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Master's Dissertation
DOI
https://doi.org/10.11606/D.87.2013.tde-05112013-113438
Document
Author
Full name
Cristiane Tambascia Pereira
Institute/School/College
Knowledge Area
Date of Defense
Published
São Paulo, 2013
Supervisor
Committee
Fernandes, Andrea Balan (President)
Carvalho, Cristiane Rodrigues Guzzo
Ferreira, Rita de Cassia Cafe
Title in Portuguese
Estudos funcionais e estruturais para a caracterização da via de captação e assimilação de sulfato em Xanthomonas axonopodis pv. citri.
Keywords in Portuguese
Bioinformática
Oligonucleotídeos
Proteínas recombinantes
Xanthomonas
Abstract in Portuguese
Em Escherichia coli, a assimilação de sulfato e sua redução a sulfeto é mediada por um transportador do tipo ABC codificado pelos genes sbpcysWUA e várias enzimas codificadas por cysNCDGHIJ. Embora a importância deste sistema tenha sido largamente demonstrada em outros organismos, não existem relatos na literatura sobre a via na bactéria fitopatogênica Xanthomonas axonopodis pv. citri (X. citri). Neste trabalho, utilizando uma abordagem funcional baseada em análises de bioinformática, proteômica e gene repórter, mostramos que a via de sulfato está presente e ativa em X. citri. Ainda, a partir da expressão e produção em larga escala da proteína ligadora de sulfato Sbp, realizamos ensaios de biofísica que evidenciaram a interação da proteína com sulfato e o aumento da estabilidade térmica e estrutural, obtendo-se cristais. O trabalho apresenta as primeiras evidências da funcionalidade desta via em X. citri durante o crescimento in vitro e infecção in vivo e abre pespectivas de estudos para compreensão do papel deste íon na fisiologia do microrganismo.
Title in English
Structural and functional studies for characterization of the sulfate uptake and assimilation pathway from Xanthomonas axonopodis pv. citri.
Keywords in English
Bioinformatics
Oligonucleotides
Recombinant proteins
Xanthomonas
Abstract in English
In Escherichia coli, the capture and reduction of sulfate to sulfide is mediated by an ABC-type transporter encoded by genes sbpcysWUA and several enzymes encoded by cysNCDGHIJ. Although the importance of this system has been widely demonstrated in other organisms, there are no reports in the literature related to the way that sulfate is assimilated and reduced in plant pathogen Xanthomonas axonopodis pv. citri (X. citri). In this work, using a functional approach based on bioinformatics analysis, proteomics and gene reporter, we show that the way is present and active in X. citri. Indeed, the sulfate binding protein was expressed and produced in large scale for biophysical assays demonstrating the interaction of the protein with sulfate and increased thermal stability and crystals was produced. The study presents the first evidence of the functionality of this pathway in X. citri during growth in vitro and in vivo infection and opens perspectives studies to understand the role of this ion in the physiology of the microorganism.
 
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Publishing Date
2014-01-16
 
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