3-Deoxy-D-manno-octulosonate 8-phosphate (KDO8P) synthase is the enzyme responsible for catalyzing the first reaction in the biosynthesis of KDO. KDO is an essential component in the cell wall of Gram-negative bacteria and plants. This compound is not present in mammals; therefore the enzymes responsible for its biosynthesis are potential targets for the development of new antibiotic agents. KDO8P synthase catalyzes the condensation reaction between phosphoenol pyruvate (PEP) and D-arabinose 5-phosphate (A5P) to form KDO8P. Two types of KDO8P synthase have been identified; a metal-dependent type and a non metal-dependent type. KDO8P synthase from the organism Chlorobium tepidum (Cte) has been partially purified and partially characterized. In line with predictions based on sequence alone, the activity of this enzyme is dependent on the presence of a divalent metal ion and is sensitive to the presence of the metal chelating agent EDTA. Cte KDO8P synthase was found to have the highest activity in the presence of Mn2+ or Cd2+. KDO8P synthase from the organism Acidithiobacillus ferrooxidans (Afe) has also been cloned, purified and biochemically characterized. Afe KDO8P synthase was also found to be a metallo enzyme and the catalytic activity is highest in the presence of Mn2+ or Co2+. Afe KDO8P synthase was found to exist as a tetramer in solution and is most active within the pH range of 6.8 to 7.5 and within a temperature range of 35 ºC to 40 ºC. Sequence analysis suggests that this enzyme has characteristics conserved throughout the metallo and the non-metallo KDO8P synthases and is closely related to the metal-dependent 3-deoxy-D-arabino-heptulosonate 7-phosphate (DAH7P) synthases. The role of several active-site residues of Afe KDO8P synthase has been investigated. A C21N mutant of Afe KDO8P synthase was found to retain 0.5% of wildtype activity and did not require a divalent metal ion for catalytic activity. This suggests that the metallo and non-metallo KDO8P synthases have similar catalytic mechanisms.

Thesis (Ph. D.) -- University of Texas Southwestern Medical Center at Dallas, 2003. / Vita. Bibliography: 162-207.

Thesis (Ph. D.)--University of Washington, 1999. / Vita. Includes bibliographical references (leaves 112-144).

