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published_or_final_version / Microbiology / Master / Master of Philosophy

Oysters are filter-feeding bivalves, which filter water for nutrients and often accumulate contaminants and human pathogens such as Vibrio parahaemolyticus and Vibrio vulnificus naturally occurring in the marine environment. These naturally occurring pathogens have been frequently isolated from raw shellfish, particularly oyster, in the United States and are recognized as the leading causes of human gastroenteritis associated with seafood consumption. Human illness caused by consumption of raw oyster contaminated with V. parahaemolyticus and Vibrio vulnificus typically results in reduced sales of oysters and a consequent significant financial burden for the producers. The United States produces more than 27 million pounds of oysters each year with a large portion of them being produced from the coastal water of the Gulf of Mexico. It is estimated that 20 million Americans eat raw shellfish and consumption of raw oyster is responsible for about 95% of all deaths associated with seafood consumption in the U.S., making raw oysters one of the most hazardous seafoods. Several post-harvest processes, including low temperature pasteurization, freezing, high pressure processing and irradiation, have been reported capable of reducing Vibrio contamination in raw oysters. However, most of them require either a significant amount of initial investment or operation costs, and oysters are often killed during processing. Cost-effective post-harvest processing for reducing V. parahaemolyticus in raw oysters without significant adverse effects on the oysters remains to be developed. This study was conducted to determine impacts of low-temperature (15, 10 and 5°C) depuration and frozen storage on reducing V. parahaemolyticus and V. vulnificus in raw oysters. Depuration of the Gulf oyster (Crassostrea virginica) with electrolyzed oxidizing (EO) water (chlorine, 30 ppm; pH 2.82; oxidation-reduction potential, 1,131mV) containing 3% NaCl was found ineffective on reducing both V. parahaemolyticus and V. vulnificus in the oysters. Reductions of V. parahaemolyticus and V. vulnificus in oyster after 48 h of EO water depuration at 22°C were limited to 0.7 and 1.4 log MPN/g, respectively. Depuration with EO water at lower temperatures did not enhance reductions of Vibrio in the oysters. Greater reductions of V. parahaemolyticus (1.2 log MPN/g) and V. vulnificus (2.0 log MPN/g) were observed when the oysters were depurated with artificial seawater (ASW) at room temperature (22°C) for 48 h. Decreasing temperature of ASW to 15°C for depuration significantly increased the reductions of V. parahaemolyticus and V. vulnificus to 2.1 and 2.9 log MPN/g, respectively, after 48 h of process. However, depuration of oyster in ASW at 10 and 5°C were found less effective than at 15°C in reducing Vibrio in the Gulf oysters. An extended depuration with ASW at 15°C for 96 h was capable of achieving 2.6 and 3.3 log MPN/g of reductions of V. parahaemolyticus and V. vulnificus, respectively, in the Gulf oysters. Study of effects of frozen storage at -10, -23 and -30°C on reducing V. parahaemolyticus in raw half-shell Pacific oyster (Crassostrea gigas) found that the population of the bacterium decreased faster in oysters stored at -10 than at -23 or -30°C. Holding half-shell Pacific oyster at -10°C for three months or at -23°C for four months was capable of achieving a greater than 3-log (MPN/g) reduction of V. parahaemolyticus in the Pacific oyster. / Graduation date: 2008

Diss. (sammanfattning) Stockholm : Karol inst., 2003. / Härtill 4 uppsatser.

Diss. (sammanfattning) Göteborg : Univ. , 2009. / Härtill 3 uppsatser.

