Molecular and functional characterization of the chemotactic genes in the PCBs-degrader Pseudomonas pseudoalcaligenes KF707

Although bacteria represent the simplest form of life on Earth, they have a great impact on all living beings. For example the degrader bacterium Pseudomonas pseudoalcaligenes KF707 is used in bioremediation procedures for the recovery of polluted sites. Indeed, KF707 strain is know for its ability to degrade biphenyl and polychlorinated biphenyls - to which is chemotactically attracted - and to tolerate the oxydative stress due to toxic metal oxyanions such as tellurite and selenite. Moreover, in bioremediation processes, target compounds can be easily accessible to KF707 through biofilm formation. All these considerations suggest that KF707 is such a unique microorganism and this Thesis work has been focused on determining the molecular nature of some of the peculiar physiological traits of this strain. The genome project provided a large set of informations: putative genes involved in the degradation of aromatic and toxic compounds and associated to stress response were identified. Notably, multiple chemotactic operons and cheA genes were also found. Deleted mutants in the cheA genes were constructed and their role in motility, chemotaxis and biofilm formation were assessed and compared to those previously attributed to a cheA1 gene in a KF707 mutant constructed by a mini-Tn5 transposon insertion and which was impaired in motility and biofilm development. The results of this present Thesis work, taken together, were interpreted to suggest that in Pseudomonas pseudoalcaligenes KF707 strain, multiple factors are involved in these networks and they might play different roles depending on the environmental conditions. The ability of KF707 strain to produce signal molecules possibly involved in cell-to-cell communication, was also investigated: lack of a lux-like QS system - which is conversely widely present in Gram negative bacteria – keeps open the question about the actual molecular nature of KF707 quorum sensing mechanism.

Identificación y análisis funcional de genes implicados en la homeostasis de níquel en la bacteria endosimbiótica de leguminosas Rhizobium leguminosarum

La asociación Rhizobium-leguminosa constituye una interacción planta-microorganismo particularmente beneficiosa a nivel medioambiental debido a su capacidad promotora del crecimiento vegetal en condiciones de deficiencia de nitrógeno. Se ha demostrado que una excesiva concentración de metales pesados en el suelo afecta negativamente la competitividad bacteriana y al desarrollo de interacciones diazotróficas eficientes (Chaudri et al., 2000; Pereira et al., 2006). Por otro lado, el suministro de metales como Fe, Mo, Ni o Cu es fundamental para la biosíntesis de enzimas bacterianas relacionadas con el proceso de fijación de nitrógeno que ocurre en el interior de los nódulos de las leguminosas (Moreau et al., 1995). Con objeto de identificar sistemas génicos implicados en la homeostasis de níquel en bacterias endosimbióticas, se ha llevado a cabo una mutagénesis mediante inserción aleatoria de un minitransposón derivado de Tn5 en Rhizobium leguminosarum bv. viciae UPM1137, una cepa capaz de resistir elevadas concentraciones de níquel y cobalto. Como resultado de esta mutagénesis se han obtenido 14 mutantes incapaces de crecer en medios suplementados con NiCl2. La localización de la inserción en estos mutantes muestra que una elevada proporción de los genes afectados codifican proteínas de membrana o proteínas secretadas. En paralelo, se ha obtenido la secuencia del genoma de la cepa UPM1137, lo que permite realizar estudios in silico comparando los genomas disponibles de varias cepas de R. leguminosarum bv. viciae, que presentan una menor sensibilidad a metales. El análisis bioinformático de los genomas secuenciados y la caracterización fenotípica de los mutantes obtenidos permitirá identificar potenciales sistemas de resistencia y su contribución a la homeostasis de metales.

Identification and functional characterization of Rhizobium leguminosarum bv. viciae genetic systems involved in nickel homeostasis.

A collection of Rhizobium leguminosarum bv. viciae strains isolated from ultramafic and contaminated soils in Italy and Germany, respectively, was analyzed for resistance to nickel and cobalt ions. These assays led to the identification of strain UPM1137, which is able to grow at high concentrations of nickel and cobalt. In order to identify genetic systems involved in the homeostasis to these metals, a random mutagenesis was carried out in UPM1137 by inserting a Tn5-derivative minitransposon. As a result 4313 transconjugants were obtained, being 39 of them (0.90%) unable to grow at 1.5 mM NiCl2. The identification of the transposon insertion site in these mutants showed that the disrupted genes encode proteins belonging to different functional categories, where the secreted and membrane proteins were the most numerous. The analysis of heavy metal resistance and phenotypes in symbiotic and free –living cells will define the contribution of these genes to metal homeostasis.

