Memoria para optar al Título Profesional de Médico Veterinario / Conocer la condición sanitaria de animales silvestres en cautiverio permite determinar el riesgo a enfermar al que se encuentran expuestos estos animales, pudiendo incluso producirse mortalidad en aquellos casos más extremos. Por otro lado, estos antecedentes también son necesarios para poder determinar el riesgo de infección de enfermedades zoonóticas al que se encuentra expuesta la población humana, que asiste a los recintos donde se encuentran estos animales. Esta información permite establecer las medidas sanitarias necesarias para asegurar el buen estado de salud tanto de los animales como de aquellos humanos que permanezcan en contacto directo o indirecto con ellos. El objetivo de este trabajo fue detectar Salmonella enterica en muestras de animales pertenecientes al Parque Zoológico Buin Zoo (Región Metropolitana, Chile), para lo cual se colectaron tórulas rectales desde 201 mamíferos y tórulas cloacales desde 121 aves. Las tórulas fueron cultivadas y luego sometidas a PCR del gen invA. Dentro de la clase Mammalia se muestrearon 51 animales pertenecientes al orden Carnivora, 33 al orden Primates y 117 al orden Ungulata, lográndose aislar S. enterica a partir del 10,44% de las muestras, de las cuales un 42,21% perteneció al orden Ungulata. Dentro de la clase Aves, se obtuvieron muestras a partir de 9 animales pertenecientes al orden Pelecaniformes, 5 al orden Charadriiformes y 107 al orden Anseriformes, aislándose S. enterica a partir del 0,83% de las muestras / Financiamiento: Departamento de Conservación e Investigación del Parque Zoológico Buin Zoo y Proyecto Fondecyt No. 11110398

Memoria para optar al Título Profesional de Médico Veterinario / La aparición de la resistencia antimicrobiana por parte de bacterias zoonóticas es una preocupación actual en la salud pública. Salmonella enterica es un patógeno emergente que ha cobrado mayor importancia en este último tiempo y se desconoce su impacto en especies acuáticas. El objetivo de este estudio fue determinar fenotipos de resistencia a antibióticos en cepas de Salmonella enterica y su asociación con distintos hospederos en Chile. Se analizaron 111 cepas correspondientes 49 de aves acuáticas, 30 de aves comerciales y 32 humanas. Se utilizó el método de difusión en placa Kirby Bauer según las normas recomendadas por el Clinical Laboratory Standards Institute (2007) para determinar los fenotipos de resistencia. Los antibióticos utilizados fueron 10: enrofloxacino, amoxicilina - ácido clavulánico, cefotaxima, gentamicina, tetraciclina, sulfametazol - trimetoprim, ceftofiur, cefradina, ampicilina y cefradroxilo. Para analizar la asociación de los fenotipos de resistencia con su hospedero de origen se utilizó el programa Infostat versión 2010. Del total de cepas de S. enterica analizadas se encontraron 42 cepas resistentes en aves acuáticas, 16 en aves comerciales y 10 en humanos. Dentro de las cepas de aves acuáticas 33 demostraron resistencia al antimicrobiano tetraciclina. En el caso de aves comerciales y humanos los valores absolutos de resistencia no fueron significativos. En relación a la asociación de los fenotipos de resistencia con su hospedero de origen, se demostró en aves acuáticas en relación a 4 agentes antimicrobianos: Tetraciclina, amoxicilina + ácido clavulánico, ampicilina y gentamicina. De acuerdo al estudio realizado, se demuestra el efecto del uso de antimicrobianos en la fauna silvestre como es el caso de las aves acuáticas y de qué manera se ve desfavorecido el medio ambiente con los altos niveles de resistencia. Los resultados sugieren que las infecciones con Salmonella en aves acuáticas podría tener un gran impacto en la salud pública y animal. / Proyecto Fondecyt 11110398

Memoria para optar al Título Profesional de Médico Veterinario / Salmonella corresponde a una de las principales causas de enfermedades gastroentéricas, principalmente por la ingestión de alimentos de origen animal contaminados. Para detectar la presencia de Salmonella en alimentos existe el método tradicional y métodos alternativos. El método tradicional es bastante extenso y laborioso, retrasando la obtención de resultados. Los métodos alternativos permiten obtener resultados más rápidamente, aun cuando, deben ser validados y verificados frente a un método tradicional. Este estudio tiene por finalidad verificar al interior de la Unidad de Bacteriología Pecuaria del Servicio Agrícola y Ganadero (SAG) un nuevo método alternativo denominado VIDAS® UP SALMONELLA para la detección de Salmonella spp. en dos matrices del ámbito alimentario, como lo son esponja sobre carcasa de bovino y enjuague de carcasa de pollo. Para ello se analizaron, en forma simultánea, 52 muestras de cada matriz por el método alternativo y por el método tradicional, de tal manera de obtener valores de sensibilidad relativa, especificidad relativa y exactitud relativa del método alternativo, y la concordancia entre ambos métodos mediante el índice Kappa. Para la matriz esponja sobre carcasa de bovino se obtuvo una sensibilidad relativa de 100%, especificidad relativa de 100% y exactitud relativa de 100%. Mientras que en la matriz enjuague de carcasa de pollo los valores fueron de 80,7%, 100% y 90,4% respectivamente. La concordancia alcanzada fue de 1,0 y 0,81 para esponja sobre carcasa de bovino y enjuague de carcasa de pollo respectivamente. Los resultados permiten concluir que este nuevo método alternativo obtiene criterios muy buenos en comparación con su contraparte tradicional, entregándole a la Unidad de Bacteriología Pecuaria una nueva herramienta de análisis