CARVALHO, Edirsana Maria Ribeiro de. Quantificação e identificação de Vibrio spp. na hemolinfa de camarões Litopenaeus vannamei cultivados em fazendas no Estado do Ceará. 2009. 89 f. : Dissertação (mestrado) - Universidade Federal do Ceará, Centro de Ciências Agrárias, Departamento de Engenharia de Pesca, Fortaleza-CE, 2009 / Submitted by Nádja Goes (nmoraissoares@gmail.com) on 2016-07-12T14:11:24Z No. of bitstreams: 1 2009_dis_emrcarvalho.pdf: 2284309 bytes, checksum: 922a46195a8b0319c114f69b1a9eefd9 (MD5) / Approved for entry into archive by Nádja Goes (nmoraissoares@gmail.com) on 2016-07-12T14:11:51Z (GMT) No. of bitstreams: 1 2009_dis_emrcarvalho.pdf: 2284309 bytes, checksum: 922a46195a8b0319c114f69b1a9eefd9 (MD5) / Made available in DSpace on 2016-07-12T14:11:51Z (GMT). No. of bitstreams: 1 2009_dis_emrcarvalho.pdf: 2284309 bytes, checksum: 922a46195a8b0319c114f69b1a9eefd9 (MD5) Previous issue date: 2009 / Most of shrimp bacterial infections are caused by Vibrio bacteria genus. This study aims to quantify identify Vibrio in the hemolymph of the marine shrimp Litopenaeus vannamei cultivated in farms in State of Cear, Brazil. Related is also the time of coagulation of hemolymph with counts of Vibrio. Sixteen samples were performed on four farms (A, B, C and D), eight shrimps with 4 g, and eight with eight grams shrimps. The collections were made in seasonal periods: rainy and drought. A total of 480 samples, and 240 to shrimps with 4 g and 8 g each, respectively. The values in the Standard Plate Count (SPC) of total vibrio, Vibrio Suc+ and Suc- in the hemolymph of shrimp samples with 4 g, in the rainy season ranged from: 2. 74 x 10⁴ to 28.70 x 10⁶ CFU/mL (est.), <6.0 to 14.64 x 106CFU/mL and <6.0 to 12.72 x 106 CFU/mL, respectively. For hemolymph of shrimp with 8 g of the SPC and the total Vibrio Suc+ and Sucranged from 1.68 x 10⁴ to 15.18 x 10⁶ CFU/mL (est.), <6.0 to 1.8 x 105 CFU/mL and <6.0 to 15.18 x 106CFU/mL (est). During the period of drought for these values of shrimp haemolymph of four grams, were: 9.0 x 10² (est.) to 5.40 x 104 CFU mL total Vibrio, <6.0 to 1.71 x 10⁴CFU/mL (Suc-) and <6.0 to 5.40 x 104 CFU/mL (Suc+). For shrimp with 8 g a total Vibrio count was 9.0 x 10² (est.) CFU/mL to 2.0 x 10⁷, <6.0 to 4.02 x 106 CFU/mL (Suc-) and <6.0 to 2.0 x 10⁷CFU/mL (est.) (Suc+). The results show that the rates of colonies of Vibrio Suc- and Suc+ were higher in the rainy season than in the drought. There was no relationship between the time of coagulation and the counts of Vibrio in the hemolymph of shrimps. The species that predominated in the period were: V. coralliilyticus, V. parahaemolyticus, Vibrio pelagius II, Vibrio alginolyticus, Vibrio mediterranei, Vibrio mimicus, Vibrio vulnificus B1 / As infecções bacterianas em camarões são causadas, freqüentemente, por bactérias do gênero Vibrio O presente estudo teve por objetivo quantificar e identificar Vibrio na hemolinfa do camarão marinho Litopenaeus vannamei cultivado em fazendas no Estado do Ceará, Brasil. Relacionou-se também o tempo de coagulação das hemolinfas com as contagens de Vibrio. Foram realizadas 16 coletas em quatro fazendas (A, B, C e D), sendo oito para camarões com 4 g, e oito para camarões com oito gramas. As coletas foram realizadas nos períodos sazonais: chuvoso e estiagem. Perfazendo um total de 480 amostras, sendo 240 para camarões com 4 g e 8 g cada, respectivamente. Os valores da Contagem Padrão em Placas (CPP) de víbrios totais, de víbrios Sac+ e Sac-, nas amostras de hemolinfa dos camarões com 4g, no período chuvoso, variaram de: 2,74 x 104 a 28,70 x 106 UFC/mL (est.); de < 6,0 a 14,64 x 106UFC/mL e de < 6,0 a 12,72 x 106 UFC/mL, respectivamente. Para a hemolinfa dos camarões com 8 g os valores obtidos foram: 1,68 x 104 a 15,18 x 106 UFC/mL (est.), de < 6,0 a 1,8 x 105 UFC/mL e de < 6,0 a 15,18 x 106UFC/mL (est.), respectivamente. No período de estiagem esses valores para a hemolinfa dos camarões de quatro gramas, foram: 9,0 x 102 (est.) a 5,40 x 104 UFC/mL víbrio total; < 6,0 a 1,71 x 10⁴UFC/mL (Sac–) e de < 6,0 a 5,40 x 104 UFC/mL (Sac+). Para os camarões com 8 g a contagem de Víbrio total foi de 9,0 x 102 (est.) a 2,0 x 107, de < 6,0 a 4,02 x 106 UFC/mL (Sac-) e de < 6,0 a 2,0 x 10⁷ UFC/mL (est.) (Sac +). Os resultados mostram que os índices de colônias de Vibrio Sac- e Sac+ foram maiores no período chuvoso do que no de estiagem. Não houve relação entre o tempo de coagulação e as contagens de Vibrio na hemolinfa dos camarões. As espécies que predominaram nos períodos estudados foram: V. coralliilyticus, V. parahaemolyticus, Vibrio pelagius II, Vibrio alginolyticus, Vibrio mediterranei, Vibrio mimicus, Vibrio vulnificus B1.

