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Applied and Environmental Microbiology, October 2003, p. 5812-5818, Vol. 69, No. 10
0099-2240/03/$08.00+0     DOI: 10.1128/AEM.69.10.5812-5818.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.

Establishment of an Arbitrary PCR for Rapid Identification of Tn917 Insertion Sites in Staphylococcus epidermidis: Characterization of Biofilm-Negative and Nonmucoid Mutants

Johannes K.-M. Knobloch,* Max Nedelmann, Kathrin Kiel, Katrin Bartscht, Matthias A. Horstkotte, Sabine Dobinsky, Holger Rohde, and Dietrich Mack

Institut für Medizinische Mikrobiologie und Immunologie, Universitätsklinikum Hamburg-Eppendorf, D-20246 Hamburg, Germany

Received 14 March 2003/ Accepted 15 July 2003

Transposon mutagenesis with the Enterococcus faecalis transposon Tn917 is a genetic approach frequently used to identify genes related with specific phenotypes in gram-positive bacteria. We established an arbitrary PCR for the rapid and easy identification of Tn917 insertion sites in Staphylococcus epidermidis with six independent, well-characterized biofilm-negative Tn917 transposon mutants, which were clustered in the icaADBC gene locus or harbor Tn917 in the regulatory gene rsbU. For all six of these mutants, short chromosomal DNA fragments flanking both transposon ends could be amplified. All fragments were sufficient to correctly identify the Tn917 insertion sites in the published S. epidermidis genomes. By using this technique, the Tn917 insertion sites of three not-yet-characterized biofilm-negative or nonmucoid mutants were identified. In the biofilm-negative and nonmucoid mutant M12, Tn917 is inserted into a gene homologous to the regulatory gene purR of Bacillus subtilis and Staphylococcus aureus. The Tn917 insertions of the nonmucoid but biofilm-positive mutants M16 and M20 are located in genes homologous to components of the phosphoenolpyruvate-sugar phosphotransferase system (PTS) of B. subtilis, S. aureus, and Staphylococcus carnosus, indicating an influence of the PTS on the mucoid phenotype in S. epidermidis.

* Corresponding author. Mailing address: Institut für Medizinische Mikrobiologie und Immunologie, Universitätsklinikum Hamburg-Eppendorf, Martinistr. 52, D-20246 Hamburg, Germany. Phone: 49-40-42803-3147. Fax: 49-40-42803-4881. E-mail: knobloch{at}uke.uni-hamburg.de.

Applied and Environmental Microbiology, October 2003, p. 5812-5818, Vol. 69, No. 10
0099-2240/03/$08.00+0     DOI: 10.1128/AEM.69.10.5812-5818.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.



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