The autophagy pathway is an essential component of the innate immune response, capable of rapidly targeting intracellular bacteria, which are subsequently degraded by lysosomal enzymes. Recent work has begun to elucidate the regulatory signalling for autophagy induction in response to pathogenic bacteria. However, the initial signalling regulating autophagy induction in response to the detection of pathogens remains largely unclear. Here we report that AMPK, an important upstream activator of the autophagy pathway, is rapidly stimulated upon detection of pathogenic bacteria, prior to bacterial invasion. Bacterial recognition is initially achieved through detection of outer membrane vesicles (OMVs). Additionally, we show that AMPK signalling relieves mTORC1-mediated repression of the autophagy pathway in response to Salmonella infection, positioning the cell for a rapid induction of autophagy. Surprisingly, we found that the activation of AMPK and inhibition of mTORC1 in response to extracellular Salmonella are not accompanied by an induction of bulk autophagy. However, upon Salmonella invasion AMPK signalling is required for efficient and selective targeting of bacteria-containing vesicles by the autophagy pathway through activation of pro-autophagic kinase complexes. Collectively, these results demonstrate a key role for AMPK signalling in coordinating the rapid autophagic response prior to invasion of pathogenic bacteria.

Die in dieser Arbeit etablierte Insertions-Duplikations-Mutagenese IDM ermöglicht es, das Genom von pathogenen Bakterien zu mutagenisieren und die so generierte Mutantenbank im high-throughput-Format auf Gene zu untersuchen, die unter bestimmten Bedingungen für das infektiöse Potential oder für das Überleben dieser Keime von Bedeutung sind. Die Grundlage von IDM bildet ein konditional replizierender Vektor, in den eine Genbank des Wirtsorganismus kloniert wird und der unter nicht-permissiven Replikationsbedingungen mittels homologer Rekombination ins Chromosom integriert und dadurch einen Gen-Knockout bedingt. Das IDM-Verfahren weist gegenüber der Transposon-Mutagenese den Vorteil auf, dass das Genom nach dem Zufallsprinzip saturierend mutagenisiert werden kann und dass keine hot spots für die Insertion auftreten. Darüber hinaus kann der mutierte Genlocus nach Screening der Mutanten schnell per PCR identifiziert werden, indem die Exzision des Vektors induziert und das klonierte, homologe Fragment sequenziert wird. Die Insertion des Vektors ins Chromosom und damit der Gen-Knockout ist selbst ohne Selektionsdruck sehr stabil, so dass die Mutanten im Zellkultur- oder Tier-System untersucht werden können. IDM wurde im Rahmen dieser Arbeit erfolgreich auf Salmonella enterica Serovar typhimurium und Listeria monocytogenes angewandt. Die Applikation von IDM auf S. typhimurium hatte zum Ziel, Gene zu identifizieren, deren Produkte für das Überleben dieses Gram-negativen Keims in Vollmedium unter Laborbedingungen essentiell sind. Ausgehend von 14.000 S. typhimurium Fragmentbank-Klonen konnten durch Induktion der Integration des Vektors 262 Klone identifiziert werden, für welche die Mutation zu einem lethalen Phänotyp führte. 116 der 262 entsprechenden Proteine konnte durch IDM erstmalig eine essentielle Funktion für die Vitalität von S. typhimurium zugewiesen werden. Darunter befinden sich sowohl Proteine, die homolog sind zu Proteinen anderer klinisch-relevanter Keime, als auch Proteine, die Salmonella-spezifisch sind. Der größte Teil der identifizierten Proteine ist in die Speicherung und Weitergabe von Information (Transkription, Translation, DNA-Reparatur etc.) involviert, viele sind allerdings auch Proteine unbekannter Funktion. Die Essentialität der durch IDM identifizierten Gene konnte durch die Konstruktion von konditional lethalen Mutanten bestätigt werden. IDM ist demnach das erste Mutagenese-Verfahren, welches das essentielle Gen-Set von S. typhimurium für das Überleben in Vollmedium zu definieren vermochte. Basierend auf den IDM Daten konnte es auf 511 Gene, d.h. auf 11 % des Gesamt-Genoms beziffert werden. Bei der Applikation von IDM auf L. monocytogenes lag der Fokus auf der Identifizierung von Genen, die für das Überleben dieses Gram-positiven Bakteriums im Zytosol von eukaryontischen Zellen von Bedeutung sind. Im Screening von bis dato 720 der 1491 L. monocytogenes Insertionsmutanten auf ein attenuiertes Replikationsverhalten in Caco-2 Zellen konnten 69 Mutanten selektioniert werden. In diesen Mutanten sind Gene ausgeknockt, deren Produkte hauptsächlich wichtige Funktionen in der Nährstoffbereitstellung, in der Energiesynthese und im Metabolismus inne haben. Mit der Insertions-Duplikations-Mutagenese IDM steht ein molekulares Werkzeug zur Verfügung, welches für die Identifzierung neuer targets für sowohl Breitband- als auch Spezies-spezifische Antiinfektiva eingesetzt werden kann und welches unbekannten Proteinen eine biologische Funktionen zuweisen kann. / Using insertion-duplication-mutagenesis IDM, that has been established in this work, it is possible to mutate the genome of pathogenic bacteria and to generate a mutant bank. This bank can be screened in a high throughput format on genes that are relevant under certain conditions for