El objetivo del presente trabajo fue hacer un recuento de Vibrio parahaemolyticus Kanagawa positivo a partir de 50 muestras de pescados, moluscos y crustáceos crudos procedentes de terminales pesqueros, muelles y supermercados de consumo en Lima Metropolitana y Callao entre noviembre de 1999 y abril de 2000. El análisis microbiológico se realizó de acuerdo a la metodología recomendada por el Manual Bacteriológico Analítico (BAM, 7 ed.). Se halló Vibrio parahaemolyticus Kanagawa positivo en 4 muestras aislándose 5 cepas con las características bioquímicas correspondientes de un total de 568 cepas sospechosas. Los valores hallados de Número Más Probable (NMP) fueron en pescados y crustáceos de 3/g cada uno y en moluscos un valor mínimo de 3/g y un máximo de 7,4/g. Las muestras que presentaron Vibrio parahaemolyticus Kanagawa positivo (n=4) representaron un 8% del total de especies marinas (n=50), siendo los porcentajes hallados en pescados 2% (n=1), moluscos 4% (n=2) y crustáceos 2% (n=1). Palabras Clave: Vibrio parahaemolyticus, pescados, crustáceos, moluscos, Kanagawa positivo, NMP, Lima Metropolitana y Callao. / The aim of this research was performing a count of Vibrio parahaemolyticus Kanagawa positive out of 50 samples including raw fish, mollusks and crustaceans collected from fishermen’s wharf, fisheries, and supermarkets of edible character in Metropolitan Lima and Callao between november 1999 and april 2000. The microbiological analysis was performed according to Bacteriological Analytical Manual (BAM, 7 ed.). Vibrio parahaemolyticus Kanagawa positive was found in 4 samples from 5 strains with biochemical features that met those of Vibrio parahaemolyticus out of a total of 568 analized strains. The Most Probable Number (NMP) values found are as follows: Fish and crustaceans 3/g each, while molluscs had a minimum value of 3/g and a maximum of 7,4/g. Vibrio parahaemolyticus Kanagawa positive samples (n=4) represented 8% out of the total marine samples (n=50), being the percentages found in fish 2% (n=1), mollusks 4% (n=2) and crustaceans 2% (n=1) out of the total number of samples as well. Key Words: Vibrio parahaemolyticus, fish, mollusks, crustaceans, Kanagawa positive, MPN, Metropolitan Lima and Callao.

