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Master's Dissertation
DOI
10.11606/D.42.2015.tde-10122015-101302
Document
Author
Full name
Nicolas Federico Villamil Munevar
Institute/School/College
Knowledge Area
Date of Defense
Published
São Paulo, 2015
Supervisor
Committee
Spira, Beny (President)
Baldini, Regina Lúcia
Galvao, Teca Calcagno
Title in Portuguese
Análise do metabolismo de polifosfato e do operon pst em Pseudomonas aeruginosa.
Keywords in Portuguese
phoU
ppk
ppx
Pseudomonas aeruginosa
Fosfato
Operon
Operon pst
Pho box
Polifosfato
Regulação gênica
Regulon Pho
Transportador Pst
Abstract in Portuguese
O operon pst de P. aeruginosa codifica um transportador de fosfato de alta afinida-de e também a proteína PhoU que, em conjunto, atuam como repressores da ex-pressão do regulon Pho dessa espécie. A atividade de PhoU está também associada ao metabolismo de polifosfato (poliP), dado que mutantes phoU nulos apresentam um vasto acúmulo do biopolímero. Ensaios de β-galactosidase mostraram uma alteração na expressão dos genes ppk e ppx, envolvidos no metabolismo de poliP, no mutante phoU. Observou-se que na cepa selvagem, a transcrição de ppk e de ppx não responde às limitações de Pi ou de nitrogênio, sendo esses genes altamente expressos em condições normais de crescimento. Além disso, determinou-se que ppk é co-transcrito com o gene hemB, os quais formam, portanto, um operon. O operon pst também foi analisado. Foi identificado por ensaios de northern blot o transcrito do primeiro gene do operon, pstS, que codifica uma proteína periplasmática. Também, foi identificado um promotor imediatamente a montante de phoU, o gene mais distal do operon, que permitiria sua expressão em condições normais do crescimento bacteriano. Por fim, determinou-se por ensaios de EMSA que as duas sequências consenso Pho box presentes no operon pst são completamente funcionais.
Title in English
Analysis of the metabolism of polyphosphate and of the pst operon in Pseudomonas aeruginosa.
Keywords in English
phoU
ppk
ppx
Pseudomonas aeruginosa
pst operon
Gene regulation
Operon
Pho box
Pho regulon
Polyphosphate
Pst transporter
Abstract in English
The pst operon in P. aeruginosa encodes a high-affinity phosphate transporter and the PhoU protein, which together act as repressors of Pho regulon of this species. The PhoU activity is also related with polyphosphate (polyP) metabolism, since phoU null mutants have a large accumulation of the biopolymer. β-galactosidase assays allowed to confirm a change in the expression of ppk and ppx genes, in-volved in PolyP metabolism, in the phoU mutant. It was also evidenced that in the wild type strain, the ppk and ppx transcription does not respond to Pi or nitrogen starvation, and that these genes are highly expressed under conditions of normal growth. In addition, it was determined that ppk is co-transcribed with hemB, a gene involved in the synthesis of porphyrins, and they constitute therefore an operon. The pst operon was also examined. Was identified by northern blot the transcript of the first gene in the operon, pstS, which encodes a periplasmic protein. Also, a promoter was identified immediately upstream of phoU, the most distal gene in the operon, allowing its expression in normal conditions of bacterial growth. Finally, it was determined by EMSA that the two consensus sequences Pho box present in the pst operon are fully functional.
 
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Release Date
2017-12-10
Publishing Date
2015-12-11
 
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