by Lee Kai Fai, Calvin. / Thesis (Ph.D.)--Chinese University of Hong Kong, 1995. / Includes bibliographical references (leaves 159-167). / ACKNOWLEDGMENTS --- p.i / ABSTRACT --- p.ii / CONTENTS --- p.iv / ABBREVIATIONS --- p.xi / Chapter CHAPTER ONE --- General Introduction --- p.1 / Chapter 1.1 --- The Phenomenon of Host-controlled Restriction --- p.1 / Chapter 1.2 --- Classification of Restriction and Modification Systems --- p.2 / Chapter 1.2.1 --- Type I Restriction-Modification Systems --- p.2 / Chapter 1.2.2 --- Type II Restriction-Modification Systems --- p.3 / Chapter 1.2.3 --- Type III Restriction-Modification Systems --- p.4 / Chapter 1.2.4 --- Type IV Restriction-Modification Systems --- p.5 / Chapter 1.3 --- Occurrence of Restriction-Modification Systems --- p.6 / Chapter 1.4 --- Effect of Methylation --- p.7 / Chapter 1.5 --- Alternation of Recognition Specificities --- p.7 / Chapter 1.5.1 --- Cross Protection by DNA Methyltransferase --- p.8 / Chapter 1.5.2 --- A-Assisted Restriction Endonuclease (RARE) Cleavage --- p.9 / Chapter 1.5.3 --- Site-specific Cleavage mediated by Triple-helix formation --- p.9 / Chapter 1.5.4 --- Site-specific Cleavage of Duplex DNA with a λ repressor- Staphylococcal Nuclease Hybrid --- p.10 / Chapter 1.5.5 --- Achilles' heel Cleavage --- p.10 / Chapter 1.5.6 --- Chimeric Restriction Endonuclease --- p.11 / Chapter 1.6 --- Cloning of Restriction and Modification Systems --- p.11 / Chapter 1.6.1 --- Selection based on Modification --- p.11 / Chapter 1.6.2 --- Other Cloning Strategies --- p.12 / Chapter 1.6.2.1 --- Sub-Cloning of Plasmids --- p.12 / Chapter 1.6.2.2 --- Selection based on Restriction --- p.13 / Chapter 1.6.2.3 --- Multi-step Cloning --- p.13 / Chapter 1.6.2.4 --- Cloning in AP1-200 and AP1-200-9 strain --- p.13 / Chapter 1.6.2.5 --- Direct Cloning of Restriction gene by 'endo-blue' method --- p.14 / Chapter 1.7 --- Genetic Location of Restriction-Modification Systems --- p.14 / Chapter 1.8 --- Sequences of Restriction-Modification Systems --- p.15 / Chapter 1.9 --- Catalytic Properties of Type II Restriction-Modification Systems --- p.17 / Chapter 1.10 --- Crystallography of Type II Restriction and Modification Enzymes --- p.19 / Chapter 1.11 --- Evolution of Type II Restriction and Modification Enzymes --- p.22 / Chapter 1.12 --- Aim of Study --- p.23 / Chapter CHAPTER TWO --- Materials and Methods --- p.24 / Chapter 2.1 --- Bacterial Strains --- p.24 / Chapter 2.2 --- General Techniques --- p.25 / Chapter 2.2.1 --- Phenol/Chloroform Extraction --- p.25 / Chapter 2.2.2 --- Ethanol Precipitation --- p.25 / Chapter 2.2.3 --- Spectrophotometry --- p.25 / Chapter 2.2.4 --- Restriction digestion of DNA --- p.26 / Chapter 2.2.5 --- Agarose Gel Electrophoresis of DNA --- p.26 / Chapter 2.2.6 --- Recovery of DNA fragment from Agarose gel --- p.26 / Chapter 2.2.7 --- Minipreparation of Plasmid --- p.27 / Chapter 2.2.8 --- Large-Scale Preparation of Plasmid DNA --- p.28 / Chapter 2.2.8A --- By Equilibrium Centrifugation in Cesium Chloride- Ethidium Bromide Gradient --- p.28 / Chapter 2.2.8B --- By Using Qiagen-tip 100 Cartridge --- p.29 / Chapter 2.2.9 --- Preparation of Competent Cells --- p.30 / Chapter 2.2.10 --- Transformation of Competent Cells --- p.31 / Chapter 2.2.11 --- Screening of Recombinant Plasmids --- p.32 / Chapter 2.2.11A --- Using Selective media --- p.32 / Chapter 2.2.11B --- Rapid Alkaline Lysis Method --- p.32 / Chapter 2.2.12 --- Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis (SDS-PAGE) --- p.33 / Chapter 2.2.13 --- Size Exclusion Chromatography --- p.34 / Chapter 2.2.14 --- Electroblotting of Protein on Polyvinylidene Difluoride (PVDF) membrane --- p.35 / Chapter 2.2.15 --- Isoelectric Focusing (EEF) --- p.36 / Chapter 2.2.16 --- Protein Assay --- p.37 / Chapter 2.3 --- DNA Sequencing --- p.37 / Chapter 2.3.1 --- Isolation of a template DNA --- p.38 / Chapter 2.3.2 --- DNA Denaturation and Annealing Reaction --- p.38 / Chapter 2.3.3 --- Labeling and Termination Reaction --- p.38 / Chapter 2.3.4 --- DNA Sequencing Electrophoresis --- p.39 / Chapter 2.3.5 --- Autoradiography --- p.40 / Chapter CHAPTER THREE --- Purification and Characterization of Restriction Endonuclease from Escherichia coli HK31 --- p.41 / Chapter 3.1 --- Introduction --- p.41 / Chapter 3.2 --- Materials and Methods --- p.42 / Chapter 3.2.1 --- Preparation of Crude enzyme Extract --- p.42 / Chapter 3.2.2 --- Purification of R.EcoHK31I --- p.42 / Chapter 3.2.3 --- Characterization of Restriction endonuclease --- p.43 / Chapter 3.2.3.1 --- Enzyme Activity assay --- p.43 / Chapter 3.2.3.2 --- "Optimal pH, Temperature, Metal Ion and Salt concentration of R.EcoHK31I" --- p.43 / Chapter 3.2.3.3 --- Assay for the Purity of R.EcoHK31I --- p.43 / Chapter 3.2.3.4 --- Determination of Recognition Specificity --- p.44 / Chapter 3.2.3.5 --- Determination of the Cleavage Specificity --- p.44 / Chapter 3.3 --- Results and Discussion --- p.45 / Chapter 3.3.1 --- Purification ofR.EcoHK31I from Escherichia coli HK31 --- p.45 / Chapter 3.3.2 --- "Optimal pH,Temperature, Metal ions and Salt concentration of R.EcoHK31I" --- p.46 / Chapter 3.3.3 --- Unit Definition --- p.51 / Chapter 3.3.4 --- Purity of the R.EcoHK31I --- p.51 / Chapter 3.3.5 --- Recognition Site of the R.EcoHK31I --- p.51 / Chapter 3.3.6 --- Sensitivity of the R.EcoHK31I to dcm Methylation --- p.52 / Chapter 3.3.7 --- Cleavage Specificity of R.EcoHK31I --- p.52 / Chapter CHAPTER FOUR --- Cloning of EcoEK31I Restriction and Modification (R-M) System from Escherichia coli HK31 --- p.57 / Chapter 4.1 --- Introduction --- p.57 / Chapter 4.2 --- Materials and Methods --- p.58 / Chapter 4.2.1 --- Extraction of genomic DNA from E. coli HK31 --- p.58 / Chapter 4.2.2 --- Extraction of Extra-Chromosomal DNA from E. coli HK31 --- p.59 / Chapter 4.2.3 --- Restriction Digestion of the Total DNA --- p.59 / Chapter 4.2.4 --- Preparation of Linearized and Dephosphorylated Vector --- p.60 / Chapter 4.2.5 --- Fill-in Reaction --- p.60 / Chapter 4.2.6 --- Ligation between Vector and Digested Chromosomal DNA --- p.61 / Chapter 4.2.7 --- Selection of Clones Harboring Methyltransferase gene --- p.61 / Chapter 4.2.8 --- Screening of the Survival Clones --- p.62 / Chapter 4.3 --- Results --- p.62 / Chapter 4.3.1 --- Construction of Genomic Libraries --- p.62 / Chapter 4.3.2 --- Selection of the Methyltransferase Gene --- p.66 / Chapter 4.3.3 --- In vitro Detection of R.EcoHK31I activity --- p.67 / Chapter 4.3.4 --- Functional Localization of EcoHK31I --- p.67 / Chapter 4.3.5 --- Subcloning of the Complete EcoHK31I R-M System --- p.72 / Chapter 4.4 --- Discussion --- p.72 / Chapter 4.4.1 --- Construction of Genomic Libraries --- p.72 / Chapter 4.4.2 --- Cloning of EcoHK31I Restriction and Modification System --- p.75 / Chapter 4.4.2.1 --- Selecting Endonuclease --- p.75 / Chapter 4.4.2.2 --- Detection of Restriction Endonuclease Activity --- p.76 / Chapter 4.4.3 --- Functional Localization of the R-M System --- p.76 / Chapter CHAPTER FIVE --- The Nucleotide Sequences of the EcoHK31I R-M System --- p.78 / Chapter 5.1 --- Introduction --- p.78 / Chapter 5.2 --- Materials and Methods --- p.79 / Chapter 5.2.1 --- Sequencing Strategies --- p.79 / Chapter 5.2.2 --- DNA Sequencing --- p.80 / Chapter 5.2.3 --- Sequence Analysis --- p.80 / Chapter 5.3 --- Results and Discussion --- p.80 / Chapter 5.3.1 --- Nucleotide Sequences and Deduced Amino Acid sequences --- p.80 / Chapter 5.3.2 --- Comparison of Amino Acid Sequences --- p.85 / Chapter CHAPTER SIX --- Purification and Characterization of EcoHK31I Methyltransferase from E. coli K802 [pEcoHK31E] --- p.91 / Chapter 6.1 --- Introduction --- p.91 / Chapter 6.2 --- Materials and Methods --- p.92 / Chapter 6.2.1 --- Preparation of Crude enzyme Extract --- p.92 / Chapter 6.2.2 --- Purification of M.EcoHK31I --- p.92 / Chapter 6.2.3 --- Characterization of EcoHK31I Methyltransferase --- p.93 / Chapter 6.2.3.1 --- Enzyme Activity assay --- p.93 / Chapter 6.2.3.2 --- Determination of Methylation specificity --- p.93 / Chapter 6.2.3.3 --- Determination of Molecular weight of M.EcoHK31I --- p.94 / Chapter 6.2.3.4 --- Determination ofM.EcoHK31I Kinetics --- p.94 / Chapter 6.3 --- Results and Discussion --- p.96 / Chapter 6.3.1 --- Purification of EcoHK31I Methyltransferase --- p.96 / Chapter 6.3.2 --- M.EcoHK31I Modification Specificity --- p.99 / Chapter 6.3.3 --- "Determination of Molecular Weight ofM,EcoHK31I" --- p.99 / Chapter 6.3.4 --- Catalytic Properties of EcoHK31I Methyltransferase --- p.103 / Chapter 6.3.5 --- A Novel m5C-MTase M.EcoHK31I --- p.103 / Chapter CHAPTER SEVEN --- Over-expression and Characterization of EcoHK31I Restriction and Modification Enzymes --- p.106 / Chapter 7.1 --- Introduction --- p.106 / Chapter 7.1.1 --- Expression Vector pTrc series --- p.107 / Chapter 7.1.2 --- Expression Vector pET series --- p.107 / Chapter 7.2 --- Materials and Methods --- p.109 / Chapter 7.2.1 --- General technique --- p.109 / Chapter 7.2.2 --- Polymerase Chain Reaction --- p.110 / Chapter 7.2.3 --- Construction of plysSM13 --- p.110 / Chapter 7.2.4 --- Construction of pTrc99A-R36 --- p.110 / Chapter 7.2.5 --- Construction of pET3a-M38 --- p.111 / Chapter 7.2.6 --- Construction of pET3a-C23 --- p.111 / Chapter 7.2.7 --- Expression of Recombinant Proteins in E. coli hosts --- p.115 / Chapter 7.2.8 --- Purification of Recombinant R.EcoHK31I --- p.115 / Chapter 7.2.9 --- Determination of Molecular Weight of Recombinant R. EcoHK31I --- p.115 / Chapter 7.2.10 --- Polyclonal Antibodies against R.EcoHK31I --- p.116 / Chapter 7.2.11 --- Western Blotting --- p.116 / Chapter 7.2.12 --- Purification of Recombinant M.EcoHK31I polypeptide α --- p.117 / Chapter 7.2.13 --- Purification of Recombinant M.EcoHK31I polypeptide β --- p.118 / Chapter 7.2.14 --- In vitro Complementation Methylation Activity --- p.118 / Chapter 7.2.15 --- Incorporation of [3H]-AdoMet to non-methylated Lambda DNA --- p.119 / Chapter 7.3 --- Results and Discussion --- p.119 / Chapter 7.3.1 --- Expression of Recombinant R. EcoHK31I --- p.119 / Chapter 7.3.2 --- Purification and Characterization of Recombinant R.EcoHK31I --- p.120 / Chapter 7.3.2.1 --- Purification of Recombinant R.EcoHK31I --- p.120 / Chapter 7.3.2.2 --- Characterization of Recombinant R.EcoHK31I --- p.122 / Chapter 7.3.2.2.1 --- Molecular Weight and Isoelectric point of the Recombinant R.EcoHK31I --- p.122 / Chapter 7.3.2.2.2 --- Antibodies to Recombinant R.EcoHK31I --- p.125 / Chapter 7.3.3 --- Expression and Purification of M.EcoHK31Ipolypeptide α --- p.127 / Chapter 7.3.4 --- Expression and Purification of M.EcoHK31I polypeptide β --- p.127 / Chapter 7.3.5 --- Characterization of M.EcoHK31I polypeptides a and β --- p.129 / Chapter 7.3.5.1 --- Molecular Weight Determination --- p.129 / Chapter 7.3.5.2 --- Isoelectric Point Determination --- p.132 / Chapter 7.3.5.3 --- In vivo and in vitro Methylation Activity --- p.132 / Chapter CHAPTER EIGHT --- Generation and Activity Assay of Q193G Mutein of M.EcoHK31I Polypeptide a --- p.138 / Chapter 8.1 --- Introduction --- p.138 / Chapter 8.2 --- Materials and Methods --- p.139 / Chapter 8.2.1 --- Construction of pET3a-M38 (Q193G) --- p.139 / Chapter 8.2.2 --- Expression and Purification of Q193G protein --- p.140 / Chapter 8.2.3 --- In vivo and in vitro methylation activity of Q193G Mutein --- p.140 / Chapter 8.3 --- Results and Discussion --- p.145 / Chapter 8.3.1 --- "Construction, Expression and Purification of Q193G Mutein" --- p.145 / Chapter 8.3.2 --- Determination of Molecular Weight and Isoelectric point of Q193G --- p.145 / Chapter 8.3.3 --- In vivo and in vitro methylation activity of Q193G Mutein --- p.145 / Chapter 8.3.4 --- Recognition Specificity of Q193G Mutein --- p.147 / Chapter CHAPTER NINE --- General Discussion --- p.151 / REFERENCES --- p.159 / APPENDIX A --- p.168

由於潔淨用水日漸短缺，科學家著力研究各種水淨化方法，其中以光催化技術作水淨化處理為可行的方法之一。光催化是以半導體光催化劑在光照射下所產生的活性物種（reactive oxidative species）進行消毒，其中的失活原理、各活性物種的作用和活性物種對細菌的攻擊方位，雖然已有廣範的研究，但當中仍有不清之處，比如說過氧化氫(H₂O₂)在光催化失活的作用便是其中之一，在光催化系統中所產生的H₂O₂濃度一般較低，因此其對細菌失活的效能仍然存有爭議。 / 本研究設計一種新的反應器去研究H₂O₂在連續供應模式中的失活動力學。在 8 mM 的H₂O₂下，10⁵的大腸桿菌(Escherichia coli)在8小時內完全失活。而在 2 mM 的H₂O₂ 下，並無出現顯著失活，由於該濃度遠遠高於一般光催化系統所產生的濃度（<50 μM），因此可以推斷，即使一般光催化系統所產生的H₂O₂是連續供應，也不會使細菌失活。然而在光照的情況下，其失活動力學大為不同，在強光照射(200 mW cm⁻²)下，H₂O₂的失活效率顯著增強，證明光照和過氧化氫之間存有協同效應。這現象亦出現於光預處理過(light pretreated)的大腸桿菌，進一步證實了光照改變細菌的生理機能，從而使其易於被H₂O₂失活。 / 其後我們使用RNA測序(RNA sequencing)去檢測的大腸桿菌的基因表達水平在光照下的變化，以便研究光照和H₂O₂之間的協同作用的機理。大多數涉及抵抗氧化的基因，包括過氧化氫酶(catalase, CAT)和超氧化物歧化酶(superoxide dismutase,SOD)的表達、DNA修復及細菌內的鐵含調控等等，其mRNA 水平沒有顯著的增加或減少，只有dps、fes和sodB有明顯的變化。此外，還有幾種調控細胞內的銅合量（cutA和cueR）和細胞膜組成（ompA、ompC、resx和gnsB）的基因在光照下產生顯著變化。 經RNA測序後，我們選定了10個目標基因，並選擇相對的大腸桿菌變異體(mutants)，對比他們和母體(E. coli BW25113)經過光預處理後被H₂O₂的失活效能。雖然這次研究並未找到相關基因，但研究結果表示，光照和H₂O₂的協同效應，應該是光照增加細胞膜的通透性和提高細菌內Fenton劑含量，使細菌內的羥基自由基(·OH)的濃度增加，因此加強對細菌DNA的損傷。 / 最後，我們亦比較了AgBr/Ag/Bi₂WO₆在不同的光源的照射下的對大腸桿菌的光催化失活效率。雖然發光二極管(light emitting diode)和熒光管都常用於室內照明，但AgBr/Ag/Bi₂WO₆的細菌失活效率在兩者的光照下表現出顯著的差異，而不同的發射波長下的細菌失活效率和AgBr/Ag/Bi₂WO₆光學吸收表現出良好的相關性。此外，相對其他顏色的發光二極管，綠色發光二極管照射下在犧牲劑研究(scavenger study)的結果大為不同，進一步表明了光照的發射波長(emissionwavelength)對光催化失活機制的影響。 / 本研究揭示了H₂O₂和光照在光催化失活中的重要性，並演示了H₂O₂和光照射之間的協同作用，也闡明了光照的屬性如何影響光催化下各活性物種的產生。本研究不僅提供了一個新的角度去探討的光照、H₂O₂和細菌的生理狀態在光催化失活中的重要性，也提供了新的方向和方法去研究光催化失活機制的。 / Due to the increasing concern for the need of clean drinking water, different methods for water purification have been developed. Photocatalysis, which makes use of semiconductor photocatalyst for the generation of reactive charged and oxidative species (ROSs) under light irradiation, is one of the most promising methods for water disinfection. The mechanisms of the photocatalytic inactivation have been extensively investigated. Different factors, including the roles of ROSs and the ROSs target site(s) of bacterial cell, were elaborated by different studies. However, there are still controversial issues on the role of H₂O₂ in photocatalytic inactivation. The effectiveness of the low concentration of H₂O₂ in the bacterial inactivation process is still under question. / This study designs a new reactor to study the kinetic of H₂O₂ inactivation in continuous supply mode. Complete inactivation of 5-log Escherichia coli within 8 h is achieved when 8 mM of H₂O₂ is applied. No significant inactivation was observed when 2 mM H₂O₂ is applied, this concentration of H₂O₂ is much higher than that detected in common photocatalytic system (< 50 μM). The results show that H₂O₂ produced by common photocatalytic system is not harmful to bacterial cell, even they are produced continuously. However, when light irradiation of 200 mW cm⁻² , using Xenon lamp as lighting source, was applied to the system, the inactivation efficiency of H₂O₂ was significantly enhanced, which demonstrate the synergistic effect between the light irradiation and H₂O₂. The enhancement of inactivation by H₂O₂ can also be observed with light pretreated E. coli K-12, further confirms that light irradiation alter the physiology of the bacterial cell which increases their sensitivity to H₂O₂. / In order to find out the mechanism(s) of the synergism between the light irradiation and H₂O₂, RNA sequencing (RNA-Seq) was used to reveal the change of gene expression level of the E. coli under light irradiation. The mRNA level of most of the genes involve in catalase (CAT) and superoxide dismutase (SOD) expression, DNA repairing and intracellular iron regulation did not have significant increase or decrease. Only dps, fes and sodB showed significantly changes. Moreover, some genes that related to regulation of intracellular copper (cutA and cueR) and membrane composition (ompA, ompC, resX and gnsB) also showed significantly changes under light irradiation. After the RNA-Seq, ten genes were chosen as the possible target genes that related to the mechanism(s). Then the inactivation of E. coli BW25113 (parental strain) and the isogenic deleted mutants by H₂O₂ with light pretreatment were conducted and compared. Although the gene(s) that directly involved in the mechanisms of the synergy between H₂O₂ and light irradiation are not identified in the study, the results show that genes that are important to bacterial defense of oxidative damages, such those responsible for CAT and SOD expression and DNA repairing, are not involved in the mechanism(s). Increase of cell permeability and intracellular Fenton’s reagent content should be the main causes for the enhancement of H₂O₂ under light irradiation. / Finally, the inactivation efficiency of E. coli K-12 using AgBr/Ag/Bi₂WO₆ under different lighting sources is compared. The results show that inactivation efficiency under different emission wavelength are highly correlated with the optical absorption of the AgBr/Ag/Bi₂WO₆. Photocatalytic inactivation under two indoor lighting sources, LED lamps and Fluorescence tubes, also showed significant difference. The result of scavenger study under green LED lamps is completely different from those under other colour of LED lamps, indicates that emission wavelength also has great influence in photocatalytic inactivation mechanisms. / This study reveals the roles of H₂O₂ and light irradiation in photocatalytic inactivation and demonstrates the synergism between the H₂O₂ and light irradiation. The influence of the properties of light irradiation, including the light intensity and major emission wavelength, on the ROSs production by photocatalyst is also reported as well. This study not only provides a new perspective to the importance of light irradiation, H₂O₂ and the physiology of bacteria in photocatalytic inactivation, but a new approach in the investigation of photocatalytic inactivation mechanisms as well. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Ng Tsz Wai. / Thesis (Ph.D.) Chinese University of Hong Kong, 2014. / Includes bibliographical references (leaves 111-131). / Abstracts also in Chinese.