the infectious potential and the survival of these germs. IDM is based on a conditionally replicating vector which integrates into the chromosome under non-permissive replication conditions via homologous recombination after having cloned a gene bank from the host organism into it. The vector integration causes a knockout of the respective gene. In contrast to transposon mutagenesis, the IDM approach has the advantage that the whole genome is mutated and that no mutation hot spots occur. Furthermore, after having screened the mutant bank, the mutated gene locus can be identified very quickly by applicating PCR: excision of the vector is induced and the cloned, homologous fragment is sequenced. Integration of the vector into the chromosome and therefore the gene knockout is very stable even without any selection so that the mutants can be examined in the cell culture and animal model. In this work IDM has been applicated successfully to Salmonella enterica Servovar typhimurium and Listeria monocytogenes. Application of IDM to S. typhimurium aimed to identify genes whose products are essential for the survival of that Gram negative germ in rich medium under laboratory conditions. Starting with 14.000 S. typhimurium fragment-bank clones, induction of integration of the vector led to the identification of 262 clones for which the mutation resulted in a lethal phenotype. For 116 of the 262 respective proteins, this is the first data that assigns to these proteins an essential function for viability of S. typhimurium. Amongst them are proteins that are homologue to proteins of other clinically relevant germs, as well as proteins that are Salmonella specific. Most of the identified proteins are involved in information storage and processing (transcription, translation, DNA repair etc.), but many are proteins of unknown function. The essentiality of the identified genes could be confirmed by construction of conditionally lethal mutants. Hence, IDM is the first mutagenesis approach that reached to define the essential gene-set of S. typhimurium for survival in rich medium. Based on the IDM data it could be estimated at 511 genes, that means at 11 % of the whole genome. The application of IDM to L. monocytogenes focussed on the identification of genes that play an important role for the survival of that Gram-positive bacterium in the cytosol of eucaryotic cells. Until now 720 of the 1491 L. monocytogenes mutants have been screened on a attenuated replication behaviour in Caco-2 cells and 69 mutants have been selected. These mutants harbour mutations in genes whose products mainly hold important functions in nutrient uptake, energy synthesis and metabolism. The insertion-duplication-mutagenesis IDM has proven to be a suitable molecular tool that can be used for the identification of novel targets for both broad spectrum and species-specific antibiotics and that can assign a biological function to unknown proteins.

MSc (Med), Epidemiology and Biostatistics, Faculty of Health Sciences, University of the Witwatersrand / Introduction Given the increasing prevalence of non-typhoidal salmonella in humans, especially as an opportunistic illness associated with HIV, enhanced surveillance for non-typhoidal salmonella (NTS), including screening for antibiotic resistance, is conducted annually in South Africa. We aimed to determine whether there is an association between trimethoprim-sulfamethoxazole (TMP-SMX) prophylaxis and multi-drug resistant NTS infection, to establish whether various factors modify the relationship between TMP-SMX resistance and invasive NTS infection, to examine whether these associations vary by province, and to quantify the resistance rates of NTS to a range of antibiotics. Methods This study was a secondary analysis of enhanced surveillance data on NTS collected between 2003 and 2005. We used descriptive methods to assess the prevalence of NTS by year, province and serotype, and to determine the prevalence of four MDR patterns. Univariate and multivariate regression models were used to investigate the relationships between TMP-SMX prophylaxis and MDR NTS. Univariate logistic regression was used to assess the relationship between invasive NTS and TMP-SMX resistance. Results TMP-SMX prophylaxis is associated with the ACKSSuT pattern (OR 1.91, 95% CI 1.14 – 3.19, p=0.0080) and the AKSSuT MDR pattern (OR 2.00, 95% CI 1.26 – 3.15, p=0.0015). Being on TMP-SMX prophylaxis is associated with an increased odds of having at least one of the four MDR patterns investigated (OR 1.43, 95% CI 1.00 – 2.04, p=0.0388). We also found high rates of resistance to all antibiotics tested except for ciprofloxacin and imipenem. The highest resistance rate was observed for sulfamethoxazole (>75.85%). S. enterica Isangi isolates showed the highest levels of resistance, with 94.43% having at least one MDR pattern. Other factors significantly associated with MDR NTS were ESBL production, prior treatment with antibiotics, HIV status and resistance to TMP-SMX. Discussion and conclusions Isolates from patients on TMP-SMX prophylaxis were associated with an increased odds of having the ACKSSuT and AKSSuT MDR patterns, not taking into account other explanatory factors. These associations did not remain significant when possible confounders were taken into account. Despite the threat of increased multi-drug resistance, TMP-SMX prophylaxis remains important in certain clinical settings.

