Investigation of the genetic location of the protease genes in Pseudomonas fluorescens strains T24 and T25

Wells, Trudy Annette (1997) Investigation of the genetic location of the protease genes in Pseudomonas fluorescens strains T24 and T25. Masters thesis, Memorial University of Newfoundland.

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Abstract

Economic and industrial interest in prokaryotic extracellular proteases has been the impetus for the study of extracellular proteases. Pseudomonas fluorescens strains T24 and T25, have been recognized for their ability to produce extracellular proteases, and the presence of plasmid DNA in these strains led to the hypothesis that the protease genes may be located on the plasmid DNA. To determine whether or not the protease genes were located on the plasmid DNAs of the two strains, various experiments, including conjugation, transformation, and curing via heat treatment were employed. While protease positive transconjugants and transformants were not detected in the conjugation and transformation experiments, respectively, the curing experiment suggested the plasmid location of the protease genes. -- Partial restriction endonuclease digestion maps were created for the plasmids, pT24 and pT25. The single restriction endonuclease digests for each of the plasmids was then probed with a nonradioactive, digoxigenin-labeled construct, pUT 8, reported to contain a protease gene. The pUT 8 construct hybridized specifically with a 3.75 kbp Sal I fragment and two Pst I fragments (sizes 3.4 kbp and 1.72 kbp) from the pT24 and pT25 plasmids, providing a second line of evidence for the plasmid origin of the protease genes. Regions similar to the pUT8 construct located on the pT24 and pT25 plasmids were then ligated to the cloning vector pUC18, and used to transform Escherichia coli. While thousands of colonies were screened, protease positive transformants were not detected. The pUT 8 probe was also used to probe the genomic DNA from P. ffuorescens strains T24 and T25. The probe did not hybridize with digests of genomic DNA from either Pseudomonas strain, thus providing a third line of evidence for the plasmid origin of the protease genes.

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