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Title: The isolation and characterisation of conditional (Out ts) and null (Out⁻) secretion mutants of Erwinia carotovora subspecies carotovora (SCRI193)
Author: Housby, J. Nicholas
ISNI:       0000 0001 3582 3275
Awarding Body: University of Warwick
Current Institution: University of Warwick
Date of Award: 1993
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Ecc strain SCRI193 secretes a wide range of extracellular degradative enzymes. Examples of these include pectate lyase (Pel), cellulase (Cel) and protease (Prt). Secretion mutants, Outˉ (Pelˉ, Celˉ, Prt+) that accumulate Pel and Cel periplasmically have previously been isolated by transposon and chemical mutagenesis. Protease secretion was always unaffected. Genetic and molecular analysis has revealed a cluster of genes (out) that are essential for the secretion of Pel and Cel in Ecc. In order to eventually investigate the putative periplasmic intermediate in the natural secretion process this study has employed localised mutagenesis, using hydroxylamine, of the out cluster, in order to isolate conditional secretory mutants. Tn5 was successfully linked 3' to the out cluster. Using the Ecc generalised transducing phage, øKP, localised mutagenesis yielded 17 Out' mutants (Pelˉ, Celˉ, Prt+) that accumulated Pel and Cel periplasmically. These 17 mutants included 2 conditional secretory mutants (Out15), HJN1003 and HJN1004, that were shown to accumulate Pel and Cel periplasmically at the restrictive temperature (33°C) but were wild type for secretion at the permissive temperature (26°C) and one auxotroph that was defined as histidine requiring. Each mutation was shown to be linked to the transposon Tn5 and most were subsequently shown, by cosmid complementation, to be within the out cluster. The two Outts secretion mutants, HJN1003 and HJN1004, were mapped to outE and outL respectively. PCR amplification, cloning and sequence analysis has revealed two lesions in outL, from the Outts strain HJN1004 and one lesion in the Outˉ strain HJN1008. Attempts to perform pulse chase experiments proved to be difficult and suggestions have been made to overcome these problems.
Supervisor: Not available Sponsor: Science and Engineering Research Council
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available
Keywords: QP Physiology ; QR Microbiology