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Title: Identification and characterisation of a novel gene algK from the alginate biosynthetic cluster of Pseudomonas aeruginosa
Author: Aarons, Simon J.
Awarding Body: University of Edinburgh
Current Institution: University of Edinburgh
Date of Award: 1996
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In P. aeruginosa, the majority of genes involved in alginate synthesis and export are clustered as a polycistronic operon at 34 minutes on the chromosome. Within this operon are two uncharacterised regions, thought to encode the final unidentified steps in alginate exopolysaccharide production. The characterisation of one of these two regions is reported here. The region between alg44 (which encodes a protein of unknown function) and algE (which encodes an outer membrane, alginate-permeable pore) was subcloned and sequenced. Sequence analysis revealed a continuous open reading frame of 1475 base pairs which encodes a putative polypeptide of 475 amino acids with a predicted molecular weight of 52,470Da. This genetic locus was termed algK and a computer search through the available databases did not reveal any obvious homologous proteins to the predicted AlgK polypeptide. However, computer analysis did predict that AlgK may have an N-terminal lipoprotein signal sequence for extracytoplasmic secretion. To confirm the sequence data, protein expression surveys in both E. coli and P. aeruginosa strains was performed. Results from these experiments confirmed both the presence of the open reading frame for algK and the approximate molecular weight of the AlgK polypeptide. Expression studies of algK within E. coli and P. aeruginosa in the presence of [35S]methionine also suggested that the protein was post-translationally modified, consistent with the view that cleavage and removal of a signal peptide was occurring. The topological organisation and subcellular localisation of AlgK within E. coli has been analysed experimentally with the aid of 3' deletions of algK which result in in-frame fusions to mature β-lactamase (the level of ampicillin resistance conveyed by the fusion proteins reflects on their subcellular locations). This data predicted that mature AlgK had an entirely periplasmic location (although potentially anchored to a membrane) and also provides supporting evidence for the cleavage of the AlgK pre-protein. As a further part of the study, the algK gene within the P. aeruginosa chromosome of a mucoid P. aeruginosa strain was insertionally inactivated using a suicide vector. The resulting mutant was found to display a non-mucoid phenotype. Complementation analysis of this mutant failed to reveal whether the AlgK protein is obligately required for alginate exopolysaccharide production in P. aeruginosa.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available