| Summary: | 碩士 === 中山醫學院 === 生物化學研究所 === 87 === Abstract
The protein translocation machinery in Gram-negative bacteria are classified into three types. The type II pathway or the so-called general secretion pathway (GSP) is the most common one. The outer membrane GSP secretion machinery is composed of twelve to thirteen gene products designated as GspCDEFGHIJKLMNO. The N-termini of GspGHIJ are highly homologous to the N-terminus of PilA pilus, and were considered as the same family. However, the pili structure of GspGHIJ structure had never been identified or isolated. In this study, we used biochemical and genetic methods to systematically investigate the GspGHIJ pili structure in the outer membrane protein secretion system of Xanthomonas campestris. We had prepared anti-XpsG antibody and xpsG knockout mutant strain. XpsG protein was found to present in the soluble and insoluble fraction of lysed cells. Gel filtration chromatography analysis showed that the XpsG from membrane fraction was in the dimer form and from soluble fraction was in a higher order form. In the presence of high concentration of detergent DOC, the higher-order form dissociated to dimers. Second gel filtration analysis of the dissociated dimer in the absence of DOC revealed that the proteins could not revert to the higher-order form, indicating that the formation of the higher order from might require the participation of other factors. The soluble high-order form was stable at 80℃ and did not be dissociated. Under low concentration of DOC, the structure did not dissociate at pH 8 but dissociated at pH10.3. The XpsG-containing, high-order structures observed in this study could be the pilus-like structure proposed for the pilin-like protein family.
The five-conserved aspartate residues of XpsG protein at the 70, 103, 121, 128 and 139 positions were substituted to glutamate by site-directed mutagenesis. By -amylase secretion function assay, only mutant D103E was found that could not compensate the secretion ability of XC1713 and could interfere the secretion ability of XC1701. Using gel filtration chromatography analyses, no difference of elution profiles were found between wild type and mutant XpsG proteins in soluble and membrane forms. These results indicated that D103 of XpsG protein might be play a special function on protein secretion mechanism and this function was not related to the multimer function.
In conclusion of this study, XpsG protein might form pilus-like structure, and the soluble higher-order form of XpsG protein might be cleavaged from membrane pilus-like structure.
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