Introduction: Nontyphoidal Salmonella (NTS) has been associated with HIV from the outset of the HIV pandemic. The few NTS studies done in Africa and America have not documented the impact of HIV on clinical-microbiologic features and mortality in patients with NTS infection. This study determined the association between HIV serostatus and mortality proportion, clinical presentation, length of hospital stay, frequency of invasive NTS infection recurrence, NTS serotypes and estimated the population attributable fraction of mortality due to HIV among patients with invasive NTS infection in South Africa. Methods: Secondary data from enteric diseases national surveillance in South Africa from 2003 to 2006 were analysed as a cross sectional study. A total of 1 398 subjects with known HIV serostatus were obtained after data cleaning. Data analysis was done in Stata using chi squared test for categorical variables and Wilcoxon rank sum test / Kruskal- Wallis test for continuous variables. Logistic regression models were used to quantify the associations, and adjust for confounders and effect modification. Population attributable fraction was calculated to quantify the impact of HIV on mortality. Results: Majority (82.26%) of patients were HIV positive. The frequency pattern of HIV positive serostatus in different age groups coincided with that of invasive NTS. The overall mortality was 32.00%. HIV positive patients had a higher proportion (35.79 %) of mortality than HIV negative patients (15.55 %) (P<0.001). Fifty five percent of deaths in this study population were attributed to HIV infection. In multivariate models, HIV positive patients were more likely than HIV negative patients to die (OR = 2.50, 95% CI 1.69- 3.70), to develop lower respiratory tract infection (LRTI) (OR = 1.89, 95% CI,1.34- 2.65), to have recurrence of invasive NTS (OR = 3.90, 95% CI 1.41-10.77), to stay less than 16 days in hospitals (OR = 1.61, 95% CI, 1.08-2.40) and to be infected with Salmonella serotype Typhimurium infection (OR = 2.59, 95% CI 1.91-3.51). There were no significant differences in temperature, cardiac arrest, meningitis and site of specimen isolation (p>0.05). Discussion: The major limitation to this study was poor data quality of the surveillance system, including missing HIV serostatus hence the findings cannot be generalized to patients with unknown HIV status. Conclusion: HIV infection is common among patients with invasive NTS and is associated with excess mortality, LRTI, fewer than 16 days of hospital stay, recurrent invasive NTS infection and Salmonella Typhimurium. It is important for clinicians to rule out HIV infection in patients with invasive NTS especially those presenting with LRTI and Salmonella Typhimurium infection in addition to recurrent NTS infection, which is a wellknown feature associated with HIV. Recommendation: Since these patients received antimicrobials and had considerable mortality, the first line treatment of invasive NTS should be reviewed especially to HIV positive patients by investigating resistance patterns and conducting a clinical trial of newer and effective antimicrobials.

by Tam Fung-ping. / Thesis (M.Phil.)--Chinese University of Hong Kong, 1993. / Includes bibliographical references (leaves 254-264). / Chapter I. --- Title page --- p.I / Chapter II. --- Abstract --- p.II / Chapter III. --- Acknowlegements --- p.III / Chapter IV. --- Table of contents --- p.IV / Chapter V. --- List of tables --- p.V / Chapter VI. --- List of figures --- p.VI / Chapter VII. --- Abbreviations --- p.VII / Chapter Chapter 1. --- Literature Reviews / Chapter 1.1 --- Modes of energy generation in facultative bacteria --- p.1 / Chapter 1.1.1 --- Difference in energy generation mechanism between respiratory and fermentative pathways --- p.2 / Chapter 1.1.2 --- Difference in carbon metabolism during anaerobiosis --- p.6 / Chapter 1.2 --- Repression and derepression of genes during anaerobiosis --- p.8 / Chapter 1.3 --- Global regulatory network for respiratory control --- p.8 / Chapter 1.3.1 --- Fnr-regulated gene expression --- p.10 / Chapter 1.3.2 --- NarL-regulated gene expression --- p.11 / Chapter 1.3.3 --- Crp-regulated gene expression --- p.12 / Chapter 1.3.4 --- ArcA-regulated gene expression --- p.13 / Chapter 1.3.5 --- Overlapping control of gene expression --- p.14 / Chapter 1.3.6 --- Regulatory mechanism of respiratory control --- p.16 / Chapter 1.4 --- Other regulatory