Levels of Glycine Betaine in Growing Cells and Spores of Bacillus Species and Lack of Effect of Glycine Betaine on Dormant Spore Resistance

Abstract

Bacteria of various Bacillus species are able to grow in media with very high osmotic strength in part due to the accumulation of low-molecular-weight osmolytes such as glycine betaine (GB). Cells of Bacillus species grown in rich and minimal media contained low levels of GB, but GB levels were 4-to 60-fold higher in cells grown in media with high salt. GB levels in Bacillus subtilis cells grown in minimal medium were increased ϳ7-fold by GB in the medium and 60-fold by GB plus high salt. GB was present in spores of Bacillus species prepared in media with or without high salt but at lower levels than in comparable growing cells. With spores prepared in media with high salt, GB levels were highest in B. subtilis spores and >20-fold lower in B. cereus and B. megaterium spores. Athough GB levels in B. subtilis spores were elevated 15-to 30-fold by GB plus high salt in sporulation media, GB levels did not affect spore resistance. GB levels were similar in wild-type B. subtilis spores and spores that lacked major small, acid-soluble spore proteins but were much lower in spores that lacked dipicolinic acid.

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

“…One notable finding in this work was that storage of newly harvested spores at 4°in either water or spent sporulation medium resulted in no notable changes in 3PGA or ribonucleotide levels and no generation of detectable ATP although incubation for extremely long times could have led to changes reflecting extremely slow metabolism. We also note that spores contain unphosphorylated low-molecular-weight compounds that disappear in spore germination (33,34), and perhaps these compounds are metabolized more rapidly in dormant spores. It is also possible that there was some rRNA degradation during spore storage at 4°C in water or spent medium…”

“…The glass tube was rinsed with 1 ml of cold water, and the rinse was transferred to a microcentrifuge tube. The microcentrifuge tubes were centrifuged for 1 min at 14,000 ϫ g; all supernatant fluids were pooled and applied to a 1.5-ml Chelex (Na form) column to remove divalent cations, especially paramagnetic Mn 2ϩ as described previously (22), and the column was washed with 1 ml of water. This process was carried out in duplicate for samples taken after various times of incubation at 4, 37, or 50°C for 7 or 25 days.…”

“…The purified spores were suspended in 4°C water at an optical density at 600 nm (OD 600 ) of 50, and multiple 12-ml aliquots were prepared. Two tubes were immediately used (labeled the zero time sample) to obtain small molecules using procedures described previously (5,10,12,(20)(21)(22), as follows. (i) To remove any growing cells or germinated spores, the spores were centrifuged through a high-density solution of 60% Histodenz (Sigma Chemical Co., St. Louis, MO) since under these conditions dormant spores pellet, and germinated spores, growing and sporulating cells, and cell debris float (23).…”

Role of SpoVA Proteins in Release of Dipicolinic Acid during Germination of Bacillus subtilis Spores Triggered by Dodecylamine or Lysozyme

“…For analysis of other molecules released during dodecylamine germination, spores of strain FB113 (cwlJ sleB) at an OD 600 of 2 were germinated at 45°C with 0.5 mM dodecylamine in 10 mM KPO 4 buffer (pH 7.7). At various times, samples (10 ml) were centrifuged, the supernatant fluid was passed through a 1-ml Chelex column at 23°C to remove divalent cations, in particular Mn 2ϩ , and the runthrough fraction was lyophilized (10). The pellet fraction was boiled in 1 ml water for 20 min, cooled on ice for 15 min, and centrifuged, and the supernatant fluid was treated and lyophilized as described above.…”

“…The solutions were then subjected to nuclear magnetic resonance (NMR) spectroscopy in a 500-MHz Varian Inova spectrometer. The pulse program used as well as data acquisition, processing, and display were all described previously (10). Levels of glutamic acid, a small molecule present in significant amounts in spores (12,22), were determined by a comparison of the peak heights of signals from selected protons in this compounds to the peak heights due to known concentrations of glutamic acid added to spore extracts before Chelex chromatography.…”