La investigación tuvo como objetivo incorporar una metodología de identificación rápida conocida como ARDRA (Amplified Ribosomal DNA Restriction Analysis) para identificar especies del género Vibrio. Se estandarizó la técnica con cepas referenciales. Luego, se aislaron cepas bacterianas asociadas con el cultivo de Litopenaeus vannamei. Posteriormente, se realizaron pruebas bioquímicas para encontrar cepas candidatas de pertenecer al género Vibrio. Al finalizar esta primera etapa, la técnica ARDRA estandarizada, fue aplicada en las cepas candidatas, confirmando de esta manera la factibilidad de la metodología bajo las condiciones estudiadas. En una segunda etapa, se secuenció la región 16S rDNA para confirmar e identificar las cepas candidatas por análisis filogenético. Se reportaron tres especies diferentes con alta similitud pertenecientes al Vibrio core group (Vibrio communis, Vibrio harveyi y Vibrio parahaemolyticus). Con estos resultados, fue posible diseñar una identificación rápida por ARDRA para identificar el Vibrio core group y la especie Vibrio communis. La metodología de diseño del ARDRA fue soportado por una valoración diagnóstica bioinformática, obteniendo de esta evaluación, una sensibilidad y una especificidad de 97,1 y 76,9%, respectivamente para la identificación del Vibrio core group, mientras que para identificar la especie Vibrio communis, se obtuvo una sensibilidad y una especificidad de 100 y 97,4%, respectivamente. Finalmente, se ha demostrado que es posible identificar ciertas especies del genero Vibrio asociados con la acuicultura de Litopenaeus vannamei, por ARDRA y esta metodología de identificación, tiene la ventaja de ser mucho más rápido y económico en comparación con la identificación por análisis filogenético, teniendo a su vez la desventaja de ser dependiente del uso del secuenciamiento en un primer momento para el diseño del ARDRA. Palabras clave: Litopenaeus vannamei, ARDRA, Vibrio communis, Vibrio harveyi, Vibrio parahaemolyticus, Vibrio core group, evaluación diagnóstica bioinformática. / --- The research aimed to incorporate a quick identification methodology known as ARDRA (Amplified Ribosomal DNA Restriction Analysis) to identify Vibrio species. Technique was standardized with reference strains. Then, bacterial strains were isolated associated with the cultivation of Litopenaeus vannamei. Subsequently, biochemical tests were performed to find candidate strains belonging to the genus Vibrio. Upon completion of this first stage, the standard technique (ARDRA) was applied for candidate strains, thus confirming the feasibility of the method under the conditions studied. In a second step, the 16S rDNA region sequenced to confirm and identify candidate strains for phylogenetic analysis. Three different species were reported with high similarity belonging to the Vibrio core group (Vibrio communis, Vibrio harveyi and Vibrio parahaemolyticus). With these results, it was possible to design a quick identification by ARDRA to identify the Vibrio core group and Vibrio communis. The design methodology ARDRA was supported by a bioinformatics diagnostic assessment, obtaining this evaluation, a sensitivity and specificity of 97,1 and 76.9% respectively for the identification of Vibrio core group, while identifying the species Vibrio communis, yielded a sensitivity and specificity of 100 and 97,4%, respectively. Finally, it has proved possible to identify certain Vibrio species associated with aquaculture Litopenaeus vannamei, by ARDRA identification and this methodology has the advantage of being much faster and cheaper compared with the identification by phylogenetic analysis, having in turn, the disadvantage of being dependent on the use of the sequencing at first for ARDRA design. Keywords: Litopenaeus vannamei, ARDRA, Vibrio communis, Vibrio harveyi, Vibrio parahaemolyticus, Vibrio core group, bioinformatic diagnostic evaluation.

Although the importance of cation/proton antiporters in cellular physiology is well recognized and widely studied, many antiport systems remain underinvestigated. In this work, I report the phenotypic and biochemical effects of deletion of the cytoplasmic C-terminal tail of the NhaP2 antiporter from Vibrio cholerae (Vc-NhaP2). Namely, deletion of the C-terminal tail results in diminished K+/H+ and Na+/H+ antiport activity, as well as a 5-fold decrease in affinity for its major substrate, K+ (measured as the apparent Km at pH 7.5). Furthermore, reconstitution of antiport activity in the truncation mutant upon addition of exogenous C-terminal tail is demonstrated. Currently, the only known mechanism of antiport is for NhaA, which lacks a cytoplasmic tail. Therefore, these results suggest that NhaP2 may employ a novel mechanism of antiport in which the cytoplasmic tail is directly or indirectly involved.

