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The two-component system controlling inducible glycopeptide resistance in Enterococci
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Quigley, Andrew Michael (2010) The two-component system controlling inducible glycopeptide resistance in Enterococci. PhD thesis, University of Warwick.
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Official URL: http://webcat.warwick.ac.uk/record=b2334515~S15
Abstract
VanS and VanR form the two-component regulatory system that controls inducible
glycopeptide resistance in Enterococci. Upon induction VanS, a sensor histidine
kinase, phosphorylates itself on a conserved histidine residue. This phosphate is
transferred to a conserved aspartate residue on VanR. The phosphorylated form of
VanR is the transcriptional activator of the vanAHX genes which, when expressed,
directly confer vancomycin resistance. VanS also possesses VanR phosphatase
activity, providing a mechanism by which to repress vanAHX transcription.
This thesis describes approaches used towards the crystallisation of VanS and VanR.
These are based upon previous crystallisation studies resulting in full-length VanS
crystals which diffracted to 8Å, as well as a cytoplasmic structure of an analogous
histidine kinase from Thermatoga maritima (Marina et al., 2005). Full-length and
truncated forms of VanSA and full-length VanRA were cloned, expressed in E.coli
and purified for crystallisation studies. Autokinase activity was biochemically
characterised using radiolabelling and spectrophotomic assays, in tandem with a
novel application of mass spectrometry. Site-directed mutagenesis of VanSA, led to
the observation that ATP hydrolysis may occur independently of the autokinase
function of VanSA. Adenosine 5’ tetraphosphate was also discovered, as a novel
product of VanSA. Based upon these data an expanded model for VanS autokinase
activity has been proposed. This may be expanded to include the phosphotransfer
and phosphatase mechanisms and validated through the measurement of associated
product formation. Finally a new mechanism for the control of the VanRS two component
system has been proposed. Future studies will validate and expand this
model.
This work has significantly increased our knowledge of this system providing the
tools and foundations that will lead to the elucidation of the way that this two component
system functions. This has the potential for the development of novel
inhibitors that either complement or supersede existing therapies.
Item Type: | Thesis (PhD) | ||||
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Subjects: | Q Science > QR Microbiology | ||||
Library of Congress Subject Headings (LCSH): | Enterococcus -- Research, Glycopeptides -- Research, Mass spectrometry | ||||
Official Date: | February 2010 | ||||
Dates: |
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Institution: | University of Warwick | ||||
Theses Department: | Department of Biological Sciences | ||||
Thesis Type: | PhD | ||||
Publication Status: | Unpublished | ||||
Supervisor(s)/Advisor: | Roper, David | ||||
Sponsors: | Biotechnology and Biological Sciences Research Council (Great Britain) (BBSRC) | ||||
Extent: | 310 leaves : ill., charts | ||||
Language: | eng |
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