Salmonella enterica is an enteric pathogen with a broad host tropism that can cause disease ranging from self-limited gastroenteritis to enteric fever. The evolution of S. enterica as a pathogen is driven by the horizontal acquisition of genes that promote virulence and survival within host immune cells, as well as the coordinated regulation of these and ancestral genes by two-component systems (TCS). TCS integrate environmental cues with the transcriptional reprogramming of bacteria, and in the case of Salmonella, result in niche-specific gene expression in response to anti-bacterial cues produced by the host. The TCS SsrA-SsrB in S. enterica is considered the master regulator for intracellular virulence, where SsrA is a sensor kinase that triggers the activation of the DNA binding protein SsrB. The full suite of genes regulated by SsrB in S. enterica, as well as the cues that activate this TCS, have not been fully characterized. Here, we demonstrated that horizontally acquired and ancestral genes in the S. enterica genome have evolved to be regulated by SsrB, and the repression of a set of ancestral genes involved in flagellar motility promotes evasion of the host immune system. Additionally, we identified the production of reactive oxygen species (ROS) by host immune cells as a signal that can activate a cluster of genes regulated by the SsrA-SsrB TCS, likely mediated by SsrA sensing of these ROS. Together, these results expand our understanding of the complex interplay between the pathogen S. enterica and the host that results in bacterial infections. / Thesis / Doctor of Philosophy (PhD) / Salmonella enterica (S. enterica) is a species of bacteria that can cause food poisoning in various animals, including humans, through consumption of contaminated food and water. During an infection, host cells activate numerous defense mechanisms to prevent disease. S. enterica has evolved to turn specific genes on or off in response, resulting in modifications to bacterial and host cell behaviour that promote infection. The timing of these genetic changes is controlled by proteins that can sense specific environmental signals and adjust gene expression accordingly. The specific signals sensed by S. enterica that allow for adaptive gene expression within the host, and the types of genes that are regulated to promote survival, have not been fully identified. Here, we show that S. enterica evolved to repress genes involved in flagellar motility to hide from the host immune response. We further demonstrate that S. enterica can sense anti-bacterial molecules produced by the host, called reactive oxygen species, to trigger specific changes in gene expression. Together, this work reveals novel aspects for the molecular basis of Salmonella enterica pathogenesis.

Escherichia coli transporter protein AcrB and its homologues are the inner membrane components of the Resistance-Nodulation-Division (RND) family efflux pumps in Gram-negative bacteria. It is well accepted that soluble proteins are only marginally stable, but such insight is missing for membrane proteins. The lack of stability data, including thermodynamic stability and oligomer association affinity is a result of intrinsic difficulties in working with membrane proteins. In addition, the degradation of soluble proteins in E. coli has been extensively studied whereas the degradation process of membrane proteins remains unclear. A focus of my thesis is the validation and development of methods used to measure the thermo- and oligomeric- stability of membrane proteins. I investigated the mechanism of a popular thermal-stability assay developed specifically for the study of membrane proteins uses a thiol-specific probe, 7-diethylamino-3-(4-maleimidophenyl)-4-methylcoumarin (CPM). I found that, contrary to current understanding, the presence of a sulfhydryl group was not a prerequisite for the CPM thermal stability assay. The observed fluorescence increase is likely caused by binding of the fluorophore to hydrophobic patches exposed upon protein unfolding. I then applied these methods in the study of three projects. In the first project, I investigated how suppressor mutations restore the function of AcrBP223G, in which the Pro223 to Gly mutation compromised the function of AcrB via disrupting AcrB trimerization. The results suggested that the function loss resulted from compromised AcrB trimerization could be restored through various mechanisms involving the compensation of trimer stability and substrate binding. In the second project, I created two AcrB fusion proteins, with C-terminal yellow fluorescence protein (YFP) and cyan fluorescence protein (CFP), respectively. YFP and CFP form a fluorescence resonance energy transfer (FRET) pair. Using this pair of fusion proteins, I studied AcrB assembly both in detergent micelles and in lipid bilayers. A positive cooperativity was observed in kinetic studies of association of AcrB trimer. Reconstitution experiment revealed that the association showed a higher FRET efficiency and faster association rate in liposome than in DDM. In the last project, I developed a fluorescence method to study the degradation of AcrB-ssrA by the ClpXP system. Comparing to the degradation of GFP-ssrA, degradation of AcrB-CFP-ssrA showed a lower maximum velocity and tighter binding to the enzymes with a positive cooperativity.