systems in respiratory control --- p.19 / Chapter 1.5 --- The puzzle of regulatory network in anaerobiosis --- p.22 / Chapter 1.6 --- ArcA-ArcB system in Escherichia coli --- p.24 / Chapter 1.6.1 --- Arc A and ArcB for aerobic respiratory control --- p.24 / Chapter 1.6.2 --- arcA/dye/msp/fex/sfrA/cpxC gene are on identical genetic locus --- p.26 / Chapter 1.6.3 --- Arc function and Sfr function of Arc A protein are separately regulated --- p.28 / Chapter 1.6.4 --- ArcB-ArcA as sensor regulator in two component system for respiratory control --- p.29 / Chapter 1.7 --- Objectives and strategies of present study --- p.37 / Chapter Chapter 2. --- Materials / Chapter 2.1 --- Bacterial strains --- p.41 / Chapter 2.2 --- Culture mediums --- p.44 / Chapter 2.3 --- "Buffers, chemicals and antibiotics" --- p.46 / Chapter 2.4 --- DNA primers --- p.53 / Chapter Chapter 3. --- Primer extension analysis for locating the transcriptional start point of anaerobic inducible arcA in pFS --- p.34 / Chapter 3.1 --- Introduction --- p.55 / Chapter 3.2 --- Methods --- p.57 / Chapter 3.2.1 --- Preparation of total RNA --- p.59 / Chapter 3.2.2 --- Formaldeyde agarose gel electrophoresis of RNA --- p.60 / Chapter 3.2.3 --- Spectrometric estimation of RNA --- p.61 / Chapter 3.2.4 --- End-labelling of arcAusp primer with 32P --- p.62 / Chapter 3.2.5 --- Precipitation of arcAusp primer with samples RNA --- p.63 / Chapter 3.2.6 --- Primer extension reaction --- p.63 / Chapter 3.3 --- Results / Chapter 3.3.1 --- Preparation of RNA --- p.67 / Chapter 3.3.2 --- Determination of transcription start site by primer extension --- p.67 / Chapter 3.4 --- Discussions --- p.76 / Chapter 3.4.1 --- Selective activations of aerobic and anaerobic transcripts in response to oxygen level --- p.76 / Chapter 3.4.2 --- The arcA promoter is a sigma-70 dependent promoter --- p.77 / Chapter 3.4.3 --- Experimental design --- p.77 / Chapter Chapter 4. --- In vitro chemical mutagensis for finding some important regulatory elements of arcA in pFS --- p.34 / Chapter 4.1 --- Introduction / Chapter 4.2 --- Methods --- p.84 / Chapter 4.2.1 --- Large scale preparation of pFS34 plasmid --- p.84 / Chapter 4.2.2 --- PCR-mediated chemical mutagenesis of pFS34 --- p.86 / Chapter 4.2.3 --- Restriction enzyme digestion of PCR-amplified arcA insert after phenol extraction --- p.90 / Chapter 4.2.4 --- Large scale preparation of vector pFZYl and restriction enzyme digestion --- p.91 / Chapter 4.2.5 --- Ligation of EcoRI-SalI digested pFS34 fragment and vector pFZYl --- p.91 / Chapter 4.2.6 --- Preparation of electrotcompetent cell Salmonella typhymurium JR502 and electro-transformation --- p.92 / Chapter 4.2.7 --- Screening of transformed clones by LB-amp50-xgal plates --- p.93 / Chapter 4.2.8 --- Screening of recombinants colonies by Polymerase chain reaction (PCR) --- p.94 / Chapter 4.2.9 --- Screening of single-point mutated clones by PCR-single stranded conformational polymorphism (PCR-SSCP) technique --- p.96 / Chapter 4.2.10 --- Screening of mutated pFS34 clones with altered promoter activities byβ-gal assay --- p.98 / Chapter 4.2.11 --- Sequencing of mutated clones --- p.101 / Chapter 4.2.11.1 --- Recombinant M13 single-stranded sequencing of the mutated clones --- p.101 / Chapter 4.2.11.2 --- pUC18 double-stranded DNA sequencing of mutated clones --- p.105 / Chapter 4.3 --- Results --- p.108 / Chapter 4.3. --- l PCR-mediated chemical mutagenesis of pFS34 --- p.108 / Chapter 4.3.2 --- Screening of transformed clones by LB-amp50-xgal plate --- p.112 / Chapter 4.3.3 --- Screening of recombinants colonies by polymerase chain reaction (PCR) --- p.112 / Chapter 4.3.4 --- Screening of single-point mutated clones by PCR-single stranded conformational polymorphism (PCR-SSCP) technique --- p.114 / Chapter 4.3.5 --- Screening of mutated pFS34 clones with altered promoter activities byβ-gal assay --- p.117 / Chapter 4.3.6 --- Sequencing of mutated clones --- p.123 / Chapter 4.4 --- Discussions --- p.135 / Chapter 4.4.1 --- The possible mechanisms in anaerobic transcription --- p.135 / Chapter 4.4.2 --- The possible mechanisms in aerobic transcription --- p.143 / Chapter 4.4.3 --- Experimental design --- p.146 / Chapter Chapter 5 --- Investigation of the effect of integration host factor (IHF) and autoregulation on the expression of pFS34 / Chapter 5.1 --- Introduction --- p.152 / Chapter 5.2 --- Methods --- p.154 / Chapter 5.2.1 --- Construction of