Molecular genetic studies on pathogenicity of genus Bacteroides.

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1. A gene encoding neuraminidase of Bacteroides fragilis strain YCH46 was cloned into the cosmid vector pHC79. The neuraminidase gene nanH was subcloned from the cosmid and was located within a 1.8-kilobase-pair XhoI-KpnI restriction endbnuclease fragment. DNA sequencing of the insert revealed an open reading frame which encodes 296 amino acids. The predicted protein sequence had no marked similarity to known bacterial neuraminidase sequences. A 5.4-kb BamHI-SaII fragment containing the nanH gene was cloned into the Escherichia coli-Bacteroides shuttle vector pVAL-1. The resulting plasmid, pKK105, was mobilized from E. coli strain HB101 to Bacteroides uniformis strain BU1001 by the conjugative plasmid R751.The neuraminidase activity in crude cell extracts from strain BU1001 carrying pKK105 was found to be 1600-fold greater than that found in strain HB101 carrying the lame plasmid.2. High voltage electroporation was applied to transformation of B. thetaiotaomicron strain 5482 using the shuttle plasmid pVAL-1. With the optimized procedure, B. thetaiotaomicorn was transformed with an efficiency up to 3xlO^5 transformants per mug pVAL-1 plasmid DNA.3. A plasmid vector pUCST5 carrying a lethal gene pnd was constructed for the use of positive-selection of recombinant plasmids.