The studies reported in this thesis describe the work done in the area of translation initiation where a previously unknown role of multiple copies of initiator tRNA in E. coli has been reported. Also the role of SsrA resume codon in resumption of translation, until not clearly known has been reported here. Chapter -1 discusses the relevant literature in understanding translation and initiator tRNA selection on the ribosome during initiation. It also discusses the literature pertaining to the aspect of release of stalled ribosomal complexes by SsrA. This is followed by the next chapter (chapter- 2) which discusses the materials and methods used throughout the study. Chapter- 3 describes the studies leading to the role of multiple copies of initiator tRNA in E. coli in governing the fidelity of initiator tRNA selection on the P site of the ribosome. This is followed by Chapter-4 which describes the role of the resume codon of the SsrA in governing the efficiency of trans-translation in releasing the stalled ribosomal complexes. The summaries of the chapters 3 and chapter 4 are briefly described below. i) Role of conserved 3GC base pairs of initiator tRNA in the initiator-elongator tRNA discrimination. Translation initiation is the first step in the very important and highly conserved biological process of protein biosynthesis. The process involves many steps, a wide array of protein factors at each specialized step and a large ribonucleoprotein particle; the ribosome to decode the information of the mRNA template into biologically active proteins. The process of initiation is still unclear largely due to fewer reports of available structural data. One of the very interesting questions that people have been trying to address is how the initiator tRNA is selected on the P- site of the ribosome and what is the importance of the conserved three GC base pairs in the anticodon stem of the initiator tRNA. Here in this study, I have studied this question by using the classical genetic technique of generating and characterizing the mutant initiator tRNA defective at the step of initiation. I have identified and analyzed the suppressors which are capable of rescuing this defect in initiation. The study involves two such E. coli suppressor strains (named D4 and D27). These suppressors can initiate translation from a reporter CAT mRNA with amber codon, independent of the presence of the three consecutive GC base pairs in the anticodon stem of initiator tRNAs. Mapping of the mutations revealed that the mutants are defective in expression of the tRNA1fMet (metZVW) gene locus which encodes the initiator tRNA. Both the suppressors (D4 and D27) also allow initiation with elongator tRNA species in E. coli. Taken together, the results show that E. coli when deficient in the initiator tRNA concentration can lead to initiation with elongator tRNA species. ii) The Role of SsrA/tmRNA in ribosome recycling and rescue. Occasionally during the process of translation, the ribosomes stall on the mRNA before the polypeptide synthesis is complete. This situation is detrimental to the organism because of the sequestration of the tRNAs as ‘peptidyl tRNAs’ and the ribosomes. In E. coli one of the pathways to rescue stalled ribosomes involves disassembly of these stalled complexes to release peptidyl tRNAs which are then recycled by peptidyl tRNA hydrolase (Pth), an essiential enzyme in E. coli. The other pathway which is not essential in E. coli but is conserved in all prokaryotes involves SsrA or tmRNA (transfer messenger RNA). The tmRNA is charged with alanine and recognizes the stalled ribosomal complexes and acts as tRNA to bind the A-site. It also functions as mRNA by adding a undecapeptide (which is actually a tag for degradation by cellular proteases) to the existing polypeptide and there is normal resumption of the translation. In most sequences of SsrA ORF, the first codon of the ORF, called as resume codon, is conserved. I wanted to understand the importance of the conservation of the resume codon. Towards this end I randomly mutated the resume codon and studied the effect of the altered resume codon in the rescue of stalled ribosomal complexes. The effect of over-expression of these mutants was investigated in the rescue of the Pthts defect since it is known that the overexpression of SsrA rescues the temperature sensitive phenotype of the Pthts strain and so causes less accumulation of peptidyl–tRNA in E. coli .The effect for these mutants has also been studied by the growth of hybrid λimmP22 phages. I also used AGA minigene system to study the effect of various mutants which has been shown to sequester tRNAArg (UCU) in the ribosomal P-site, translation of this minigene causes toxicity to E. coli. I have tried to study the effect of the SsrA mutants in rescue of toxicity caused by the minigene. Overall, the observations indicate that the conservation of the resume codon is important in E. coli and having mutated resume codon probably leads to deficient trans-translation during one or the other growth conditions.

