Thèse diffusée initialement dans le cadre d'un projet pilote des Presses de l'Université de Montréal/Centre d'édition numérique UdeM (1997-2008) avec l'autorisation de l'auteur. / Thèse numérisée par la Direction des bibliothèques de l'Université de Montréal.

The MCM helicase plays an important role in eukaryotic DNA replication, unwinding double stranded DNA ahead of the replication fork. MCM is a hetero-hexamer consisting of the six related proteins, Mcm2-Mcm7. The distantly related MCM-binding protein (MCM-BP) was first identified in a screen for proteins interacting with MCM2-7 in human cells and was found to specifically interact with Mcm3-7 but not Mcm2. It is conserved in most eukaryotes and seems to play an important role in DNA replication but its exact function is not clear yet. This study contributes to the understanding of the fission yeast homologue of MCM-BP, named Mcb1, but also of MCM-BP in general. Results presented in this thesis document the initial biochemical characterisation of the complex Mcb1 forms with Mcm proteins, the MCMᴹᶜᵇ¹ complex. Interactions of Mcb1 with Mcm proteins, potential interaction sites between the proteins and the size of the complex were analysed using a variety of methods, including tandem affinity purification, co-immunoprecipitation, sucrose gradients and in vitro pull-down assays. Sequence analysis and structure prediction were utilised to gain some insight into Mcb1 and MCM-BP ancestry and structure. Results presented here indicate that fission yeast Mcb1 shares homology with Mcm proteins and forms a complex with Mcm3-Mcm7 but not Mcm2 and thus replaces the latter in an alternative high molecular weight complex that is likely to have an MCM-like appearance. Deletion of mcb1⁺ showed that Mcb1 is essential in fission yeast. To examine the cellular function of the protein, temperature-sensitive mutants were generated. Inactivation of Mcb1 leads to an increase in DNA damage and cell cycle arrest in G2-phase depending on the activation of the Chk1 dependent DNA damage checkpoint. Similar observations were made when Mcb1 was overexpressed, indicating that certain levels of the protein are important for accurate DNA replication. Construction of truncated versions of Mcb1 suggested that almost the full-length protein is needed for proper function.

No description available.

La surexpression rétrovirale du facteur de transcription HOXB4 résulte en une expansion sélective des cellules souches hématopoïétiques (CSH) in vitro et in vivo et ce, sans induire de leucémie. Par contre, la demi-vie intracellulaire de la protéine est de seulement une heure et le fait que la protéine disparaît du milieu de culture après environ 4 heures représente un obstacle majeur à l’utilisation clinique de la protéine HOXB4. Trois mutants HOXB4 ayant une substitution d`un seul acides aminés (AA) parmi les 31 premiers AA ont démontré une augmentation de la stabilité de la protéine. Nous avons donc évalué l’effet de HOXB4 et de ses trois mutants sur la production de cellules progénitrices myéloïdes. L’expression ectopique de HOXB4 sauvage (s-HOXB4) et HOXB4 mutant (m-HOXB4) a un effet comparable sur la fréquence des cellules progénitrices myéloïdes en essai clonogénique. Par contre, la capacité de prolifération des cellules progénitrices myéloïdes qui surexpriment s-HOXB4 et 1423 m-HOXB4 a été supérieure à celle des cellules contrôles (GFP seul) et des deux autres mutants. De plus, malgré le fait que toutes les variantes de HOXB4 confèrent une capacité d’autorenouvellement similaire aux cellules progénitrices multipotents (GEMM), la production des progéniteurs granulocytaires (CFU-G) est compromise lorsque les cellules surexpriment 1426 et 1427 m-HOXB4. D’autre part, la densité cellulaire des colonies myéloïdes qui surexpriment ces deux mutants est diminuée, ce qui suggère que ces mutations ont non seulement augmenté sa stabilité, mais potentiellement affecté certaines fonctions biologiques de s-HOXB4. Enfin, 1423 m-HOXB4 semble n’avoir perdu aucune fonction de s-HOXB4 dans nos évaluations clonogéniques in vitro, ce qui fait de ce mutant une molécule intéressante pour des applications cliniques d’expansion des cellules progénitrices hématopoïétiques. / Over-expression of the homeobox transcription factor HOXB4 results in a selective expansion of HSC in vitro and in vivo. However, the intracellular half-life of the protein is only one hour, which represents an important obstacle for its