Different strains of Vibrio parahaemolyticus (Vp) in broth cultures and Vp-inoculated live Pacific oysters (Crassostrea gigas) were subjected to high pressure processing (HPP) at 241, 276, 310, and 345 MPa. Results showed Vp numbers were reduced by HPP in both pure culture and whole oysters. Vp inactivation was dependent on time and pressure. Optimum conditions for reducing Vp in pure culture and oysters to non-detectable levels were achieved at 345 MPa for 30 and 90 s, respectively. Resistance variations were detected between Vp in pure culture and in oysters. HPP proved to be an efficient means of reducing Vp in oysters. The decimal reduction rates of Vp 03:K6 strain was compared with clinical ATCC 17802 and environmental AST Vp strains in pure culture and Pacific oysters under high pressure processing (HPP) at 276 and 310 MPa for different treatment times based on Calik and others (2001) data. Results showed that 03:K6 strain had relatively higher decimal reduction rates when compared to two other strains. The 03:K6 strain in oyster had the highest D-value of 1.77 min under 276 MPa HPP treatment. Reduction trends presented by Vp 03:K6 strain must be considered when making commercial HPP setting decisions for reduction of Vp. / Graduation date: 2002

Objetivos. Determinar las características epidemiológicas y clínicas de la gastroenteritis causada por Vibrio parahaemolyticus del grupo pandémico en el Perú. Materiales y métodos. Se examinó las historias clínicas y registros de laboratorio de cien casos de gastroenteritis en los cuales se aisló V. parahaemolyticus del grupo pandémico y no pandémico. Se recolectó información epidemiológica y clínica y se realizó el análisis estadístico de los datos para evaluar si la gravedad de la enfermedad se asoció con la presencia de las cepas del grupo pandémico. Resultados. Se logró colectar información epidemiológica en 85% de los casos e información clínica sólo en 37% de los casos, principalmente de los hospitalizados. Los casos del grupo pandémico tuvieron una mayor probabilidad de tener deposiciones líquidas (96,3% frente a 62,5%, p<0,05), presentar deshidratación moderada o grave (100% frente a 60%, p<0,05) y requerir atención hospitalaria (98% frente a 42,9%, p<0,0001). Fue más probable aislar una cepa pandémica en personas de 30 o más años de edad (63% frente a 39,5%, p<0,05). Conclusiones. El Vibrio parahaemolyticus del grupo pandémico causa enfermedad gastrointestinal de mayor gravedad que las cepas no pandémicas, con mayor probabilidad de requerir atención hospitalaria. Basados en este reporte, se recomienda incluir la identificación de V. parahaemolyticus en el diagnóstico etiológico de agentes causantes de gastroenteritis grave en el sistema de salud del Perú. / Objective. To determine the epidemiological and clinic characteristics of gastroenteritis caused by Vibrio parahaemolyticus strains of the pandemic group in Peru. Material and methods. Clinical and laboratory records were searched in 100 cases of gastroenteritis caused by V parahaemolyticus, either of the pandemic or non pandemic group. Clinical and epidemiological data were collected and statistical analysis was done to evaluate if the severity of illness was associated with the pandemic group. Results. Epidemiological data were collected in 85% of cases, and clinical data were only available in 37% of cases, mainly on those hospitalized. Cases associated with the pandemic strains had a higher probability of liquid stools (96.3% vs. 62.5%, p<0.05), moderate or severe dehydration (100% vs. 60%, p<0.05), and hospital care (98% vs. 42.9%, p<0.0001). Cases aged thirty or older were associated with the pandemic strains (63% vs. 39.5%, p<0.05). Conclusions. Vibrio parahaemolyticus of the pandemic group causes more severe gastrointestinal disease than none pandemic strains, with higher probability of requiring hospital care. Based on this report, it is advisable to include the identification of V. parahaemolyticus in the etiological diagnosis of agents causing severe gastroenteritis in the Peruvian health system.

