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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

item Whitehead, Terence

Submitted to: Current Microbiology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: May 9, 1997
Publication Date: N/A

Interpretive Summary: Bacteria that grow without oxygen are normal inhabitants of the mouth and intestinal tract. Certain strains of these bacteria can cause diseases, such as abscesses and periodontal disease. Genetic studies of these bacteria are important in determining the cause of disease. This report details the development of a novel "reporter gene," which can indicate when nparticular genes in a bacterium are "turned on" by environmental factors. This reporter gene should allow scientists to determine what genes are important for causation of diseases by certain bacteria and what avenues are available for fighting these diseases.

Technical Abstract: Members of the genera Bacteroides and Porphyromonas are common inhabitants of the human intestinal and oral microflora, and certain species are capable of causing disease states in humans. Genetic studies of these organisms are important for determining factors involved in the development of such 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 xylan, a major component of fiber in the diet. A gene encoding for a bifunctional xylosidase/arabinosidase (XA) enzyme was previously cloned in our laboratory from B. ovatus V975 as part of a xylan-inducible operon. The XA gene was isolated by polymerase chain reaction and subcloned into the E. coli plasmid pBluescript II KS+. 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 was subcloned into E. coli/Bacteroides shuttle vectors and introduced by conjugation into different Bacteroides species and Porphyromonas gingivalis. The results of transcriptional fusions in Bacteroides species and E. coli were evaluated. The characteristics 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 enzymatic assays. In addition, bacterial colonies can be screened directly on agar plates by fluorescence using methylumbelliferyl derivatives as substrates for either enzymatic activity.

Last Modified: 4/22/2015
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