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H. Zhu, T. C. R. Conibear, F. Stapleton, M. D. P. Willcox; Genetic Comparison Between Virulent and Avirulent Strains of Pseudomonas aeruginosa Isolated From the Eye. Invest. Ophthalmol. Vis. Sci. 2007;48(13):2658.
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© ARVO (1962-2015); The Authors (2016-present)
Pseudomonas aeruginosa is the major causes of visual loss following microbial keratitis, induced by ocular trauma or contact lens wear. P. aeruginosa 6294 is a virulent ocular strain isolated from a case of microbial keratitis. Strain Paer3 was cultured from an asymptomatic contact lens wearer and is avirulent in a murine keratitis model. The aim of this study was to identify genetic elements found in P. aeruginosa 6294 and not in Paer3.
A modified suppression subtractive hybridisation (SSH) technique was used to examine and compare genomes between P. aeruginosa virulent strain 6294 and avirulent strain Paer3. Digested genomic DNA from 6294 was ligated with two distinct adaptor sequences. Hybridisation was allowed to occur between 6294 DNA and an excess of Paer3 DNA. This DNA was then subjected to a PCR reaction that selectively amplifies only fragments containing one of each adaptor sequence. These amplicons were cloned and screened by southern blot. Fragments that were unique to 6294 were then sequenced using ABI Prism 3700 platform. DNA sequences were submitted to BLAST search to determine homology to known sequences.
A total of 144 clones were screened for specificity to 6294. Thirty-one of DNA fragment inserts were identified to be present in strain 6294 but not in Paer3. Eleven fragments were sequenced and showed no similarity to each other, host or vector DNA. Seven of the 11 sequences showed significant similarity to the sequences from the published PAO1 database, including the quorum sensing regulator lasI, a probable bacteriophage integrase, 3 hypothetical proteins, a probable hydroxylase large subunit and a benzoylformate decarboxylase. One sequence was most similar to a sugar MFS transporter protein from P. putida. Other three fragments showed no significant similarity to any published DNA sequence.
This study has demonstrated SSH to be a powerful and reliable technique to examine and compare genomes between two strains. The identified genes that present in strain 6294 but not in strain Paer3 may be potentially responsible for the variation of observed virulence phenotypes.
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