exert their regulatory effect. On the similar line, the phosphorylation defective SK (KdpDH642Q) had least affinity with KdpE suggesting that perhaps this mutant SK, fails to interact with the RR. We have also shown that both the kdpD and kdpE genes are in the same operon and are up regulated in potassium ions limitation and osmotic stress conditions. Overall, using the biochemical approaches, we have established that Rv1027c–Rv1028c operon of M. tuberculosis encodes a functional and a typical KdpDE two component signal transduction system. Using the similar biochemical and biophysical approaches, we have demonstrated that NarS-NarL proteins constitute a functional TCS and His241 and Asp61 are the phosphorylatable residues. In contrast to KdpDE which shows typical behaviour of TCS, NarSL TCS showed atypical behaviour. Malhotra and group’s work on NarSL suggested that there is cross-regulation between NarS/NarL and DevS/DosT/DevR systems. We addressed this possibility on three separate levels, by examining (i) the cross-phosphorylation of DevR and NarL RRs by non-cognate sensor kinases NarS and DevS/DosT respectively, (ii) the interaction between DevR and NarL RR proteins, and (iii) examining the effect of DevR-NarL interactions on their DNA binding properties. Our studies ruled out the presence of any physiologically relevant phosphorylation mediated cross-talk between NarS/NarL and DevS/DosT/DevR. We identified that the cross talk between these TCSs could be explained on the basis of interaction between NarL and DevR RRs and their subsequent binding to the target gene promoter regions for concerted regulation of gene expression. We also identified that DevR activation is critical for cooperative action with NarL. This process comes out as a novel mechanism of gene regulation via heteromerization of RRs. We hypothesized that formation of NarL-DevR heteromers may arise because of high sequence similarities. Conclusively, our study provides insights into the functionality of M. tuberculosis NarL/NarS TCS and regulatory function of NarL protein which acts in concert with another RR, DevR. Overall, NarS-NarL system showed an atypical, novel mode of gene regulation involving RR heteromerization. Subsequent to the basic biochemical characterization of NarSL and KdpDE system, the genome wide phosphotransfer profiling was done to identify the cross-connections between TCSs. Remarkably, we found that specificity was the exception rather than the rule. While only three of the TCS pairs were completely specific, all the other nine TCS pairs exhibited crosstalk, including a few that were highly promiscuous. We classified the interactions as specific, one-to-many, and many-to-one signaling circuits. We also profiled all the RRs including the orphans for their ability to accept phosphoryl group from a low molecular weight donor, acetyl phosphate, and interestingly found that only two RRs DevR and NarL were capable of accepting phosphoryl group from such a donor. Interestingly, none of the orphan RRs accepted phosphoryl group from any donor, neither SKs nor low molecular weight phospho donors, warranting further analysis of their roles and presence in the M. tuberculosis genome. Our exhaustive map of the crosstalk between the TCSs of M. tuberculosis sets the stage for a renewed view of TCS signaling and proposes a dispersive-integrative landscape for TCS signaling rather than one of insulation. As an extension of our basic characterization work of NarSL TCS, we also attempted to understand the localization pattern of NarS sensor kinase in M. smegmatis cells using fluorescence approaches. It is known that many bacterial receptors including sensor kinases form clusters or show specific localization patterns inside the cell. We found that NarS shows distinct cellular localization pattern. However, the functional significance of this localization pattern is not obvious yet and warrants further investigations. We also developed a few non-radioactive methods to study interaction between two component systems to overcome the limitations associated with radioactive experiments in studying TCSs. We developed fluorescence resonance energy transfer (FRET) to study in vitro interaction between two component proteins which was sensitive to the phosphorylation status of the proteins. Using fluorescently tagged SKs and RRs, we determined a change in FRET for KdpDE and NarSL TCS pairs in vitro. Our study thus also provides an alternative approach to study TCS signaling, using an easier, non-radioactive and high throughput approach. In summary, our study presents the evidence of an alternative paradigm of bacterial signaling, where significant crosstalk between the underlying TCSs prevails. The new paradigm is expected to have important implications in our understanding of the virulence and pathogenesis of bacterial infections. Overall, our studies (i) allowed the establishment of functionality of all paired TCSs encoded in the genome of M. tuberculosis including NarSL and KdpDE TCSs, (ii) identified the novel mechanism of gene regulation by NarL RR and DevR, (iii) demonstrated the existence of TCS signaling which is contrary to the existing notion of specificity (iv) showed the distinct localization pattern of NarS and (v) developed non-radioactive approaches to study two component interactions.

