Item – Theses Canada

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OCLC number
46582682
Link(s) to full text
LAC copy
LAC copy
Author
Goecke, Michelle Elisa,1973-
Title
A study of the regulation of expression of dsbA from Salmonella enterica serovar Typhimurium.
Degree
M. Sc. -- Queen's University at Kingston, 1998
Publisher
Ottawa : National Library of Canada = Bibliothèque nationale du Canada, [1999]
Description
2 microfiches.
Notes
Includes bibliographical references.
Abstract
Correct disulfide bond formation is required for proper protein folding and stability of disulfide bond-containing proteins. For extracytoplasmic proteins, disulfide bond formation occurs in the periplasm of Gram-negative organisms, and is catalyzed by the disulfide oxidoreductase DsbA. In E. coli, it has been shown that dsbA has two promoters separated by about 1 kb. The distal promoter is regulated by the Cpx signal transduction system. The regulation of dsbA in Salmonella enterica serovar Typhimurium is currently unknown. This study was undertaken to examine the regulation of expression of dsbA in this organism. Northern analyses revealed the presence of two distinct dsbA mRNA transcripts differing in size by approximately 200 base pairs. This suggests that, for S. typhimurium dsbA, transcription may initiate from two distinct promoters. Under different conditions, the abundance of each transcript varies. The smaller transcript is more abundant when oxygen is limiting or in an htrA null strain. In minimal E glucose media pH 7.6 both transcripts appear more abundant compared to transcripts from an LB culture. Using a S. typhimurium dsbA::lacZ transcriptional fusion to examine activity of the proximal promoter, it was elucidated that expression of dsbA is growth phase regulated, with maximum induction of expression occurring at the onset of stationary phase. Furthermore, this phenomenon was shown not to be dependent upon RpoS or SlyA, two factors involved in the regulation of transcription during stationary phase. In a S. typhimurium dsbA null strain this stationary phase induction was even greater than in the wild-type, whereas in an E. coli strain, dsbA expression increased at approximately the same rate during both logarithmic and stationary phase. Under conditions of low pH and oxygen limitation, stationary phase induction of dsbA expression did not occur. Western blotting showed that steady-state levels of DsbA did not vary significantly throughout the phases of growth, nor were steady-state levels different in overnight static cultures versus aerated cultures. Clearly, this work has provided evidence that suggests that S. typhimurium dsbA is regulated, and this regulation is different from that seen in E.coli. These findings support the idea that S. typhimurium dsbA has two promoters, and that the proximal promoter is maximally induced by an as yet unidentified factor at the onset of stationary phase.
ISBN
0612282007
9780612282001