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United States Department of Agriculture

Agricultural Research Service

Title: Development of a Bifunctional Xylosidase/arabinosidase Gene As a Reporter Gene for the Gram-Negative Anaerobes Bacteroides and Porphyromonas and Escherichia Coli

Author
item Whitehead, Terence

Submitted to: American Society for Microbiology
Publication Type: Abstract Only
Publication Acceptance Date: May 23, 1996
Publication Date: N/A

Technical Abstract: Members of the genera Bacteroides and Porphyromonas are capable of causing disease in humans. Genetic studies on these organisms are important for determining factors involved in the development of these diseases. A reporter gene for transcriptional fusions may prove useful for studies of gene regulation in these organisms. Bacteroides ovatus is a normal inhabitant of the human intestinal tract and is one of the few Bacteroides species capable of degrading xylans, one of the major components of fiber in the diet. A gene encoding for a bifunctional xylosidase/arabinosidase (XA) has previously been cloned in our laboratory from B. ovatus V975 as part of a xylan-inducible operon. The XA gene has been isolated from the operon by polymerase chain reaction cloning and subcloned into the E. coli plasmid pBlueScript. The XA gene is under transcriptional regulation in E. coli by the lac promoter, and both activities can be induced with IPTG. The XA gene has been subcloned into E. coli/Bacteroides shuttle vectors and is being introduced into different Bacteroides species and Porphyromonas gingivalis by conjugation. The results of transcriptional fusions in these organisms will be evaluated. The advantages of the XA reporter system are the total lack of arabinosidase and xylosidase activities in most Bacteroides species, Porphyromonas gingivalis, and E. coli, and the ease of enzyme assays. In addition, bacterial colonies can be screened directly on agar plates by fluorescence using methylumbelliferyl derivatives as substrates for either enzyme activity.

Last Modified: 12/22/2014
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