Submitted by Cássia Santos (cassia.bcufg@gmail.com) on 2015-10-26T14:23:53Z No. of bitstreams: 2 Dissertação - Mayara Regina Pereira - 2015.pdf: 2344518 bytes, checksum: 121abb85c9406f07753d362631b51eda (MD5) license_rdf: 23148 bytes, checksum: 9da0b6dfac957114c6a7714714b86306 (MD5) / Approved for entry into archive by Luciana Ferreira (lucgeral@gmail.com) on 2015-10-26T14:46:04Z (GMT) No. of bitstreams: 2 Dissertação - Mayara Regina Pereira - 2015.pdf: 2344518 bytes, checksum: 121abb85c9406f07753d362631b51eda (MD5) license_rdf: 23148 bytes, checksum: 9da0b6dfac957114c6a7714714b86306 (MD5) / Made available in DSpace on 2015-10-26T14:46:05Z (GMT). No. of bitstreams: 2 Dissertação - Mayara Regina Pereira - 2015.pdf: 2344518 bytes, checksum: 121abb85c9406f07753d362631b51eda (MD5) license_rdf: 23148 bytes, checksum: 9da0b6dfac957114c6a7714714b86306 (MD5) Previous issue date: 2015-04-28 / Fundação de Amparo à Pesquisa do Estado de Goiás - FAPEG / Environmental surfaces and items for non-critical health health facilities are in important reservoirs of pathogenic and multiresistant bacteria. This study aimed to analyze the contamination of environmental surfaces and products for non-critical health by gram-negative rods (GNR) as a risk factor for the health of workers in the dressing rooms in básica.Trata is an epidemiological study attention the analytical type, held from May 2013 to June 2014 in five dressing rooms. The collection was through swabs with subsequent microbiological evaluation of the specimens. Of the 61 sites analyzed, 11 (18.0%) had positive culture for BGN with 13 microorganisms isolated. Of these, eight (61.5%) were identified as belonging to the family Enterobacteriaceae: two Serratia ficaria, Serratia odorifera two biogroup II, an Escherichia vulneris, one Enterobacter aerogenes, Citrobacter diversus and one aglomerans Pantoea, which showed resistance, ampicillin (50.0%), cefotaxime (50.0%), cefoxitin (37.5%), carbapenems (37.5%), amoxicillin / clavulanic acid (12.5%). As for the gram-negative non-fermenting rods (GNNFR), we identified five (38.5%) bacteria: Four Stenotrophomonas maltophilia and Pseudomonas stutizeri. All GNNFR were sensitive to carbapenems. It is concluded that the microbiological profile of isolates higher prevalence of Enterobacteriaceae, including emerging species of epidemiological importance to infections. These findings in the ambience after processing represent a risk factor of these agents for health workers. As for the factor of risk for workers against contamination by gram negative environmental surfaces and products for health stands out eventual colonization and infection of workers in the event of imunocomprometimentos future, besides acting as a disseminator of these micro-organisms. / Superfícies ambientais e artigos para saúde não críticos de estabelecimentos de saúde constituem-se importantes reservatórios de bactérias patogênicas e multirresistentes. Este estudo teve como objetivo, analisar a contaminação de superfícies ambientais e produtos para saúde não críticos por bastonetes gram-negativos (BGN) como fator de risco para a saúde de trabalhadores das salas de curativos na atenção primária a saúde. Trata-se de um estudo epidemiológico, do tipo analítico, realizado de maio de 2013 a junho de 2014 em cinco salas de curativos. A coleta se deu por meio de swabs com subsequente avaliação microbiológica dos espécimes. Dos 61 sítios analisados, 11 (18,0%) apresentaram cultura positiva para BGN, com 13 micro-organismos isolados. Desses, oito (61,5%) foram identificados como pertencentes à família Enterobacteriaceae: duas Serratia ficaria, duas Serratia odorifera biogrupo II, uma Escherichia vulneris, uma Enterobacter aerogenes, uma Citrobacter diversus e uma Pantoea aglomerans, os quais apresentaram resistência à ampicilina (50,0%), cefotaxima (50,0%), cefoxitina (37,5%), carbapenens (37,5%), amoxacilina/ácido clavulânico (12,5%). Quanto aos bastonetes gram-negativos não fermentadores (BGNNF), identificaram-se cinco (38,5%) bactérias: quatro Stenotrophomonas maltophilia e uma Pseudomonas stutizeri. Todos os BGNNF foram sensíveis aos carbapenens. Concluiu-se que o perfil microbiológico dos isolados demonstrou maior prevalência de Enterobacteriaceae, incluindo espécies emergentes, com importância epidemiológica para as infecções. Tais achados na ambiência, após seu processamento, representam um fator de risco desses agentes para os trabalhadores da saúde. Quanto ao fator de riscos para trabalhadores frente a contaminação por gram negativos de superfícies ambientais e produtos para saúde destaca-se a colonização e eventual infecção do trabalhador em caso de futuros imunocomprometimentos, além de atuar como veiculador desses micro-organismos.

Submitted by Luciana Ferreira (lucgeral@gmail.com) on 2016-08-25T15:41:01Z No. of bitstreams: 2 Dissertação - Késia Cristina de Oliveira Batista - 2016.pdf: 2882952 bytes, checksum: 8ca4241645e2fbd94d861045795c419e (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) / Approved for entry into archive by Luciana Ferreira (lucgeral@gmail.com) on 2016-08-25T15:41:26Z (GMT) No. of bitstreams: 2 Dissertação - Késia Cristina de Oliveira Batista - 2016.pdf: 2882952 bytes, checksum: 8ca4241645e2fbd94d861045795c419e (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) / Made available in DSpace on 2016-08-25T15:41:26Z (GMT). No. of bitstreams: 2 Dissertação - Késia Cristina de Oliveira Batista - 2016.pdf: 2882952 bytes, checksum: 8ca4241645e2fbd94d861045795c419e (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) Previous issue date: 2016-03-28 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - CAPES / This was an epidemiological, cross-sectional analytical study, performed in a Higher Education Institution (HEI) of Goiás/Brazil, with data collection from July to October 2014. Data were collected through a structured questionnaire and biological material collection (nasal swab). The level of nasal cavity colonization of DS teachers by Enterobacteriaceae was considered high (22.0%), with 14.6% of professionals being colonized by multiresistant microorganisms. These results were associated with inadequate personal habits and provide evidence for broadening the discussion of this issue among DS and directing strategies for controlling the biological risk for dentistry workers. / Trata-se de um estudo epidemiológico, transversal e analítico, realizado em uma Instituição de Ensino Superior (IES) de Goiás/Brasil, com coleta dos dados de julho a outubro de 2014. Os dados foram obtidos mediante aplicação de questionário estruturado e coleta de material biológico (swab nasal). A colonização da cavidade nasal dos CD docentes por Enterobacteriaceae foi considerada elevada (22,0%), sendo que 14,6% dos profissionais estavam colonizados por micro-organismos multirresistentes. Os resultados encontrados foram associados a hábitos pessoais inadequados e oferecem evidências para ampliar a discussão desta temática entre CD e direcionar estratégias para o controle do risco biológico aos trabalhadores em odontologia.