Dissertação (mestrado)—Universidade de Brasília, Faculdade de Agronomia e Medicina Veterinária, Programa de Pós-Graduação em Saúde Animal, 2016. / Micro-organismos patogênicos podem contaminar os alimentos por meio da manipulação, higienização e controle ambiental insatisfatórios. Além da adoção das Boas Práticas, o uso de antimicrobianos em alimentos constituiu um passo importante para o controle de doenças infecciosas. Nos sistemas em que ocorre migração do composto ativo para o alimento devem ser considerados apenas aqueles que são aprovados como aditivos alimentares. O objetivo deste estudo foi avaliar a influência da concentração de sal na ação da natamicina sobre micro-organismos patogênicos visto que este conservante tem sido utilizado em banhos de imersão em diversos laticínios no país. Cepas de Candida albicans, Escherichia coli e Staphylococcus aureus foram inoculadas em diferentes concentrações salinas e em água peptonada e receberam tratamentos com natamicina. Estas soluções foram mantidas a temperatura de 12°C e o comportamento dos micro-organismos avaliados com 0, 24 e 48 horas (T0, T1 e T2). Cada micro-organismo foi avaliado isoladamente bem como a associação de C. albicans e E. coli. Nas condições propostas pela pesquisa, foi possível concluir que a natamicina 0,025% não apresenta eficácia sobre Candida albicans inoculada em concentrações salinas abaixo de 5%. Os resultados obtidos nas contagens de E. coli sugerem que a natamicina pode interferir no seu desenvolvimento mesmo em concentrações que podem ser consideradas baixas (0,1%) e em condições de salinidade de 7,5% a 10%. A associação da natamicina com cloreto de sódio potencializa a sua ação antimicrobiana podendo representar economia e o seu uso ser ampliado pelas indústrias. / Pathogenic microorganisms can contaminate food through manipulation cleaning and environmental control unsatisfactory. Besides the adoption of good practice, the use of antimicrobials in food established an important step for the control of infectious diseases. In systems which there are migration from the active ingredient to the food they should be regarded only those compounds that are approved as food additives. The purpose of this study was to evaluate the influence of the concentration of salt in the action of natamycin over pathogenic micro-organisms since this preservative has been used in immersion baths in several dairy products in the country. Strains of Candida albicans, Escherichia coli and Staphylococcus aureus were inoculated in different salt concentrations and in peptone water and were treated with natamycin. These solutions were maintained at temperature of 12 ° C and the behavior of micro-organisms assessed at 0, 24 and 48 hours (T0, T1 and T2). Each microorganism was evaluated singly as well as the combination of C. albicans and E. coli. As proposed by the survey it was concluded that the 0.025 % natamycin has no effect on Candida albicans inoculated in salt concentrations below 5 %. The results obtained from the E. coli counts suggests that natamycin can interfere with their development even at concentrations that may be considered low ( 0.1 %) and saline conditions of 7.5 % to 10 %. The combination of natamycin with sodium chloride enhances its antimicrobial activity may represent the economy and its use be extended by industries.

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Orientador: José Moacir Marin / Banca: Clóvis Wesley Oliveira de Souza / Banca: José Eduardo Zaia / Banca: Alessandra Aparecida Medeiros / Banca: Lúcia Maria Carareto Alves / Resumo: Escherichia coli é a bactéria mais comum entre os patógenos entéricos causadores de doenças intestinais. As diferentes classes de E. coli causadoras de diarréia são reconhecidas através dos fatores de virulência que elas apresentam. As E. coli produtoras de Shiga toxina (STEC), especialmente o sorotipo O157:H7 tem sido associado a diversas doenças no ser humano. Além do sorotipo O157:H7, vários outros sorogrupos não-O157 também estão associados a infecções em humanos. Estas bactérias podem ser recuperadas de muitos animais, mas o gado bovino é reconhecido como o seu mais importante reservatório natural. Para análise da sobrevivência de cepas STEC não-O157 em sistemas de compostagem, inicialmente foram coletadas fezes de três vacas saudáveis que apresentaram E. coli portando o gene stx2, característico de cepas STEC. Foram montados dois sistemas de compostagem: o primeiro foi realizado em vala de 60cmd, no qual E. coli apresentando o gene stx2 foi eliminada após 8, 25 e 30 dias nas temperaturas de 40, 42 e 38°C, respectivamente; o segundo sistema foi realizado sobre o solo em um monte em forma de pirâmide com 1md, no qual as bactérias foram eliminadas após 4, 4 e 7 dias nas temperaturas de 65, 56 e 52°C, respectivamente. A temperatura alcançada durante a compostagem e os microrganismos presentes no esterco parecem ser os responsáveis pela eliminação do patógeno nos sistemas de compostagem, o qual pode ser útil para a redução da carga patogênica presente no esterco destinado para aplicações no solo. / Abstract: Escherichia coli is the most common bacteria among the enteric pathogens able to cause intestinal disease. Several classes of diarrhea-causing E. coli are recognized on the basis of their virulence factors production. Shiga-like toxigenic E. coli (STEC), especially serotype O157:H7, have been associated with many diseases in human beings. Besides sorotype O157:H7, many others non-O157 sorogroups have also been associated with human infections. These bacterias can be isolated from a range of animals, but cattle is generally recognized as the major natural source. To analyze the survival of non-O157 STEC strains in composting system, first was collected faeces from three healthy cows that contain E. coli STEC cells carrying the stx2 gene. Two composting systems were used: the first one was a cave with 60cmd were the E. coli STEC cells with stx2 gene were eliminated after 8, 25 and 30 days at 40, 42 and 38°C, respectively; the second one was a heap pyramid system with 1md, where the cells were eliminated after 4, 4, 7 days at 65, 56 and 52°C, respectively. The reached temperature in the composting systems and the indigenous microorganisms present in the manure seems to contribute to pathogen elimination, what may be a useful means of reducing the pathogen load of manure destined for soil application. / Doutor

Thesis (Ph.D.)--Massachusetts Institute of Technology, Dept. of Biology, 1981. / MICROFICHE COPY AVAILABLE IN ARCHIVES AND SCIENCE. / Bibliography: leaves 174-182. / by Daniel Jeffrey Capon. / Ph.D.

