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S-layer biogenesis studies in Caulobacter crescentus : RsaA anchoring and the localization of the S-layer-associated protease Ford, Matthew James
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.
Item Metadata
Title |
S-layer biogenesis studies in Caulobacter crescentus : RsaA anchoring and the localization of the S-layer-associated protease
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Creator | |
Publisher |
University of British Columbia
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Date Issued |
2006
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Description |
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.
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Genre | |
Type | |
Language |
eng
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Date Available |
2010-01-05
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Provider |
Vancouver : University of British Columbia Library
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Rights |
For non-commercial purposes only, such as research, private study and education. Additional conditions apply, see Terms of Use https://open.library.ubc.ca/terms_of_use.
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DOI |
10.14288/1.0092468
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URI | |
Degree | |
Program | |
Affiliation | |
Degree Grantor |
University of British Columbia
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Graduation Date |
2006-05
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Campus | |
Scholarly Level |
Graduate
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Aggregated Source Repository |
DSpace
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Rights
For non-commercial purposes only, such as research, private study and education. Additional conditions apply, see Terms of Use https://open.library.ubc.ca/terms_of_use.