Korza George, Abini-Agbomson Stephen, Setlow Barbara, Shen Aimee, Setlow Peter
Department of Molecular Biology and Biophysics, UConn Health, Farmington, CT, United States of America.
Department of Molecular Biology and Microbiology, Tufts University School of Medicine, Boston, MA, United States of America.
PLoS One. 2017 Aug 29;12(8):e0182656. doi: 10.1371/journal.pone.0182656. eCollection 2017.
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.
芽孢杆菌属的休眠孢子缺乏三磷酸腺苷（ATP）和烟酰胺腺嘌呤二核苷酸（NADH），仅含有少量其他常见的低分子量能量储备，包括3-磷酸甘油酸（3PGA）和谷氨酸。最近，有报道称枯草芽孢杆菌孢子含有约30微摩尔/克干重的L-苹果酸，其也可作为能量储备。在本研究中，使用酶促测定法和对通过几种不同方法制备的提取物进行的13C-核磁共振（13C-NMR），测定了枯草芽孢杆菌、蜡样芽孢杆菌、巨大芽孢杆菌和艰难梭菌休眠孢子核心中的L-苹果酸水平。这些测定发现，蜡样芽孢杆菌和巨大芽孢杆菌孢子中的L-苹果酸水平≤0.5微摩尔/克干重，枯草芽孢杆菌孢子中的L-苹果酸水平≤1微摩尔/克干重，巨大芽孢杆菌和枯草芽孢杆菌孢子中的L-乳酸和丙酮酸水平<0.5微摩尔/克干重。艰难梭菌孢子中的L-苹果酸水平≤1微摩尔/克干重，而3PGA水平约为7微摩尔/克；后者的值通过31P-NMR测定，介于先前测定的巨大芽孢杆菌和枯草芽孢杆菌孢子中的3PGA水平之间。对孢子提取物的13C-NMR分析进一步表明，巨大芽孢杆菌、枯草芽孢杆菌和艰难梭菌在孢子核心中含有大量的碳酸盐/碳酸氢盐。干孢子中含量>3微摩尔/克的低分子量含碳小分子有：i）巨大芽孢杆菌、枯草芽孢杆菌和艰难梭菌中的吡啶二羧酸、碳酸盐/碳酸氢盐和3PGA；ii）巨大芽孢杆菌和枯草芽孢杆菌中的谷氨酸；iii）枯草芽孢杆菌中的精氨酸；iv）所有这三个物种中至少有一种未鉴定的化合物。
