Construction And Function Of The Shigella Dysenteriae A1 Iron, Shua Single, Double Mutant Analysis

The main focus of this project is to employ knock-out technique for functional gene research in Shigella dysenteriae. we selected the suicide plasmid pCVD442 of RecA recombination system as the knock-out strategy. pCVD442 is a very low copy plasmid and is difficult to conduct gene operation directly on it. We innovatively combined Gateway technique with construction of recombinant plasmid. Using this reformative method we successfully constructed three mutants. One mutant MTS-1 was yielded by a part of IroN deletion,in which Kan~r gene replaced the part, the other mutant MTS-2 was yielded by Shu A::Cat insertion , and the mutant MTS which were knockout these two genes in the same ways.The features of mutants were measured through common media cultivation, cultured cell and animal level. There was no difference in optical density between the wild type and mutants in iron-repleted L broth. However, growth of mutants in L broth containing 150μM DIP(iron chelator) was decreased relatively to the wild type after 4 hours , and addition of Fe to the cultures stimulated the growth of the mutants to wild-type level without DIP. The effects of mutations were tested by determining the numbers of CFU recovered after infection. The results demonstrated that there was no difference for intracellular survival and multiplication in HeLa and U937 cell between mutants and wild type, as well as the ability to form plaques on a HeLa cell monolayer. But the mutants appeared to be slightly sensitive to cation depletion by DIP in cell culture media. On the other hand, mutants led to similar keratoconjuctivitis in guinea pig to the wild type. These data suggested that IroN and ShuA genes are related to iron transport in Shigella dysenteriae 51197. As another iron transport system can compensate for the lack of IroN and ShuA, the mutant didn't exhibit apparent virulent decrease.By microarray, we compared the transcriptional profiling of mutants with SD51197 in iron-repleted and iron-depleted media. In general, genes of mutants MTS seem more sensitive to iron limitation. Analysis of the expression profiling data displayed that the up-regulated genes in MTS were more than that in SD51197, and the up-regulated genes mainly focused on the functions such as membrane transportation, amino acid biosynthesis and un-classified functions in both strains. On the other hand, the down-regulated genes mainly involved in energy metabolism and carbohydrate metabolism, which may be related to the decrease in growth under iron-limited condition. As expected, many genes encoding proteins involved in iron transport increased their expression levels. What's more, the number and range of up-regulated genes in mutant were greater than that in SD51197. This result testified again that iron transport encoded by sit operon is important in growth.we selected the suicide plasmid pCVD442 combined with Gateway technique ,This knock-out method was proved to be effective and high-throughput The dissertation provides a useful research method for functional genomics researches in Shigella, and it also supplies a possible way for research on unknown genes.