Vibrio cholerae, der Erreger der Cholera, ist ein Gram-negatives, fakultativ pathogenes Bakterium. In dieser Arbeit konnte die V. cholerae Oberflächenstruktur identifiziert werden, an die der temperente V.cholerae-Phage K139 adsorbiert. Phagenbindungs-Studien mit gereinigtem Lipopolysaccharid (LPS) ergaben, daß das O-Antigen der Serogruppe O1 den Phagenrezeptor darstellt. Zusätzlich wurden phagenresistente Mutanten des transluzenten O1 El Tor Inaba Stammes P27459 nach Inkubation mit einem lytischen K139-Derivat isoliert. Analysen des LPS-Laufverhaltens in Polyacrylamid-Gelen (PAA) zeigten, daß viele der Spontanmutanten defekte LPS-Moleküle synthetisierten, die entweder im O-Antigen, im Kernoligosaccharid oder in beidem betroffen waren.Phagenresistente Mutanten mit offensichtlich unverändertem LPS bildeten entweder transluzente oder opake Kolonien. Weiterhin wurden ausgewählte spontan phagenresistente Stämme genetisch analysiert. O-Antigen Mutanten wurden in Southernblot-Analysen mit spezifischen, gegen das bereits gut charakterisierte O-Antigen-Biosynthese-Gencluster (rfb) gerichtete Sonden untersucht. Zwei der O-Antigen negativen Stämme waren durch Insertion des IS-Elementes IS1004 in das rfb-Gencluster entstanden. Spontan phagenresistente Mutanten mit verändertem Kernoligosaccharid ohne O-Antigen (R-LPS-Mutanten) sind wahrscheinlich im Kernoligosaccharid-Biosynthese-Gencluster (waa) mutiert, das in der V. cholerae Datenbank identifiziert wurde. waaF, das für die Heptosyl-II-Transferase kodiert, wurde durch genetische Manipulation inaktiviert und zeigte im PAA-Gel das gleiche Migrationsverhalten wie zwei spontan phagenresistente Mutanten. In den Spontanmutanten konnte jedoch im Gegensatz zu der konstruierten Mutante durch ein WaaF-exprimierendes Plasmid lediglich das Kernoligosaccharid, nicht aber das O-Antigen wiederhergestellt werden. Weitere genetische Analysen ergaben, daß eine der Spontanmutanten 546 bp deletiert hatte, die Teile von waaF und waaL betrafen, letzteres kodiert dabei vermutlich für die O-Antigen-Ligase. Spontanmutanten mit intaktem O-Antigen aber verändertem Kernoligosaccharid konnten als galU-Mutanten charakterisiert werden, die auch im Galaktosekatabolismus beeinträchtigt waren. Zusätzlich wurden zwei weitere gal-Gene, galE und galK, durch genetische Manipulation inaktiviert. Diese Mutanten konnten ebenfalls keine Galaktose mehr verstoffwechseln, synthetisierten aber ein intaktes LPS. In Gegenwart hoher Galaktosekonzentrationen wurde in galU- und galE- Mutanten aufgrund der Defekte im Gal-Stoffwechsel Lyse beobachtet. Zusätzlich wurde die Rolle von galU und galE in der Biofilmbildung untersucht. Da der transluzente Wildtyp (Wt) im Gegensatz zu Opakvarianten keinen Biofilm bilden konnte, wurden galE und galU auch in einer Opakvariante inaktiviert. galU- und galE-Mutationen erzeugten in der Opakvariante wieder eine transluzente Koloniemorphologie und einen biofilm-negativen Phänotyp an abiotischen Oberflächen. Diese Daten deuten an, daß die Synthese von UDP-Galaktose ausgehend von UDP-Glukose für die Synthese des Exopolysaccharides (VPS) notwendig ist. Virulenzstudien in neugeborenen Mäusen ergaben, daß O-Antigen negative Stämme sowie galU-Mutanten sehr viel schlechter und R-LPS-Mutanten nicht mehr im Dünndarm kolonisieren konnten. Da galE und galEK-Mutanten ebenso gut wie der Wt kolonisierten, konnte ausgeschlossen werden, daß toxische Galaktose-Effekte für den Kolonisierungsdefekt der galU-Mutante verantwortlich waren. Zusätzlich wurde die Überlebensfähigkeit der LPS-Mutanten in Gegenwart von verschiedenen Substanzen, die nachweislich im menschlichen Dünndarm vorkommen, unter „in