A construction of cDNA library and categorization of genes by subtraction in prokaryote - A gene cloning of new known gene associated with pathogenic factors in P. gingivalis-

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A purpose of this study were a search of new pathogenic genes and a analysis of the biological function of the coded protein in Porpyromonas gingivalis. The effects of hemin, growth or oxygen to gene expression were examined. Then, the cDNA library was constructed and the new known genes were categorized. For purification of mRNA, the 16S and 23S rRNA were subtracted from a total RNA by using the biotin-labeled PCR product which is coded 16S-23S rRNA gene and streptoavidin magnet beads. The 5S rRNA and tRNA were removed by using a cDNA column with size-selection. After subtraction with tester cDNA and biotin-labeled control cDNA, the resulting cDNA solution was inserted into the plasmid and transformated into E. coli. The inserted fragments were analyzed the DNA sequence and searched the homology. Moreover, the homologous genes were identified by RT-PCR or Northern-blotting. A 40k-Da outer membrane-associated protein gene, which was cloned previously in our laboratory, was detected in subtracted cDNA library between early-log phase and late-log phase. Hence, we constructed the P. gingivalis mutant by insertion mutagenesis and characterized. The mutant has led to diminished remarkably aggregation with Gram-positive bacteria, the autoaggregation, adherence to human epithelial cells, and activities of hemagglutination, hemolysin, protease like RGP and KGP. The results in SDS-PAGE and Western-blotting analysis were suggested the fimbriae of the mutant did not expressed on the cell surface. So, the reduction of these pathogenicity in the mutant might be occurred by change of cell surface components like outer membrane proteins and fimbriae and so on. In conclusion, this subtraction technology should have a wide range of application to the detection of the pathogenicity in prokaryote.