Burkholderia cepacia is a common soil bacterium of significance in agriculture and bioremediation. B. cepacia is also an opportunistic pathogen of humans causing highly communicable pulmonary infections in cystic fibrosis and immunocompromized patients. The pyrB gene encoding ATCase was cloned and ATCase was purified by the glutathione S-transferase gene fusion system. The ATCase in B. cepacia has been previously classified as a class A enzyme by Bethell and Jones. ATCase activity gels showed that B. cepacia contained a holoenzyme pyrBC complex of 550 kDa comprised of 47 kDa pyrB and 45 kDa pyrC subunits. In the course of purifying the enzyme, trimeric subunits of 140 kDa and 120 kDa were observed as well as a unique proteolysis of the enzyme. The 47 kDa ATCase subunits were cleaved to 40 kDa proteins, which still demonstrated high activity as trimers. The proteolysis site is between Ser74 and Val75 residues. To confirm this, we converted the Ser74 residue to an Ala and to an Arg by site-directed mutagenesis. After this primary sequence changed, the proteolysis of ATCase was not observed. To further investigate the characteristics of B. cepacia pyrB gene, a pyrB knock-out (pyrB-) was constructed by in vitro mutagenesis. In the assay, the 550 kDa holoenzyme and 140 kDa and 120 kDa trimers disappeared and were replaced with a previously unseen 480 kDa holoenzyme pyrB- strain. The results suggest that B. cepacia has two genes that encode ATCase. ATC1 is constitutive and ATC2 is expressed only in the absence of ATC1 activity. To check for the virulence of these two strains, a eukaryotic model virulence test was performed using Caenorhabditis elegans (C. elegans). The pyrB1+pyrB2+ (wild type) B cepacia killed the nematode but pyrB1-pyrB2+ B. cepacia had lost its virulence against C. elegans. This suggests that ATC1 (pyrB1) is involved in virulence in B.cepacia and ATC2 (pyrB2) is not.

