| Bacterial flagellum is a lash-like appendage locatingin the surface of the bacteria. It is bacterial locomotive organellepropelling cells through liquid envirionment to look for more favorable growing site. And flagellum also play important roles in the process of infection produced by some intestinal pathogenic bacteria.More and more new antimicrobial agents choosebacterial flagella synthesis pathway as the target.The flagellum is a huge protein complex consisting of at least three parts:the basal body, the hook, and the filament. For self-assembly of the bacterial flagellum, most of the component proteins synthesized in the cytoplasm are exported by the flagellar type III export apparatus to the growing, distal end. Genes associated with flagella synthesis are strictly controlled. The flagellar genes can be divided into three classes according totheir transcriptional hierarchy. The class1flhDC operon is at the top of the hierarchy, whose expression is required for the transcription of all other flagellar operons. The proteins translated byflhDCoperon, FlhD and FlhC can form the FlhD4C2complex that activate the expression of the class2genes encoding components of the hook-basal body (HBB) and some flagellar-specific regulators sigma factor δ28. The class3genes are responsible for filament formation, motor function, and chemotaxis.Flagellar protein export is highly organized and well controlled in every step of the flagellar assembly process. Flagella gene regulation involves the level of DNA, mRNA level and protein level. FlhD4C2complex functions as the master regulatorthat binds to the class2flagellarpromoters and to its own promoter to induce downstream flagellargene expression. We have known that FliT not noly is a chaperone specific for the filament-capping proteinFliD, but it could bind to the FlhD4C2complex, thereby inhibiting the activationof the class2promoters.We first find the fragment of FliD interacting with the FliT protein. To discover the fragment of FliDassociating with FliT, four truncations of FliD are cloned,1-155aa,1-215aa,155-end,215-end. Pull down assay show that only the215-end fragment of FliD could interact with FliT.To get the crystal of FliT-FliD complex, we co-transformed FliT94and FliD(215-end) to BL21(DE3). And we purified the FliT-FliD complex using SourceQ and superdex200. The complex was digested by trypsin for30min at zero before used for crystal growing. Luckly, we got the crystal of FliT-FliD complex.Although the structure of FliT has reported, how FliT interacts with FliD/FlhDC is not clear so far. So five mutations of FliT are made, and we hope to understand the mechanism using pull down assay. Fortunately, our experiments show that the30th amino acid of FliT play a very important role in the interactions of FliT with FliD.The second part of our research is about Z0021in E.coliO157:H7. Escherichia coli O157:H7is a gastrointestinal pathogen that has become a serious public health concern, as it is associated withoutbreaks and severe diseases such as hemolytic-uremic syndrome.And the Z0021is located in01-1,which is a putative fimbria-encoding genomic island. Deletion of Z0021increased the motility of E. coli O157:H7, which correlated with an increase in flagellin production and enhanced assembly offlagella on the cell surface. In contrast, overexpression of20021inhibited motility.We have known that Z0021exert its regulatoryeffects downstream of the transcription and translation of flhDC but prior to the activation of class Ⅱ/Ⅲ promoters.Since the structure of Z0021has not reported, we try to make clone and purify it. Unfortunately, we could not get the crystal of Z0021. The bacterial swimming motility assay show that both the overexpression of Z0021and Z0021-23-end could repress the motility of Mg1655. So we suppose that Z0021play its function in the cytoplasm, not the periplasmic space. |
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