Functional Genomic Analysis and Protein Expression in Lactobacillus

by Duong, Tri

Abstract (Summary)
The lactic acid bacteria (LAB) are important in the production of food, industrial chemicals, and bulk ingredients. LAB, particularly probiotic lactobacilli, occupy important niches in the gastrointestinal tracts of humans and animals and are increasingly recognized as modulators of human and animal health. Major advances have been made in the genomic characterization of LAB facilitating their expanded use in bioprocessing and health. Trehalose is a cryoprotectant used to protect starter cultures from damage caused by freezing and lyophilization. Characterization of the tre locus of Lactobacillus acidophilus NCFM identified a trehalose PTS transporter, trehalose-6-phosphate hydrolase and a transcriptional regulator. Knockout mutants were used to determine that uptake and hydrolysis of trehalose is required for cryoprotection in L. acidophilus. Analysis of the FOS, lac and tre operons and pgm gene of L. acidophilus identified a number of putative promoter and repressor elements which were used to construct a series of expression vectors for use in lactobacilli. ?-glucuronidase reporter assays showed FOS, lac, and tre based vectors to be highly inducible by their specific carbohydrate and repressed by glucose. A construct based on the phosphoglycerate mutase (pgm) promoter was constitutively highly expressed. The development of these expression vectors is intended to support several novel applications including the delivery of vaccines and biotherapeutics by intestinal lactobacilli. The oxalate-degrading capabilities of lactobacilli have been studied with much interest in their potential use in a probiotic strategy for the management of hyperoxaluria and urinary stone disease. We describe the construction of a plasmid for the overexpression of the L. acidophilus NCFM oxalate degradation proteins, Frc and Oxc, and characterize its effect on oxalate degradation activity of L. acidophilus and Lactobacillus gasseri. This construct was able improve oxalate degradation by L. gasseri ATCC 33323 and complement an L. acidophilus Frc knockout mutant. Dendritic cells (DC) are antigen presenting cells found at mucosal surfaces that are important in directing acquired immunity. In this study, we construct an expression vector for recombinant DC-targeted Bacillus anthracis protective antigen for oral delivery by Lactobacillus acidophilus as a potential vaccine strategy against anthrax and evaluate its protective capability using a mouse model.
Bibliographical Information:

Advisor:Robert M. Kelly; Dahlia M. Nielsen; Amy M. Grunden; Todd R. Klaenhammer

School:North Carolina State University

School Location:USA - North Carolina

Source Type:Master's Thesis

Keywords:functional genomics


Date of Publication:12/12/2007

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