The purpose of this research was to identify and characterize novel molecular mechanisms in copper homeostasis. Pseudomonas putida KT2440 is a soil bacterium studied for its potential use in bioremediation of soils contaminated with aromatic organic contaminants. The cinAQ operon was analyzed. cinAQ is transcribed in presence of copper. The product of cinA is a periplasmic azurin-like protein with a methionine and histidine rich region, characterized by a high redox potential (456 ±4 mV). CinQ was shown to be a pyridine nucleotide-dependent nitrile oxidoreductase that catalyzes the reduction of preQ₀ to preQ₁, the first committed step in the biosynthetic pathway leading to the production of the unusual nucleotide queuosine. Gene disruption of cinQ in Pseudomonas putida KT2440 and in Pseudomonas aeruginosa PAO1 did not result in a significant increase in copper sensitivity on disk assays. Furthermore, a P. putida KT2440 cinA mutant also did not present a greater sensitivity to copper on disk assays while cinA mutants in Pseudomonas aeruginosa PAO1 presented increased toxicity to copper compared to the wild-type. CinA is by sequence similarity proposed to be an electron shuttle, and was shown to be upregulated in the presence of copper. Increasing CinA levels in the periplasm after copper stress may represent a mechanism used to regenerate the multicopper oxidase CopA (involved in Cu(I) to Cu(II) oxidation). Alternatively, CinA could act as an electron shuttle that takes part in an alternative electron transport chain once redox active copper is available, or it could represent a periplasmic copper chaperon. CinQ is involved in the biosynthesis of the rare hyper-modified nucleotide queuosine, found in the wobble position of several tRNAs, and required to avoid the readthrough of the stop codon UAG. Transcription of cinAQ was shown to be under the control of the two component system CinR-CinS. CinS is a histidine kinase, with a sensor domain located in the periplasm. CinR is the cognate response regulator that activates transcription of genes upon phosphorylation from CinS. The CinR-CinS two component system was shown to be responsive to 0.5 LM copper. CinS displayed very high metal specificity and elicited a response only in the presence of copper and silver, but not other metals. Modeling of the CinS protein structure, performed using Swiss Model and using the periplasmic sensor DcuS from Escherichia coli as a template, identified a potential copper binding site, containing H37 and H147. Sequence alignment of copper sensing histidine kinases further identified other conserved residues in the periplasmic domain. Site-Directed Mutagenesis was used to generate CinS mutants that were tested for their ability to activate the cinAQ promoter in presence of Cu. When challenged with copper CinS mutant H37R and H147R had an almost 10 fold reduction in copper sensitivity compared to the wild-type, indicating a possible role in Cu coordination. Other CinS mutants responded similarly to the wild-type in the presence of 10 μM of Cu.

tRNA molecules contain more than 80 chemically unique nucleotide base modifications that contribute to the chemical and physical diversity of RNAs as well as add to the overall fitness of the cell. For instance, base modifications have been shown to play a critical role in tRNA molecules by improving the fidelity and efficiency of translation. Most of this work has been carried out extensively in Gram-negative bacteria, however, the role of modified bases in tRNAs as they relate to thermostability, structure, and transcriptional regulation in Gram-positive bacteria, such as Bacillus subtilis and Bacillus anthracis, are not well characterized. Infections by Gram-positive bacteria that have become more resistant to established drug regiments are on the rise, making Gram-positive bacteria a serious threat to public safety. My thesis work examined what role partial base modification of the tyrosyl-anticodon stem-loops (ASLTyr ) of B. subtilis and B. anthracis have on thermostability, structure, and transcriptional regulation. The ASLTyr molecules have three modified residues which include Queuine (Q34), 2-thiomethyl-N6-dimethylallyl (ms2i6A37), and pseudouridine (Y39). Differential Scanning Calorimetry (DSC) and UV melting were employed to examine the thermodynamic effects of partial modification on ASLTyr stability. The DSC and UV data indicated that the Y39 and i6A37 modifications improved the molecular stability of the ASL. To examine the effects of partial base modification on ASLTyr structure, NMR spectroscopy was employed. The NMR data indicated that the unmodified and [Y39]-ASLTyr form a protonated C-A+ Watson-Crick-like base pair instead of the canonical bifurcated C-A+ interaction. Additionally, the loop regions of the unmodified and [Y39]-ASLTyr molecules were well ordered. Interestingly, the [i6A37]- and [i6A37; Y39]- ASLTyr molecules did not form a protonated C-A+ base pair and the bases of the loop region were not well ordered. The NMR data also suggested that the unmodified and partially modified molecules do not adopt the canonical U-turn structure. The structures of the unmodified, [Y39]-, and [i6A37;Y39]-ASLTyr molecules did not depend on the presence of Mg2+, but the structure of the [i6A37]-ASLTyr molecule did depend on the presence of multivalent cations. Finally, to determine the repercussions that partial modification has on physiology and tRNA mediated transcriptional regulation in B. anthracis, antibiotic sensitivity tests, growth curves, and quantitative real-time polymerase chain reaction (qRT-PCR) were employed. Strains deficient in ms2 showed comparable growth to the parent strain when cultured in defined media, but Q deficient strains did not. The loss of ms2i6A37 conferred resistance to spectinomycin and ciprofloxacin, whereas the loss of Q34 resulted in sensitivity to erythromycin. Changes in the ratio full-length to truncated transcripts of the tyrS1 and tyrS2 genes were used to monitor tRNA mediated transcriptional regulation. The qRT-PCR data suggested that tyrS1 and tyrS2 are T-box regulated and that the loss of ms2i6A37 and Q34 might affect the interaction of the tRNATyr molecule with the specifier sequence, which is located in the 5’-untranscribed region (UTR) of the messenger RNA (mRNA).