clinical use. Three HOXB4 mutants with single amino acid (AA) substitution in the first 31 AA have demonstrated increased intracellular stability of the protein. We evaluated the effect of wild type (wt) and mutant (m) HOXB4 on expansion and differentiation of myeloid progenitors. Ectopic expressions of wt- and m- HOXB4 had a comparable effect on the frequency of myeloid progenitors in semi-solid assay. However, the proliferative capacity of myeloid progenitors expressing wt-HOXB4 and 1423 m-HOXB4 was considerably better than that of control (GFP only) and other two mutants. Additionally, while all HOXB4 variants conferred similar self-renewal capacity to multipotent (GEMM) progenitors, the production of single-lineage granulocytic progenitors (CFU-G) was severely compromised when cells were expressing 1426 and 1427 m-HOXB4. Conversely, the cellularity of myeloid colonies was also diminished with these mutants suggesting, that both mutations affect certain biologic function(s) of wt-HOX4 in addition to increasing its stability. On the other hand, 1423 m-HOXB4 did not seem to lose any wt-HOXB4 functions tested in in vitro assays making this mutant an interesting target for further investigation.

O cádmio (Cd) é um metal pesado encontrado naturalmente no meio ambiente em níveis da ordem de 0,5 mg.L-1. No entanto, atividades humanas têm aumentado sua concentração na biosfera, por meio de efluentes industriais não tratados. Essa elevação pode causar estresse oxidativo nas plantas, conferindo aberrações cromossômicas, danos ao DNA e alterações nos microtúbulos das células. Pesquisas têm avaliado os efeitos do Cd em diversas espécies de plantas. Entretanto, o tomateiro ainda não tem sido alvo de tais pesquisas mesmo sendo considerada uma planta modelo em outras áreas. Dessa forma, justifica-se a condução de experimentos que avaliem a genotoxicidade do Cd nesta espécie através de abordagens baseadas na citogenética. Este estudo foi conduzido para avaliar efeitos genotóxicos do Cd em células meristemáticas de raízes de tomateiro do cultivar Micro-Tom através da investigação de aberrações cromossômicas, grau de danos causados ao DNA e anormalidades das fibras do fuso mitótico e do citoesqueleto. Evidenciou-se que a partir da dose mínima de Cd empregada (variou de 0,018 a 13 mg.L-1), foram observadas aberrações cromossômicas, tais como quebras, perdas, adesões e pontes. Ainda empregando-se as mesmas doses de Cd, constataram-se danos ao DNA por meio do ensaio de cometa. Porém, de acordo com a dose de 3,7 mg.L-1 de Cd, os microtúbulos de ?-tubulinas sofreram modificações em sua estrutura que alteram a organização do citoesqueleto celular, do fuso mitótico e da citocinese. Em geral, o tomateiro apresentou sensibilidade em baixas concentrações de Cd, o que possibilita considerá-lo como uma importante planta modelo com potencial para estudos de genotoxicidade aplicados ao monitoramento ambiental e testes de compostos tóxicos. / Cadmium (Cd) is a heavy metal found naturally in the environment at low levels (around 0.5 mg.L-1). Some human activities have increased its concentration in the biosphere due to untreated industrial effluents. This increase may induce oxidative stress in plants and potentially causing chromosomal aberrations, DNA damage and changes in the cell microtubules. Researchers have evaluated the Cd effects in several plant species. However, tomato has not been the target of these researches even though it is considered as a plant model in others areas. Therefore, experiments are needed to assess the genotoxicity of Cd in this plant species through cytogenetical approaches. The present work was carried out to assess the Cd genotoxic effects on root-meristem cells of Micro-Tom based on chromosomal aberrations, DNA damage and abnormal mitotic spindle fibers and cytoskeleton. Low Cd concentrations (ranging from 0.018 to 13 mg.L-1) were able to induce chromosomal aberrations such as breaks, losses, stickiness and bridges. At the same concentrations, DNA damage was evidenced using the comet assay. Concentrations of 3.7 mg.L-1 Cd induced structural modifications of the microtubules of ?-tubulin that altered the organization of the cytoskeleton, the mitotic spindle fibers and cytokinesis. In general, tomato presented sensitivity to low concentrations of Cd. These results suggest that tomato is also a good model for studies of genotoxicity to be applied on environmental monitoring and toxic compounds assays.

