Mechanism of the hydrolysis of 4‐methylumbelliferyl‐ β ‐ d ‐glucoside by germinating and outgrowing spores of Bacillus species

Aims:  To determine the mechanism of the hydrolysis of 4‐methylumbelliferyl‐ β ‐ d ‐glucopyranoside ( β ‐MUG) by germinating and outgrowing spores of Bacillus species. Methods and Results:  Spores of B. atrophaeus (formerly B. subtilis var. niger, Fritze and Pukall 2001) are used as biological indicators of the efficacy of ethylene oxide sterilization by measurement of β ‐MUG hydrolysis during spore germination and outgrowth. It was previously shown that β ‐MUG is hydrolysed to 4‐methylumbelliferone (MU) during the germination and outgrowth of B. atrophaeus spores (Chandrapati and Woodson 2003), and this was also the case with spores of B. subtilis 168. Germination of spores of either B. atrophaeus or B. subtilis with chloramphenicol reduced β ‐MUG hydrolysis by almost 99%, indicating that proteins needed for rapid β ‐MUG hydrolysis are synthesized during spore outgrowth. However, the residual β ‐MUG hydrolysis during spore germination with chloramphenicol indicated that dormant spores contain low levels of proteins needed for β ‐MUG uptake and hydrolysis. With B. subtilis 168 spores that lacked several general proteins of the phosphotransferase system (PTS) for sugar uptake, β ‐MUG hydrolysis during spore germination and outgrowth was decreased >99·9%. This indicated that β ‐MUG is taken up by the PTS, resulting in the intracellular accumulation of the phosphorylated form of β ‐MUG, β ‐MUG‐6‐phosphate ( β ‐MUG‐P). This was further demonstrated by the lack of detectable glucosidase activity on β ‐MUG in dormant, germinated and outgrowing spore extracts, while phosphoglucosidase active on β ‐MUG‐P was readily detected. Dormant B. subtilis 168 spores had low levels of at least four phosphoglucosidases active on β ‐MUG‐P: BglA, BglH, BglC (originally called YckE) and BglD (originally called YdhP). These enzymes were also detected in spores germinating and outgrowing with β ‐MUG, but levels of BglH were the highest, as this enzyme's synthesis was induced ca 100‐fold during spore outgrowth in the presence of β ‐MUG. Deletion of the genes coding for BglA, BglH, BglC and BglD reduced β ‐MUG hydrolysis by germinating and outgrowing spores of B. subtilis 168 at least 99·7%. Assay of glucosidases active on β ‐MUG or β ‐MUG‐P in extracts of dormant and outgrowing spores of B. atrophaeus revealed no enzyme active on β ‐MUG and one enzyme that comprised ≥90% of the phosphoglucosidase active on β ‐MUG‐P. Partial purification and amino‐terminal sequence analysis of this phosphoglucosidase identified this enzyme as BglH. Conclusions:  Generation of MU from β ‐MUG by germinating and outgrowing spores of B. atrophaeus and B. subtilis is mediated by the PTS‐driven uptake and phosphorylation of β ‐MUG, followed by phosphoglucosidase action on the intracellular β ‐MUG‐P. The major phosphoglucosidase catalyzing MU generation from β ‐MUG‐P in spores of both species is probably BglH. Significance and Impact of the Study:  This work provides new insight into the mechanism of uptake and hydrolysis of β ‐MUG by germinating and outgrowing spores of Bacillus species, in particular B. atrophaeus . The research reported here provides a biological basis for a Rapid Readout Biological Indicator that is used to monitor the efficacy of ethylene oxide sterilization.