Cloning of genes affecting capsule expression in Staphylococcus aureus strain M

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Abstract
Staphylococcus aureus strain M produces large amounts of capsular polysaccharide. It produces a non-encapsulated variant at a frequency of 0.01% at 37 degrees C. At high temperature (43 degrees C), the frequency of capsule loss was shown to be 1-38%. A 19 kb plasmid and a prophage were found to be carried by the M strain, but curing of these elements did not affect capsular production. To clone the capsular (cap) genes, a plasmid library of S. aureus M was constructed directly in S. aureus RN4200. The library was then infected with phage 80 alpha. After transduction of the phage lysates to a Cap- mutant derived from M strain, a recombinant plasmid was obtained which complemented the mutant to a Cap+ phenotype. Chromosomal walking experiments were used to clone additional nearby cap genes. Complementation tests using a collection of Cap- mutants showed that most of the mutants were complemented by a 19.4 kb DNA fragment, suggesting that the majority of the cap genes affecting capsule production are clustered together.