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United States Department of Agriculture

Agricultural Research Service

Title: Development of a Novel Bifunctional Bacteroides Gene As a Transcriptional Reporter Gene for Gram-Negative Anaerobes and Escherichia Coli

Authors
item Whitehead, Terence
item Xing, W - UNIV NEBRASKA
item Morrison, M - UNIV NEBRASKA

Submitted to: Australian Society for Microbiology
Publication Type: Abstract Only
Publication Acceptance Date: October 3, 1997
Publication Date: N/A

Technical Abstract: Members of the genera Bacteroides are capable of causing disease states in humans, and genetic studies of these organisms are important for determining factors involved in the development of these diseases. A reporter gene for transcriptional fusions would be useful for studies of gene regulation in these organisms. A gene encoding for a novel bifunctional xylosidase/arabinosidase (XA) has previously been cloned in our laboratory from Bacteroides ovatus V975 as part of a xylan-inducible operon. The XA gene was isolated from the operon and inserted into the E. coli plasmid pBlueScript. The XA gene was under transcriptional regulation in E. coli by the lac promoter, and both activities could be induced with IPTG. The XA gene was subcloned into E. coli/Bacteroides shuttle vectors and introduced into different Bacteroides and Porphyromonas species by conjugation. The results of transcriptional fusions in these organisms were evaluated. The advantages of the XA reporter system are the low background or total lack of arabinosidase and xylosidase activities in most Bacteroides species, P. gingivalis, and E. coli, and the ease of performing enzymatic assays. In addition, bacterial colonies can be screened directly for gene expression on agar plates by using methylumbelliferyl derivatives as substrates.

Last Modified: 10/20/2014
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