O estresse oxidativo, causado por níveis aumentados de espécies reativas de oxigênio (ROS), pode causar danos celulares. Várias enzimas, como as subunidades da alquil-hidroperóxido redutase (AhpC e AhpF) e as superóxido dismutases (SOD), são responsáveis por remover as ROS. Os mecanismos de regulação da expressão gênica de C. crescentus para os genes ahpC, sodA, sodB e sodC, foram analisados com fusões de transcrição ao gene repórter lacZ, permitindo a quantificação da expressão por ensaios da atividade de b-galactosidase, e RT-PCR quantitativo. As culturas foram cultivadas em meio PYE ou M2 e a expressão de cada gene foi avaliada na presença de peróxido de hidrogênio (H2O2), tert-butil hidroperóxido (tBOOH), paraquat, menadiona, pirogalol, FeSO4 ou DDPi. Em C. crescentus ahpC é induzido por peróxidos e regulado por OxyR. As SODs são induzidas principalmente por superóxidos, sodB e sodC possuem indução de fase na fase estacionária e possivelmente estão sob o controle do sigma J, enquanto o gene sodA é regulado por sigma F e sigma J. / Oxidative stress, caused by increased levels of reactive oxygen species, can lead to damage in all cellular components. Several enzymes, as subunit of alkyl hydroperoxide reductase (AhpC and AhpF) and superoxide dismutases (SOD), are responsible for removing ROS. Mechanisms of gene expression of C. crescentus for genes ahpC, sodA, sodB and sodC, were evaluated with transcription fusions with the lacZ reporter gene were constructed, allowing the quantification of expression by b-galactosidase activity assays, furthermore we analyze of the gene expression by qRT-PCR assay. The cultures were grown in PYE and M2 media, and gene expression was evaluated in the presence of hydrogen peroxide (H2O2), tert-butyl hydroperoxide (tBOOH), paraquat, menadione, pyrogallol, FeSO4 or DPPi. In C. crescentus ahpC is induced by peroxides and is under the control of OxyR. The SODs are mainly induced by superoxide, sodB and sodC are induction in stationary phase and are possibly under the control of sigma J, while the sodA gene is regulated by sigma F and sigma J.

M.Sc. (Biochemistry) / Lipopolysaccharide (LPS) is a complex lipoglycan that is found in the outer membrane of Gram-negative bacteria and is composed of three regions namely the fatty acid Lipid A, a core region of short oligosaccharide chains and an O-antigen region of polysaccharides. When LPS is recognized as a microbe-/pathogen-associated molecular pattern (M/PAMP), it not only induces an innate immune response in plants but also stimulates the development of defence responses such as the immediate release of reactive oxygen species/intermediates (ROS/I), pathogen-related (PR) gene expression and activation of the hypersensitive response (HR), resulting in stronger subsequent pathogen interactions. The identification and characterisation of the elusive LPS receptor/receptor complex in plants is thus of importance, since understanding the mechanism of perception and specific signal transduction pathways will clarify, and lead to the advancement of, basal resistance in plants in order to decrease crop plant losses due to pathogen attack. In mammals, LPS binds to a LPS binding protein (LBP) to form a LPS-LBP complex which is translocated to myeloid differentiation 2 (MD2) with the presence/absence of its co-receptor, a glycosylphosphatidylinositol (GPI)-linked protein, CD14. The interaction occurs on the host membrane and triggers an inflammatory defence response through the signalling cascade activated by the interaction with Toll-like receptor 4 (TLR4). A similar LPS-receptor interaction is, however, unknown in plants. To address the LPS perception mechanism in plants, biological binding studies with regard to concentration, incubation time and temperature, affinity, specificity and saturation were conducted in Arabidopsis thaliana protoplasts using LPS labeled with Alexa 488 hydrazide. Quantum dots (Qdots), which allow non-covalent hydrophobic labeling of LPS, were further also employed in similar Arabidopsis protoplast binding studies. These studies were conducted by fluorescence determination through the use of a BD FACS Aria flow cytometer. Although Alexa-labeling does not affect the biological activity in mammalian studies, the same cannot necessarily be said for plant systems, and hence Qdots were included to address this question. The conjugation of Qdots to LPS was confirmed by transmission electron microscopy (TEM) and results illustrated higher fluorescence values as compared to Alexa-LPS fluorescence analysis. Furthermore, inhibition of the perception process is also reported using Wortmannin and Brefeldin A as suitable endo- and exocytosis inhibitors. Affinity, specificity and saturability as well as the role of endo- and exocytosis inhibition in LPS binding to protoplasts was ultimately demonstrated by both fluorophores, with the use of Qdots as a label proving to be a more sensitive strategy in comparison to the conventional Alexa 488 hydrazide label.