vitro“ Bedingungen untersucht. R-LPS und galU-Mutanten waren im Vergleich mit dem Wt sensitiver gegenüber schwachen organischen Säuren, Defensinen, dem Komplementsystem und Gallensäuren. O-Antigen negative Stämme waren dagegen weiterhin resistent gegenüber Gallensäuren und schwachen organischen Säuren aber sensitiv gegen die Komponenten des angeborenen Immunsystems. Bisher wurde für keine der LPS-Mutanten eine größere Beeinträchtigung weiterer Virulenzfaktoren, wie z.B. Motilität, Synthese der Pili TCP oder Choleratoxin-Produktion festgestellt. Auch die Zusammensetzung der Proteine in der äußeren Membran war offensichtlich nicht beeinträchtigt, allerdings wurde beobachtet, daß aus galU Mutanten in geringem Maße und aus R-LPS Mutanten in verstärktem Maße periplasmatische Proteine in den Überstand diffundieren können. Diese Ergebnisse deuten an, daß nicht nur das O-Antigen, wie bereits bekannt, sondern auch eine spezifische Kernoligosaccharid-Struktur für eine effektive Kolonisierung von V. cholerae essentiell ist. Der Grund dafür ist höchstwahrscheinlich in der Ausbildung einer stabilen äußeren Membran zu suchen, die die Persistenz in Gegenwart bakteriozider Substanzen des Dünndarms ermöglicht. / Vibrio cholerae is a Gram-negative, facultative pathogen and is the causative agent of cholera. In this study the adsorption site of the temperate phage K139 on the bacterial cell surface was determined. Phage-binding studies with purified lipopolysaccharide (LPS) showed that the O-antigen of the serogroup O1 serves as the phage receptor. In addition, phage resistant mutants of the O1 El Tor Inaba strain P27459 were screened by using a virulent isolate of phage K139. Analysis of the LPS by PAGE migration patterns revealed that most of the spontaneous mutants synthesize incomplete LPS molecules, either composed of defective O-antigen or core oligosaccharide. Phage resistent isolates with apparently normal LPS form either translucent or opaque colonies. The genetic nature of spontaneous phage resistant strains was investigated. O-antigen negative strains were investigated by Southern hybridization with probes specific for the well known O1-antigen biosynthesis cluster (rfb). Two of the investigated O-antigen negative mutants revealed insertions of element IS1004 into the rfb-gene cluster. Spontaneous phage resistant R-LPS mutants were found to synthesize an altered core without attached O-antigen. This type of mutants are most likely affected in the core oligosaccharide biosynthesis gene cluster (waa), which was identified by searching the V. cholerae genomic database. waaF, encoding heptosyl-II-transferase, was inactivated and the LPS was found to have the same migration behaviour in PAA gels than two spontaneous mutants. However, in contrast to the constructed waaF mutant complementation with waaF in trans restored only the core oligosaccharide but not the O-antigen synthesis in the spontaneous mutants. Further analysis revealed that one mutant had a deletion of 546 bp affecting waaF and waaL, the latter is thought to encode the putative O-antigen-ligase. Spontaneous mutants with intact O-antigen but altered core oligosaccharide were identified as galU mutants, which were also affected in the galactose catabolism. Other gal genes, galE and galK, were inactivated and these mutants were also found to be defective in the catabolism of exogenous galactose but synthesized an apparently normal LPS. Additionally, galU and galE mutants were found to lyse in the presence of high galactose concentrations. Furthermore the role of galU and galE in biofilm formation was investigated. In contrast to the spontaneous phage resistant