Muitas respostas moduladas pela luz durante o desenvolvimento das plantas também são reguladas por hormônios vegetais, levando à hipótese da interação entre ambos os fatores. Uma ferramenta valiosa para testar tal interação seria o uso de mutantes fotomorfogenéticos e hormonais, bem como duplos mutantes combinando ambos. Em tomateiro, embora sejam disponíveis mutantes com alterações na biossíntese de fotorreceptores e/ou na transdução do sinal da luz, bem como mutantes no metabolismo e/ou sensibilidade hormonal, esses estão presentes em cultivares diferentes, o que pode limitar seu uso de modo integrado e a construção de duplos mutantes. No presente trabalho, foram introgredidas em uma única cultivar de tomateiro, Micro-Tom (cv. MT), dezenove mutações afetando a biossíntese ou a resposta a fitocromo, bem como aos hormônios auxina (AUX), citocinina (CK), giberelina (GA), ácido abscísico (ABA), etileno (ET) e brassinoesteróides (BR). Tomando-se vantagem de tal coleção, duas respostas notadamente controladas tanto pela luz quanto por hormônios foram estudadas: alongamento e acúmulo de antocianinas em hipocótilos. Para tal, foram utilizadas as seguintes abordagens: i) tratamentos exógenos de diferentes classes hormonais em mutantes fotomorfogenéticos, ii) observação de hipocótilos de mutantes hormonais crescidos na luz e no escuro, iii) observação de duplos mutantes combinando mutações hormonais e fotomorfogenéticas. Assim, o acúmulo de antocianinas foi promovido pela CK e ABA e inibido pela GA, concordando com a redução no mutante deficiente em ABA (notabilis ou not) e no mutante hipersensível à GA (procera ou pro). Apesar do mutante com baixa sensibilidade à AUX (diageotropica ou dgt) acumular exageradamente antocianinas, a aplicação exógena não evidenciou o papel da AUX, sendo, porém, coerente com a sugestão de que esse mutante possui um balanço AUX/CK voltado para CK. Tanto a aplicação exógena quanto a avaliação nos mutantes epinastic (epi), super produção de ET, e Never ripe (Nr), baixa sensibilidade ao ET, sugerem uma função limitada desse hormônio na biossíntese de antocianinas. Na luz e no escuro, AUX, CK, ABA e ET exógenos resultaram na inibição do alongamento do hipocótilo, sendo coerente com a promoção em dgt (luz), promoção em sit (luz), inibição em epi (luz e escuro). Por outro lado, GA promoveu o alongamento corroborando a promoção em pro. Contrariando o efeito exógeno da CK, brt reduziu o alongamento na luz e no escuro. No escuro, o único mutante que apresentou alongamento do hipocótilo superior a MT foi o mutante deficiente na biossíntese do phy (aurea ou au). A utilização de duplos mutantes combinando phy- e alterações hormonais mostrou uma interação aditiva (au epi, au Nr, au dgt e au sit), sinergística (au pro) e epinástica (au brt) no acúmulo de antocianinas e alongamento do hipocótilo na luz, porém nessa última resposta, au dgt e au sit indicaram uma interação sinergística. Juntos, esses resultados indicam que, embora phy possui vias distintas da AUX, ET, ABA e GA no controle do acúmulo de antocianinas e alongamento do hipocótilo, parece que esse fotorreceptor partilha vias comuns com CK em ambas as respostas. / Many responses regulated by light during plant development are also regulated by plant hormones, suggesting an interaction between these factors. One important approach to test this hypothesis is the use of photomorphogenic and hormonal mutants and double mutant analysis. Mutants with altered photoreceptor biosynthesis, light signal transduction, hormonal metabolism and hormonal sensitivity are available in tomato. However, since they are in different cultivars, this can be a limitation for their use in a comprehensive study, as well as, for the construction of double mutants. In this work we performed the introgression of nineteen mutations in a single cultivar of tomato, Micro- Tom (cv. MT). These mutations affect biosynthesis or response to phytochrome (phy), auxin (AUX), cytokinin (CK), gibberellin (GA), abscisic acid (ABA), ethylene (ET) and brassinosteroid (BR). Using this collection of hormone mutants, we studied two responses which are controlled by light and hormones: elongation and anthocyanin accumulation in hypocotyls. For this purpose, we used three approaches: i) hormonal treatment in the photomorphogenic mutants, ii) measurement of hypocotyl lengths from hormonal mutants grown under light and dark conditions and iii) double mutant (photomorphogenic-hormonal) analysis. Anthocyanin accumulation was promoted by CK and ABA and inhibited by GA. This is in accordance with the reduction of anthocyanin accumulation in the ABA deficient mutant (not) and in the GA hypersensitive mutant (pro). Although the diageotropica (dgt), auxin-insensitive mutant, showed a high anthocyanin accumulation, the addition of auxin did not supported a role for this hormone in anthocyanin accumulation. On the other hand, this could be due to a low auxin-tocytokinin ratio presented by dgt. Data from mutants with altered metabolism and sensitivity of ethylene, epinastic (epi) and Never ripe (Nr) respectively, and from plants treated with this hormone suggest a limited role of ethylene in the anthocyanin biosynthesis. Exogenous AUX, CK, ABA and ET inhibited the hypocotyl elongation. This is coherent with the promotion of hypocotyl elongation in dgt and sit mutants under light conditions and inhibition of hypocotyl elongation in the epi mutant in the light and dark. On the other hand, GA promoted the hypocotyl elongation corroborating the same effect seen in pro. The brt mutant showed a reduced hypocotyl elongation in light and dark conditions, which contradicts the effect of exogenous cytokinin. The phytochromedeficient aurea (au) mutant was the only one to show an enhanced hypocotyl elongation in the dark compared to the wild type (MT). The combination between photomorphogenic and hormonal mutants (double mutants) showed additive (au epi, au Nr, au dgt e au sit), synergistic (au pro) and epistatic (au brt) interactions considering the anthocyanin accumulation and hypocotyl elongation. Synergistic interaction was observed in the elongation hypocotyl of the au dgt and au sit double mutants. These results indicate that phy and CK may share some signaling/metabolic pathways in the control of anthocyanin accumulation and hypocotyl elongation. On the other hand, our data do not support an interaction between phy and the hormones AUX, ET, ABA and GA in the control of hypocotyls elongation or anthocyanin accumulation.

Glucosyltransferases (GTs) are enzymes that enable transfer of glucose from an activated donor (UDP-glucose) to the acceptor substrates. A flavonol specific glucosyltransferase cloned from Citrus paradisi has strict substrate and regiospecificity (Cp3OGT). The amino acid sequence of Cp3OGT was aligned with a purported anthocyanin GT from Clitorea ternatea and a GT from Vitis vinifera that can glucosylate both flavonols and anthocyanidins. Using homology modeling to identify candidate regions followed by site directed mutagenesis, three double mutations of Cp3OGT were made. Biochemical analysis of the three mutant proteins was performed. S20G+T21S protein retained activity similar to the wildtype (WT- Kmapp-80 µM; Vmax = 16.5 pkat/µg, Mutant- Kmapp-83 µM; Vmax -11 pkat/µg) but the mutant was more thermostable compared to the WT and this mutation broadened its substrate acceptance to include the flavanone, naringenin. S290C+S319A mutant protein retained 40% activity relative to wildtype, had an optimum pH shift, but had no change in substrate specificity (Kmapp-18 µM; Vmax-0.5 pkat/µg). H154Y+Q87I protein was inactive with every class of flavonoid tested. Product identification revealed that the S20G+T21S mutant protein widened the substrate and regio-specificity of CP3OGT. Docking analysis revealed that H154 and Q87 could be involved in orienting the ligand molecules within the acceptor binding site. H363, S20, and S150 were also found to make close contact with the 7-OH, 4-OH and 3’-OH groups, respectively.

