Thèse de doctorat : Sciences de la Vie et de la Santé. Biochimie : Bordeaux 1 : 2008. / Titre provenant de l'écran-titre.

The hallmark of Gram-negative bacteria is the presence of an outer membrane (OM) surrounding the cytoplasmic membrane (here called the inner membrane [IM]) and the cell wall. The OM is a unique asymmetric bilayer with an inner leaflet consisting of phospholipid and an outer leaflet consisting of lipopolysaccharide (LPS). LPS is a large anionic molecule that typically contains six fatty acyl chains and up to several hundred sugar residues. This chemical structure explains why the OM is relatively impermeable to large hydrophobic molecules, such as detergents, bile salts, and high molecular weight antibiotics, which readily cross a normal phospholipid bilayer. LPS and the OM are essential to the viability of most Gram-negative organisms, including major human pathogens. LPS molecules are biosynthesized at the IM and subsequently exported out of the IM, across the intermembrane space (the periplasm) and through the OM to their final position at the cell surface. In Escherichia coli, the essential LPS transport proteins, LptA-G, are required for this process. This Lpt pathway includes an IM adenosine triphosphate binding cassette (ABC) transporter, LptBFG, which is associated with an additional IM protein, LptC; a periplasmic protein, LptA; and an OM complex consisting of the lipoprotein LptE and the transmembrane \(\beta\)-barrel protein LptD. All seven Lpt proteins associate as a single complex that spans the cell envelope. However, little is known about how these proteins work together to transport LPS. Here, we use in vivo and in vitro biochemical studies to probe the organization, function, and assembly of the Lpt machine. In Chapter 2, we show that LptE forms a plug within the LptD \(\beta\)-barrel and present a model for how this unusual structure can move LPS from the periplasm directly into the outer leaflet of the OM. In Chapter 3, we demonstrate that the Lpt transenvelope bridge consists of a series of structurally homologous domains – LptC, LptA, and the N-terminal domain of LptD – stacked in a head-to-tail orientation, providing a route for LPS from the IM to the OM. Finally, in Chapter 4, we connect these two sets of results by showing how the assembly of the Lpt transenvelope bridge is regulated by that of the LptD/E complex in the OM. Together, these findings explain how the functions of the Lpt proteins are coordinated to ensure delivery of LPS to the correct cellular compartment. A fundamental understanding of LPS biogenesis will contribute to the development of new therapies against Gram-negative infections.

Five strains (9L, 642A, 714, 5688T and 3384Y) of Histophilus ovis were investigated with respect to iron acquisition. All strains used ovine, bovine and goat, but not porcine or human, transferrins (Tfs) as iron sources for growth. In solid phase binding assays, total membranes from only two (9L and 642A) of the five strains, grown under iron-restricted conditions, were able to bind Tfs (ovine, bovine and goat, but not porcine or human). However, when the organisms were grown under iron-restricted conditions in the presence of bovine Tf, total membranes from all strains exhibited Tf binding (as above); competition experiments demonstrated that all three Tfs (ovine, bovine and goat) were bound by the same receptor(s). An affinity isolation procedure allowed the isolation of two putative Tf-binding polypeptides (78 and 66 kDa) from total membranes of strains 9L and 642A grown under iron-restricted conditions, and from membranes of all strains if the growth medium also contained Tf. A gene encoding a Pasteurella multocida TbpA homologue was shown to be present in each of two representative strains (9L and 3384Y); these genes were sequenced and determined to be the structural genes encoding the 78-kDa Tf-binding polypeptides. The identification of a fur homologue and a Fur box within the promoter region of tbpA in both strains indicated that Fur (and iron) is responsible for the iron-repressible nature of Tf-binding activity. Although tbpA transcripts were detected by reverse transcription (RT)-PCR with RNA isolated from strains 9L and 3384Y grown under iron-restricted conditions, with strain 3384Y, and depending on the primer pair, tbpA transcripts were detected by RT-PCR predominantly when the RNA was isolated from cells grown under conditions of iron-restriction in the presence of Tf. The presence of an additional G in the tbpA gene of strain 3384Y grown under iron-replete conditions, compared to organisms grown under iron-restricted conditions plus bovine Tf, is