Methyl-accepting chemotaxis proteins MCPs can bind one or more receptor- specific ligands. In the case of the Tar MCP of Escherichia coli (TarEc), a primary attractant ligand is aspartate. Its binding to the periplasmic domain of Tar generates a conformational change that is transmitted via helix 4 transmembrane helix 2 (TM2). An inward movement of TM2 initiates a transmembrane signal to the cytoplasmic HAMP (histidine kinases, adenyl cyclases, methyl-accepting proteins, phosphatases) domain. Baseline CheA kinase-stimulating activity and ligand-induced responses are both strongly influenced by residues at the C-terminus of transmembrane helix 2 (TM2). The cytoplasmic aromatic anchor, composed of residues Trp-209 and Tyr-210 in TarEc, is of particular importance. These residues are not highly conserved among transmembrane receptors having a HAMP domain, although there are almost always some aromatic residues in this region. The question thus becomes what properties of this aromatic anchor are necessary for proper signal transduction. In this dissertation, I studied the effect on TarEc function by substituting all possible combinations of Ala, Phe, Tyr, and Trp at positions 209 and 210. This library of TarEc variants allowed the direct assessment of the effect of the residue composition of the aromatic anchor and led to a model of how the wild-type anchor maintains the base-line signaling state in TarEc. Additional receptor variants containing double aromatic tandems and Ala substitutions for the periplasmic Trp residue were created, and the aromatic residues were also shifted in position within the six residues 207-212. Trp, Tyr, and Phe, in that order, had the greatest effect on function when they were moved to novel positions. It was also discovered that Gly-211 plays a critical role in maintaining receptor function. A model was generated that proposes that Gly-211 plays a role in maintaining the flexibility of the TM2-HAMP domain connector. The results suggest that the signaling properties of the transmembrane sensor kinases of two-component systems can be predicted by the nature of their TM2-HAMP connections. It may also be possible to modulate their activity in a controlled way by manipulating the amino acid sequences that comprise those connections.

Bacteria may live in harsh environments where they face changing and new conditions. Therefore, the ability to maintain homeostasis in cells may be vital for survival. Transition metals such as iron, zinc, and copper are essential nutrients for cell survival, but become toxic if in excess amount. In order to survive, bacteria have developed defensive mechanisms to protect themselves. Copper and silver levels need to be carefully maintained within cells to balance cellular needs with potential toxicity. This dissertation focuses on the Cus copper and silver efflux system in E. coli. The E. coli cus system is composed of two divergently transcribed operons, cusCFBA and cusRS. The cusCFBA genes encode for a tripartite metal efflux pump CusCBA and a metallochaperone CusF. The cusRS genes encode a two-component system CusS-CusR that regulates the expression of the cusCFBA genes in response to elevated levels of copper or silver in the periplasm. The histidine kinase CusS senses and binds to metals on its periplasmic sensor domain and transduces signal into the cytoplasm to further communicate with its cognate response regulator CusR through histidyl-aspartyl phosphotransfer event. CusR then outputs cellular response by activating the upregulation of the cusCFBA genes, which then turn on the CusCBA efflux pump to eliminate excess copper or silver in the periplasm. While bacterial two-component systems have been widely studied, the mechanisms of ligand-induced signal transduction by histidine kinases remain unclear. It is now known that cusS is essential for copper and silver resistance, and CusS directly binds metal ions in the periplasmic sensor domain and dimerizes upon metal binding. Thus, the goal of this research is to characterize the metal binding properties in the sensor domain, and to elucidate the signal transduction and autophosphorylation mechanisms of CusS upon metal binding. The data from this work reveal that there are two distinct metal binding sites, interface and internal binding sites, in the sensor domain of CusS, and the interface binding site is functionally more important in metal resistance in E. coli. Furthermore, metal-induced dimerization through the interface metal binding site plays an important role in CusS kinase activity. Together, these findings aid in our understanding of the molecular details in metal binding within the sensor domain of CusS. Based on these data, we propose a model for the signal transduction mechanism and histidine phosphorylation mechanism of the histidine kinase CusS.