Over the years, the view on proteases as relatively non-specific protein degradation enzymes, mainly involved in food digestion and intracellular protein turnover, has shifted and they are now recognized as key regulators of many biological processes that determine the fate of a cell. Besides their biological role, proteases have emerged as important tools in various biotechnical, industrial and medical applications. At present, there are worldwide efforts made that aim at deciphering the biological role of proteases and understanding their mechanism of action in greater detail. In addition, with the growing demand of novel protease variants adapted to specific applications, protease engineering is attracting a lot of attention. With the vision of contributing to the field of protein science, we have developed a platform for the identification of site-specific proteolysis, consisting of two intracellular genetic assays; one fluorescence-based (Paper I) and one antibiotic resistance-based (Paper IV). More specifically, the assays take advantage of genetically encoded short-lived reporter substrates that upon cleavage by a coexpressed protease confer either increased whole-cell fluorescence or antibiotic resistance to the cells in proportion to the efficiency with which the substrates are processed. Thus, the fluorescence-based assay is highly suitable for high-throughput analysis of substrate processing efficiency by flow cytometry analysis and cell sorting, while the antibiotic resistance assay can be used to monitor and identify proteolysis through (competitive) growth in selective media. By using the highly sequence specific tobacco etch virus protease (TEVp) as a model in our systems, we could show that both allowed for (i) discrimination among closely related substrate peptides (Paper I & IV) and (ii) enrichment and identification of the best performing substrate-protease combination from a background of suboptimal variants (Paper I & IV). In addition, the fluorescence-based assay was used successfully to determine the substrate specificity of TEVp by flow cytometric screening of large combinatorial substrate libraries (Paper II), and in a separate study also used as one of several methods for the characterization of different TEVp mutants engineered for improved solubility (Paper III). We believe that our assays present a new and promising path forward for high-throughput substrate profiling of proteases, directed evolution of proteases and identification of protease inhibitors, which all are areas of great biological, biotechnical and medical interest. / QC 20110516

Studies reported in this thesis address the question of how pre-termination ribosomal complexes stalled during translation of mRNA are recycled. The process of recycling of the stalled ribosomes involves many translational factors. During the course of my studies, I have uncovered new roles of SsrA (tmRNA), IF3 and ribosome recycling factor (RRF) in recycling stalled ribosomes. These findings are summarized as follows: (i) A physiological connection between tmRNA and peptidyl-tRNA hydrolase functions in Escherichia coli The bacterial ssrA gene codes for a dual function RNA, tmRNA, which possesses tRNA-like and mRNA-like regions. The tmRNA appends an oligopeptide tag to the polypeptide on the P-site tRNA by a trans-translation process that rescues ribosomes stalled on mRNAs and targets the aberrant protein for degradation. In cells, processing of the stalled ribosomes is also pioneered by drop-off of peptidyl-tRNAs. The ester bond linking the peptide to tRNA is hydrolyzed by peptidyl-tRNA hydrolase (Pth), an essential enzyme, which releases the tRNA and the aberrant peptide. As the trans-translation mechanism utilizes the peptidyl-transferase activity of the stalled ribosomes to free the tRNA (as opposed to peptidyl-tRNA drop-off), the need for Pth to recycle such tRNAs is bypassed. Thus, we hypothesized that tmRNA may rescue a defect in Pth. The findings of the experiments detailed in this thesis show that SsrA rescues a defect in Pth by reducing the peptidyl-tRNA load on Pth. (ii) Evidence for a role of initiation factor 3 in recycling ribosomal complexes stalled on mRNAs in Escherichia coli. Specific interactions between ribosome recycling factor (RRF) and EF-G mediate disassembly of post-termination ribosomal complexes for new rounds of initiation. The interactions between RRF and EF-G are also important in peptidyl-tRNA release from pre-termination complexes. Unlike the post-termination complexes (harboring tRNA), the pre-termination complexes (harboring peptidyl-tRNA) are not recycled by RRF and EF-G in vitro, suggesting participation of additional factor(s) in the process. Using a combination of biochemical and genetic approaches, we show that, 1. Inclusion of IF3 with RRF and EF-G results in recycling of the pre-termination complexes; 2. IF3 overexpression in Escherichia coli LJ14 rescues its temperature sensitive phenotype for RRF; (3) Transduction of infC135 (encoding functionally compromised IF3) in E. coli LJ14 generates a ‘synthetic severe’ phenotype; (4) The infC135 and frr1 (a promoter down RRF gene) alleles synergistically rescue a temperature sensitive mutation in peptidyl-tRNA hydrolase in E. coli; and (5) IF3 facilitates ribosome recycling by Thermus thermophilus RRF and E. coli EFG in vivo and in vitro. These lines of evidence clearly demonstrate the physiological importance of IF3 in the overall mechanism of ribosome recycling in E. coli. (iii) The role of RRF in dissociating of pre-termination ribosomal complexes stalled during elongation Translating ribosomes often stall during the repetitive steps of elongation for various reasons. The stalled ribosomes are rescued by the process of trans-translation involving tmRNA (SsrA) or by a factor mediated dissociation of the stalled ribosome into its subunits leading to the drop-off of the peptidyl-tRNA. The mechanistic details of how the factor mediated dissociation is carried out, is not well studied. Studies described in the above section have highlighted the role of RRF in dissociating stalled pre-termination complexes. However, the in vivo studies in this area have been limited for lack of defined pre-termination complexes. Two in vivo systems based on translation of AGA minigene and the ung gene (EcoUngstopless) transcripts were designed. Evidence is presented to show that translation of both of these transcripts is toxic to E. coli because of the accumulation of the transcript specific stalled pre-termination complexes. Availability of these model systems has allowed us to address the role of RRF in dissociating stalled ribosomes. We show that RRF rescues stalled ribosomes on these constructs and its overexpression can rescue the toxicity. The physiological importance of this observation is highlighted by the rescue of AGA minigene inhibitory effect on λimmP22 hybrid phage growth upon RRF overexpression.