Spores of Bacillus anthracis are the causative agents of anthrax, and are a potential threat to public health and safety. Spores can survive upon exposure to high temperatures, UV radiation, and harsh chemicals. When environmental conditions become favorable for cell growth, spores germinate and grow into vegetative cells with the help of germination enzymes, and cause diseases. The germination enzyme CwlJ1 plays an important role in degrading the cortex during the germination of B. anthracis spores. However, the specific function and catalytic activity of CwlJ1 remain elusive. Here we report for the first time the detailed in vitro mechanistic study of CwlJ1 expressed in Escherichia coli and its activity against the spore cortical fragments of B. anthracis when added exogenously. CwlJ1 was active on both decoated spores and spore cortical fragments. Through liquid chromatography-mass spectrometry analysis of the digested cortical fragments, we determined that CwlJ1 was an N-acetylmuramoyl-L-alanine amidase, which mainly recognized large segments of glycan chains in the cortex instead of the minimal structural unit tetrasaccharide. CwlJ1 does not have a binding domain, which is present in most peptidoglycan-degrading enzymes and is associated with substrate specificity; however, it was specific for muramic acid-δ-lactam-containing glycan chains and it had preference for the tetrapeptide side chains. Unlike most amidases, CwlJ1 did not contain a divalent metal cation, and it could survive at 95 °C for about an hour. This study shines some light on the mechanism of spore germination, and provides increased insight into the development of sporicidal enzyme systems for decontamination of B. anthracis and other related bacteria.

Identification of the amidase activity and in vitro characterization of the cortex lytic enzyme CwlJ1 of Bacillus anthracis spores

249th National Meeting (2015)

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