Author
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Whitehead, Terence |
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Submitted to: Society of Industrial Microbiology Annual Meeting
Publication Type: Abstract Only Publication Acceptance Date: 8/7/1997 Publication Date: N/A Citation: N/A Interpretive Summary: Technical Abstract: Members of the genera Bacteroides and Porphyromonas are capable of causing diseases in humans. Genetic evaluations of these organisms are important for determining factors involved in the development of certain 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 novel 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 was isolated from the operon by polymerase chain reaction cloning and inserted into the E. coli plasmid pBlueScript. The XA gene was placed 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 species and Porphyromonas gingivalis 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 for either enzymatic activity and determining colony fluorescence under ultraviolet light. |