Orientadores: Ranulfo Monte Alegre, Jorge Alberto Vieira Costa / Tese (doutorado) - Universidade Estadual de Campinas, Faculdade de Engenharia de Alimentos / Made available in DSpace on 2018-08-14T15:42:56Z (GMT). No. of bitstreams: 1 Castiglioni_GabrielLuis_D.pdf: 2848307 bytes, checksum: 09e1e37498a764f275a4ed9afe996844 (MD5) Previous issue date: 2009 / Resumo: Os avanços biotecnológicos na produção industrial de lipases têm proporcionado sua aplicação nos diferentes setores. Este interesse se deve à capacidade destas enzimas catalisarem algumas reações, dentre elas a transesterificação. Esta reação é reversível e resulta em altos rendimentos a partir da otimização de parâmetros experimentais, tais como, temperatura, concentração do catalisador, razão molar álcool:óleo, entre outros. Um grande número de trabalhos voltados para a produção de biodiesel por via enzimática vem sendo feito devido às vantagens a ela associada. Tendo em vista estas observações, o presente trabalho teve por objetivo o estudo da produção de lipase microbiana e sua aplicação na síntese de biodiesel. As etapas envolvidas neste projeto foram: produção de lipase por diferentes microrganismos utilizando fermentação submersa e semi-sólida; uso de diferentes reatores e meios de cultivo; escolha e caracterização da lipase que melhor apresentou vantagens em relação à produtividade e à atividade de transesterificação; estudo de imobilização e reutilização da lipase em diferentes resinas hidrofóbicas e de troca iônica; estudo da influência do tempo de reação, temperatura, pH, concentração de água, etanol e enzima, além da adição de co-solvente, goma arábica e íons metálicos na síntese do biodiesel. Efeitos positivos nos experimentos foram verificados quando se adicionou KH2PO4 e água de maceração de milho no processo de produção de lipase pela Burkholderia cepacia. Esta cepa apresentou maior potencial de produção de lipase com atividade de transesterificação do óleo de soja e etanol comparada com os demais microrganismos estudados. A caracterização da lipase demonstrou que os melhores resultados foram a 37°C e pH 8,0, seguindo modelo de inativação enzimática de primeira ordem. A resina de poli(estireno-co-divinilbenzeno) com 35% de DVB juntamente com a Amberlit IRA-900 apresentaram maior potencial de imobilização e utilização para a síntese do biodiesel. Nos experimentos de transesterificação, as variáveis estudadas apresentaram efeito na respostas de síntese de biodiesel. Os melhores resultados encontrados foram: pH igual a 6,0, temperatura em ttorno de 50ºC, uso de éter de petróleo como co-solvente, concentração de etanol próxima à quantidade estequiométrica e concentração de aproximadamente 42% de água. Por fim, na reutilização das resinas de poli(estireno-co-divinilbenzeno) com 35% de DVB e Amberlit IRA-900, foi observada uma diminuição no rendimento da reação devido à perda de parte da enzima por lixiviação. Os resultados referentes à primeira resina mostraram que a perda da atividade está relacionada ao número de usos e segue uma função de primeira ordem. A constante de perda da atividade foi de 3,017 e o tempo de meia vida do processo 18 usos. Os parâmetros cinéticos Km e Vmáx determinados na reação foram de 21,98% e 14,56%p.h-1, respectivamente. Já em relação à reutilização da lipase imobilizada na segunda resina, o tempo de meia vida foi de 6 usos. Com isso o presente trabalho apresenta uma contribuição significativa no que diz respeito ao uso de lipase de Burkholderia cepacia e agrega soluções para viabilizar o processo de transesterificação com esta enzima / Abstract: The technological advancements in the industrial production of lipases have been making its application viable in different sectors. This interest is due to these enzymes capacity to catalyze some reactions, like the transesterification. This reaction is reversible and the optimization of experimental parameters such as temperature, catalyst concentration and ethanol-to-oil molar ratio results in a high production efficiency. A great number of studies about the enzymatic production of biodiesel have been done due to its related advantages. Considering these observations, the objective of this work was to study the production of microbial lipase and its application in the synthesis of biodiesel. The stages involved in this project were: lipase production by different microorganisms using submerged and semisolid fermentation; use of different reactors and cultivation media; selection and characterization of the lipase that showed advantages related to the productivity and to the transesterification activity; study of the immobilization and reutilization of the lipase in different hydrophobic and ionic exchange resins; study of the influence of reaction time, temperature, pH, water, ethanol and enzyme concentrations and the addition of cosolvents, arabic gum and metallic ions in the synthesis of biodiesel. Positive effects were verified in the experiments when KH2PO4 and corn maceration liquor were added to the process of lipase production by Burkholderia cepacia. Compared with the other microorganisms studied, that strain showed greater potential for the production of lipase with activity of transesterification of soy oil and ethanol. The characterization of the lipase showed that the best results occurred at 37°C and pH 8.0, following the model of first order enzymatic inactivation. The poly(styrene-co-divinylbenzene) resin with 35% of DVB and Amberlit IRA-900 presented greater potential for the immobilization and synthesis of the biodiesel. In the transesterification experiments, the studied variables affected the response of the biodiesel synthesis. The best results were found at pH 6.0, temperature of 50ºC, petroleum ether as co-solvent, ethanol concentration at stequiometric values and water concentration around 42%. Finally, due to the loss of enzyme by lixiviation, it was observed a decrease in the efficiency of the reaction when the poly(styrene-codivinylbenzene) resins with 35% of DVB and Amberlit IRA-900 were reused. The results referring to the first resin showed that the loss of activity is related to the number of utilizations and it follows a first order function. The activity loss rate was 3.017 and the half-life of the process was 18 uses. The kinetic parameters Km and Vmax determined for the reaction were respectively 21.98% and 14.56%p.h-1. Referring to the reutilization of the lipase immobilized in the second resin, the half-life was 6 uses. Thus, this work presents a relevant contribution to the use of the lipase of Burkholderia cepacia and adds solutions to make the process of transesterification with this enzyme viable / Doutorado / Doutor em Engenharia de Alimentos

Tato diplomová práce se zabývala možnostmi utilizace lignocelulosového materiálu jako obnovitelného zdroje k produkci polyhydroxyalkanoátů (PHA) biotechnologickými metodami. Teoretická část práce se zaměřuje na charakterizaci rostlinné odpadní biomasy, její enzymatickou sacharifikaci a možnosti produkce a izolace hydrolytických enzymů. Dále se pak literární rešerše zabývá bakteriální produkcí PHA a možností využití lignocelulosové biomasy pro jejich produkci. V rámci experimentální části byly vybrané odpadní substráty hydrolyzovány chemickou a enzymatickou cestou. Jako odpadní substráty byly použity výlisky z jablek, hroznového vína a řepky olejné a kávová sedlina. Získané hydrolyzáty byly použity k produkci PHA bakteriálním kmenem Burkholderia cepacia. Nejslibnějším substrátem se jevily výlisky z jablek. Ukázalo se, že vybraný bakteriální kmen je schopen utilizovat odpadní substráty i bez předchozí úpravy. Supernatant po skončení kultivace jevil následující aktivity: proteasovou, lipasovou (0.47 nmol/(mL•min)), celulasovou pro CMC (6.05 nmol/(mL•min)) a filtrační papír (4.63 nmol/(mL•min)) a xylanasovou (1.71 nmol/(mL•min)). Tyto enzymy mohou představovat zajímavý vedlejší produkt výroby PHA z odpadních zemědělských materiálů. V rámci této práce byl také posouzen vliv délky kultivace a způsob hydrolýzy na výslednou produkci PHA a enzymatickou aktivitu průmyslově zajímavých enzymů.