opaque colony variant the translucent wt-strain was unable to form a biofilm, therefore the galE and galU mutations were also introduced into the opaque colony variant. galU and galE mutants of the opaque strain were translucent and unable to form a biofilm on abiotic surfaces, suggesting that the synthesis of UDP-galactose, via UDP-glucose, is essential for the biosynthesis of the exopolysaccharide. Virulence studies in infant mice indicated that O-antigen negative mutants, galU and R-LPS mutants but not galE and galEK mutants were defective in colonization of the mouse small intestine, excluding any toxic galactose effects in the gut. By investigating the sensitivity to several substances known to be present in the intestine „in vitro“, it was shown that galU and R-LPS mutants were more sensitive to weak organic acids, cationic antimicrobial peptides, the complement system and bile salts. O-antigen negative strains were found to be sensitive against gut-associated bacteriocidal substances, but they displayed significant resistance to bile salts and organic acids. No gross change in other virulence determinants such as motility, synthesis of pili TCP, choleratoxin production or outer-membrane proteins has yet been found for any of the LPS mutants. However, some periplasmic leakage for galU and significant leakage for R-LPS mutants was observed. These results suggest that not only the O-antigen, as already known, but also a specific core oligosaccharide architecture seems to be required for V. cholerae colonization, most probably in providing resistance against bacteriocidal substances.

Obwohl inzwischen über 200 verschiedene Serogruppen von V. cholerae bekannt sind, wurden Ausbrüche der Cholera hauptsächlich von Stämmen der unbekapselten Serogruppe O1 und der bekapselten Serogruppe O139 verursacht. Die Komponenten des Lipopolysaccharids (LPS) von O1 und O139, sowie die Kapsel von O139 tragen zur Kolonisierung im Gastrointestinaltrakt bei. Um die Funktion des LPS und der Kapsel als Virulenzfaktor näher zu untersuchen, wurden Adhäsionsstudien mit definierten LPS- und/ oder Kapsel-Mutanten beider pathogener Serogruppen durchgeführt. Dazu wurde die Mukus-produzierende humane Darmzelllinie HT-29-Rev MTX verwendet. Im Vergleich zum jeweiligen Wildtyp (Wt) konnte für eine O Antigen-Mutante von O1 eine Reduktion um 85%, für eine O Antigen/ Kapsel-Mutante von O139 eine Reduktion um 70% in der Adhäsionsrate festgestellt werden. Ein Beitrag von ToxR regulierten Genprodukten ist ebenfalls möglich. Weiterhin wurden mit WavJ und WavD zwei Genprodukte der Kernoligosaccharid -Biosynthese charakterisiert, welche bislang nur in dem wa*-Genclustertyp 1 der klinischen Isolate nachgewiesen worden sind. Es konnte gezeigt werden, dass beide Genprodukte an der Biosynthese des Kern OS beteiligt sind, wobei WavJ mit hoher Wahrscheinlichkeit die Heptosyl-IV-Transferase darstellt. Die wavDJ-Doppelmutanten beider Serogruppen wiesen eine erhöhte Sensitivität gegenüber Novobiocin auf. Dagegen konnte eine Attenuation der Mutanten im Mausmodell nur für die Serogruppe O139 demonstriert werden. Ein Schlüsselenzym der LPS-Biosynthese stellt die Oberflächenpolymer:Lipid A-Kern OS-Ligase (WaaL), kurz O Antigen-Ligase genannt, dar. In dieser Arbeit wurden die in der Primärstruktur stark unterschiedlichen Ligasen aus einem pathogenen (P27459) und apathogenen (V194) V. cholerae Isolat strukturell und funktionell analysiert. Es wurde gezeigt, dass die Aktivität beider Ligasen von der Anwesenheit eines N-Acetylglucosamins (GlcNAc) im Kernoligosaccharid abhängig ist. Dieser Zucker wird durch das Genprodukt WavL transferiert, welchem in dieser