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Assembly and regulation of the type III secretion system of "Yersinia enterocolitica"

Diepold, Andreas. Assembly and regulation of the type III secretion system of "Yersinia enterocolitica". 2010, PhD Thesis, University of Basel, Faculty of Science.

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Official URL: http://edoc.unibas.ch/diss/DissB_9194

Abstract

We investigated the assembly of the Yersinia enterocolitica type III secretion injectisome by grafting fluorescent proteins onto several components, YscC (outer-membrane (OM) ring), YscD (forms the inner-membrane (IM) ring together with YscJ), YscN (ATPase), and YscQ (putative C ring). The recombinant injectisomes were functional and appeared as fluorescent spots at the cell periphery. Epistasis experiments with the hybrid alleles in an array of injectisome mutants revealed a novel outside-in assembly order: whereas YscC formed spots in the absence of any other structural protein, formation of YscD foci required YscC, but not YscJ. We therefore propose that the assembly starts with YscC and proceeds through the connector YscD to YscJ, which was further corroborated by co-immunoprecipitation experiments. Completion of the membrane rings allowed the subsequent assembly of cytosolic components. YscN and YscQ attached synchronously, requiring each other, the interacting proteins YscK and YscL, but no further injectisome component for their assembly. These results show that assembly is initiated by the formation of the OM ring and progresses inwards to the IM ring and, finally, to a large cytosolic complex.
Further, we analyzed the assembly kinetics and dynamics of the cytosolic components of the Yersinia injectisome in detail, and showed that the needle subunit as well as parts of the IM export machinery can be copurified with the membrane rings using a specific combination of crosslinking and affinity purification.
Advisors:Cornelis, Guy R.
Committee Members:Jenal, Urs
Faculties and Departments:05 Faculty of Science > Departement Biozentrum > Former Organization Units Biozentrum > Molecular Microbiology (Cornelis)
Item Type:Thesis
Thesis no:9194
Bibsysno:Link to catalogue
Number of Pages:179 S.
Language:English
Identification Number:
Last Modified:30 Jun 2016 10:41
Deposited On:29 Dec 2010 10:23

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