Molecular Characterization of the Heat Shock-Regulatedgroesl Operon in Lactobacillus Zeae Strain 102S
Murphy, Cynthia M.
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https://hdl.handle.net/2142/83725
Description
Title
Molecular Characterization of the Heat Shock-Regulatedgroesl Operon in Lactobacillus Zeae Strain 102S
Author(s)
Murphy, Cynthia M.
Issue Date
1997
Doctoral Committee Chair(s)
Bruce M. Chassy
Department of Study
Food Science and Human Nutrition
Discipline
Food Science and Human Nutrition
Degree Granting Institution
University of Illinois at Urbana-Champaign
Degree Name
Ph.D.
Degree Level
Dissertation
Keyword(s)
Agriculture, Food Science and Technology
Language
eng
Abstract
Various species of lactobacilli are used in the fermentation and preservation of food and dairy products. Due to the enhanced safety of such products, and perceived health-beneficial properties attributed to the ingestion of many strains, new biotechnological applications for lactobacilli are being suggested. Although study of these organisms at the molecular level has increased in recent years, little is known about their physiological and genetic responses to environmental stresses. These studies were undertaken to evaluate the stress responses of a strain used in dairy fermentations in order to develop a valuable genetic tool, a regulated promoter for use in food-grade heterologous gene expression systems. A plasmid-free derivative of Lactobacillus zeae ATCC 393, for years the type strain Lactobacillus casei, was studied because it is well characterized. PCR amplification with degenerate primers designed to conserved regions and genomic walking were used to determine the sequence of the highly conserved shock response genes groESL (HSP60/10). A rapid RNA isolation procedure was developed, and specific heat induction conditions for the groESL operon were determined by Northern and slot blot analyses. Changes in the level of the molecular chaperone GroEL (HSP 60) were analyzed by Western blotting and found to correlate well with the changes in RNA expression levels. The specific promoter sequences involved in gene expression were also determined by primer extension. These promoter sequences can now be utilized for heat-shock regulated heterologous gene expression.
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