Arbeit die Aktivität einer N-Acetylglucosaminyltransferase zugeordnet werden konnte. Das Gen wavL wurde in allen zur Verfügung stehenden V. cholerae Isolaten nachgewiesen und stellt wahrscheinlich eine generelle Voraussetzung des Kern OS für eine O Antigen-Anheftung dar. Im Gegensatz dazu, diskriminiert die An- bzw. Abwesenheit einer Galaktose (Gal) im Kern OS die Spezifität der Ligasen von V. cholerae P27459 bzw. V194. Dabei ist die Aktivität der Galaktosyltransferase WavM, essentiell für die Aktivität der Gal-abhängigen Ligase von V194. Die Gal-unabhängige Ligase von P27459 wird hingegen durch die Anwesenheit von Gal im Kern OS inhibiert. Hybridfusionen der beiden Ligasen deuten an, dass die Erkennungsdomäne für Gal in der C-terminalen Hälfte lokalisiert ist. Erstmals wurde die Topologie einer Ligase durch PhoA- und LacZ-Fusionen analysiert. Die Suche nach konservierten Aminosäuren (AS) in verschiedenen Ligasen führte zur Identifizierung der Motive R(X3)L und H(X10)G in zwei periplasmatischen Schleife. Ein Austausch des R oder des H in diesen Motiven führte zum Verlust der Ligase-Aktiviät von WaaL aus V. cholerae und S. enterica. Damit geben diese Motive einen ersten Hinweis auf das aktive Zentrum des Enzyms. Desweiteren wurde nach möglichen O Antigen-Transportern bei V. cholerae gesucht, welche bislang noch nicht identifiziert worden waren. Über die Anpassungen von V. cholerae an aquatische Ökosysteme, insbesondere hinsichtlich der wechselnden Osmolarität, ist nahezu nichts bekannt. Durch ein in dieser Arbeit konstruiertes und etabliertes Transposonsystem konnten 3600 Mutanten erzeugt und auf Wachstumsdefekte unter hypertonischen Bedingungen untersucht werden. Eine dieser osmosensitiven Mutanten wies eine Insertion in dem Locus VCA0565 auf, welcher für eine putative Sensor-Histidinkinase kodiert. Mit dem Regulator, kodiert durch VCA0566, stellt VCA0565 das putative Zwei-Komponentensystem OsmRK dar. Transkriptomanalysen von osmR/ K-Mutanten lieferten keine Erklärung des Wachstumsdefekts unter hypertonischen Bedingungen, zeigten aber eine Vernetzung der durch OsmR/ K regulierten Gene mit dem ToxR-Regulon auf. Analysen der Außenmembran demonstrierten, dass eine Mutation von osmR/ K zu einer Repression von OmpU unter hohen Salzkonzentrationen führt. Vergleichende Experimente mit weiteren Mutanten deuteten an, dass es in osmR/ K- und toxS-Mutanten unter erhöhten Salzkonzentrationen zur Degradation von ToxR kommt. Während die Deregulation von OmpU in osmR/ K-Mutanten nur unter Salzstress zu beobachten war, führte in der toxS-Mutante auch ein Membranstress durch Zugabe von Protamin zu einer Repression von OmpU. Die zu OsmR/ K nah verwandten putativen Zwei-Komponentensysteme EnvZ/ OmpR und VCA0257/ VCA0256 hatten unter keiner der getesteten Bedingungen einen Einfluss auf die Proteine der AM. Weiterhin wurde eine C-terminale Degradation von HutA unter hypertonischen Bedingungen aufgedeckt. / Although, more than 200 serogroups of V. cholerae.were identified, however, only the strains of the non-encapsulated O1 and the encapsulated O139 serogroups were found to be responsible for cholera epidemics. The components of the LPS of O1 and O139 play a crucial role in the colonization of the gastrointestinal tract. To analyze the contribution of the LPS and the capsule in the adhesion to epithelial cells, mucus layer attachment studies using defined O antigen and/ or capsule mutants of both serogroups and the human intestinal cell line HT29-Rev MTX were performed. In case of the O antigen mutant of O1 a 85% and for the O antigen and capsule mutant of O139 a 70% reduction in the adhesion rate was determined compared to