Functional analysis of Clostridium sporogenes spore cortex lytic enzyme CwlJ

Members of the bacterial orders Bacillales and Clostridiales survive starvation by forming metabolically dormant spores, which represent the most environmentally robust cell-type found in nature. However, spores constantly monitor their environment, via receptor proteins that are localised deep within the spore, and which under suitable conditions will irreversibly trigger the process of spore germination and the resumption of vegetative growth. During this process, the depolymerisation of the thick layer of peptidoglycan (cortex) is essential in permitting hydration and the re-establishment of metabolism in the spore core. Two classes of peptidoglycan lysins are involved in cortex hydrolysis during germination: cortex-lytic enzymes (CLEs), including SleB and CwlJ, typically only recognise intact spore peptidoglycan, and are essential for initiating cortex hydrolysis; cortical fragment lytic enzymes (CFLEs), which are catalytically active against peptidoglycan fragments generated by CLE activity. In contrast to detailed studies conducted on cortex hydrolysis Bacillus, knowledge of the germination mechanisms that underpin spore germination in the Clostridia is lagging behind. In C. sporogenes milk-isolated strain UC9000, used as the non-toxigenic counterpart of C. botulinum, CLEs were identified from analyses of the genome and the functional analysis of CwlJ cortex lytic enzyme was performed. CwlJ gene was cloned into an expression vector for E. coli and protein expression examined. Expression levels were examined initially by fluorescence microscopy and SDS-PAGE analyses. CwlJ activity against decoated spores was tested and causes an OD drop of 65% when added to decoating spores. HPLC analysis of the fragments generated from CwlJ could help to identify the enzyme hydrolytic bond specificities. Achieved data could be useful to characterize the function of CwlJ in in Clostridium sporogenes germination