Chaque année, de nombreux génomes bactériens sont séquencés et il devient de plus en plus évident que la participation des îlots génomiques à la plasticité bactérienne est prépondérante. Pourtant, bien que la définition même des îlots génomiques implique qu'ils aient été acquis par transfert horizontal, le mécanisme de transfert de nombre d'entre eux demeure inconnu. L'un des mécanismes employés par les îlots génomiques pour se transférer est le transfert conjugatif. Le but de ce projet de recherche était d'étudier le mécanisme de mobilisation d'une nouvelle classe d'îlots génomiques, initialement identifiés dans les genres Vibrio et Alteromonas. Les résultats obtenus ont permis la caractérisation d'un mécanisme de mobilisation inédit reposant sur la reconnaissance par un élément intégratif et conjugatif (ICE) d'une séquence similaire à son origine de transfert sur des îlots génomiques. Grâce à cette reconnaissance spécifique, ces îlots génomiques sont mobilisables par les ICE de la famille SXT/R391, retrouvés principalement chez Vibrio. Cette étude présente l'ensemble du mécanisme de mobilisation des îlots génomiques mobilisables (MGI) par les ICE SXT/R391 à partir de l'excision du chromosome de la cellule donneuse jusqu'à l'intégration dans le chromosome de la cellule réceptrice, en passant par l'initiation du transfert au niveau de l'origine de transfert et le transfert au travers du pore de conjugaison synthétisé par l'ICE. La diversité génétique de la famille des MGI a également été étudiée et apporte des précisions sur leurs rôles et leur possible origine évolutive.

Cholera, an acute diarrheal diseases caused by the intestinal infection of the pathogenic bacterium Vibrio cholerae, continues to be a global killer in the world today. PrtV, a secreted zinc metalloprotease, is a potent cytotoxic virulence factor of V. cholerae. The 102 kDa full length multi-domain PrtV protein undergoes several N and C terminal modifications before being secreted as a 81 kDa pro-protein. The activation of the pro-protein is calcium dependent. The removal of calcium triggers auto-proteolysis to give a stable active protease with the catalytic zinc binding domain. The aim of the thesis was to study the structure and function of the PrtV protein. The results from paper I, identified the end product of the maturation of PrtV as the stable 37 kDa M6 active domain, and not a 55 kDa complex as reported earlier. Results also showed the this 37 kDa active M6 domain alone was sufficient for catalytic activity. A revised model for the maturation of PrtV was proposed. Individual domains were isolated from the PrtV protein by domain phasing methods. This included the N-terminal domain (residues 23-103), the PKD1 domain (residues 755-839), and a 25 kDa fragment (residues 589-839). The isolated domains were recombinantly over expressed as fusion proteins to increase expression and solubility. The PKD1 domain was purified to homogeneity and crystallized. The structure of the PKD1 domain reported in paper II, was solved by X-ray crystallography at an atomic resolution of 1.1 Å. From the structure, a previously unknown calcium binding site was identified at the N-terminal of the PKD1 domain. The structure also revealed two conformations for the PKD1 domain depending on free or bound calcium. From the structure, a function of the PKD1 domain as a protector of the cleavage site in the linker region between the M6 domain and the PKD1 domain in the presence of calcium was elucidated. A new model for the activation of PrtV was given. In paper III, the structure of the N-terminal domain solved by NMR spectroscopy was reported. The structure revealed two well defined helices but a third predicted helix was found to be unstructured.

