The role of small acid-soluble proteins (SASPs) in protection of spores of Clostridium botulinum against nitrous acid.

Mutant strains of Clostridium botulinum ATCC 3502 were generated using the ClosTron in four genes (CBO1789, CBO1790, CBO3048, CBO3145) identified as encoding α/ß-type SASP homologues. The spores of mutant strains in which CBO1789 or CBO1790 was inactivated demonstrated a significant increase in sensitivity to the damaging agent nitrous acid (P<0.01), a phenotype that was partially restored to wild-type in complementation studies. In contrast to nitrous acid, the spores of the CBO1789 and CBO1790 mutants showed no change in their resistance to formaldehyde and hydrogen peroxide (P>0.05), two other chemicals commonly used as components of disinfection regimes. These data indicate that the SASPs CBO1789 or CBO1790 play a significant role in resistance to nitrous acid, but not in resistance to formaldehyde or hydrogen peroxide.

Optimal spore germination in Clostridium botulinum ATCC 3502 requires the presence of functional copies of SleB and YpeB, but not CwlJ.

Germination, the process by which dormant endospores return to vegetative growth, is a critical process in the life cycle of the notorious pathogen Clostridium botulinum. Crucial is the degradation by hydrolytic enzymes of an inner peptidoglycan spore layer termed the cortex. Two mechanistically different systems of cortex lysis exist in spores of Clostridium species. C. botulinum ATCC 3502 harbours the Bacillus-like system of SleB, CwlJ and YpeB cortex lytic enzymes (CLEs). Through the construction of insertional gene knockout mutants in the sleB, cwlJ and ypeB genes of C. botulinum ATCC 3502 and the production of spores of each mutant strain, the effect on germination was assessed. This study demonstrates a reduced germination efficiency in spores carrying mutations in either sleB or ypeB with an approximate 2-fold reduction in heat resistant colony forming units (CFU/OD600) when plated on rich media. This reduction could be restored to wild-type levels by removing the spore coat and plating on media supplemented with lysozyme. It was observed that cwlJ spores displayed a similar germination efficiency as wild-type spores (P > 0.05). An optimal germinant commixture was identified to include a combination of l-alanine with sodium bicarbonate as it resulted in a 32% drop in OD600, while the additional incorporation of l-lactate resulted in a 57% decrease. Studies of the germination efficiency of spores prepared from all three CLE mutants was performed by monitoring the associated decrease in optical density but a germination defect was not observed in any of the CLE mutant strains. This was likely due to the lack of specificity of this particular assay. Taken together, these data indicate that functional copies of SleB and YpeB, but not CwlJ are required for the optimal germination of the spores of C. botulinum ATCC 3502.