Hinton, Jay C. D. (1986) Development and application of molecular genetic techniques in 'Erwinia carotovora' subsp. 'carotovora'. PhD thesis, University of Warwick.

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Abstract

Erwinia carotovora subsp. carotovora (Ecc) is an important bacterial phytopathogen, and a Member of the group of "soft rot erwinias". Methods of molecular genetic analysis were applied to Ecc strain SCRI193 to enable the future study of the process of extracellular enzyme production.

Following work with a number of transformation systems, the "Hanahan" procedure proved applicable to SCRI193, and was used to obtain up to 4 x 10⁴ transformants per μg of pBR322 DNA. The method was optimised and used for the direct introduction of in vitro-manipulated plasmids to Ecc.

Two Tn5 mutagenesis systems were developed for SCRI193. First, the pJB4JI system was used to obtain a variety of mutants, including HC500, which had a Cysˉ, Pelˉ, Celˉ, Prtˉ phenotype on assay media. Second, a pBR322::1amB⁺ plasmid was introduced to SCRI193 making the strain sensitive to λ::Tn5 infection, and allowing the isolation of several Tn5-induced mutants.

Extracellular enzyme production by SCRI193 and HC500 was studied. It appeared that the Pelˉ, Celˉ, Prtˉ phenotype of HC500 was attributable to growth under sulphur-limited conditions.

The Tn5 element and flanking DNA was cloned from HC500 and used to obtain the wild-type homologue. This wild-type cys⁺ gene "complemented” the cysB lesion of E. coli, and the significance of this is discussed.

A pectate lyase structural gene was cloned from SCKI193 and studied physically and biochemically. Alternative genetic approaches In SCRI193 involved attempts to isolate a transducing phage, and the selection of nonsense suppressor mutants.

The work presented in this thesis demonstrates that the molecular genetic tools for the detailed analysis of Ecc are now available; SCRI193 Is currently being used as a model system for the study of protein secretion.

Item Type:	Thesis (PhD)
Subjects:	Q Science > QH Natural history Q Science > QR Microbiology
Library of Congress Subject Headings (LCSH):	Erwinia carotovora -- Research, Molecular genetics -- Experiments, Extracellular enzymes -- Research, Phytopathogenic bacteria
Official Date:	March 1986
Dates:	Date Event March 1986 Submitted
Institution:	University of Warwick
Theses Department:	Department of Biological Sciences
Thesis Type:	PhD
Publication Status:	Unpublished
Supervisor(s)/Advisor:	Salmond, George
Sponsors:	Science and Engineering Research Council (Great Britain)
Extent:	1 volume (various pagings) : illustrations
Language:	eng

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