Strain MS14, exhibiting antifungal activity, was classified to belong to Burkholderia contaminans. Occidiofungin produced by strain MS14 is an octapeptide dedicated to a broad range of antifungal activities of the bacterium. The 58.2-kb genomic fragment containing 18 open reading frames (ORFs), named occidiofungin (occ) gene cluster, is required for occidiofungin production. Putative proteins encoded by five nonribosomal peptide synthetase genes (occA – occE) of the gene cluster were predicted to contain the catalytic modules responsible for the biosynthesis of occidiofungin. Transcription of all the ORFs identified in the region except ORF1 and ORF16 was regulated by both ambR1 and ambR2, the LuxR-type regulatory genes located at the left border of the cluster. The functional ambR1 gene was essential for transcription of ambR2, and constitutive expression of ambR2 did not restore the phenotype of the mutant MS14GG44(ambR1::nptII). Sequence analysis revealed that the occ gene cluster shared high similarity (99% nucleotide coverage and 91% identity) to an uncharacterized DNA region of B. ambifaria strain AMMD. The gene cluster was not found in other Burkholderia strains available in GenBank (nucleotide coverage < 24%). Analysis of G+C composition and prediction using “IslandPick” indicate that the occ gene cluster has possibly been horizontally transferred between bacteria. In addition, the absence of the gene cluster in clinical strains of Burkholderia indicates that occidiofungin is not required for potential human pathogenesis. The findings have provided insights into the development of antifungal medicines and agricultural fungicides based on occidiofungin.

The objective was to compare pentachlorophenol (PCP) degradation in contaminated groundwater by indigenous and bio-augmented (Sphingobium chlorophenolicum and Burkholderia cepacia) PCP degrading bacteria. Indigenous bacteria were identified by cloning and sequencing of 16S rDNA fragments while PCP concentrations were determined by GC-ECD. Gene expression for PCP degrading enzymes: chlorophenol 4-monooxygenase (TftD, B. cepacia) and pentachlorophenol-4-monooxygenase (pcpB, S. chlorophenolicum), was determined by RT-PCR. B. cepacia, a PCP degrading bacteria was identified as dominant indigenous bacteria. PCP concentrations correlated negatively with PCP tolerant bacteria and relative fold gene expression in treatments with air-sparging (phase2) compared to without air-sparging (phase1). PCP concentrations decreased and TftD or pcpB expressions were higher in treatments inoculated with B. cepacia (49%, 10.7 fold) or S. chlorophenolicum (32%, 7 fold), respectively, than un-inoculated (indigenous) or mixed culture inoculated treatments. Thus bio-augmentation of indigenous bacteria with B. cepacia or S. chlorophenolicum resulted in more PCP degradation than indigenous bacteria.