TnTIN and TnTAP: Mini-transposons for site-specific proteolysis in vivo

Tobacco etch virus (TEV) protease recognizes a 7-aa consensus sequence, Glu-Xaa-Xaa-Tyr-Xaa-Gln-Ser, where Xaa can be almost any amino acyl residue. Cleavage occurs between the conserved Gln and Ser residues. Because of its distinct specificity, TEV protease can be expressed in the cytoplasm without interfering with viability. Polypeptides that are not natural substrates of TEV protease are proteolyzed if they carry the appropriate cleavage site. Thus, this protease can be used to study target proteins in their natural environment in vivo, as well as in vitro. We describe two Tn5-based mini-transposons that insert TEV protease cleavage sites at random into target proteins. TnTIN introduces TEV cleavage sites into cytoplasmic proteins. TnTAP facilitates the same operation for proteins localized to the bacterial cell envelope. By using two different target proteins, SecA and TolC, we show that such modified proteins can be cleaved in vivo and in vitro by TEV protease. Possible applications of the site-specific proteolysis approach are topological studies of soluble as well as of inner and outer membrane proteins, protein inactivation, insertion mutagenesis experiments, and protein tagging.

The insect endosymbiont Sodalis glossinidius utilizes a type III secretion system for cell invasion

Sodalis glossinidius is a maternally transmitted secondary endosymbiont residing intracellularly in tissues of the tsetse flies, Glossina spp. In this study, we have used Tn5 mutagenesis and a negative selection procedure to derive a S. glossinidius mutant that is incapable of invading insect cells in vitro and is aposymbiotic when microinjected into tsetse. This mutant strain harbors Tn5 integrated into a chromosomal gene sharing high sequence identity with a type III secretion system invasion gene (invC) previously identified in Salmonella enterica. With the use of degenerate PCR, we have amplified a further six Sodalis inv/spa genes sharing high sequence identity with type III secretion system genes encoded by Salmonella pathogenicity island 1. Phylogenetic reconstructions based on the inv/spa genes of Sodalis and other members of the family Enterobacteriaceae have consistently identified a well-supported clade containing Sodalis and the enteric pathogens Shigella and Salmonella. These results suggest that Sodalis may have evolved from an ancestor with a parasitic intracellular lifestyle, possibly a latter-day entomopathogen. These observations lend credence to a hypothesis suggesting that vertically transmitted mutualistic endosymbionts evolve from horizontally transmitted parasites through a parasitism–mutualism continuum.

Further Studies of the Role of Cyclic β-Glucans in Symbiosis. An ndvC Mutant of Bradyrhizobium japonicum Synthesizes Cyclodecakis-(1→3)-β-Glucosyl1

The cyclic β-(1→3),β-(1→6)-d-glucan synthesis locus of Bradyrhizobium japonicum is composed of at least two genes, ndvB and ndvC. Mutation in either gene affects glucan synthesis, as well as the ability of the bacterium to establish a successful symbiotic interaction with the legume host soybean (Glycine max). B. japonicum strain AB-14 (ndvB::Tn5) does not synthesize β-glucans, and strain AB-1 (ndvC::Tn5) synthesizes a cyclic β-glucan lacking β-(1→6)-glycosidic bonds. We determined that the structure of the glucan synthesized by strain AB-1 is cyclodecakis-(1→3)-β-d-glucosyl, a cyclic β-(1→3)-linked decasaccharide in which one of the residues is substituted in the 6 position with β-laminaribiose. Cyclodecakis-(1→3)-β-d-glucosyl did not suppress the fungal β-glucan-induced plant defense response in soybean cotyledons and had much lower affinity for the putative membrane receptor protein than cyclic β-(1→3),β-(1→6)-glucans produced by wild-type B. japonicum. This is consistent with the hypothesis presented previously that the wild-type cyclic β-glucans may function as suppressors of a host defense response.

The chaperone/usher pathways of Pseudomonas aeruginosa: Identification of fimbrial gene clusters (cup) and their involvement in biofilm formation

Pseudomonas aeruginosa, an important opportunistic human pathogen, persists in certain tissues in the form of specialized bacterial communities, referred to as biofilm. The biofilm is formed through series of interactions between cells and adherence to surfaces, resulting in an organized structure. By screening a library of Tn5 insertions in a nonpiliated P. aeruginosa strain, we identified genes involved in early stages of biofilm formation. One class of mutations identified in this study mapped in a cluster of genes specifying the components of a chaperone/usher pathway that is involved in assembly of fimbrial subunits in other microorganisms. These genes, not previously described in P. aeruginosa, were named cupA1–A5. Additional chaperone/usher systems (CupB and CupC) have been also identified in the genome of P. aeruginosa PAO1; however, they do not appear to play a role in adhesion under the conditions where the CupA system is expressed and functions in surface adherence. The identification of these putative adhesins on the cell surface of P. aeruginosa suggests that this organism possess a wide range of factors that function in biofilm formation. These structures appear to be differentially regulated and may function at distinct stages of biofilm formation, or in specific environments colonized by this organism.

Endogenous mutagenesis by an insertion sequence element identifies Aeromonas salmonicida AbcA as an ATP-binding cassette transport protein required for biogenesis of smooth lipopolysaccharide.

