In Escherichia coli, FhuA, together with the energy-transducing TonB-ExbB-ExbD complex, mediates the uptake of hydroxamate-type siderophores across the outer membrane. In addition to transporting ferrichrome, FhuA functions as the primary receptor for several bacteriophages, antibiotics, and colicin M. To comprehend how FhuA binds its cognate ligands, and to establish the molecular basis of active transport, the three-dimensional structure must be determined. In pursuit of this goal, an affinity tag was inserted into the fhuA gene at a known surface-exposed location. The recombinant FhuA protein was overexpressed and purified by metal-chelate chromatography. Sparse matrix screening established parameters for the growth of well-ordered crystals of FhuA. The crystallographic structure of FhuA and its complex with ferricrocin were solved at 2.50 and 2.70 A by multiwavelength anomalous dispersion. The crystallographic structure reveals that FhuA is a monomeric integral outer membrane protein that is composed of two domains. The C-terminal domain, a 22-stranded antiparallel beta-barrel, spans the outer membrane. Positioned within the beta-barrel is the N-terminal 'cork' domain, which is formed by a mixed four-stranded beta-sheet with four interspersed alpha-helices. Upon binding of ferricrocin, conformational changes are propagated from the extracellular pocket to the periplasmic pocket of FhuA; a transmembrane signal indicating the liganded status of the receptor. Structural analysis combined with sequence homologies and mutagenesis data are used to propose a mechanism for TonB-dependent siderophore-mediated iron acquisition. / Noncovalently associated with the membrane-embedded surface of FhuA is a single lipopolysaccharide molecule. Following the examination of electrostatic protein-lipid contacts, a structure-based algorithm revealed a conserved four-residue LPS-binding motif that is present among known LPS-binding proteins. / An expansion of these structural studies was the solution of the crystallographic structures of additional liganded complexes. The three-dimensional structures of FhuA in complex with the antibiotics albomycin and rifamycin CGP 4832 were determined at 3.10 and 2.90 A, respectively. In addition to probing the plasticity and accessibility of the ligand-binding site, these results establish a structural platform for the design of novel antibiotics. Moreover, insights into the physical association of FhuA with TonB can be derived from these data.

The genus Mycobacterium is best recognized for its pathogens M. tuberculosis and M. leprae, the etiologic agents of Tuberculosis and Leprosy. Sequencing of their genomes has revealed an evolutionary process of reductive genomics. Another common species, the M. avium complex (MAC), consists of both environmental isolates that can cause opportunistic infection in humans as well as pathogenic isolates that cause disease primarily in birds and livestock. The basis of this variation in disease phenotypes is unknown. Two genome sequences representing a pathogen of cattle, M. avium subsp. paratuberculosis (MAP) and an opportunistic isolate from a human (M. avium subsp. hominissuis) have served as the foundation for the comparative genomics of MAC. This is complicated by a level of genetic variability one log greater than found within the M. tuberculosis complex (MTBC), and by the existence of other MAC subsets beyond the two sequenced strains. In this thesis, I set out to define the phylogenetic relationships of the various members of MAC and explore the evolutionary processes that led to the emergence of the pathogenic species MAP. An identification scheme was developed to unambiguously brand subsets of MAC, a tool lacking in the past thus hampering data interpretation. Expansion to a multilocus sequence analysis (MLSA) system revealed that the MAC consists of a highly variable group with most of the genotypes belonging to what is considered to be the environmental subset. However, both the avian and MAP pathogens manifested as two separate clones that have independently evolved from their larger subset. The distribution and directionality (insertion or deletion) of large se / Le genre Mycobacterium est mieux reconnu pour ses espèces pathogènes M. tuberculosis et M. leprae, les agents étiologiques de la tuberculose et de la lèpre. Le séquençage de leur génome a indiqué un processus évolutionnaire de réduction génomique. Des autres espèces communes, le complexe M. avium (MAC) est composé de souches environnementales qui peuvent causer des infections opportunistes chez l'homme aussi bien que de souches pathogènes qui causent la maladie principalement chez les oiseaux et le bétail. La base de cette variation phénotypique est inconnue. Deux séquences génomiques représentant le pathogène de bétail M. avium subsp. paratuberculosis (MAP) et un isolat opportuniste d'humain (M. avium subsp. hominissuis) ont servi de base pour la génomique comparative du MAC. Ceci est compliqué par un niveau de variabilité génétique d'un ordre logarithmique plus grand que celui qui se trouve dans le complexe de M. tuberculosis (MTBC), et par l'existence d'autres sous-ensembles de MAC au-delà des deux souches séquençées. Dans cette thèse, je cherche à définir les rapports phylogénétiques des divers membres du MAC et à explorer les processus évolutionnaires qui ont mené à l'apparition de l'espèce pathogène MAP. Une technique d'identification a été développée pour déterminer clairement les sous-ensembles de MAC, un outil dont l'absence par le passé limitait l'analyse des données. La possibilité d'utiliser un système d'analyse par séquençage multi-locus (MLSA) a révélé que le MAC est composé d'un groupe hautement variable avec la plus grande partie des génotypes appartenant à ce que l'on considère comme étant le$