Burkholderia pseudomallei is a gram-negative opportunistic soil pathogen that causes the life-threatening disease melioidosis. It is endemic in Northern Australia and Southeast Asia but can be found throughout many other regions in the world. Diabetes mellitus is a predisposing risk factor for infection with this organism and it has been demonstrated that diabetic males are particularly susceptible to severe infection. Previous research suggested that monocytes isolated from the whole blood of diabetic males demonstrated a decreased ability to produce the proinflammatory cytokines IL-1β and IL-8. We hypothesized that monocyte-derived macrophages from diabetic males would also secrete lower levels of pro-inflammatory cytokines and that this difference between gender and diabetic state would be more pronounced compared to those seen previously with monocytes. Twenty volunteer with type I diabetes mellitus (ten males and ten females), along with twenty healthy age- and gender-matched controls donated blood for this study. Monocytes were collected from whole blood and allowed to differentiate into macrophages with the use of human recombinant granulocyte-macrophage colony-stimulating factor (GM-CSF). Macrophages were then divided into groups and infected with B. pseudomallei, B. thailandensis (a closely related by non-pathogenic bacterium that inhabits similar niches), and E. coli. An uninfected control was used as well. At six hours post-infection, mRNA was collected from all cells and qPCR was performed to determine cytokine expression levels. All mRNA values collected from cells which had been infected with bacterial agents were normalized against the corresponding concentrations of mRNA from mock-infected cells. Mean log fold increases in both IL-1β and IL-8 were computed and compared. Preliminary testing showed decreased levels of both IL-1β and IL-8 from B. pseudomallei-infected macrophages isolated from a diabetic male compared to the healthy, age-matched male control. Surprisingly, results from all forty donors demonstrated that gender and diabetic state were not significant factors in the proinflammatory responses of macrophages infected with B. pseudomallei, although further testing is needed to determine if these results were influenced by experimental parameters.

No description available.

No description available.

A xilose é um dos principais componentes dos materiais lignocelulósicos, os quais são de grande interesse para produção de bioprodutos como etanol e polihidroxialcanoatos (PHA). Visando melhorar o consumo de xilose em Burkholderia sacchari, uma grande produtora de PHA, os seguintes genes codificadores de xilose isomerase foram nela inseridos e avaliados: xylABs, xylABc, xylAPl, xylABp e xylABx, respectivamente de B. sacchari, B. cenocepacia, Photorhabdus luminescens, B. phymatum e B. xenovorans. Foi ainda sintetizado o gene de B. sacchari (xylA*) no qual foram inseridas modificações descritas na literatura como capazes de aumentar o consumo de xilose em outros organismos. As linhagens recombinantes de B. sacchari abrigando os genes xylABs e xylA* tiveram um aumento de aproximadamente 30%, e aquelas abrigando os genes xylABp e xylABx de 23%, no consumo de xilose quando comparadas com a linhagem controle. Essas quatro linhagens recombinantes foram aquelas que conseguiram produzir maior quantidade de P3HB, aproximadamente 70% a mais do que linhagem controle. / Xylose is a major component of lignocellulosic materials, which are of great interest for the production of bio-products, such as ethanol and polyhydroxyalkanoates (PHA). To improve the consumption of xylose in Burkholderia sacchari, a major PHA producer, the following genes, encoding xylose isomerase, were introduced in these bacteria: xylABs, xylABc, xylAPl, xylABp and xylABx, respectively from B. sacchari, B. cenocepacia, Photorhabdus luminescens, B. phymatum e B. xenovorans. The gene of B. sacchari (xylA*) was also synthesized with several modifications described in the literature as able to increase the consumption of xylose in other organisms. Recombinant strains harboring B. sacchari xylABs and xylA* gene had an increase of approximately 30% in the xylose consumption compared to the control strain, and those harboring xylABx and xylABp gene an increase of 23%. These four recombinant strains were those that were able to produce more P3HB, approximately 70% more than the control strain.

