Establishment of an arbitrary PCR for rapid identification of Tn917 insertion sites in Staphylococcus epidermidis: characterization of biofilm-negative and nonmucoid mutants.
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.
Knobloch
Johannes K-M
JK
Institut für Medizinische Mikrobiologie und Immunologie, Universitätsklinikum Hamburg-Eppendorf, D-20246 Hamburg, Germany. knobloch@uke.uni-hamburg.de
Nedelmann
Max
M
Kiel
Kathrin
K
Bartscht
Katrin
K
Horstkotte
Matthias A
MA
Dobinsky
Sabine
S
Rohde
Holger
H
Mack
Dietrich
D
D001422:Bacterial Adhesion / Q000235:genetics; D001426:Bacterial Proteins / Q000235:genetics* / Q000378:metabolism; D001483:Base Sequence; D018441:Biofilms; D004251:DNA Transposable Elements / Q000235:genetics*; D006801:Humans; D008969:Molecular Sequence Data; D016254:Mutagenesis, Insertional; D009154:Mutation; D017351:N-Acetylglucosaminyltransferases / Q000235:genetics; D010641:Phenotype; D010744:Phosphoric Monoester Hydrolases / Q000235:genetics; D016133:Polymerase Chain Reaction / Q000379:methods*; D013212:Staphylococcus epidermidis / Q000235:genetics* / Q000254:growth & development
D001426:Bacterial Proteins; D004251:DNA Transposable Elements; D017351:N-Acetylglucosaminyltransferases; C037649:N-acetyllactosaminide beta-1,6-N-acetylglucosaminyltransferase; D010744:Phosphoric Monoester Hydrolases; C091162:RsbU protein, Bacillus subtilis