Este trabalho descreve a construção de plasmídeos para expressão e ancoragem de proteínas de alta afinidade a íons Hg2+ e As5+. Os genes merR e arsR de C. metallidurans foram inseridos no vetor que contém o sistema para expressão e ancoragem de proteínas heterólogas em bactérias Gram-negativas originando os plasmídeos pCM-Hg e pCM-As. MerR e ArsR foram produzidas sob comando do promotor pan. E. coli recombinantes apresentaram resistência 100% superior a Hg2+ e As5+. C. metallidurans/pCM-As apresentou MIC > Na3As02 1000 mM sendo a Gram-negativa com maior capacidade de sobrevivência a íons As5+. Os plasmídeos elevaram a sobrevivência das bactérias estudadas, podendo ser usados para aumentar índices de sobrevivência e fornecer viabilidade a outras cepas. Células recombinantes apresentaram capacidade de adsorver Hg2+ ou As5+ do meio em níveis superiores às linhagens selvagens. As bactérias descritas são excelentes candidatas para biorremediação. Este trabalho apresenta pela primeira vez a ancoragem da proteína ArsR na superfície celular de um micro-organismo. / This work describes the construction of plasmids for expression and anchoring of high affinity proteins to Hg2+ or As5+ ions. C. metallidurans merR and arsR genes were inserted into the vector which contains the system for expression and anchoring of heterologous proteins in Gram-negative bacterias origining the plasmids pCM-Hg and pCM-As. MerR and ArsR were produced under pan promoter command. Recombinant E. coli showed resistance 100% higher to Hg2+ and As5+. C. metallidurans/pCM-As showed MIC > Na3As02 1000 mM being the most resistance Gram-negative able to survive in As5+ sites. The plasmids increased the studied Gram-negatives bacterial surviving and they can be utilized in other strains to increase the surviving levels and supply viability. Recombinant cells showed ability for adsorption of Hg2+ or As5+ from the media in enhanced levels as compared to the wild type. The described bacterias are excellent candidates for bioremediation. This work presents for the first time the cell surface display of ArsR protein on a microorganism.