O pEO5, que codifica hemolisina, foi isolado de uma amostra de EPEC do sorotipo O26. Este plasmídio mostrou ser conjugativo e compatível com o pO157, e pelos testes de hibridização observou-se que estes plasmídios não são geneticamente relacionados. Para o estudo comparativo de similaridade foi seqüenciada uma região de 9227 pb de DNA do pEO5 que compreende todo o operon hlyCABD e suas regiões a montante e a jusante. A região do operon hemolitico (7225 pb) e a região promotora do operon foram similares às mesmas regiões do pHly152, que em uma amostra de E. coli isolada de roedor, codifica uma a hemolisina. No entanto, verificou-se a presença de elementos de inserção na região a montante do gene hlyC no pHly152. O pEO5 mostrou ser semelhante a outros plasmídios que também codificam a hemolisina em cepas de EPEC O26 de origem humana e de bovinos. A presença de estruturas semelhantes a transposons em ambas as extremidades do operon a hemolítico do pEO5 indica que esse fator de virulência provavelmente foi adquirido por transferência horizontal de genes. / The conjugative pEO5 encoding haemolysin in strains of EPEC O26 was investigated for its relationship with EHEC haemolysin-encoding of EHEC O26 and O157 strains. pEO5 was found to be compatible with EHEC virulence plasmids and did not hybridize in Southern blots with pO157, indicating that both plasmids were unrelated. A 9227 bp stretch pEO5 DNA encompassing the entire operon hlyCABD was sequenced and compared for similarity to plasmid and chromosomally inherited hly determinants. The a hly determinant of pEO5 (7252 bp) and its upstream region was most similar to corresponding sequences of pHly152, in particular, the structural a-hlyCABD and hlyR regions. pEO5 and hly of EPEC O26 strains from humans and cattle were very similar for the regions encompassing the structural a-hlyCABD. The major difference found between the hly regions of pHly152 and pEO5 is caused by the IS2 upstream of the hlyC in pHly152. The presence of transposonlike structures at both ends of hly sequence indicates that pEO5 was probably acquired by horizontal gene transfer.

Le réseau de protéines chaperons est hautement conservé dans l'ensemble du vivant. Il régule l'homéostasie des protéines au sein de la cellule en condition de croissance normale ainsi qu'en réponse à des stress environnementaux. Les chaperons membres de la famille HSP70 (Heat Shock Protein 70 kDa), famille particulièrement conservée, agissent tout au long de la biogénèse des protéines et orchestrent une pléthore de processus cellulaires liés au repliement et/ou au remodelage de protéines. Le cycle ATP-dépendant du chaperon HSP70 repose sur une étroite collaboration avec ses partenaires co-chaperons. Parmi ces co-chaperons, on distingue les membres de la famille DnaJ/HSP40 qui transfèrent les substrats vers HSP70 et stimulent son activité ATPasique, et les facteurs d'échange de nucléotides qui assurent la réinitialisation du cycle d'HSP70 permettant ainsi la libération du substrat. Au sein de la bactérie E. coli, la protéine HSP70 est appelée DnaK. Elle agit de concert avec les deux co-chaperons DnaJ et GrpE (ensemble nommés DnaKJE) afin d'assister les protéines dans leur repliement au cours de la synthèse de novo, de désagréger des protéines mal repliées, de faciliter l'adressage et le passage de protéines à travers les membranes biologiques, et de remodeler certains complexes protéiques impliqués dans des processus cellulaires variés. DnaKJE coopère efficacement avec d'autres systèmes chaperons majeurs, tels que la protéine Trigger Factor (TF) associée aux ribosomes et le complexe chaperonine GroESL, notamment pour le repliement de protéines nouvellement synthétisées dans le cytosol. De plus, une des fonctions cellulaires majeure du système DnaKJE est son implication dans la réponse au stress thermique (Heat Shock Response - HSR). DnaKJE contrôle la HSR en interagissant directement avec le facteur de transcription s32, sous-unité de l'ARN polymérase. Cette interaction facilite la dégradation de s32 par la protéase FtsH. En condition de stress, l'accumulation de protéines mal repliées au sein de la cellule entraine le recrutement de DnaKJE et par conséquent, la stabilisation de s32. Suite à cette stabilisation, une induction de la transcription de plus d'une centaine de gènes codant entre autres, pour des protéines chaperons et des protéases se met en place dans la cellule pour lutter contre le stress environnant. De ce fait, DnaK et ses co-chaperons sont considérés comme des éléments clés de la réponse cellulaire contre le collapse de l'homéostasie protéique par action directe sur des protéines mal repliées et indirecte en modulant la synthèse de nombreuses HSPs, via s32. L'étude récente de