The studies reported in this thesis address firstly, the biology of a modified nucleoside, Queuosine (Q) and secondly, the properties of mitochondrial translation initiation factor 2. A summary of the relevant literature on both these topics is presented in Chapter 1. Section I of this ‘General Introduction’ summarizes the literature on biosynthesis and physiological importance of Queuosine. Section II is a brief review of the current understanding of translation initiation in Eubacteria. Information about the mitochondrial translation initiation apparatus also features as a subsection. The next chapter (Chapter 2), describes the ‘Materials and Methods’ used throughout the experimental work presented in the thesis. It is followed by three chapters containing experimental work as described below:- i) Biosynthesis of Queuosine (Q) in Escherichia coli Q is a hypermodification of guanosine found at the wobble position of tRNAs with GUN anticodons. Q is thought to be produced via a complex multistep pathway, the details of which are not known. It was found in our laboratory that a naturally occurring strain of E. coli B105 lacked Q modification in the tRNAs. As the known enzymes of Q biosynthesis were functional in this strain, it presented us with the opportunity to uncover novel component(s) of Q biosynthetic pathway. In the present work, a genetic screen was developed to map the defect in E. coli B105 to a previously uncharacterised gene, ybaX, predicted to code for a 231 amino acid long protein with a pI of 5.6. Further genetic analyses showed that YbaX functions at a step leading to production of preQ0, the first known intermediate in the generally accepted pathway that utilizes GTP as the starting molecule. The gene ybaX has been renamed as queC. Using a combination of bioinformatics based prediction and gene knockouts, we have also been able to place two more genes, queD and queE at the initial step in Q biosynthesis, suggesting that the initial reaction of Q biosynthesis might be more complex and mechanistically different than what has been proposed earlier. ii) Caenorhabditis elegans as a Model System to Study Queuosine Metabolism in Metazoa Animals are thought to obtain Q (or its analogs) as a micronutrient from dietary sources such as gut microflora, and the corresponding base is then inserted in the substrate tRNAs by tRNA guanine transglycosylase (TGT). In animal cells, changes in the abundance of Q have been shown to correlate with diverse phenomena including stress tolerance, cell proliferation and tumor growth but the precise function of Q in animal tRNAs remains unknown. A major obstacle in the study of Q metabolism in higher organisms has been the requirement of a chemically defined medium to cause Q depletion in animals. Having discovered that E. coli B105 has a block in the initial step of Q biosynthesis, we reasoned that this strain could be used as a Q- diet for organisms like C. elegans, which naturally feed on bacteria. An analysis of C. elegans tRNA revealed that as in the other higher animals, tRNAs in the worm C. elegans, are modified by Q and its sugar derivatives. When the worms were fed on Q deficient E. coli B105, Q modification was absent from the worm tRNAs suggesting that C. elegans lacks a de novo pathway of Q biosynthesis. The inherent advantages of C. elegans as a model organism, the speed and simplicity of conferring a Q deficient phenotype on it, make it an ideal system to investigate the function of Q modification in tRNA. By microinjecting tgt-1-gfp constructs into C. elegans, we could also demonstrate that a major form of TGT is localised to the nucleus, suggesting that insertion of Q into the tRNAs could be occurring in the nucleus. iii) Dual Function of Bovine Mitochondrial Initiation Factor 2 as Initiation Factors 1 and 2 in Escherichia coli Translation initiation factors 1 and 2 (IF1 and IF2) are known as ‘universal translation initiation factors’ due to the presence of their homologs in all living organisms. Homologs of these factors are also present in the chloroplast, however, a unique situation exists in the mitochondria where IF2 homolog (IF2mt) is known to occur but an IF1 like factor is not found. We have engineered a system of E. coli knockouts to allow the study of IF2mt in a prokaryotic milieu. We found that the bovine IF2mt complements an E. coli strain wherein the gene for IF2 is knocked out, providing the first proof of a mitochondrial translation initiation factor working in a eubacterial system. This conservation of function is especially interesting in light of the recent reports revealing significant differences between the mitochondrial and eubacterial ribosomes. Further, we found that the IF2mt can also support a double knockout of IF1 and IF2 genes in E. coli, suggesting that IF2mt possesses both IF1 and IF2 like activities in E. coli. This finding offers an explanation for the lack of an IF1 like factor in mitochondria. Molecular modeling of bovine IF2mt indicated that a conserved insertion found in all mitochondrial IF2s, may form a protruding α-helix that could stabilize IF2mt on ribosomes. This insertion could in principle function as IF1 and we have explored the role of this conserved insertion both in vivo and in vitro, by generating mutants of IF2mt and EcoIF2, to lose or gain the conserved insertion respectively.