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Title: Regulation of phenotype in Pseudomonas tolaasii
Author: Baldwin, C. V. F.
Awarding Body: University of Cambridge
Current Institution: University of Cambridge
Date of Award: 2003
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Pseudomonas tolaasii is the causal agent of brown blotch disease of mushrooms. The bacterium exists in two stable phenotypic forms: the smooth, virulent wild type (1116S), which produces the toxin tolaasin; and the rough, avirulent variant (1116R), which does not produce tolaasin. The PheN-PheS two-component signal transduction pathway regulates the phenotype of these forms. Spontaneous phenotypic switching between the 1116S and 1116R is mediated by a reversible 661 bp duplication within the sensor region of the pheN gene. The role of the environment in phenotypic switching between 1116S and 1116R was studied. It was found that frequency of switching from 1116R to 1116S in colonies was enhanced with the addition of mushroom extract to the medium. Characterisation of the active component of mushroom demonstrated the signal to be heat labile, active at 1,000 fold dilution and <12,000 kDa in size. Further characterisation suggested the presence of 2 distinct signal molecules in the mushroom extract, one in the cationic fraction and one in an anionic fraction. The toxin, tolaasin, a major determinant of phenotype is produced in a cell density dependent manner. The role of N-acyl homoserine lactone (AHL) mediated quorum sensing regulation of phenotype was studied. Thin layer chromatography indicated that 1116S produces the autoinducer compound, N -(3-oxododecanoyl)-L-homoserine lactone (OdDHL), while 1116R does not produce a detectable level of AHLs, suggesting that AHL production is under the regulation of the PheN-PheS two-component signal transduction pathway. Further investigation demonstrated the presence of an unidentified secondary quorum sensing autoinducer, which is only present below an OD600 of 1.0. The addition of synthetic OdDHL to culture medium prior to inoculation with 1116S did not induce tolaasin synthesis, suggesting that either OdDHL is not involved with the regulation of tolaasin, or that a secondary level of regulation also exists.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available