Walker, Denise S. (1994) Investigation of the basis of specific secretion of cellulase CeIV by the Out apparatus of Erwinia carotovora subspecies carotovora. PhD thesis, University of Warwick.

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Abstract

Erwinia species cause soft rot in a range of economically important plants. The secretion of cellulases and pectinases, via the Out apparatus is considered a major pathogenic determinant. The Out proteins are encoded by a cluster of at least 13 genes, outC to outO, and show homology to the pullulanase secretion proteins of Klebsiella oxytoca, and secretion proteins of other plant and animal pathogens.

This study concerns the nature of the interaction between the Out apparatus and the secreted enzymes, with a view to identification of the recognition motif(s; on the secreted proteins that allow specific secretion.

Secretion deficient mutants were isolated using hydroxylamine and PCR mutagenesis. These were characterised with respect to activity and stability of the Ce1V derivatives; ability to secrete other proteins; and DNA sequence. Two classes of mutant were identified. The first class exhibited accumulation of a cata1ytica11y active but conformationally defective product which did not affect the secretion of other proteins. These were therefore predicted to be defective at the level of recognition. The second class exhibited accumulation of a stable, full size product which interfered with the secretion of pectate lyase and were therefore predicted to be blocked within the translocation apparatus. The isolation of intragenic suppressors of some of these mutants is described.
Several deleted derivatives of Ce1V were constructed, all of which were severely affected in their ability to be secreted, even when only-12 carboxy terminal amino acids were affected.

In conclusion, recognition for secretion is highly dependent on conformational integrity of both of the functional domains of which the enzyme is composed. The pathways followed by cellulase and pectinases appear to be initially separate, converging to a common translocation apparatus.

In addition, cellulase, although having limited independent macerating ability, was identified as playing a vital role in the collaborative action of exoenzymes during attack of potato tubers.

Item Type:	Thesis or Dissertation (PhD)
Subjects:	Q Science > QK Botany
Library of Congress Subject Headings (LCSH):	Erwinia carotovora, Bacterial diseases of plants, Cellulase, Secretion
Official Date:	January 1994
Dates:	Date Event January 1994 Submitted
Institution:	University of Warwick
Theses Department:	Department of Biological Sciences
Thesis Type:	PhD
Publication Status:	Unpublished
Supervisor(s)/Advisor:	Salmond, George
Sponsors:	Natural Environment Research Council (Great Britain) ; British Council
Format of File:	pdf
Extent:	xix, 168 leaves : illustrations, charts
Language:	eng

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