Na agricultura moderna, formas alternativas de alcançar maior produtividade de modo sustentável são prioridades. Entretanto, o sucesso agrícola é ainda excessivamente dependente do emprego de fertilizantes químicos e de defensivos agrícolas. A severidade de algumas doenças é um fator preocupante na produção de várias culturas. Dentre alguns patógenos de planta, destaca-se o gênero Fusarium, o qual apresenta uma expressiva importância na agricultura por ser patógeno em várias culturas de interesse econômico.. Por outro lado, a utilização de microrganismos endofíticos como agentes de biocontrole vem se tornando cada vez mais atrativa, pois, surge como uma alternativa capaz de amenizar gastos excessivos com controle químico e danos ao meio ambiente. Dentre esses microrganismos, destaca-se o gênero Burkholderia, conhecidamente capaz de produzir uma vasta gama de antimicrobianos, com diferentes níveis de especificidade. Essas bactérias vivem em interações com diversos microrganismos e plantas, em um ambiente altamente competitivo, resultando em uma fonte de metabólitos secundários, bacteriocinas dentre outros pepitideos A linhagem TC3.4.2R3 Burkholderia seminalis, isolada endofiticamente de raízes de cana-de-açúcar, é capaz de controlar vários fungos e bactérias fitopatogênicas. Apartir dessa linhagem foi construída uma biblioteca de mutantes randômicos por meio de transposon. Assim, por meio dessa biblioteca, o objetivo do trabalho foi selecionar mutantes defectivos no controle de diferentes espécies de Fusarium, identificar e caracterizar os genes nocauteados, visando o melhor entendimento do controle da fusariose. Os resultados obtidos por meio da caracterização do teste de antagonismo demonstrou que a linhagem TC3.4.2R3 selvagem produz metabólitos capazes de inibir in vitro o crescimento de Fusarium spp. A partir da biblioteca de mutantes, foram obtidos 8 mutantes defectivos para o controle de Fusarium verticilioides FV-01-CTC que não apresentaram alta especificidade quando avaliados contra outros Fusarium spp. Dentre os genes nocauteados foram identificadas sequências codificadoras para a proteína glutamato sintase, TolB e FAD. Até o momento não foi encontrado relatos sobre o glutamato como um biocontrolador de patógenos, entretanto o uso de mutantes sítio dirigido para esse gene confirmou o papel do mesmo no controle de FV-01-CTC. TolB é uma proteína relacionada ao transporte de substâncais e pode ter seu papel de controle relacioanado à secreção de metabolítos secundários, visto que in silico, no genoma da TC3.4.2R3, foram encontrados 18 clusters resposáveis pela produção de metabólitos secundários. Assim, o presente trabalho abre novas perspectivas de estudo, visto que, os mecanismos de controle da fusariose por B. seminalis TC3.4.2R3 são amplos e sinergisticos. Há uma enorme perspectiva em desvendar o papel do glutamato e TolB no controle de fitopatógenos. Os mutantes caracterizados, pode ser fonte de novos estudos para o complexo entendimento da interação microrganismo-planta-patógeno, visto que há a possibilidade de verificar por exemplo o papel do glutamato proveniente da linhagem TC3.4.2R3 na promoção de crescimento vegetal. / In modern agriculture, alternative ways of achieving high productivity with sustainability are priority. However, agricultural success is still excessively dependent on the use of chemical fertilizers and pesticides. The severity of some diseases are worrying factor in the production of differents crops. Among plant pathogens, the genus Fusarium stands out, which has an important role in agriculture being disease causer in several crops such as: sugarcane, corn, passion fruit, tomato, banana, rice, and others. Fusarium can be found inhabiting soil in the most diverse geographical regions of the world, especially in tropical and subtropical climates. On the other hand, studies using endophytic microorganisms as biocontrol agents has become increasingly attractive, since they appear as an alternative to chemical control. Among these microorganisms, the genus Burkholderia stands out. This genis is able to produce a wide range of antimicrobials, with different levels of specificity. Moreover, Burkholderia has been received special attention because its potential plant growth promoter, bioremediation agents and recent studies aimed at biocontrol of diseases. These bacteria live in interactions with diverse microorganisms and plants in a highly competitive environment representing significant source of new bioactive secondary metabolites, bacteriocins among other pepitides. The B. seminalis strain TC3.4.2R3, endofitically isolated from sugarcane roots, is able to control diverse phytopathogenic fungi and bacteria. By Tn5 randomic mutations, it was obtained a library of TC3.4.2R3 mutantes. Thus, using this library, the objective of this work was select defective mutants to control of Fusarium spp., with the aimed the better understand the fusariose control by identificayion and characteriazation of knockouted genes. The results obtained through the characterization of the antagonism assays allowed us to conclude that wild-type TC3.4.2R3 produces many metabolites with antifungal activity under in vitro conditions, being able to control the growth Fusarium spp. From the mutants library we found 8 defectives mutants to control of F. verticilioides FV-01-CTC. Among the knockouted genes we found sequences enconding glutamato syntase, TolB and FAD. To date no reports have been found on glutamate as a pathogen biocontroller, however the use of mutant site directed to this gene confirmed the role of the same in the control of FV-01-CTC. TolB is a protein related to the transport of substances and may have its biocontrol role related to the secretion of secondary metabolites, since in silico analisys in the genome of TC3.4.2R3, 18 clusters were found as responsible for the production of secondary metabolites. Thus, the present work opens new perspectives of study, confirming that the mechanisms of control of fusariosis by B. seminalis TC3.4.2R3 are ample and synergistic. There is a huge prospect in unraveling the role of glutamate and TolB in controlling of plant pathogens. The characterized mutants may be the source of new studies for understanding the complex interaction among microorganism-plant-pathogen, since it is possible to verify, for example, the role of glutamate from the TC3.4.2R3 strain in the plant growth-promotion.