Levels of L-malate and other low molecular weight metabolites in spores of Bacillus species and Clostridium difficile

Abstract

Dormant spores of Bacillus species lack ATP and NADH and contain notable levels of only a few other common low mol wt energy reserves, including 3-phosphoglyceric acid (3PGA), and glutamic acid. Recently, Bacillus subtilis spores were reported to contain ~ 30 μmol of L-malate/g dry wt, which also could serve as an energy reserve. In present work, L-malate levels were determined in the core of dormant spores of B. subtilis, Bacillus cereus, Bacillus megaterium and Clostridium difficile, using both an enzymatic assay and 13C-NMR on extracts prepared by several different methods. These assays found that levels of L-malate in B. cereus and B. megaterium spores were 0.5 μmol/g dry wt, and 1 μmol/g dry wt in B. subtilis spores, and levels of L-lactate and pyruvate in B. megaterium and B. subtilis spores were < 0.5 μmol/g dry wt. Levels of L-malate in C. difficile spores were 1 μmol/g dry wt, while levels of 3PGA were ~ 7 μmol/g; the latter value was determined by 31P-NMR, and is in between the 3PGA levels in B. megaterium and B. subtilis spores determined previously. 13C-NMR analysis of spore extracts further showed that B. megaterium, B. subtilis and C. difficile contained significant levels of carbonate/bicarbonate in the spore core. Low mol wt carbon-containing small molecules present at > 3 μmol/g dry spores are: i) dipicolinic acid, carbonate/bicarbonate and 3PGA in B. megaterium, B. subtilis and C. difficile; ii) glutamate in B. megaterium and B. subtilis; iii) arginine in B. subtilis; and iv) at least one unidentified compound in all three species.