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-beta-D-glucopyranoside (beta-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 beta-MUG hydrolysis during spore germination and outgrowth. It was previously shown that beta-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 beta-MUG hydrolysis by almost 99%, indicating that proteins needed for rapid beta-MUG hydrolysis are synthesized during spore outgrowth. However, the residual beta-MUG hydrolysis during spore germination with chloramphenicol indicated that dormant spores contain low levels of proteins needed for beta-MUG uptake and hydrolysis. With B. subtilis 168 spores that lacked several general proteins of the phosphotransferase system (PTS) for sugar uptake, beta-MUG hydrolysis during spore germination and outgrowth was decreased >99.9%. This indicated that beta-MUG is taken up by the PTS, resulting in the intracellular accumulation of the phosphorylated form of beta-MUG, beta-MUG-6-phosphate (beta-MUG-P). This was further demonstrated by the lack of detectable glucosidase activity on beta-MUG in dormant, germinated and outgrowing spore extracts, while phosphoglucosidase active on beta-MUG-P was readily detected. Dormant B. subtilis 168 spores had low levels of at least four phosphoglucosidases active on beta-MUG-P: BglA, BglH, BglC (originally called YckE) and BglD (originally called YdhP). These enzymes were also detected in spores germinating and outgrowing with beta-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 beta-MUG. Deletion of the genes coding for BglA, BglH, BglC and BglD reduced beta-MUG hydrolysis by germinating and outgrowing spores of B. subtilis 168 at least 99.7%. Assay of glucosidases active on beta-MUG or beta-MUG-P in extracts of dormant and outgrowing spores of B. atrophaeus revealed no enzyme active on beta-MUG and one enzyme that comprised > or =90% of the phosphoglucosidase active on beta-MUG-P. Partial purification and amino-terminal sequence analysis of this phosphoglucosidase identified this enzyme as BglH. CONCLUSIONS Generation of MU from beta-MUG by germinating and outgrowing spores of B. atrophaeus and B. subtilis is mediated by the PTS-driven uptake and phosphorylation of beta-MUG, followed by phosphoglucosidase action on the intracellular beta-MUG-P. The major phosphoglucosidase catalyzing MU generation from beta-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 beta-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.