Análise da resistência a cobre e zinco sobre o crescimento e expressão gênica em Xylella fastidiosa em condições de biofilme
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Data
2007-07-27
Autores
Rodrigues, Carolina Munari [UNESP]
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Editor
Universidade Estadual Paulista (Unesp)
Resumo
Baseado nas informações geradas após o seqüenciamento do genoma, foi desenvolvido uma série de meios de cultura de composição definida (XDM1, XDM2, XDM3, XDM4 e XDM5). Portanto, um dos objetivos do presente trabalho foi estabelecer a curva de crescimento da X. fastidiosa no meio definido XDM2. Foi utilizada a estirpe 9a5c de X. fastidiosa mantida nesse mesmo meio de cultura. A medida da taxa de crescimento bacteriano foi realizada através de leituras de densidade óptica durante dezesseis dias com intervalos de 48 horas. A avaliação da viabilidade celular foi realizada através da contagem de unidade formadora de colônia (UFC) por diluição seriada. As duas avaliações apresentaram uma alta correlação (R2= 0,91) verificada através de regressão exponencial. O início da fase estacionária foi obtido após 10 dias de crescimento. De posse dos dados de UFC, foi possível calcular o tempo de geração da X. fastidiosa no meio XDM2, de aproximadamente 21 horas. Comparando o tempo de geração obtido na curva de crescimento em meio PW (11,37h), foi comprovado que a bactéria apresenta um crescimento mais rápido em meio PW do que no meio definido XDM2. Outro aspecto interessante revelado no estudo do genoma funcional da X. fastidiosa está relacionado à expressão de genes associados à patogenicidade. O principal mecanismo de patogenicidade é a formação de biofilme no xilema da planta hospedeira conduzindo ao bloqueio dos vasos do xilema. Análise da expressão diferencial de genes desse fitopatógeno em condição de virulência e durante a formação do biofilme revela padrões de genes associados à adaptação e competitividade no ambiente do hospedeiro. Esses genes possivelmente são ativados no biofilme maduro e são essenciais para sua manutenção na planta. Além disso, sabe-se que bactérias que formam biofilme apresentam resistência crescente a compostos...
Based on informations generated after the genome sequencing, a series of culture media of defined composition were developed (XDM1, XDM2, XDM3, XDM4 e XDM5). Therefore, one of the objectives of the present work was to establish the growth curve of X. fastidiosa in the defined medium XDM2. For this, we utilized the 9a5c strain of X. fastidiosa maintained in this medium. The measurement of the bacterial growth rate was done by optical density analysis during sixteen days with intervals of 48 hours. The evaluation of cellular viability was carried out counting the colony units forming (CFU) by serial dilution. Both evaluations presented a high correlation (R2 = 0,91) verified by exponential regression. The beginning of the stationary phase was observed after ten days of growth. With the data of CFU, it was possible to calculate the generation time of X. fastidiosa in XDM2 medium, estimated to be of approximated 21 hours. Comparing the generation time obtained for the growth curve in PW medium (11,37 hours), we proved that the bacteria grow faster in PW compared with the defined medium XDM2. Another interesting aspect reveled by the study of functional genome of X. fastidiosa is related to the expression of genes associated with pathogenicity. The main mechanism of its pathogenicity is the formation of a biofilm in the xylem of the host plant, causing the blockage of the xylem vessels. Analysis of the differential expression of genes of this phytophatogen in condition of virulence and during the biofilm formation reveals standards associated to adaptation and competition in the host environment. These genes are possibly activated in the mature biofilm and are essential for its maintenance in the plant. Besides, it is known that bacteria that form biofilm present elevated resistance to antimicrobial compounds while biofilm is structured, like antibiotics, heavy metals... (Complete abstract click electronic access below)
Based on informations generated after the genome sequencing, a series of culture media of defined composition were developed (XDM1, XDM2, XDM3, XDM4 e XDM5). Therefore, one of the objectives of the present work was to establish the growth curve of X. fastidiosa in the defined medium XDM2. For this, we utilized the 9a5c strain of X. fastidiosa maintained in this medium. The measurement of the bacterial growth rate was done by optical density analysis during sixteen days with intervals of 48 hours. The evaluation of cellular viability was carried out counting the colony units forming (CFU) by serial dilution. Both evaluations presented a high correlation (R2 = 0,91) verified by exponential regression. The beginning of the stationary phase was observed after ten days of growth. With the data of CFU, it was possible to calculate the generation time of X. fastidiosa in XDM2 medium, estimated to be of approximated 21 hours. Comparing the generation time obtained for the growth curve in PW medium (11,37 hours), we proved that the bacteria grow faster in PW compared with the defined medium XDM2. Another interesting aspect reveled by the study of functional genome of X. fastidiosa is related to the expression of genes associated with pathogenicity. The main mechanism of its pathogenicity is the formation of a biofilm in the xylem of the host plant, causing the blockage of the xylem vessels. Analysis of the differential expression of genes of this phytophatogen in condition of virulence and during the biofilm formation reveals standards associated to adaptation and competition in the host environment. These genes are possibly activated in the mature biofilm and are essential for its maintenance in the plant. Besides, it is known that bacteria that form biofilm present elevated resistance to antimicrobial compounds while biofilm is structured, like antibiotics, heavy metals... (Complete abstract click electronic access below)
Descrição
Palavras-chave
Genética, Drogas - Resistencia em microorganismos, Antimicrobial compounds
Como citar
RODRIGUES, Carolina Munari. Análise da resistência a cobre e zinco sobre o crescimento e expressão gênica em Xylella fastidiosa em condições de biofilme. 2007. 101 f. Dissertação (mestrado) - Universidade Estadual Paulista, Instituto de Biociências de Botucatu, 2007.