Escherichia coli mutant --- p.155 / Chapter 5.2.2 --- PCR check of mutant for the presence of pFS34 and pFZYl plasmid --- p.157 / Chapter 5.2.3 --- β-galactosidase assay of aerobic and anaerobic activities change of pFS34 --- p.157 / Chapter 5.3 --- Results / Chapter 5.3.1 --- Effect of integration factor (IHF) on pFS34 --- p.158 / Chapter 5.3.1.1 --- PCR analysis of E. coli. himA and himD mutant for the presence of pFS34 and pFZYl plasmid --- p.158 / Chapter 5.3.1.2 --- β-galatosidase assay of aerobic and anaerobic activities of pFS34 in E. coli. himA and himD mutant --- p.158 / Chapter 5.3.2 --- Autoregultion on expression of pFS34 --- p.162 / Chapter 5.3.2.1 --- PCR analysis of E. coli. arcA mutant for the presence of pFS34 plasmid --- p.162 / Chapter 5.3.2.2 --- β-galctosidase assay of aerobic and anaerobic activities of pFS34 (arcA-lacZ) in E. coli. arcA mutant --- p.162 / Chapter 5.4 --- Discussions --- p.167 / Chapter 5.4.1 --- Effect of IHF on aerobic and anaerobic expression of arcA --- p.167 / Chapter 5.4.1.1 --- Possible regulatory mechanism of IHF on aerobic transcription --- p.167 / Chapter 5.4.1.2 --- Possible regulatory mechanism of IHF on anaerobic transcription --- p.170 / Chapter 5.4.1.3 --- Affinity binding of IHF depends on topological state of arcA --- p.172 / Chapter 5.4.1.4 --- Possible role of IHF in global regulation of anaerobiosis --- p.173 / Chapter 5.4.1.5 --- Experimental design --- p.174 / Chapter 5.4.2 --- Autoregulatory expression of arcA in pFS34 --- p.176 / Chapter Chapter 6. --- PCR walking of arcA from Salmonella typhimurium LT2 / Chapter 6.1 --- Introduction --- p.177 / Chapter 6.2 --- Methods --- p.186 / Chapter 6.2.1 --- Preparation of chromosomal DNA from Salmonella typhimurium LT2 --- p.186 / Chapter 6.2.2 --- Amplification of genomic arcA by linear PCR with arcAcds primer --- p.187 / Chapter 6.2.3 --- Low stringency PCR amplification of single-stranded arcA gene fragment and genomic DNA with anchor- random primer (delC-32R & delC-34R) --- p.188 / Chapter 6.2.4 --- High stringency PCR amplification with arcAcds primer and delC-23 primer --- p.189 / Chapter 6.2.5 --- High stringency PCR amplification with arcAusp2 and delC-23 primer --- p.190 / Chapter 6.2.6 --- "High stringency PCR amplification with delC-23 primer only, arcAusp2 primer only and mixture of delC-23 and arcAusp2 primer" --- p.191 / Chapter 6.2.7 --- High stringency PCR amplification with arcAusp2 only and Sau3A restriction enzyme digestion of PCR products --- p.192 / Chapter 6.2.8 --- Cloning of PCR walking products into pUC18 and heat shock transforming into E.coli. JM83 --- p.193 / Chapter 6.2.9 --- Confirmation of inserts in the clones and estimation of inserts size by PCR --- p.194 / Chapter 6.2.10 --- Dideoxy sequencing of PCR walking arcA fragments in pUC18 --- p.194 / Chapter 6.2.11 --- Subcloning of arcA fragment into pFZYl and PCR analysis for insertion of one insert with proper orientation --- p.195 / Chapter 6.2.12 --- arcA-galactosiadase assay of PCR walking arcA fragment-lacZ fusion --- p.196 / Chapter 6.3 --- Results --- p.198 / Chapter 6.3.1 --- Preparation of chromosomal DNA from Salmonella typhimurium LT2 --- p.198 / Chapter 6.3.2 --- Amplification of genomic arcA by linear PCR with arcAcds primer --- p.198 / Chapter 6.3.3 --- Low stringency PCR amplification of single-stranded arcA gene fragment and genomic DNA with anchor- random primer (delC-32R and delC-34R) --- p.200 / Chapter 6.3.4 --- High stringency PCR amplification with arcAcds primer and delC-23 primer --- p.200 / Chapter 6.3.5 --- High stringency PCR amplification with arcAusp2 、 primer and delC-23 prime --- p.203 / Chapter 6.3.6 --- "High stringency PCR amplification with delC-23 primer only, arcAusp2 primer only and mixture of delC-23 and arcAusp2 primer to check for flanking ends of bands" --- p.205 / Chapter 6.3.7 --- High stringency PCR amplification with arcAusp2 primer and Sau3A restriction enzyme digestion of PCR products --- p.207 / Chapter 6.3.8 --- Cloning of PCR walking products into pUC18 and heat-shock transforming into E. coli. JM83 --- p.210 / Chapter 6.3.9 --- Confirmation of inserts in the clones and estimation of inserts size by PCR --- p.210 / Chapter 6.3.10 --- Dideoxy sequencing of arc A PCR walking fragment: :pUC18 --- p.210 / Chapter 6.3.11 --- Subcloning of arcA fragment into pFZYl and PCR check for right insertion of single insert with proper orientation --- p.226 / Chapter 6.3.12 --- β-galactosidase assay --- p.232 / Chapter 6.4 --- Discussions --- p.227 / Chapter 6.4.1 --- PCR based gene walking strategy --- p.227 / Chapter 6.4.2 --- Confirmation of cloned arcA gene in pFS34 was a geniune arcA gene of S. typhimurium --- p.240 / Chapter 6.4.3 --- Promoter activity of further upstream arcA clones - AU87::pFZYl --- p.241 / Chapter Chapter 7. --- Overall Discussion --- p.244 / Chapter 7.1 --- Summary --- p.244 / Chapter 7.2 --- Proposed Model of regulation of arcA in Salmonella typhimurium --- p.249 / Chapter 7.3 --- Further Studies --- p.251 / References --- p.254