La probabilité des fautes transitoires augmente avec l'évolution des technologies. Ceci a suscité un intérêt croissant pour les méthodes prédictives d'analyse des comportements erronés d'un circuit. Ce travail porte sur l'étude de deux aspects complémentaires : l'injection de fautes dans des circuits décrits au niveau RTL et l'analyse des résultats obtenus à l'issue de campagnes d'injection.<br />Nous présentons une nouvelle approche pour la génération de mutants, permettant l'instrumentation d'un circuit pour des modèles de fautes hétérogènes. Pendant la définition d'une campagne d'injection de fautes, le flot d'analyse que nous avons proposé permet au concepteur d'introduire, dans le même circuit, des inversions de bits uniques (SEU) ou multiples (MBF), ou encore des transitions erronées. En outre, nous avons visé une génération de mutant la plus efficace selon plusieurs contraintes qui incluent (1) la modification simple et automatique de la description initiale du circuit, (2) l'optimisation des entrées additionnelles pour le contrôle d'injection et (3) la réduction du surcoût matériel après synthèse pour une bonne compatibilité avec des campagnes d'injection de fautes basées sur l'émulation.<br />Dans le flot d'analyse, un modèle comportemental est généré permettant au concepteur d'identifier les chemins de propagation d'erreurs dans le circuit. Une telle analyse vise à identifier, très tôt dans le flot de conception, les modes de défaillance inacceptables d'un circuit afin de modifier immédiatement sa description et ainsi améliorer sa robustesse.<br />Nous présentons des résultats obtenus suite à des injections multi niveaux dans des descriptions VHDL de circuits numériques. Ces résultats démontrent qu'une campagne d'injection réalisée très tôt dans le processus de conception, sur une description encore très éloignée de l'implémentation finale, peut donner des informations très utiles sur les caractéristiques de sûreté d'un circuit.

X-ray crystallography is a powerful method for determining the three-dimensional structures of biological macromolecules at atomic resolution. Crystallography can reliably provide the answer to many structure related questions, from global folds to atomic details of bonding. Crystallographic techniques find wide applications in understanding macromolecular assembly, enzyme mechanism, mode of activation of enzymes, substrate-specificity, ligand-binding properties, domain movement etc. The knowledge of accurate molecular structures is also a prerequisite for rational drug design and for structure based functional studies to aid the development of effective therapeutic agents. The current thesis can be broadly divided into two major parts. The first four chapters deal with assembly studies that have been carried out on Sesbania mosaic virus and the next two chapters describe the structure and function of a stationary phase survival protein, SurE from Salmonella typhimurium. In both studies X-ray crystallographic techniques have been used extensively for the structural studies. Viruses are obligate parasites with a proteinaceous capsid enclosing the genetic material. For genetic economy, several copies of capsid proteins self assemble to form complex virus capsids. Due to their intricate symmetric structures, viruses are considered as minute marvels of molecular architecture and study of virus structures serve as a paradigm for solutions to problems concerning macromolecular assembly and function in general. Crystallography provides a means of visualizing intact virus particles as well as their isolated constituent proteins and enzymes at near-atomic resolution, and is thus an extraordinarily powerful tool for understanding the function of these biological systems. Protein-protein interactions, protein-nucleic acid interactions, metal-ion mediated interactions, interactions between capsid proteins and auxillary or scaffolding proteins and particle maturation or post processing of capsid protein subunits are various elements that play a role in capsid assembly. Many structural and sequential motifs have been proposed as important conformational switches of capsid assembly. A functional analysis of these motifs by way of mutations in the capsid protein and structural studies of these mutants can provide further insight into capsid assembly pathways. Interaction between capsid protein subunits can determine the size and robustness of the capsid. Analysis of protein-protein interactions can help in understanding the principles of self-assembly. Arresting capsid assembly by disrupting intersubunit interactions and trapping the assembly intermediates will be helpful to delineate the changes that happen in capsid protein during the course of assembly and understand assembly pathways. Sesbania mosaic virus (SeMV) is a plant virus with a positive sense single-stranded RNA genome and belongs to the Sobemovirus genus. The protein and nucleic acids of SeMV can be separated and reassembled in vitro. Also, expression of the coat protein (CP) gene of SeMV in E. coli leads to the formation of virus like particles (VLPs). Therefore, SeMV is an excellent model system to