Analysis of Metabolism in Dormant Spores of Bacillus Species by ³¹ P Nuclear Magnetic Resonance Analysis of Low-Molecular-Weight Compounds

Abstract

This work was undertaken to obtain information on levels of metabolism in dormant spores of Bacillus species incubated for weeks at physiological temperatures. Spores of Bacillus megaterium and Bacillus subtilis strains were harvested shortly after release from sporangia and incubated under various conditions, and dormant spore metabolism was monitored by 31 P nuclear magnetic resonance (NMR) analysis of molecules including 3-phosphoglyceric acid (3PGA) and ribonucleotides. Incubation for up to 30 days at 4, 37, or 50°C in water, at 37 or 50°C in buffer to raise the spore core pH from ϳ 6.3 to 7.8, or at 4°C in spent sporulation medium caused no significant changes in ribonucleotide or 3PGA levels. Stage I germinated spores of Bacillus megaterium that had slightly increased core water content and a core pH of 7.8 also did not degrade 3PGA and accumulated no ribonucleotides, including ATP, during incubation for 8 days at 37°C in buffered saline. In contrast, spores incubated for up to 30 days at 37 or 50°C in spent sporulation medium degraded significant amounts of 3PGA and accumulated ribonucleotides, indicative of RNA degradation, and these processes were increased in B. megaterium spores with a core pH of ϳ7.8. However, no ATP was accumulated in these spores. These data indicate that spores of Bacillus species stored in water or buffer at low or high temperatures exhibited minimal, if any, metabolism of endogenous compounds, even when the spore core pH was 7.8 and core water content was increased somewhat. However, there was some metabolism in spores stored in spent sporulation medium. Spores of various Bacillus species are generally referred to as metabolically dormant as metabolism of these spores of both exogenous and endogenous compounds is extremely low (1, 2). However, there are reports that there is metabolic activity in these supposedly dormant spores, including oxidation of exogenous compounds such as glucose (3) and degradation of endogenous rRNA and even transcription, when B. subtilis spores are incubated for a number of days at physiological temperatures (4). In the latter two processes, this metabolism was suggested to take place shortly after spores were released from sporangia and to be important in adaptation of the spores to the environments in which they were released and incubated.There have been relatively few detailed studies of metabolism of endogenous compounds in dormant spores although it is known that spores of both Bacillus and Clostridium species have minimal levels, if any, of normal high-energy compounds such as nucleoside triphosphates and reduced pyridine nucleotides (1). However, spores do have significant levels of ribonucleoside monophosphates, with AMP being the most abundant, as well as much smaller amounts of ADP. More importantly, spores have rather significant levels of 3-phosphoglyceric acid (3PGA), a potential rapid source of ATP, and spore 3PGA levels are generally significantly higher than those of AMP. Spores of Bacillus species also have significant levels of the ...

Changes in Bacillus Spore Small Molecules, rRNA, Germination, and Outgrowth after Extended Sublethal Exposure to Various Temperatures: Evidence that Protein Synthesis Is Not Essential for Spore Germination

“…Following all incubations, spore preparations were centrifuged and washed with water and final pellets were suspended in 0.2 ml of 20% HistoDenz (Sigma Chemical Co., St. Louis, MO), applied to the top of 1.9 ml of 50 or 55% HistoDenz in water for B. subtilis and B. megaterium spores, respectively, and centrifuged at 4°C at ϳ8,000 ϫ g for 10 min. Under these centrifugation conditions, dormant spores that retain their DPA pellet while spores that have lost DPA and are germinated, dead, or both float (11,19,20). Spore pellets from these centrifugations were isolated, washed several times with 1.5 ml of water to remove HistoDenz, suspended in water at an OD 600 of ϳ5, and stored at 4°C prior to analyses.…”

“…The inocula used to prepare spores of PS4432 contained 0.5% xylose, but no xylose was present in or on sporulation medium (12,15). In some experiments, B. subtilis spores were prepared in 2ϫSG liquid medium, harvested immediately after most sporangia had lysed, and purified as described previously (11). The spent medium from liquid sporulation was filter sterilized and stored at 4°C.…”

“…Spores of these strains were prepared in liquid supplemented nutrient broth at 30°C, and spores were purified as described previously (11), as was spent sporulation medium, and spores and spent sporulation medium were stored as described above. All of the spores used in this work were free (Ͼ98%) from growing or sporulating cells, germinated spores, or cell debris, as shown by phase-contrast microscopy and by the uniformity of all spore pellets at the final purification steps.…”

“…In particular, spores have ϳ0.1% of the ATP levels of growing cells, although the levels of free adenine nucleotides are similar to those in growing cells (6). Other studies have shown that (i) detectable ATP generation takes place only after completion of spore germination, (ii) spore germination can take place relatively normally even if ATP production from spores' endogenous energy reserves is blocked, (iii) there is no evidence of significant metabolism of spores' endogenous energy reserves until after completion of spore germination, and (iv) protein synthesis is also minimal, if it takes place at all, until after completion of spore germination (6)(7)(8)(9)(10)(11).…”

The Exosporium Layer of Bacterial Spores: a Connection to the Environment and the Infected Host

“…The spore is a survival rather than a replicative mechanism, as only a single spore develops within the cytoplasm of the sporulating cell (an endospore within a mother cell). The spore is released from the mother cell by lysis, and the mature spore exists as a dormant form of the cell that lacks metabolic activity (1,2). Spores can persist in the environment for long periods.…”