Abscisic acid (ABA) is a key plant hormone regulating agronomically important processes including seed maturation and dormancy, stomatal opening and closure, along with the transcriptional and physiological response of plants to abiotic and biotic stresses. The current study sought to functionally characterize members of an ABA-responsive gene family encoding GRAM (Glucosyltransferases, Rab-like GTPase activators and Myotubularins) domain proteins in Arabidopsis thaliana. Utilizing reverse genetics loss- and gain-of-function lines associated with GEM-RELATED 5 (GER5) were obtained, which displayed several defects in reproductive development. Gene expression profiling, RNA in situ hybridization and immunohistochemical techniques were utilized to evaluate GER5 and two closely related GRAM genes, GEM-RELATED 1 (GER1) and GLABRA2 EXPRESSION MODULATOR (GEM) in reproductive structures. Microarray profiling of seeds from ger5-2 mutants and wild-type plants revealed transcriptional changes in carbohydrate metabolism, hormone signaling and catabolic processes accompanied seed development defects of ger5-2 mutants. Seed germination assays further revealed ger5-2 mutants exhibited reduced sensitivity to ABA. In assessing GER5, GER1 and GEM as putative ABA-response genes, a second study evaluated the expression of GRAM, AuTophaGy-related (ATG), and ABA-response genes in source and sink organs exposed to abiotic stress or within mutant backgrounds deficient in sugar signaling. Monodansylcadaverine (MDC) staining was also utilized to localize autophagosomes or autophagic bodies within vegetative or reproductive organs during plant development, or in response to carbon starvation or abiotic stress. In a third study transcriptional differences in ABA metabolism, transport and homeostasis were examined within reproductive organs (cauline leaves, inflorescence meristem, developing siliques) exposed to cold and heat stress. This study revealed reproductive organs are characterized by unique patterns of ABA metabolism which differ from tissues typically associated with classical ABA responses. Together, these studies indicate GER5, an uncharacterized ABA-responsive GRAM domain gene, plays a novel role in the reproductive development of plants and that ABA metabolism and signaling are uniquely regulated in reproductive organs.

Pseudomonas aeruginosa es un bacilo gram-negativo, patógeno oportunista, ampliamente distribuído en la naturaleza. Se investigó la presencia de dicho microorganismo en el bioterio convencional de la sección Leucemia Experimental del Instituto de Investigaciones Hematológicas "Mariano Castex" de la Academia Nacional de Medicina de Buenos Aires. La finalidad del estudio fue establecer las medidas necesarias para su erradicación. Se detectó la presencia del microorganismo en el ambiente (jaulas, estanterías, en un pequeño porcentaje de los animales, y en uno de los 4 miembros de personal). Se estableció la asociación entre endoparasitosis y colonización intestinal por P. aeruginosa. Los niveles séricos de IgG anti-P. aeruginosa estaban aumentados en animales portadores de P. aeruginosa. Se tomaron medidas tendientes a la erradicación del microorganismo. Se cambió el desinfectante usado en el bioterio y se erradicaron los ectoparásitos por tratamiento con piretrina y limonero, y los endoparásitos con piperacina. Se mejoró el estatus sanitario de los animales y se calificó a la colonia de ratones de la Academia Nacional de Medicina como convencional monitoreada grado II.

No description available.

