Efficient Inhibition of Germination of Coat-Deficient Bacterial Spores by Multivalent Metal Cations, Including Terbium (Tb ³⁺ )

Abstract

Release of dipicolinic acid (DPA) and its fluorescence with terbium (Tb 3؉ ) allow rapid measurement of the germination and viability of spores of Bacillus and Clostridium species. However, germination of coat-deficient Bacillus spores was strongly inhibited by Tb 3؉ and some other multivalent cations. Tb 3؉ also inhibited germination of coat-deficient Clostridium perfringens spores.Many Bacillus and Clostridium species can form dormant, resistant spores under conditions unfavorable for growth (24,25). These spores can resist extreme conditions and survive for years in the absence of nutrients. However, when nutrients return, these spores can germinate and become growing cells (16,23,25). Some spore-forming bacteria can also cause food spoilage, food poisoning, and disease, and spores of Bacillus anthracis are a potential bioterrorism agent (9, 13, 25). Therefore, rapid, sensitive assays for bacterial spores have applied importance.An assay has been developed to detect low spore concentrations, based on spores' high concentration (ϳ10% [dry weight]) of pyridine-2,6-dicarboxylic acid (dipicolinic acid [DPA]) (9). DPA forms a strong 1:1 highly fluorescent complex with terbium (Tb 3ϩ ) ions, and spore quantification by Tb 3ϩ -DPA fluorescence takes only a few minutes (4, 12). DPA is also released in the first minutes of spore germination (23), and since germination is essential for spore viability, Tb 3ϩ -DPA fluorescence has been used to detect spore germination, and there is a good correlation between DPA release and spore viability (4,26,28,29,31,32). However, we report a flaw in the latter assay of spore viability, as Tb 3ϩ and some other multivalent metal cations strongly inhibit germination of coat-deficient spores. The coats comprise the outermost layer of spores of many species, including Bacillus subtilis, and protect spores against many agents, including enzymes and reactive chemicals (8,10,11,24).The B. subtilis strains used are isogenic derivatives of a laboratory 168 strain and are (i) PS533 (wild type), carrying plasmid pUB110, encoding resistance to kanamycin (22); (ii) PS3328 (⌬cotE mutant), in which much of the cotE coding sequence was replaced with a tetracycline resistance (Tc r ) cassette (15); and (iii) PS3738 (⌬safA mutant), which carries a Tc r cassette replacing much of the safA coding sequence (10). These strains were sporulated at 37°C on 2ϫ Schaeffer's-glucose (SG) agar plates without antibiotics, and spores were harvested and cleaned as described previously (14). The Bacillus cereus strain used was strain T obtained from H. O. Halvorson, and its spores were prepared at 30°C in a defined liquid medium and were purified as described previously (3). The Bacillus megaterium strain used was strain ATCC 12782, and its spores were prepared at 30°C in liquid supplemented nutrient broth and were harvested and cleaned as described previously (5). The Clostridium perfringens strain used was MRS101 (⌬cpe::catP mutant) in which the enterotoxin gene (cpe) in the C. perfringens food poisoning isolate S...

High Salinity Alters the Germination Behavior of Bacillus subtilis Spores with Nutrient and Nonnutrient Germinants

“…Thus, the next potential NaCl inhibition targets would be the yet-unidentified ion channels involved in rapid cation efflux (29,34,39) and/or the DPA excretion channels that seem to be at least in part encoded by the spoVA operon, although their organization and mode of action is not yet understood (17,34,40,41). In a previous study reporting inhibitory effects of multivalent metal ions on the germination of decoated B. subtilis spores, the authors proposed that these ions form complexes with DPA, which could then act as a plug for the DPA channels (42). Whether this would also be possible for monovalent ions is unclear, but ionic interactions would certainly be conceivable.…”

Water and Small-Molecule Permeation of Dormant Bacillus subtilis Spores

“…The results described above indicated that coat defects have significant effects on permeation of molecules, including water, into spores. To examine if this is also the case for other small molecules that are thought to exert their effects by acting at a spore's inner membrane just outside the spore's core, we examined spores' permeation by two compounds, Tb 3ϩ and dodecylamine, that can influ- ence spores' return to life in the process of germination (27,29,34). TbCl 3 at ϳ50 M is often used to monitor the progress of spore germination, by measuring DPA release via Tb-DPA complex formation (28).…”

“…TbCl 3 at ϳ50 M is often used to monitor the progress of spore germination, by measuring DPA release via Tb-DPA complex formation (28). TbCl 3 at 50 M only minimally inhibits the germination of intact spores but completely inhibits the germination of severely coat-defective spores (29). The mechanism for this inhibition has not been definitively established, but it has been suggested that Tb 3ϩ binds to DPA being released from the protein channel in spores' IM through which DPA is released and the Tb-DPA complex blocks this channel completely.…”

“…Again, 180-l aliquots of germination mixtures were added to 20 l of 500 M TbCl 3 and the fluorescence was read as described above. The amount of total DPA in all spores used for germination experiments was determined by boiling spores for 30 min, centrifuging, and measuring DPA in the supernatant fluid by its fluorescence with Tb 3ϩ as described previously (28,29). These total DPA values were used to determine the percentages of spore germination in all germination experiments.…”

Identification of New Proteins That Modulate the Germination of Spores of Bacillus Species

“…In a few experiments, spores were germinated as described above but without Tb 3ϩ present from the initiation of germination. Instead, at various times after germination was initiated, aliquots of the germinating culture were centrifuged, the supernatant fluid was made 50 M in TbCl 3 and Tb-DPA fluorescence was measured as described previously (22). Spore germination was also routinely monitored at the end of germination incubations by phase-contrast microscopy.…”