Mutagenesis of Propionibacterium acnes and analysis of two CAMP factor knock-out mutants.
P. acnes is a skin commensal that is frequently associated with inflammatory diseases such as acne vulgaris. Despite the availability of the genome sequence functional studies on P. acnes are scarce due to a lack of methods for genetic manipulation of this bacterium. Here we present an insertional mutagenesis approach for the inactivation of specific P. acnes genes. The gene of interest can be disrupted and replaced with an erythromycin-resistance cassette by employing homologous recombination. We used this method to generate knock-out mutants of camp2 (PPA0687) and camp4 (PPA1231), encoding CAMP factor homologs with predicted co-hemolytic activities. The successful inactivation of the two genes was confirmed by PCR and Western blotting experiments using specific anti-CAMP2/CAMP4 sera. The Δcamp2 but not the Δcamp4 mutant exhibited reduced hemolytic activity in the CAMP reaction with sheep erythrocytes, indicating that CAMP2 is the major active co-hemolytic factor of P. acnes. The biological relevance of the CAMP factors remains unclear as disruption of camp2 or camp4 did not significantly alter the transcriptome response of HaCaT cells to P. acnes. The here presented insertional mutagenesis approach will facilitate future studies on P. acnes.
Sörensen
Meike
M
Max Planck Institute for Infection Biology, Department of Molecular Biology, D-10117 Berlin, Germany.
Mak
Tim N
TN
Hurwitz
Robert
R
Ogilvie
Lesley A
LA
Mollenkopf
Hans J
HJ
Meyer
Thomas F
TF
Brüggemann
Holger
H
D000818:Animals; D000900:Anti-Bacterial Agents / Q000494:pharmacology; D001426:Bacterial Proteins / Q000235:genetics*; D015153:Blotting, Western; D004912:Erythrocytes / Q000187:drug effects; D004917:Erythromycin / Q000494:pharmacology; D055786:Gene Knockout Techniques / Q000379:methods*; D006460:Hemolysin Proteins / Q000235:genetics*; D006461:Hemolysis; D051379:Mice; D016254:Mutagenesis, Insertional / Q000379:methods*; D016133:Polymerase Chain Reaction; D011425:Propionibacterium acnes / Q000235:genetics*; D011995:Recombination, Genetic; D012641:Selection, Genetic; D012756:Sheep
D000900:Anti-Bacterial Agents; D001426:Bacterial Proteins; D006460:Hemolysin Proteins; D004917:Erythromycin