Antibiotic resistance in multidrug resistant bacteria cause high mortality rates worldwide, where there has been over 1,000,000 deaths reported as of the year 2019. Antibiotics were thought to be the cure for fighting infectious diseases and preventing further spreading of infection. This became a major problem due to bacteria evolving and developing mechanisms for resistance. The purpose of this review was to see if there are differences in the resistance mechanism of gram negative and gram positive bacteria, focusing mainly on the six most common multidrug resistant pathogenic bacteria; Staphylococcus aureus, Pseudomonas aeruginosa, Enterococcus faecium, Acinetobacter baumannii, Klebsiella pneumoniae and Streptococcus pneumoniae. The results show that there is a difference in the resistance mechanism between gram positive and gram negative multidrug resistant bacteria. The difference in resistant mechanisms is due to the cell wall compositions of gram negative and gram positive bacteria. The main difference as to why the gram negative bacteria have more resistance is due to the outer membrane. Antibiotics have a hard time to diffuse through and into the cell, that is they can easily decrease their outer membrane permeability. Gram positive bacteria lack an outer membrane which makes them become more susceptible to antibiotics. The most common antibiotic resistance mechanisms in gram negative bacteria are outer membrane mechanisms such as lipid A and lipopolysaccharide modification as well as mutations in porin channels. On the other hand, the most common resistance mechanisms for gram positive bacteria are point mutations especially in penicillin binding proteins as well mutations in the rpoB gene. One important gram positive bacteria is Methicillin resistant Staphylococcus aureus, which developed a new mechanism against antibiotics, a missense mutation and mutation on the promoter region in penicillin binding protein 4. Recently new research has come forward showing that N-chlorotaurine (NCT) inhibits resistance in both gram positive and gram negative multidrug resistant bacteria. The research on NCT is still fairly new and only time will tell if this method of inhibiting resistance will be used in the future. This review highlights the importance and concern of multidrug resistance bacteria, especially due to bacteria being able to rapidly evolve when antibiotics are used incorrectly. It is important to understand the differences in resistance between gram negative and gram positive bacteria and how resistance spreads. This knowledge can be used to develop antibiotics that treat infections. It is however still a challenge to overcome resistance in multidrug resistant bacteria due to evolutionary adaptation especially through horizontal gene transfer, where resistant bacteria can adapt to changing conditions. / Antibiotikaresistens hos multiresistenta gramnegativa och grampositiva bakterier orsakar hög dödlighet över hela världen, där det har rapporterats över 1,000,000 dödsfall för år 2019. Antibiotika ansågs vara botemedlet för att bekämpa infektionssjukdomar och förhindra ytterligare spridning av infektioner. Detta blev ett stort problem på grund av att bakterier utvecklades mekanismer för resistens, vilket gör att de kan överleva när de behandlas med antibiotika. Syftet med denna studien är att se om det finns skillnader i resistensmekanismener för gramnegativa och grampositiva bakterier, med fokus på de sex vanligaste multiresistenta bakterierna; Staphylococcus aureus, Pseudomonas aeruginosa, Enterococcus faecium, Acinetobacter baumannii, Klebsiella pneumoniae och Streptococcus pneumoniae. Resultaten visar att det finns en skillnad i resistensmekanismen mellan grampositiva och gramnegativa multiresistenta bakterier. Skillnaden i resistenta mekanismer beror på cellväggssammansättningen av gramnegativa och grampositiva bakterier. Den största skillnaden till varför de gramnegativa bakterierna har mer resistens beror på det yttre membranet. Antibiotika har svårt att penetrera genom och in i cellen genom att minska deras yttre membranpermeabilitet. Grampositiva bakterier saknar ett yttre membran som gör att de blir mer mottagliga för antibiotika. De vanligaste antibiotikaresistensmekanismerna hos gramnegativa bakterier är yttre membranmekanismer som lipid A och lipopolysackaridmodifiering samt mutationer i porinkanaler. De vanligaste resistensmekanismerna för grampositiva bakterier är punktmutationer, särskilt i penicillinbindande proteiner samt mutationer i rpoB genen. En viktig grampositiv bakterie är Meticillin-resistent Staphylococcus aureus, som utvecklade en ny mekanism mot antibiotika, en missense-mutation och mutation på promotorregionen i penicillinbindande protein 4. Nyligen har ny forskning kommit fram som visar att N-klorotaurin (NCT) hämmar resistens i både grampositiva och gramnegativa multiresistenta bakterier. Forskningen om NCT är fortfarande ny och bara tiden kommer att utvisa om denna metod för att hämma resistens kommer att användas i framtiden. Den här studien belyser vikten och oron för multidresistena bakterier, särskilt på grund av att bakterier snabbt kan utveckla antibiotikaresistens när antibiotika används på fel sätt. Det är viktigt att förstå skillnaderna i resistens mellan gramnegativa och grampositiva bakterier och hur resistens sprids inom resistenta bakterier. Denna kunskap kan användas för att utveckla antibiotika som behandlar infektioner orsakade av både gramnegativa och grampositiva bakterier. Det är fortfarande en utmaning att övervinna resistens hos multiresistenta bakterier på grund av evolutionär anpassning särskilt genom horisontell genöverföring, där resistenta bakterier kan anpassa sig till förändrande förhållanden.

京都大学 / 新制・課程博士 / 博士(農学) / 甲第23952号 / 農博第2501号 / 新制||農||1091(附属図書館) / 学位論文||R4||N5387(農学部図書室) / 京都大学大学院農学研究科応用生命科学専攻 / (主査)教授 栗原 達夫, 教授 小川 順, 教授 阪井 康能 / 学位規則第4条第1項該当 / Doctor of Agricultural Science / Kyoto University / DGAM