The gram-positive bacterium Streptococcus sanguinis is a member of human indigenous oral microbialflora and has long been recognized as a key player in the bacterial colonization of the mouth. S. sanguinis is also the most common viridians streptococcal species implicated in infective endocarditis. Although many studies have focused on two-component systems in closely related Streptococcus species such as S. mutans, S. pneumoniae and S. gordonii; the mechanism of the response regulator in S. sanguinis is still unknown. The ability of S. sanguinis to adapt and thrive in hostile environments suggests this bacterium is capable of sensing and responding to various environmental stimuli. The present study clearly demonstrates that a number of RR genes, SSA_0204, SSA_0217, SSA_1810, SSA_1794, and SSA_1842, in S. sanguinis are essential to the recognition and response to various environmental stresses. Results from this study also identified genes SSA_0260, SSA_0261, and SSA-0262, involved in acidic tolerance and suppressed by SSA_0204 response regulator.

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.

The rhizobia-legume symbiosis is the most agriculturally significant source of naturally fixed nitrogen, accounting for almost 25% of all biologically available nitrogen. Rhizobia-legume compatibility restrictions impose limits on symbiotic nitrogen fixation. In many cases, the molecular basis for symbiotic compatibility is not fully understood. The signals required for establishing a symbiotic partnership between nitrogen-fixing bacteria (e.g. Sinorhizobium meliloti) and leguminous plants (e.g. Medicago truncatula) have been partially characterized at the molecular level. The first stage of successful root colonization is competitive occupation of the rhizosphere (which is poorly understood). Here, the bacteria introduce themselves as potential symbiotic partners through the secretion of glycolipid "Nod" factors. In response, the host facilitates a more exclusive mode of colonization by the formation of a root nodule – a new organ capable of hosting dense intracellular populations of symbiotic rhizobia for nitrogen fixation. This dissertation reports the exhaustive identification of S. meliloti genes that permit competitive colonization of the M. truncatula rhizosphere, and includes a mechanistic study of one particular bacterial signaling pathway that is crucial for both rhizosphere colonization and nodulation. I have made use of Tn-seq technology, which relies on deep sequencing of large transposon mutant libraries to monitor S. meliloti genotypes that increase or decrease in relative abundance after competition in the rhizosphere. This work included the collaborative development of a new computational pipeline for performing Tn-seq analysis. Our analysis implicates a large ensemble of bacterial genes and pathways promoting rhizosphere colonization, provides hints about how the host plant shapes this environment, and opens the door for mechanistic studies about how changes in the rhizosphere are sensed and interpreted by the microbial community. Notable among these sensory pathways is a three-protein signaling system, consisting of FeuQ, FeuP, and FeuN, which are important for both rhizosphere colonization and nodule invasion by S. meliloti. The membrane-bound sensor kinase FeuQ can either positively or negatively influence downstream transcription of target genes by modulating the phosphorylation state of the transcriptional activator FeuP. FeuN, a small periplasmic protein, inhibits the positive mode of FeuPQ signaling by its direct interaction with the extracellular region of FeuQ. FeuN is essential for S. meliloti viability, underscoring the vital importance of controlling the activity of downstream genes. In summary, I have employed several powerful genetic, genomic, computational, and biochemical approaches to uncover a network of genes and pathways that coordinate root colonization and nodulation functions.

Bacteria commonly use two-component sensors to sense and respond to their environment. The Gram-negative opportunistic pathogen Pseudomonas aeruginosa has one of the largest sets of two-component sensors known in bacteria, which likely contributes to its ability to adapt to diverse environments, including the human host. Several of these sensors such as GacS have been shown to play a role in the regulation of virulence in this pathogen. However, the role of the majority of sensors remains unknown. In this thesis I show that the two-component sensor KinB is required for full P. aeruginosa virulence in the recently characterized model host Danio rerio. I found that KinB regulates several virulence-associated phenotypes in P. aeruginosa including pyocyanin and elastase production and motility. I show that KinB regulates these phenotypes through the global sigma factor AlgU, which plays a critical role in the repression of P. aeruginosa acute virulence factors and through its cognate response regulator, AlgB, albeit in a non-canonical manner. KinB’s primary role in the regulation of acute virulence is to act as a phosphatase to dephosphorylate AlgB.