The Gram-negative pathogen Vibrio cholerae uses variety of regulatory molecules to modulate expression of virulence factors. One important regulatory element of microorganisms is small non-coding RNAs (sRNAs), which control various cell functions such as expression of cell membrane proteins, mRNA decay and riboswitches. In this thesis studies, we demonstrated the roles of the sRNAs VrrA in regulation of outer membrane protein expression, biofilm formation and expression of ribosome binding proteins. In addition, we showed that VrrB, a newly discovered sRNA, played a role in amino acid dependent starvation survival of V. cholerae and might functioned as a riboswitch. VrrA, a 140-nt sRNAs in V. cholerae, was controlled by the alternative sigma factor σE. The outer membrane protein, OmpT is known to be regulated by environmental signals such as pH and temperature via the ToxR regulon and carbon source signals via the cAMP–CRP complex. Our studies provide new insight into the regulation of OmpT by signals received via the σE regulon through VrrA. We demonstrated that VrrA down-regulate ompT translation by base-pairing with the 5′ region of the ompT mRNA in a Hfq (RNA chaperone protein) dependent manner. V. cholerae biofilms contain three matrix proteins—RbmA, RbmC and Bap1—and exopolysaccharide. While much is known about exopolysaccharide regulation, little is known about the mechanisms by which the matrix protein components of biofilms are regulated. In our studies, we demonstrated that VrrA negatively regulated rbmC translation by pairing to the 5' untranslated region of the rbmC transcript and that this regulation was not stringently dependent on Hfq. In V. cholerae, VC0706 (Vrp) and VC2530 proteins are homologous to ribosome-associated inhibitor A (RaiA) and hibernation promoting factor (HPF) of Escherichia coli, respectively. HPF facilitates stationary phase survival through ribosome hibernation. We showed that VrrA repressed Vrp protein expression by base-pairing to the 5´ region of vrp mRNA and that this regulation required Hfq. We also showed that Vrp was highly expressed during stationary phase growth and associated with the ribosomes of V. cholerae. We further demonstrated that Vrp and VC2530 were important for V. cholerae starvation survival under nutrient-deficient conditions. While VC2530 was down-regulated in bacterial cells lacking vrrA, mutation of vrp resulted in increased expression of VC2530. Riboswitches are an important class of regulators in bacteria, which are most often located in the 5' untranslated region (5´ UTR) of bacterial mRNA. In this study, we discovered the novel non-coding sRNA, VrrB located at the 5´ UTR of a downstream gene encoding Vibrio auxotropic factor A (VafA) for phenylalanine. In V. cholerae, reduced production of VafA was observed in the presence of phenylalanine and phenylpyruvate in the culture media. Some analogs of phenylalanine and phenylpyruvate could also modulate the expression of VafA. Furthermore, bacterial cells lacking the vrrB gene exhibited high production of VafA, suggesting that VrrB might function as a riboswitch that controls VafA expression.

Quorum sensing is a cell density-dependent bacterial gene regulatory mechanism used for the expression of colonization-related genes. The symbiotic relationship between the luminescent bacterium Vibrio fischeri and the Hawaiian bobtail squid Euprymna scolopes serves as a model system to study the molecular processes underlying bacterial colonization. This system is especially well-suited for the investigation of the impact of quorum sensing on colonization because (i) it is an easily accessible, natural, two-species colonization model, and (ii) quorum sensing regulates luminescence expression in V. fischeri, which allows the non-invasive detection of quorum-sensing activity both in culture and in symbiosis. While the impact of one of V. fischeri's quorum-sensing systems, lux, on luminescence expression and symbiotic competence has been extensively studied, little was known about other putative systems. The results of this study demonstrate that the V. fischeri ain system is essential for both maximal luminescence expression and symbiotic competence. The ain system predominantly induces luminescence expression at intermediate cell densities, which occur in culture, while the lux system is responsible for luminescence expression at the high cell densities found in symbiosis, suggesting the sequential induction of luminescence gene expression by these two systems. Furthermore, the ain quorum sensing system is important for the processes underlying colonization initiation, while the impact of the lux system is apparent only in later stages of the symbiosis, indicating distinct functions of these two systems during the colonization process. A global transcriptome. analysis of quorum-sensing mutants revealed that ain quorum sensing represses motility gene expression, providing a likely explanation for the initiation defect. Although it has been known that many bacterial species possess multiple quorum-sensing systems, this is the first study demonstrating that two quorum-sensing systems are employed to specifically regulate functions important at distinct cell densities occurring during the colonization process.

Bibliography: leaves 169-208 / ix, 208 leaves : ill ; 30 cm. / Title page, contents and abstract only. The complete thesis in print form is available from the University Library. / Thesis (Ph.D.)--University of Adelaide, Dept. of Microbiology and Immunology, 1988

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

Bibliography: leaves 143-172 / vi, 172 leaves, [25] leaves of plates : ill ; 30 cm. / Title page, contents and abstract only. The complete thesis in print form is available from the University Library. / Thesis (Ph.D.)--University of Adelaide, Dept. of Microbiology and Immunology, 1989