by Cheung, Man Wai William. / Thesis (M.Phil.)--Chinese University of Hong Kong, 1992. / Includes bibliographical references (leaves 113-123). / ABSTRACT --- p.i / ACKNOWLEDGMENTS --- p.ii / DEDICATION --- p.iii / TABLE OF CONTENTS --- p.iv / LIST OF FIGURES --- p.viii / LIST OF TABLES --- p.x / Chapter 1. --- Introduction --- p.1 / Chapter 1.1. --- General Introduction --- p.1 / Chapter 1.2. --- Purpose of Study --- p.3 / Chapter 2. --- Literature Review --- p.7 / Chapter 2.1. --- Central Pathways of Aerobic and Anaerobic Carbon Catabolism --- p.7 / Chapter 2.2. --- Global Regulation of Gene Expression by Oxygen --- p.10 / Chapter 2.2.1. --- Two approaches for the studies --- p.10 / Chapter 2.2.2. --- FNR regulation --- p.12 / Chapter 2.2.3. --- ArcAB regulation --- p.19 / Chapter 2.2.3.1. --- arcA --- p.19 / Chapter 2.2.3.2. --- arcB --- p.20 / Chapter 2.2.3.3. --- A member of the Two- Components regulatory systems --- p.21 / Chapter 2.3. --- Molecular Analysis of Promoters --- p.26 / Chapter 2.3.1. --- S1 mapping --- p.29 / Chapter 2.3.2. --- Primer extension --- p.29 / Chapter 2.3.3. --- DNaseI footprinting --- p.30 / Chapter 2.3.4. --- Mutational analysis of promoters --- p.32 / Chapter 3. --- Materials and Methods --- p.35 / Chapter 3.1. --- Bacterial strains and Plasmids --- p.35 / Chapter 3.2. --- Media --- p.35 / Chapter 3.3. --- Solutions --- p.38 / Chapter 3.4. --- Small Scale Preparation of Plasmid DNA --- p.40 / Chapter 3.5. --- Large Scale Preparation of Plasmid DNA --- p.41 / Chapter 3.5.1. --- Growth of bacterial culture --- p.41 / Chapter 3.5.2. --- Lysis by alkali --- p.43 / Chapter 3.5.3. --- Purification of closed circular DNA by cesium chloride gradient equilibrium centrifugation --- p.44 / Chapter 3.5.4. --- Digestion of DNA with restriction endonucleases --- p.45 / Chapter 3.6. --- Analysis of DNA Samples with Agarose Gel Electrophoresis --- p.45 / Chapter 3.7. --- Cloning of DNA Fragments from Nest-deleted M13mpl8 Clones to pFZYl --- p.47 / Chapter 3.8. --- Introduction of Plasmids into Cells --- p.48 / Chapter 3.8.1. --- Heat shock transformation --- p.48 / Chapter 3.8.1.1. --- Preparation of competent cells (I) --- p.48 / Chapter 3.8.1.2. --- Preparation of competent cells (II) --- p.49 / Chapter 3.8.2. --- High efficiency transformation by electroporation --- p.50 / Chapter 3.8.2.1. --- Preparation of electro- competent cells --- p.50 / Chapter 3.8.2.2. --- Electro-transformation --- p.51 / Chapter 3.9. --- DNA Sequencing by Chain Termination Method --- p.51 / Chapter 3.9.1. --- Preparation of single-stranded M13 templates for sequencing reaction --- p.51 / Chapter 3.9.2. --- Sequencing reactions using single- stranded templates --- p.53 / Chapter 3.9.3. --- Preparation of polyacrylamide gel for sequencing --- p.54 / Chapter 3.9.4. --- Electrophoresis of the DNA samples --- p.55 / Chapter 3.10. --- Construction of Nested Clones by Exonuclease III Unidirectional Deletions --- p.55 / Chapter 3.10.1. --- Unidirectional nested deletion of M13mpl8 clones --- p.55 / Chapter 3.10.2. --- Screening of nested clones by Direct gel electrophoresis --- p.56 / Chapter 3.10.3. --- Screening of nested clones of M13mpl8 and pFZYl by Polymerase Chain Reaction --- p.57 / Chapter 3.11. --- β-galactosidase Assay --- p.59 / Chapter 3.12. --- Primer Extension --- p.60 / Chapter 3.12.1. --- Preparation of total RNA from Gram- negative bacteria --- p.60 / Chapter 3.12.2. --- Labelling the 5' end of the oligonucleotides --- p.61 / Chapter 3.12.3. --- Hybridization and primer extension --- p.62 / Chapter 4. --- Result --- p.63 / Chapter 4.1. --- Subcloning of arcA promoter into M13mpl8/19 --- p.63 / Chapter 4.2. --- Sequencing of p34一18i and p3419i using M13 Sequencing primers (-47) and ArcA-cds Primers --- p.63 / Chapter 4.3. --- Unidirectional Nested Deletion of p3418i using Exonuclease