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The frequency of transformation of rat cells obtained with recombinant plasmid DNAs which contained an intact early region of the polyoma genome (Bam clones) was not significantly different from those which carry an interrupted early region (Eco RI clones) or even part of the early region (Hind III-1 fragment transformant). The viral DNA within several Bam and Eco RI clones as well as fragment transformants was examined by Southern blotting. These cell lines contain integrated viral sequences which sometimes are tandemly arranged. We also found that the presence of large T antigen in transformed cells does not significantly alter the frequency at which tandem integration occurs. Furthermore, Rat-1 cells in culture were transformed with cellular DNA isolated from one Hind III-1 fragment transformant. These studies showed that the oncogenic potential of the viral DNA is maintained after integration. These findings demonstrate that only part of the polyoma early region is required to initiate and maintain transformation.

A library of cDNA clones was prepared from measles virus infected cells. Six classes of viral specific cDNA sequences were identified by a combination of Northern and Southern blot hybridization analysis. / Clones corresponding to measles virus NP, P/C and M genes were initially identified by hybridization selection translation experiments. Restriction mapping analysis and comparison with the recently published nucleic acid sequences have confirmed the identities of these clones. / The cloned sequences corresponding to the NP and M genes were subcloned into a plasmid vector which contains the Salmonella phage SP6 promoter sequence. These constructions facilitated the in vitro synthesis of RNA. This cloned RNA was subsequently translated in vitro, giving rise to the measles virus NP and M proteins. In addition to the full length NP and M proteins, smaller peptides were observed in this in vitro expression system. The series of smaller peptides made from the cloned NP RNA appear to correlate with NP-related peptides which are seen in extracts prepared from infected cells. / The other three classes of viral specific clones could not be conclusively identified by hybridization selection translation experiments. However, northern blot hybridization analysis enabled a tentative assignment of these three cloned sequences to the measles virus HA, F and L genes. The clone corresponding to the F protein was subsequently expressed in vitro using the SP6 transcription vector system described above.

Cell surface T antigen, detected by a radioimmune assay using ('125)I-labeled S. aureus protein A and antibodies against either authentic T antigen or D2 T antigen, was found in SV40-transformed and -infected cells and in Ad2('+)D2-infected cells. / To determine how the target antigen of this assay is related to SV40 large T antigen and D2 T antigen and whether or not it is an integral membrane protein, plasma membranes from cell lines infected with SV40 or Ad2('+)D2 were purified. Protein blotting with polyclonal and monoclonal antibody overlay was used to identify a specific protein of 90 kd in membranes from SV40-transformed and lytically infected cells that was indistinguishable from nuclear T antigen. The blotting procedure and overlay with polyclonal anti-D2 T antibody showed that plasma membranes from HeLa cells infected with Ad2('+)D2 contained a 100 kd protein that was also indistinguishable from its nuclear counterpart. Unlike SV40 membrane T antigen, membrane D2 T antigen was produced in large quantities. Up to 40% of membrane D2 T antigen remained tightly associated with membranes under alkaline conditions (to pH 11.5). It could not be totally solubilized by treatment with ionic detergents or nonionic detergents or both. Contamination of membrane D2 T antigen by nuclear D2 T antigen in purified plasma membrane preparations was less than 5 percent as demonstrated by reconstitution experiments. / Velocity sedimentation analysis showed that both nuclear and membrane D2 T antigen existed as heterogeneous molecules of predominantly 5 to 7 S with smaller amounts of 14 to 16 S and 23 S when centrifuged under low salt conditions. The higher S value forms could be dissociated to the 5 to 7 S forms when centrifuged in the presence of high salt concentrations. Sephacryl S-300 chromatography in high salt in the presence of Brij-99 showed that the nuclear form was dissociated to low molecular weight species while the membrane form eluted as a complex of high molecular weight. This indicates that the membrane form of D2 T antigen contains a hydrophobic region not present in the nuclear form and suggests a difference between the two not hitherto described.

Infection of L cells with reovirus results in a gradual inhibition of host protein synthesis and a concomitant increase in viral specific protein synthesis. Previous in vitro studies using cell-free extracts from uninfected and infected cells suggested that the observed pattern of protein synthesis in infected cells is the consequence of a transition of the host translational apparatus from cap dependence to cap independence. To test this hypothesis, it was necessary to demonstrate that mRNA being translated at late times in infected cells was uncapped. Progeny subviral particles synthesize the bulk of the viral mRNA present in infected cells at late times after infection. Therefore, progeny subviral particles were isolated and the mRNA synthesized by these particles was subjected to 5' terminal analysis. The results of these experiments indicated that progeny subviral particles synthesize uncapped mRNA with the 5' terminal structure pGpC... . Examination of the 5' termini of reovirus mRNA's associated with polyribosomes at different times post infection clearly showed that uncapped viral mRNAs (pG... terminated) were translated with increasing predominance as a function of time after infection. Thus, at late times only uncapped viral mRNAs were translated, while capped host mRNAs, although still present in infected cells, were excluded from polyribosomes. It was concluded from these findings that the mechanism for takeover of protein synthesis by reovirus involves a transition to cap independent translation coupled to the synthesis of large quantities of uncapped viral mRNAs. Further studies indicated that crude initiation factor preparations from uninfected L cells restored the ability of cell-free extracts from infected cells to translate capped mRNAs. Crude initiation factor preparations from late infected cells did not have restoring activity. Numerous studies have shown that cap binding proteins present among eucaryotic initiation factors facilitate transla