Analysis of an Aeromonas salmonicida A layer-deficient/O polysaccharide-deficient mutant carrying a Tn5 insertion in the structural gene for A protein (vapA) showed that the abcA gene immediately downstream of vapA had been interrupted by the endogenous insertion sequence element ISAS1. Immunoelectron microscopy showed that O polysaccharides did not accumulate at the inner membrane-cytoplasm interface of this mutant. abcA encodes an unusual protein; it carries both an amino-terminal ATP-binding cassette (ABC) domain showing high sequence similarity to ABC proteins implicated in the transport of certain capsular and O polysaccharides and a carboxyl-terminal potential DNA-binding domain, which distinguishes AbcA from other polysaccharide transport proteins in structural and evolutionary terms. The smooth lipopolysaccharide phenotype was restored by complementation with abcA but not by abcA carrying site-directed mutations in the sequence encoding the ATP-binding site of the protein. The genetic organization of the A. salmonicida ABC polysaccharide system differs from other bacteria. abcA also differs in apparently being required for both O-polysaccharide synthesis and in energizing the transport of O polysaccharides to the cell surface.

Disulfide oxidoreductase activity of Shigella flexneri is required for release of Ipa proteins and invasion of epithelial cells.

Secretion of IpaB, IpaC, and IpaD proteins of Shigella flexneri, essential for the invasion of epithelial cells, requires a number of proteins encoded by the spa and mxi loci on the large plasmid. Introduction of dsbA::Tn5 into S.flexneri from Escherichia coli K-12 reduced invasiveness, which resulted from a decrease in the capacity to release IpaB, IpaC, and IpaD proteins into the external medium. Examination of the surface-presented Ipa proteins of the dsbA mutant, however, revealed Ipa proteins at levels similar to those on wild-type cells. Since the defective phenotype was similar to that of the spa32 mutant of S. flexneri and the Spa32 sequence possessed two Cys residues, the effect of dsbA mutation of the folding structure of Spa32 under reducing conditions and on the surface expression of Spa32 was investigated. The results indicated that Spa32 was a disulfide-containing protein whose correctly folded structure was required for its presentation on the outer membrane. Indeed, replacing either one of the two Cys residues in Spa32 with Ser by site-directed mutagenesis reduced its capacity to release Ipa proteins into the external medium and led to the accumulation of Spa32 protein in the periplasm. These results indicated that the DsbA protein performs an essential function during the invasion of mammalian cells, by facilitating transport of the Spa32 protein across the outer membrane.

tonB3 is required for normal twitching motility and extracellular assembly of type IV pili

Three mutants with Tn5-B21 insertion in tonB3 (PA0406) of Pseudomonas aeruginosa exhibited defective twitching motility and reduced assembly of extracellular pili. These defects could be complemented with wild-type tonB3.

Molecular regulation of iron uptake in pseudomonas aeruginosa

The microbial demand for iron is often met by the elaboration of siderophores into the surrounding medium and expression of cognate outer membrane receptors for the ferric siderophore complexes. Conditions of iron limitation, such as those encountered in vivo, cause Pseudomonas aeruginosa to express two high-affinity iron-uptake systems based on pyoverdin and pyochelin. These systems will operate both in the organism's natural habitat, soil and water, where the solubility of iron at neutral pH is extremely low, and in the human host where the availability of free iron is too low to sustain bacterial growth due to the iron-binding glycoproteins transferrin and lactoferrin. Cross-feeding and radiolabelled iron uptake experiments demonstrated that pyoverdin biosynthesis and uptake were highly heterogeneous amongst P.aeruginosa strains, that growth either in the presence of pyoverdin or pyochelin resulted in induction of specific IROMPs, and that induction of iron uptake is siderophore-specific. The P.aeruginosa Tn5 mutant PH1 is deficient in ferripyoverdin uptake and resistant to pyocin Sa, suggesting that the site of interaction of pyocin Sa is a ferripyoverdin receptor. Additional Tn5 mutants appeared to exploit different strategies to achieve pyocin Sa-resistance, involving modifications in expression of pyoverdin-mediated iron uptake, indicating that complex regulatory systems exist to enable these organisms to compete effectively for iron. Modulation of expression of IROMPs prompted a study of the mechanism of uptake of a semi-synthetic C(7) α-formamido substituted cephalosporin BRL 41897A. Sensitivity to this agent correlated with expression of the 75 kDa ferri-pyochelin receptor and demonstrated the potential of high-affinity iron uptake systems for targeting of novel antibiotics. Studies with ferri-pyoverdin uptake-deficient mutant PH1 indicated that expression of outer membrane protein G (OprG), which is usually expressed under iron-rich conditions and repressed under iron-deficient conditions, was perturbed. Attempts were made to clone the oprG gene using a degenerate probe based on the N-terminal amino acid sequence. A strongly hybridising HindIll restriction fragment was cloned and sequenced, but failed to reveal an open reading frame correspondmg to OprG. However, there appears to be good evidence that a part of the gene codmg for the hydrophilic membrane-associated ATP-binding component of a hitherto uncharacterised periplasmic- binding-protein-dependent transport system has been isolated. The full organisation and sequence of the operon, and substrate for this putative transport system, are yet: to be elucidated,

Linker-scanning analysis of the HIV-1: integrase protein

Mémoire numérisé par la Direction des bibliothèques de l'Université de Montréal.

Linker-scanning analysis of the HIV-1: integrase protein

Mémoire numérisé par la Direction des bibliothèques de l'Université de Montréal.