III --- p.65 / Chapter 4.3.1. --- Large scale preparation of p3A18i DNA for Exonuclease III unidirectional nested deletion --- p.65 / Chapter 4.3.2. --- Construction of 3' and 5' overhangs --- p.65 / Chapter 4.3.3. --- Exonuclease III digestion --- p.67 / Chapter 4.3.4. --- Repairing of the 3' and 5' overhangs to generate blunt ends --- p.67 / Chapter 4.3.5. --- Blunt-end ligation of the nested deletion M13mpl8 subclones p3418i --- p.67 / Chapter 4.3.6. --- Transformation --- p.69 / Chapter 4.3.7. --- Screening of nest-deleted p3418i clones by Direct Gel --- p.71 / Chapter 4.3.8. --- Screening of nested deletion p3418i clones by PCR Screening --- p.73 / Chapter 4.3.9. --- Sequencing of the nested deletion p3418i clones --- p.76 / Chapter 4.4. --- Cloning of Nested Deletion DNA Fragments from M13mpl8 into pFZYl --- p.80 / Chapter 4.4.1. --- Screening of pFZYl clones using PCR Screening --- p.80 / Chapter 4.5. --- Expression of Nest-Deleted arcA Promoter Clones in E. coli MC1061-5 --- p.87 / Chapter 4.6. --- Expression of Nest-Deleted arcA Promoter Clones in S. typhimurium JR501 --- p.89 / Chapter 4.7. --- Primer Extension --- p.89 / Chapter 5. --- Discussion --- p.93 / Chapter 5.1. --- Sequencing of arcA Promoter --- p.93 / Chapter 5.2. --- Unidirectional Nested Deletion of p3A18i using Exonuclease III --- p.94 / Chapter 5.3. --- Screening of Nest-deletion p3418i Subclones --- p.95 / Chapter 5.4 --- Cloning of Nest-deleted DNA Fragments from M13mpl8 Subclones into pFZYl --- p.99 / Chapter 5.5. --- Screening of Nest-deleted pFZYl Subclones of p3418i --- p.101 / Chapter 5.6. --- The Effect of 5' Unidirectional Nested Deletion on the Expression of the Cloned arcA promoter in E. coli M1061-5 and S typhimurium JR501 --- p.102 / Chapter 5.7. --- Primer Extension --- p.102 / Chapter 5.8. --- Sequence Analysis of the Cloned arcA Promoter --- p.104 / Chapter 6. --- Conclusion and Further Studies --- p.111 / Chapter 7. --- Reference Cited --- p.113

RNA-dependent control of gene expression is crucial for bacterial adaptation to environmental stresses, such as fluctuations in ambient temperature. In the enteric pathogen Salmonella Typhimurium, a drastic downshift in temperature immediately triggers the “cold shock response” in which selective expression of cold shock proteins (CSPs) aids acclimatisation. The major cold shock protein, CspA, and some of its homologues function as RNA chaperones and play critical roles in destabilising aberrant RNA secondary structures that form at reduced temperatures. However, the precise roles and targets of this protein family remain unclear. With the aim of generating a genome-wide map of protein-RNA interactions, in vivo UV cross-linking and analysis of cDNA (CRAC) was performed. This novel, high-throughput technique allows identification of all RNA targets for a particular protein, which in this case was the cold-induced protein CspA, and constitutively expressed CspE. CRAC results reveal a remarkable number and diversity in the RNA targets of these CSPs. For example, CspA targets approximately 25% of the RNA encoded by the Salmonella genome. CspA and CspE were shown to target mRNAs encoding proteins involved in metabolism, stress, cell division and RNA turnover, as well as a number of mRNAs that are cold shock-inducible. Bioinformatic analyses have shown that mainly protein coding regions are targeted and, interestingly, 5’ untranslated regions (UTR) and small RNAs, which often play roles as regulators of translational control. There also appears to be a reproducible pattern of repeated binding along mRNA transcripts, suggesting a role for these Csps in maintaining mRNAs in a linear conformation, which is required for efficient translation. To validate targets, phenotypic analyses were performed, including growth studies during amino acid starvation, and the response to heat shock and UV DNA damage. These experiments confirmed involvement of the paralogues, and further bioinformatic analysis revealed that these proteins were targeting key regulatory regions on some specific targets. A more in-depth analysis was carried out on one target – the general stress response sigma factor RpoS (σS) and a model of CspA paralogue involvement in regulating the mRNA of this target is presented. Overall, the in vivo data from this study suggests that these cold shock proteins are crucial for modulating key cellular processes beyond that which their name implies.