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Doctoral Thesis
DOI
https://doi.org/10.11606/T.46.1999.tde-30052008-113113
Document
Author
Full name
Regina Lúcia Baldini
E-mail
Institute/School/College
Knowledge Area
Date of Defense
Published
São Paulo, 1999
Supervisor
Committee
Gomes, Suely Lopes (President)
Maldonado, Gabriel Padilla
Menck, Carlos Frederico Martins
Oliveira, Carla Columbano de
Santelli, Roberto Vicente
Title in Portuguese
Expressão do operon de choque térmico groESL durante o ciclo celular de Caulobacter crescentus
Keywords in Portuguese
Caulobacter crescentus
Choque térmico
Ciclo celular
GroEL
GroES
Abstract in Portuguese
Caulobacter crescentus é uma bactéria Gram-negativa de vida livre cujo ciclo celular depende de eventos de diferenciação celular. A célula pré-divisional assimétrica dá origem a duas células filhas morfológica e funcionalmente distintas: a célula-talo e a célula móvel. A expressão do operon groESL é regulada por choque térmico e durante o ciclo celular a temperaturas normais, sendo a transcrição máxima na célula pré-divisional, com níveis baixos na célula-talo. Numa linhagem superexpressando σ32, os níveis do mRNA e da proteína GroEL estão aumentados, indicando que a transcrição ocorre a partir de um promotor ativado por σ32. A região regulatória também apresenta uma sequência repetida invertida, CIRCE, em que mutações de ponto aumentam a transcrição apenas a temperaturas normais de crescimento, indicando o papel inibitório desse elemento. Fusões de transcrição groESL-/acZ com mutações em CIRCE deixam de apresentar regulação temporal, bem como a síntese de GroEL numa linhagem hrcA-, em que o gene codificando o provável repressor que se liga em CIRCE está interrompido. Estes resultados indicam que o sistem CIRCE/HrcA está envolvido com a regulação da expressão de groESL durante o ciclo celular. Tentativas de se construir uma linhagem com groEL interrompido não tiveram sucesso, indicando ser este um gene essencial em todas as temperaturas. Um mutante condicional de groESL foi construído por recombinação homóloga e, em condições restritivas, o crescimento é inibido, os níveis de DnaK aumentam e as células se tomam filamentosas, porém não foi observada lise celular. As proteínas essenciais que são dependentes de GroEL/GroES para atingirem sua conformação funcional ainda não foram determinadas.
Title in English
Cell cycle expression of the heat shock groESL operon in Caulobacter crescentus
Keywords in English
Caulobacter crescentus
Cell cycle
GroEL
GroES
Heat shock
Abstract in English
Caulobacter crescentus is an aquatic free-living Gram-negative bacterium whose cell cycle depends on cell differentiation. The asymmetrical predivisional cell gives rise to two morphologically and functionally different daughter cells: the swarmer cell an the stalked cell. The expression of the groESL operon is induced by heat shock and is cell cycle controlled at normal temperatures, with maximal transcription in the predivisional cell and very low levels in the stalked cell. In this work, it was demonstrated that, in a strain overexpressing σ32, the levels of groESL transcripts and the synthesis of GroEL are increased, confirming that this factor is responsible for the transcriptional activation of the σ32 -like promoter of this operon, that also presents a inverted repeat called CIRCE in its regulatory region. groESL-lacZ transcription fusions with point mutations in CIRCE indicated a negative role of this cis-acting element only at normal growth temperatures, with a minor effect on heat shock induction. In addition, the expression of these fusions are no longer cell-cycle regulated, as well as GroEL synthesis in a strain which does not have the HrcA protein, the putative repressor that binds CIRCE, indicating that the CIRCE-HrcA system are involved in cell cycle regulation of groESL in C. crescentus. It was also shown that groEL is an essential gene at normal growth temperatures, since a strain with groEL disrupted is not viable. A conditional mutant was obtained by homologous recombination and in restrictive conditions growth is inhibited, DnaK levels are increased and the cells become filamentous, but no celllysis was observed. The proteins that require GroEL/GroES for proper folding have not been identified yet.
 
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Publishing Date
2008-06-05
 
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