Abscisic acid (ABA) is a key plant hormone regulating agronomically important processes including seed maturation and dormancy, stomatal opening and closure, along with the transcriptional and physiological response of plants to abiotic and biotic stresses. The current study sought to functionally characterize members of an ABA-responsive gene family encoding GRAM (Glucosyltransferases, Rab-like GTPase activators and Myotubularins) domain proteins in Arabidopsis thaliana. Utilizing reverse genetics loss- and gain-of-function lines associated with GEM-RELATED 5 (GER5) were obtained, which displayed several defects in reproductive development. Gene expression profiling, RNA in situ hybridization and immunohistochemical techniques were utilized to evaluate GER5 and two closely related GRAM genes, GEM-RELATED 1 (GER1) and GLABRA2 EXPRESSION MODULATOR (GEM) in reproductive structures. Microarray profiling of seeds from ger5-2 mutants and wild-type plants revealed transcriptional changes in carbohydrate metabolism, hormone signaling and catabolic processes accompanied seed development defects of ger5-2 mutants. Seed germination assays further revealed ger5-2 mutants exhibited reduced sensitivity to ABA. In assessing GER5, GER1 and GEM as putative ABA-response genes, a second study evaluated the expression of GRAM, AuTophaGy-related (ATG), and ABA-response genes in source and sink organs exposed to abiotic stress or within mutant backgrounds deficient in sugar signaling. Monodansylcadaverine (MDC) staining was also utilized to localize autophagosomes or autophagic bodies within vegetative or reproductive organs during plant development, or in response to carbon starvation or abiotic stress. In a third study transcriptional differences in ABA metabolism, transport and homeostasis were examined within reproductive organs (cauline leaves, inflorescence meristem, developing siliques) exposed to cold and heat stress. This study revealed reproductive organs are characterized by unique patterns of ABA metabolism which differ from tissues typically associated with classical ABA responses. Together, these studies indicate GER5, an uncharacterized ABA-responsive GRAM domain gene, plays a novel role in the reproductive development of plants and that ABA metabolism and signaling are uniquely regulated in reproductive organs.

Staphylococcus aureus is a Gram positive potentially pathogenic bacterium which has a propensity to gather resistance determinants. Mupirocin is a novel topical antibiotic active against many Gram positive species, including staphylococci and effective in the treatment and prevention of staphylococcal infections. Mupirocin acts by competitively inhibiting the charging of isoleucyl tRNA-synthetase (IRS) with isoleucine. Resistance has been observed in Staphylococcus aureus and coagulase negative staphylococci. Intermediate-level resistance (MIC >8μg ml^-1 >512μg ml^-1) is thought to be due to spontaneous mutations in the native IRS. High-level resistance (>512μg ml^-1) is conferred by a second IRS protein, encoded by mupA which has a much lower affinity for mupirocin than isoleucine. The mupirocin resistance gene (mupA) is usually found on a 4.05kb EcoRI fragment of plasmids of otherwise varied EcoRI restriction pattern which are easily transferred between strains by filter mating. Prior to the onset of these studies, mupirocin resistance had not been found linked to another resistance determinant. Initial investigations intended to identify mechanisms of gene flux resident on mupA plasmids revealed a family of related mupA plasmids, the p3356 family which includes three plasmid types: p3356, p3356D and p3358. p3356 contains a single copy mupA flanked by direct repeats of the staphylococcal insertion sequence IS257. p3356D is identical to p3356 except for the duplication of a "mupA-IS257" cassette in tandem repeat. p3358 is related to p3356D by the insertion of a pT181-like plasmid (tetracycline resistant) accompanied by the duplication of an IS257 in direct repeat to flank the inserted pT181; thus p3358 is the first documented example of linked resistance between mupirocin and another resistance determinant, namely tetracycline. IS257 has been implicated as the recombinogenic site in the gene duplication event involved in the evolution of p3356D from p3356. IS257 co-integrative transposition has been demonstrated to allow the co-integration of pOX7 with p3356 to generate a p3358-type plasmid in which pT181 is replaced by pOX7. Therefore, it is concluded that IS257 transposition and recombination is a mechanism by which staphylococcal replicons can evolve to form multiply resistant replicons.

Corrigenda pasted onto front fly-leaf. / Bibliography: leaves 247-286. / xv, 286 leaves : ill. (some col.) ; 30 cm. / Title page, contents and abstract only. The complete thesis in print form is available from the University Library. / This thesis examines an aspect of the pathogenesis of a model extracellular enteric pathogen, the causative agent of human cholera. The export of TcpA (Toxin-Coregulated Pilus) and assembly of the TCP is explored as a paradigm of macromolecular export in Gram negative bacteria. TcpA is examined in detail in an attempt to define strictly conserved regions between species. The TCP of the emergent 0139 (Bengal) serotype is demonstrated to be of El Tor type. The possibily that proteases such as the soluble haemagglutinin (SHA) may have a detachase role centring on TCP dispersal/TcpA degradation is also discussed. / Thesis (Ph.D.)--University of Adelaide, Dept. of Microbiology, 1997

Thesis (Ph.D.)--McMaster University, 2006. / Supervisor: E.D. Brown. Includes bibliographical references (leaves 95-104).