study the assembly pathways that lead to the formation of complex virus shells. Earlier structural and functional studies on the native virus and the recombinant capsid protein and its various mutants have revealed the following: SeMV is a T=3 virus with chemically identical A-, B- and C-subunits occupying quasi equivalent positions in the icosahedral asymmetric unit of the virus particle. The A-type subunits form pentamers at the five-fold, and the B- and C- type subunits form hexamers at the icosahedral three-fold axes. The amino terminus of the polypeptide is ordered from residue 72 in the A- and B- subunits whereas it is ordered from residue 44 in the C-subunit. The disordered segment in all the subunits has an arginine rich motif (N-ARM). The segment ordered only in C-subunits has a -annulus structure that promotes intersubunit interactions at the quasi six-fold and a -segment (A). The virus is stabilized by protein-protein, protein–RNA and Ca2+ mediated protein-protein interactions. Virus like particles (VLPs) formed by the expression of full length CP encapsidate 23 S E. coli rRNA and CP mRNA. Expression of a deletion mutant lacking the N-terminal 65 residues (rCP∆N65) which results in the removal of the N-ARM, the -annulus and the A leads to the formation of stable T=1 particles. The -annulus, which was earlier believed to be an important molecular switch controlling the assembly of T=3 VLPs was found to be dispensable. The N-ARM, though important for RNA encapsidation, was not essential for capsid assembly . Depletion of Ca2+ ions led to slight swelling of virus particles and significantly reduced stability. Extensive studies on the VLPs suggested that the assembly is most likely initiated by the dimers of the capsid protein. Following a brief account of the historical highlights in the field of structural virology, a review of current literature on the available crystal structures of viruses and various assembly studies on viruses that have been carried out with emphasis on role of nucleic acid mediated interactions, protein-protein interactions and role of specific residues and ion-mediated interactions in assembly are presented in Chapter I of the thesis. A separate section in this chapter deals with the disassembly experiments that have led to the formation of smaller oligomers of spherical viruses. This chapter also gives an account of the earlier work that has been carried out on SeMV, which is the model system of study for the present thesis. Chapter II describes in detail the structural studies on the β-annulus deletion mutant of SeMV. A unique feature of several T = 3 icosahedral viruses is the presence of a structure called the β-annulus formed by extensive hydrogen bonding between protein subunits related by icosahedral three-fold axis of symmetry. This unique structure has been suggested as a molecular switch that determines the T = 3 capsid assembly. In order to examine the importance of the β-annulus, a deletion mutant of Sesbania mosaic virus coat protein in which residues 48–59 involved in the formation of the β-annulus were deleted retaining the rest of the residues in the amino terminal segment (rCP (Δ48–59)) was constructed. When expressed in Escherichia coli, the mutant protein assembled into virus like particles of size close to that of the wild type virus particles. The purified capsids were crystallized and their three dimensional structure was determined at 3.6Å resolution by X-ray crystallography. The mutant capsid structure closely resembled that of the native virus particles. However, surprisingly, the structure revealed that the assembly of the particles has proceeded without the formation of the β-annulus. Therefore, the β-annulus is not essential for T = 3 capsid assembly as speculated earlier and may be formed as a consequence of the particle assembly. This is the first structural demonstration that the virus particle morphology with and without the β-annulus could be closely similar. Chapter III begins with a detailed description of the interfacial residue mutations that have been carried out in SeMV with the aim of disrupting assembly and trapping an assembly intermediate. These mutations were performed in rCP as well as rCP∆N65 gene. Among these, a single point mutation of a Trp 170 to a charged residue (either Glu or Lys) arrested virus assembly and resulted in stable dimers of the capsid protein. The chapter also gives an account of the biophysical characterization of these mutants. rCP∆N65 dimer mutants showed a characteristic 230 nm peak in CD spectral studies which may be due to the interactions of a stretch of aromatic residues in the capsid protein. The isolated dimers were more susceptible to trypsin cleavage compared to the assembled capsids due to the exposed basic amino terminus. Thermal melting studies showed that the isolated dimer mutants were much less stable when compared to the assembled capsids, probably due to the loss of intersubunit interactions and Ca2+ mediated interactions. The structure of one of the isolated dimer mutant- rCP∆N65W170K was solved to a resolution of 2.65Å. Chapter IV describes the crystal structure