Gene replacement in Staphylococcus carnosus and Staphylococcus xylosus

Abstract

A system for high-efficiency gene replacement in Staphylococcus carnosus and Staphylococcus xylosus has been developed, that is based on temperature-sensitive Escherichia coli-Staphylococcus shuttle vectors for fragment delivery and erythromycin resistance cassettes to facilitate selection of genomic copies of disrupted genes. The approach was tested by constructing a phosphotransferase-deficient mutant of S. carnosus and an S. xylosus mutant strain unable to utilize sucrose. Allelic replacements were observed at rather high frequencies, ranging from approximately 10% for the ptsI gene in S. carnosus up to 50% for the scrB gene in S. xylosus. These differences most likely reflect the length of homology rather than strain-specific variations in recombination efficiencies. Apart from the staphylococcal species tested in this study, the system appears to be applicable in other staphylococci.

The adhesive properties of the Staphylococcus lugdunensis multifunctional autolysin AtlL and its role in biofilm formation and internalization

“…Species identity of clinical S. lugdunensis isolates was proven phenotypically according to standard procedures (Becker and von Eiff, 2011) and molecularly by GenoType ® Staphylococcus (Hain Lifescience, Nehren, Germany). The atlL-deficient S. lugdunensis mutant was constructed by using the plasmids pQE30 (Qiagen, Hilden, Germany), pEC4, and pBT9 (Brückner, 1997). For the complementation of the atlLdeficient mutant, the vector pCU1 was used (Kozlowski et al, 1987).…”

Molecular analysis of the thymidine-auxotrophic small colony variant phenotype of Staphylococcus aureus

“…The restriction-negative S. aureus strain RN4220 (Kreiswirth et al, 1983) was used as intermediate host for transferring DNA constructs from E. coli strain DH5a to S. aureus SH1000 (Horsburgh et al, 2002). The plasmids pBT2 (Bru¨ckner, 1997) and pHPS9 (Haima et al, 1990) were employed as cloning vectors. Both plasmids are shuttle and expression vectors able to replicate in E. coli and staphylococci.…”

Rot and Agr system modulate fibrinogen-binding ability mainly by regulating clfB expression in Staphylococcus aureus NCTC8325

“…The media were solidified with 1.5% (wt/vol) agar, if needed. The mutants were constructed using a method as previously described [39]. For complementing the mutants, the target gene and its promoter from S. aureus NCTC8325 were amplified by polymerase chain reaction (PCR).…”