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S-layer biogenesis studies in Caulobacter crescentus : RsaA anchoring and the localization of the S-layer-associated protease

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Title: S-layer biogenesis studies in Caulobacter crescentus : RsaA anchoring and the localization of the S-layer-associated protease
Author: Ford, Matthew James
Degree: Master of Science - MSc
Program: Microbiology and Immunology
Copyright Date: 2006
Issue Date: 2010-01-05
Series/Report no. UBC Retrospective Theses Digitization Project [http://www.library.ubc.ca/archives/retro_theses/]
Abstract: Despite the widespread occurrence of bacterial S-layers, little is known about the mechanisms of attachment to the cell surface, especially in the case of Gram-negative organisms. The S-layer of Caulobacter crescentus is composed of a single protein, RsaA. After export, RsaA assembles into a hexagonal crystalline array that covers the bacterium. In this array, some RsaA monomers are directly attached to the cell surface, while others are surface anchored only by interacting to other RsaA monomers. Since truncations (9) and mutations (8) in the RsaA N-terminus result in S-layer shedding into the culture medium, we hypothesized that the N-terminus of RsaA anchors the monomer to the cell surface. However, since disruption of the RsaA N-terminus and disruption of the putative RsaA subunit-subunit interaction domain both result in the same phenotype (S-layer shedding), when a particular mutation results in a shedding phenotype, it is difficult to know whether RsaA anchoring or RsaA subunit-subunit interaction has been perturbed. To tease apart these issues, we have developed an assay where small RsaA fragments are incubated with S-layer-negative cells to assess the ability of the fragments to re-attach. In doing so we found that the RsaA anchoring region lies in the first ~ 225 amino acids, that this RsaA anchoring region requires a smooth lipopolysaccharide molecule found on the outer membrane, and that even minor perturbations within the first ~ 225 amino acids of RsaA cause loss of anchoring. Mutations that lie outside of the RsaA anchoring region but still result in the shedding phenotype are likely disrupting RsaA-RsaA subunit-subunit interactions rather than directly disrupting RsaA anchoring. As a by-product of these anchoring studies, we have recent preliminary data that Sap, an S-layer editing protease, is likely to be an extracellular membrane-bound protease, rather than an intracellular protease as previously proposed. Additionally, we have found that Sap is likely to be secreted to the cell surface via the same Type I secretion transporter that the S-layer protein utilizes.
Affiliation: Science, Faculty of
URI: http://hdl.handle.net/2429/17530
Scholarly Level: Graduate

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