analysis of the rCP∆N65W170K mutant dimer and compares its structure with the dimers of native virus, T=3 and T=1 VLPs. A number of structural changes occur especially in the loop and interfacial regions during the course of assembly. The dimer in solution was “more relaxed” than the dimer that initiates assembly. Ca2+ ion is not bound and consequently the C-terminal residues are disordered. The FG loop, which interacts with RNA, was found to be flexible and adopts a different conformation in the unassembled dimer. The present thesis also deals with the structural and functional studies of a phosphatase, SurE, the stationary phase survival protein from Salmonella typhimurium. Chapter V provides a general introduction on Salmonella, which is a mesophilic food borne pathogen, its general features, classification and stress responses. This chapter also gives an account of stationary phase in bacteria and stress responses. A brief description about phosphatases and their classification is also presented in this chapter. Following this, a review of the current literature on the structural, biochemical and functional role of stress related proteins and phylogenetic and enzymatic studies of various homologues of SurE are described in detail. Chapter VI deals with the detailed crystal structure analysis of SurE, the first stationary phase survival protein from a mesophilic organism. SurE, of Salmonella typhimurium forms part of a stress survival operon regulated by the stationary phase RNA polymerase alternative sigma factor. SurE is known to improve bacterial viability during stress conditions. It functions as a phosphatase specific to nucleoside monophosphates. Here we report the X-ray crystal structure of SurE from Salmonella typhimurium (St SurE). The protein crystallized in two forms- orthorhombic F222 and monoclinic C2. The two structures were determined to resolutions of 1.7Å and 2.7Å, respectively. The protein exists as a domain swapped dimer. The residue Asp 230 is involved in several interactions that are probably crucial for domain swapping. A divalent metal ion is found at the active site of the enzyme, which is consistent with the divalent metal-ion dependent activity of the enzyme. Interactions of the conserved DD motif present at the N-terminus with the phosphate and the Mg2+ present in the active site suggest that these residues play an important role in enzyme activity. The divalent metal ion specificity and the kinetic constants of SurE were determined using the generic phosphatase substrate- para- Nitro Phenyl Phosphate. The enzyme was inactive in the absence of divalent cations and was most active in the presence of Mg2+. Thermal denaturation studies showed that St SurE is much less stable compared to its homologues and an attempt was made to understand the molecular basis of the lower thermal stability based on solvation free energy. The thesis concludes with a brief summary of the entire work that have been presented and future prospects. The various crystallographic, biochemical and biophysical techniques employed in the investigations are described under the section experimental techniques in Appendix I and the NCS matrices used in the structure solution of the β-annulus deletion mutant are listed in Appendix II.

Prokaryotic cells such as Escherichia coli use glucose as their preferred carbon source. In the absence of glucose, these cells resort to other sources to generate glucose and this process of de novo synthesis of glucose is termed gluconeogenesis. Phosphoenolpyruvate carboxykinase (Pck) is one of the three enzymes important in regulating gluconeogenesis. It converts oxaloacetic acid (OAA) from the Krebs cycle to phosphoenolpyruvate (PEP), a glycolytic intermediate. The Pck structural gene (pckA) is regulated by catabolite repression. There is a 100-fold induction of pckA-lacZ fusions at the onset of stationary phase concurrent with induction of glycogen synthesis. Mutants affecting the expression of pckA were analysed to shed some light on the mechanism of its genetic regulation.<p>Spontaneous mutants isolated with Pck- (lack of PEP carboxykinase activity) and Suc- (inability to utilise succinate as carbon source) phenotypes were previously characterised as atpG mutants defective in the ã subunit of ATP synthase.<p>In this work we find by reverse transcriptase and real time quantitative PCR that levels of pckA mRNA are normal in the atpG mutants and that the defects in expression of pckA are therefore likely at the level of translation, protein assembly and/or protein degradation. As expected, ATP synthase activity and proton pumping in inside-out membrane vesicles were defective in these atpG mutants. It is likely that one of these defects is affecting regulation or expression of the pckA gene. It was observed that atpG mutants were defective in calcium-dependent transformation although they could be made competent for electroporation. The atpG mutants were also defective for growth of P1 bacteriophage although they could serve as recipients for P1-dependent generalised transduction. These latter phenotypes are also likely due to defects in energy metabolism.