Model organism Caenorhabditis elegans is a 1 mm long, soil-dwelling hermaphrodite nematode, which feeds on bacteria. Contributing to a peptidomic project, procedures to compare the peptide content of different life stages have been developed for monitoring protein expression, processing and turnover. Sequential step and continuous gradient RP-HPLC fractionation and mass spectrometry have proved effective. / A class of cysteine-rich peptide previously discovered in this laboratory has been studied. Three chromosome V genes encode such peptides. The small, cysteine-rich, and secreted character is reminiscent of some cysteine-containing antimicrobial peptides, notably mytilins and mytilins (Mytilus galloprovincialis ), Ascaris suum antibacterial factor (ASABF) and its C. elegans homologues (ABF-1, ABF-2). ABF-2 is antimicrobial against Gram positive bacteria. / To obtain sufficient peptide for structural and functional studies, recombinant genes have been expressed in yeast, to produce native and epitope-tagged forms. Their activity remains unknown, but ABF2 has also been expressed and is active.

The dormancy (DosR) regulon of Mycobacterium tuberculosis is normally induced in response to hypoxia or nitric oxide (NO). Intriguingly, in the absence of stimuli, the DosR regulon is constitutively over-expressed in Beijing lineage strains (72). Expression of the DosR regulon is controlled by the DosR/DosS/DosT two-component system (TCS). Analysis of Beijing (n=26) and non-Beijing (n=50) isolates revealed a frameshift mutation in the dosT gene that is restricted to the most recently evolved Beijing sub-lineages (groups 2 to 5). The presence of this mutation is positively correlated to the level of dosR expression under normal growth conditions. However, expression of mutant dosT in a non-Beijing strain did not increase dosR expression and complementation of Beijing strains with wild-type dosT did not reverse the DosR regulon phenotype, together indicating that the dosT mutation is not directly responsible for DosR regulon over-expression in Beijing strains. When Beijing isolates were exposed to a NO-donor known to induce dosR expression, dosT mutant strains exhibited a decreased response to NO compared to wild-type dosT strains, while strains complemented with wild-type dosT had an increased response to a NO-donor. The dosT frameshift appears to be a compensatory mutation that may act to limit further induction of the DosR regulon in response to stimuli like NO. This work shows that M. tuberculosis strains are variable in their response to signals encountered during host infection and further characterizes the unique biochemical features of the Beijing lineage. / Le régulon de dormance (DosR) de Mycobacterium tuberculosis est normalement induit en réponse à l'hypoxie ou à l'oxide nitrique (NO). Étonnament, parmi les souches bactériennes de la lignée Beijing, le régulon DosR est surexprimé même en l'absence de stimulus (72). L'expression du régulon DosR est contrôlée par le système à deux composantes DosR/DosS/DosT. L'analyse d'isolats des lignées Beijing (n=26) ou non-Beijing (n=50) a révélé une mutation décalant le cadre de lecture du gène dosT parmi les lignées Beijing les plus récentes (sous-lignées 2 à 5). La présence de cette mutation est positivement corrélée avec le niveau d'expression du gène dosR en condition de culture normale. Cependant, l'expression du gène dosT muté dans une souche de la lignée non-Beijing n'a pas résulté en une augmentation de l'expression du gène dosR et l'introduction du gène dosT type sauvage dans une souche de la lignée Beijing n'a pas permis de diminuer la surexpression du régulon DosR. Ces deux observations indiquent que la mutation du gène dosT n'est pas directement responsable de la surexpression du régulon DosR dans la lignée Beijing. Lorsque des isolats de la lignée Beijing furent exposés à un doneur de NO reconnu comme étant un inducteur de l'expression du gène dosR, les souches possédant le gène dosT muté démontrèrent une réponse diminuée au NO comparée aux souches possédant un gène dosT type sauvage. Réciproquement, les souches complémentées avec le gène dosT type sauvage démontrèrent une réponse plus importante au NO. La mutation déphasante de dosT semble être une mutation compensatoire afin de limiter l'induction incontrôlée du régulon DosR en réponse à un stimulus comme le NO. La présente étude a démontré que les souches de M. tuberculosis ne répondent pas toutes de la même façon aux signaux reçus lors d'une infection, par exemple, et elle a permis d'identifier certaines charactéristiques