l'intéractome de DnaK révèle qu'au moins 50% des enzymes impliquées au sein du métabolisme central (MC) de la cellule interagissent avec DnaK à température physiologique. A travers l'analyse d'une banque de suppresseurs multi-copie, nous avons identifié six gènes associés au MC : ackA, ldhA, lpd, pykF, talB et csrC qui lorsqu'ils sont surexprimés, permettent de restaurer partiellement le défaut de croissance d'une souche mutante n'exprimant pas les chaperons DnaK et Trigger Factor (deltatig deltadnaKJ). Remarquablement, la surexpression d'ackA, talB et csrC supprime également le défaut de croissance d'un mutant dnaK à haute température, ce qui suggère une implication importante de DnaK au niveau du MC. Dans ce projet, l'implication de DnaK dans le fonctionnement du métabolisme carboné a été établi par une analyse métabolique combinant analyses macro-cinétiques (suivi de croissance, analyse de la consommation des substrats et de la production de produits du métabolisme) sur différentes sources de carbones seules ou en mélange et analyses micro-cinétiques (flux métaboliques par marquage 13C). Finalement, ces travaux apportent différentes hypothèses quant au rôle de DnaK dans le contrôle du MC, directement ou indirectement via la régulation de la HSR, en réponse à une défaillance de l'homéostasie protéique ou d'une carence nutritionnelle. / Intricate networks of highly conserved molecular chaperone machines govern cellular protein homeostasis, both under lenient and more stressful growth conditions. Members of the highly conserved HSP70 family of molecular chaperones are key players in this process, acting at nearly every step in protein biogenesis. The ATP-dependent chaperone cycle of HSP70 chaperones relies upon the cooperation with a cohort of essential cochaperones, including DnaJ/HSP40 family members that recruit the chaperone to specific substrate and/or cellular localization and stimulate its ATPase activity, and nucleotide exchange factors, which insure proper resetting of the chaperone cycle and the resulting substrate release. In the bacterium Escherichia coli, the multifunctional HSP70 chaperone, named DnaK, acts in concert with its cochaperones DnaJ and GrpE (all together referred as DnaKJE) to efficiently, assist de novo protein folding, protein disaggregation, protein targeting and translocation through biological membranes, and protein complexes remodeling leading to multiple cellular activities. Remarkably, previous works also showed that DnaKJE can efficiently cooperate with other major cytosolic chaperones, including the ribosome-bound Trigger Factor (TF) and the chaperonin GroESL, especially during the folding of newly-synthesized cytosolic proteins. In addition, one of the key cellular functions of DnaKJE in E. coli is the regulation of the heat shock response (HSR). In this case, DnaKJE controls the HSR by interacting directly with the heat shock sigma factor s32 subunit of the RNA polymerase to facilitate it degradation by the FtsH protease. Under stress condition, DnaKJE is recruited to accumulating misfolded proteins, leading to an increased stability of s32 and the subsequent induction of more than hundred heat shock proteins. Therefore, DnaK, and its cochaperones are central components of the cellular response to proteostasis collapse, both by acting directly on misfolded proteins and by modulating the synthesis a plethora of heat shock chaperones and proteases. The recently described in vivo interactome of DnaK in E. coli revealed that at least 50% of the central metabolism enzymes interact with DnaK at physiological temperature. Remarkably, through a multicopy suppression analysis we have now identified six genes of the central metabolism (CM), namely ackA, ldhA, lpd, pykF, talB and csrC, which when overexpressed partially suppress the growth defect of the sensitive double mutant lacking DnaK and Trigger Factor (deltatig deltadnaKJ ), with half of them, namely ackA, talB and csrC, additionally suppressing the growth defect of the single ?dnaKJ mutation at high temperature, thus strongly suggesting a major role of DnaK in this process. Using a combination of growth assays on specific carbon sources entering the CM at various metabolic nodes with NMR analyses for characterizing the carbon source assimilation, identifying and quantifying the metabolism by-products and determining metabolic flux rearrangements, we show that DnaKJE impacts the responsiveness of the central metabolism by acting either directly at the level of the CM or along the first step of substrate assimilation. How does the multifunctional DnaK chaperone modulate the CM, either directly or indirectly via the control of the HSR, in response to proteostasis failure or nutrient starvation is discussed.