wild type. It is likely that ToxR regulated gene products also contribute to the adhesion, since a toxR-mutant of O1 showed a 3-fold reduction in the adhesion rate. In addition the two gene products of the core oligosaccharide biosynthesis, WavJ and WavD, were characterized. So far the corresponding genes could only be found in the wa*-gene cluster type 1 of clinical isolates. It could be demonstrated, that single and double knockout mutants have an effect on core oligosaccharide biosynthesis in both serogroups. Based on bioinformatical data it is likely that WavJ represents the heptosyl-IV-transferase. Double mutants in wavJ and wavD of both serogroups showed an attenuated growth in the presence of novobiocin, whereas only the mutants in O139 demonstrated reduced colonization in the in vivo mouse model. The surface polymer:lipid A-core ligase (WaaL), also called the O antigen ligase, is a key enzyme in the LPS biosynthesis of Gram- bacteria. Part of this work focused on the structural and functional characteristics associated with the recognition of the core oligosaccharide of two distantly related ligases of a virulent (P27459) and an environmental (V194) V. cholerae isolate. It was demonstrated that the activity of both ligases is dependent on the presence of N-acetylglucosamine, which is attached to the core oligosaccharide by the WavL glycosyltransferase. The gene wavL could be found in all V. cholerae isolates so far. In contrast, an additional sugar substitution, i.e. galactose, which is transfered by the WavM galactosyltransferase, discriminates the core oligosaccharide specificity of the ligases of P27459 and V194. The activity of WavM is essential for the activity of the galactose-dependent ligase of V194, whereas it hinders the galactose-independent ligase of P27459 to transfer the O antigen onto the core oligosaccharide. WaaL protein hybrids between galactose dependent and non-dependent ligases indicate that the galactose recognition site is located in the C-terminal half. Using PhoA and LacZ fusions the topology of the ligase of P27459 was determined. Amino acid sequence alignments of WaaL proteins identified the distinct conserved motifs R(X3)L and H(X10)G in two periplasmic loops. By site directed mutagenesis of the histidine and arginine residues within these motifs, an abortism of O antigen transfer reaction for WaaLs of V. cholerae and Salmonella enterica was found. Furthermore the putative O antigen-transport systems of V. cholerae were investigated. In this work a new transposon system was constructed and established, resulting in 3600 mutants, which were screened for growth defects under hypertonic conditions. One of these mutants had an insertion in locus VCA0565, which encodes a putative sensor histidine kinase. In combination with the transcriptional regulator, encoded by VCA0566, they represent the putative two-component system OsmRK. Comparing the transcriptom of osmR/ K-mutants to the wild type revealed no explanation for the osmosensitive phenotype, but showed some interaction between the regulon of OsmR/ K and ToxR. Analysis of the outer membrane demonstrated, that a mutation in osmR/ K results in a repression of OmpU under hypertonic conditions. Comparative experiments, including additional mutants indicated a degradation of ToxR in osmR/ K- and toxS-mutants in presence of high salt concentrations. In contrast to osmR/ K-mutants, in the toxS-mutant the repression of OmpU could be also observed by a different membrane stress caused by protamine. In addition, the analysis of the outer membrane proteins revealed a C-terminal degradation of HutA under hypertonic stress conditions.