Archaeal protein trafficking is a poorly understood process that is only beginning to unfold. Integral to this process are the various signal peptidases. Two types of archaeal signal peptidases are identified thus far: signal peptidase I (SPI) and the preflagellin peptidase.
SPI is responsible for processing the majority of secreted proteins. Sequence analysis of archaeal SPI enzymes indicates a Sec-11 type enzyme with two conserved Aspartic acid and a Histidine in place of the conserved Lysine in bacteria. Site directed mutagenesis and in vitro assays identified three conserved residues (Ser52, His122 and Asp148) critical for M. voltae SPI activity, distinguishing the archaeal enzyme from its bacterial and eukaryal counterparts.
The archaeal preflagellin peptidase is a type IV prepilin peptidase-like enzyme, initially characterized for its essential role in preflagellin processing prior to flagellar filament assembly. Unusual substrates have been proposed for this enzyme, including preflagellins of peculiar signal peptide lengths in certain archaeal species, as well as sugar binding proteins with extremely short signal peptides in S. solfataricus. In this thesis, in vitro comparisons of the signal peptide length requirements for the two different preflagellin peptidases, FlaK in M. voltae and PibD in S. solfataricus, are presented. While a signal peptide length cut-off of 5 amino acids was found for FlaK below which preflagellins remained unprocessed, substrates with shorter (4 and 3 aa) signal peptides were recognized and properly cleaved by PibD, suggesting a diversification of the preflagellin peptidases among the archaeal species. The ability of FlaK and PibD to complement FlaK activity in an M. maripaludis flaK mutant was evaluated.
M. maripaludis flaK is a markerless, stable mutant displaying pili as the sole cell surface appendage, providing the unique opportunity to closely study this structure without the interference of flagella. Here, purification and characterization of the archaeal pili is described for the first time in any archaeon. A putative pilus gene cluster with characteristics of type IV pilus genes was identified in M. maripaludis. In-frame deletion of the putative major pilin gene, MMP0237, resulted in a nonpiliated phenotype that could be restored by complementation, providing a direct link of this gene to piliation. / Thesis (Ph.D, Microbiology & Immunology) -- Queen's University, 2007-09-28 12:59:16.801
| Identifer | oai:union.ndltd.org:LACETR/oai:collectionscanada.gc.ca:OKQ.1974/864 |
| Date | 11 October 2007 |
| Creators | Ng, Sandy Yee Man Ng |
| Contributors | Queen's University (Kingston, Ont.). Theses (Queen's University (Kingston, Ont.)) |
| Source Sets | Library and Archives Canada ETDs Repository / Centre d'archives des thèses électroniques de Bibliothèque et Archives Canada |
| Language | English, English |
| Detected Language | English |
| Type | Thesis |
| Format | 3341093 bytes, application/pdf |
| Rights | This publication is made available by the authority of the copyright owner solely for the purpose of private study and research and may not be copied or reproduced except as permitted by the copyright laws without written authority from the copyright owner. |
| Relation | Canadian theses |
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