| Summary: | 碩士 === 中興大學 === 分子生物學研究所 === 95 === Shigella spp. causes dysentery in human, and antibiotics are usually used for therapy. Studies have shown that the virulence of the bacteria is unstable, which may be related to the virulence plasmid the bacteria contain. Our laboratory previously isolated a spontaneous avirulent mutant from a Taiwan Shigella flexneri isolate SH2576, called SH2576W1-1, and demonstrated that the mutant was generated by integration of the virulence plasmid into the chromosome of SH2576. In this study, 2-D SDS-PAGE was used for analyzing the total proteins of SH2576 and SH2576W1-1. The results show that SH2576 is different from SH2576W1-1 in two protein spots, IpaD protein and polyketide synthase modules and related protein. It is suggested that recombination between ipaD gene and the gene for polyketide synthase modules and related protein leads to genesis of SH2576W1-1. SH2576W1-1 cannot produce IpaD protein and thus is avirulent.
Studies have also shown that Shigella spp. is unstable in its drug sensitivity which may be related to the drug resistance plasmid the bacteria contain. Our laboratory previously isolated streptomycin-resistant mutants from a Taiwan S. flexneri isolate SF81. SF81 contains the virulence plasmid, the drug resistance plasmid, and four small plasmids (3 kb、4 kb、5 kb and 6 kb). Previously, two types of mutants were found. Type I mutants were generated by insertion of a Tn21-like transposon into one of the four small plasmids, while type II mutants contain a newly-formed plasmid in addition to the original four small plasmids. This study firstly continues the previous study by completing the DNA sequencing of each of the Tn21-like transposon-containing plasmids (pG1 ansd pTS1) from two type II mutants. In addition, partial sequencing was carried out with the newly-formed plasmid (pJT36) from a type II mutant. The results show that these three plasmids contain three different Tn21-like transposons.
The drug resistance plasmid of SF81 was extracted and used for making DNA library. Six different DNA clones that contain both IRL and IRR of Tn21 were chosen for testing the in vivo transposition activity of the DNA clones. Of the six clones, four were found carrying transposition activity 1-4 folds higher than the vector-only control clone.
In addition to the type I and type II mutants, another type of streptomycin-resistant plasmid mutants, type III mutants, of SF81 was found in this study. The type III mutants retain the virulence plasmid, the resistance plasmid and the four small plasmids, but the copy number of the resistance plasmid increased. The resistance plasmids of two randomly-chosen type III mutants were transferred into E. coli. Southern analysis and MIC test demonstrated that the increase in copy number of the resistance plasmid is correlated to the streptomycin resistance. For these two type III mutants, DNA regions of the resistance plasmid for origin of replication were PCR-cloned and sequenced. One identical base was found that is the only difference as compared to the sequence of the same region of the resistance plasmid in SF81. Complementation test showed that this base mutation was the cause for high copy of the resistance plasmid and streptomycin resistance in the type III mutants.
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