Characterization of Acp, a peptidoglycan hydrolase of Clostridium perfringens with N-acetylglucosaminidase activity that is implicated in cell separation and stress-induced autolysis.
This work reports the characterization of the first known peptidoglycan hydrolase (Acp) produced mainly during vegetative growth of Clostridium perfringens. Acp has a modular structure with three domains: a signal peptide domain, an N-terminal domain with repeated sequences, and a C-terminal catalytic domain. The purified recombinant catalytic domain of Acp displayed lytic activity on the cell walls of several Gram-positive bacterial species. Its hydrolytic specificity was established by analyzing the Bacillus subtilis peptidoglycan digestion products by coupling reverse phase-high-pressure liquid chromatography (RP-HPLC) and matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) analysis, which displayed an N-acetylglucosaminidase activity. The study of acp expression showed a constant expression during growth, which suggested an important role of Acp in growth of C. perfringens. Furthermore, cell fractionation and indirect immunofluorescence staining using anti-Acp antibodies revealed that Acp is located at the septal peptidoglycan of vegetative cells during exponential growth phase, indicating a role in cell separation or division of C. perfringens. A knockout acp mutant strain was obtained by using the insertion of mobile group II intron strategy (ClosTron). The microscopic examination indicated a lack of vegetative cell separation in the acp mutant strain, as well as the wild-type strain incubated with anti-Acp antibodies, demonstrating the critical role of Acp in cell separation. The comparative responses of wild-type and acp mutant strains to stresses induced by Triton X-100, bile salts, and vancomycin revealed an implication of Acp in autolysis induced by these stresses. Overall, Acp appears as a major cell wall N-acetylglucosaminidase implicated in both vegetative growth and stress-induced autolysis.
Camiade
Emilie
E
Groupe de Recherche sur les Antimicrobiens et les Micro-organismes, UPRES EA 2656, IFR 23, Université de Rouen, 22 Boulevard Gambetta, F-76183 Rouen Cedex, France.
Peltier
Johann
J
Bourgeois
Ingrid
I
Couture-Tosi
Evelyne
E
Courtin
Pascal
P
Antunes
Ana
A
Chapot-Chartier
Marie-Pierre
MP
Dupuy
Bruno
B
Pons
Jean-Louis
JL
D000118:Acetylglucosaminidase / Q000235:genetics / Q000378:metabolism / Q000502:physiology*; D000900:Anti-Bacterial Agents / Q000494:pharmacology; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism / Q000502:physiology*; D001433:Bacteriolysis / Q000187:drug effects / Q000235:genetics*; D015153:Blotting, Western; D002851:Chromatography, High Pressure Liquid; D003016:Clostridium perfringens / Q000166:cytology* / Q000187:drug effects / Q000201:enzymology* / Q000235:genetics; D004591:Electrophoresis, Polyacrylamide Gel; D016680:Genome, Bacterial / Q000235:genetics; D008826:Microbial Sensitivity Tests; D008969:Molecular Sequence Data; D016296:Mutagenesis; D009238:N-Acetylmuramoyl-L-alanine Amidase / Q000235:genetics / Q000378:metabolism / Q000502:physiology*; D017830:Octoxynol / Q000494:pharmacology; D010457:Peptidoglycan / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
D000900:Anti-Bacterial Agents; D001426:Bacterial Proteins; D010457:Peptidoglycan; D017830:Octoxynol; D000118:Acetylglucosaminidase; D009238:N-Acetylmuramoyl-L-alanine Amidase