O consumo de vegetais e de frutas tem aumentado mundialmente, bem como os surtos alimentares envolvendo esses alimentos, especialmente a alface que é o vegetal folhoso mais consumido em nível mundial. Dessa forma, o objetivo desse estudo foi realizar uma avaliação quantitativa do risco de infecções causadas por Salmonella spp. e por Escherichia coli O157:H7 a partir do consumo de alface produzida e consumida no Rio Grande do Sul, visto que esses patógenos são os mais relacionados a surtos alimentares envolvendo vegetais folhosos em nível mundial. Para melhor compreender o comportamento desses patógenos na alface, eles foram inoculados nesse vegetal separadamente e armazenados sob condições isotérmicas de 5 a 40°C para Salmonella e de 5 a 42ºC para E. coli O157:H7, bem como sob condições não isotérmicas, simulando temperaturas encontradas da colheita até a venda da alface no Rio Grande do Sul. Dados experimentais demonstraram que ambas as bactérias podem se multiplicar em todas as temperaturas examinadas. Também foi proposto um parâmetro de tempo de multiplicação insignificante (ς), o qual fornece o tempo em que a alface pode ser exposta a uma temperatura específica e não apresentar uma multiplicação expressiva. O ς foi desenvolvido com base na equação do modelo primário de Baranyi e no conceito do potencial de crescimento. ς é o valor da fase lag adicionado do tempo necessário para população microbiana aumentar 0,5 log UFC/g. O ς da alface exposta a 37 °C foi de 1,3 h, enquanto que a 5 °C foi de 3,3 dias. Além dos modelos adequados, dados de prevalência e concentração são primordiais na avaliação de risco. Assim, foi realizada uma revisão sistemática da literatura para buscar esses dados A prevalência mundial encontrada foi de 0,041 para ambos os patógenos na alface. Já a prevalência dos países desenvolvidos foi de 0,028 para Salmonella e de 0,125 para E. coli (EHEC), enquanto que nos países em desenvolvimento foi de 0,064 para Salmonella e 0,024 para E. coli (EHEC). A concentração de Salmonella em alface, em países em desenvolvimento, variou de 4,57 a 218,78 NMP/g, e para E. coli (EHEC) a concentração foi de < 3,0 NMP/g até > 1100 NMP/g. O modelo de avaliação quantitativa de risco microbiológico foi composto por nove módulos, desde o armazenamento da alface nas fazendas produtoras até o consumo. O risco médio (baseado no cenário mais comumente encontrado no Rio Grande do Sul) de infecção por Salmonella por mês foi de 0,017, enquanto que por E. coli O157:H7 foi de 0,006. Assim, de modo geral, o risco de infecção por Salmonella é maior do que por E. coli O157:H7 quando a alface é produzida e consumida nesse estado. Todos os cenários alternativos à correta higienização da alface (lavar as folhas de alface com água potável seguido de imersão em 200 ppm de cloro livre, por 15 minutos e enxaguar com água potável) aumentaram o risco. A principal redução do risco foi identificada no cenário que considerou o uso de refrigeração em todos os módulos do modelo. Análises de sensibilidade indicaram que, além da manutenção da cadeia fria e do procedimento correto de higienização, é importante reduzir a prevalência e a concentração dos patógenos na alface, a fim de diminuir o risco de infecção por essas bactérias. Por fim, a avaliação de risco desenvolvida nessa tese pode auxiliar no desenvolvimento de estratégias de intervenção para mitigar esse risco. / The consumption of vegetables and fruits has increased worldwide, as well as foodborne outbreaks involving these foods, especially lettuce that is the most consumed leafy vegetable in the world. Thus, the objective of this study was to carry out a quantitative microbial risk assessment of Salmonella spp. and Escherichia coli O157: H7 on lettuce produced and consumed in Rio Grande do Sul, since these pathogens are the most related to foodborne outbreaks involving leafy vegetables worldwide. To study the behavior of these pathogens on lettuce, they were inoculated on this vegetable separately and stored under isothermal conditions of 5 to 40 °C for Salmonella and 5 to 42 °C for E. coli O157:H7, as well as under non-isothermal conditions, simulating temperatures from the harvest until the sale of lettuce in Rio Grande do Sul. Experimental data demonstrated that both bacteria can growth at all temperatures examined. A negligible growth time parameter (ς) has also been proposed, which provides the time that lettuce can be exposed to a specific temperature and does not present an expressive growth. The ς was developed based on the equation of the Baranyi primary model and the concept of growth potential. ς is the lag phase added value of the time required for microbial population to increase 0.5 log CFU/g. The ς of lettuce exposed at 37 ºC was 1.3 h, whereas at 5 ºC it was 3.3 days. In addition, prevalence and concentration data are paramount in the risk assessment studies. Thus, a systematic review of the literature was carried out to collect these data. The global prevalence found was 0.041 for both pathogens in lettuce The prevalence of developed countries was 0.028 for Salmonella and 0.125 for E. coli (EHEC), while in developing countries it was 0.064 for Salmonella and 0.024 for E. coli (EHEC). The concentration of Salmonella in lettuce in developing countries ranged from 4.57 to 218.78 MPN/g, and for E. coli (EHEC) the concentration was < 3.0 MPN/g to > 1100 MPN/g. The quantitative microbial risk assessment model was composed by nine modules, from lettuce storage on farms to consumption. The average risk (based on the scenario most commonly found in Rio Grande do Sul) of Salmonella infection per month was 0.017, whereas for E. coli O157:H7 it was 0.006. Thus, in general, the risk of infection by Salmonella is higher than by E. coli O157:H7 when lettuce is produced and consumed in this State. All scenarios that were alternative to the correct hygiene of lettuce (washing lettuce leaves with drinking water followed by immersion in 200 ppm of free chlorine for 15 minutes and rinsing with potable water) increased the risk. The main risk reduction was identified in the scenario that considered the use of refrigeration in all modules of the model. Sensitivity analyzes indicated that, in addition to maintaining the cold chain and the correct hygienization procedure, it is important to reduce the prevalence and concentration of pathogens in lettuce, in order to reduce the risk of infection by these bacteria. Finally, the risk assessment developed in this thesis can help in the development of intervention strategies to mitigate this risk.

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