<p>Exposure to ultraviolet radiation (UVR) has been shown to cause cellular damage in cyanobacteria. In response to UVR exposure, some cyanobacteria produce scytonemin, an indole-alkaloid sunscreen capable of absorbing long-wavelength UVA radiation. Previous genomic and transcriptomic analyses have determined that the production of scytonemin is controlled by a two-component regulatory system (TCRS), encoded by Npun_F1277 and Npun_F1278 in the cyanobacterium <i>Nostoc punctiforme </i>ATCC 29133. This TCRS is thought to not only regulate scytonemin biosynthesis, but also other responses to light and UVR stimuli. To better understand the functionality of the sensor kinase (SK) Npun_F1277 and to determine if it could activate alternative UVR protection pathways, the SK was expressed in <i>Escherichia coli.</i> The first objective of this study was to observe and quantify the level of fitness conferred to <i>E. coli</i> expressing Npun_F1277 from <i>N. punctiforme </i>(strain SKE) when exposed to white light, UVA, and UVB stress. Results from these experiments do not indicate that expression of the <i>N. punctiforme</i> SK conferred an advantage to <i>E. coli</i> under white light, UVA, or UVB stress based on growth alone. Therefore, the second objective was to study the expression of regulatory genes, such as response regulators, in <i>E. coli</i> that are homologs to those associated with the SK Npun_F1277 in <i>N. punctiforme </i>using quantitative-PCR. Expression of the selected genes was measured following exposure to white light and UVA after 30 and 60 minutes as well as UVB after 15 and 30 minutes. Comparison of SKE to empty-vector (EV) control cells exposed to the same stress showed that there were significant changes in the expression of important regulatory genes (e.g. <i>recA, spoT, relA</i>) in the SKE strain. Moreover, when comparing SKE cells exposed to the same conditions above to unstressed SKE cells, a similar result was seen for SKE cells exposed to UVA and UVB as was found in the studies comparing SKE to EV cells. These results suggest that the SK Npun_F1277 may play a role in multiple defense mechanisms of <i>N. punctiforme</i> in addition to initiation of the scytonemin biosynthesis pathway. </p>

Rhizobium leguminosarum is a Gram negative, motile, nitrogen-fixing soil bacterium. Due to the scarcity of iron in the soil bacteria have developed a wide range of iron scavenging systems. The two types of iron scavenging systems used are indirect and direct. In-silico analysis of the genome identified a unique direct iron scavenging system the Hmu operon. This system has been identified in other closely related rhizobium species and is believed to be involved in utilizing heme compounds as a sole source of iron. We have attempted to characterize the role of the Hmu operon in iron utilization by monitoring the growth of R. leguminosarum ATCC 14479 in hemin supplemented media. Growth curves show that it is capable of using hemin as a sole source of iron. The outer membrane profiles were analyzed for the presence of hemin binding proteins.

Salmonella enterica is an intracellular bacterial pathogen of humans and the causative agent of the acute gastrointestinal disease, salmonellosis, and the chronic systemic infection, typhoid fever. Sensor proteins convert environmental signals, including signals detected within the host environment, into biochemical signals to control cellular responses. It has been previously established that the two component regulatory system SsrA-SsrB, consisting of the integral membrane sensor kinase protein SsrA and the cytoplasmic DNA-binding response regulator SsrB are essential for regulation of bacterial factors during systemic intracellular infection. The first chapter of this thesis describes characterization of the sensor kinase SsrA. The structure of the periplasmic sensor domain is modeled and evidence is presented that it is involved in enhancing signaling activity in response to environmental acidification encountered within the intracellular environment. A mechanism whereby protonation of histidine residues within this region in response to acidification drives conformational strain and thereby signaling is proposed. The second chapter describes identification of the DNA-binding motif of SsrB within regulated promoters as well as its regulon. Integration of experimental data with comparative genomics data resulted in identification of the palindromic heptameric DNA recognition motif of SsrB as well as identification of novel SsrB-regulated promoters. In addition, a DNA microarray analysis is described wherein the complete SsrB regulon is identified. Finally, the third chapter describes regulatory input of SsrB to the S. enterica type VI secretion system. This chapter also describes the contribution of this system to systemic dissemination of S. enterica during host infection. Altogether, these data advance understanding of how Salmonella controls factors essential for disease in response to the host environment during infection. / Thesis / Doctor of Philosophy (PhD)

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.