Germination of Clostridium cylindrosporum Spores on Medium Containing Uric Acid

M. Smith, C. Sullivan
1989 Applied and Environmental Microbiology  
Clostridium cylindrosporum spores germinated rapidly under reducing conditions when bicarbonate, uric acid, and calcium were present. Germination rates on 10 mM urate increased with increasing Ca21 (maximum rate at 5 mM Ca2+ or greater). Germination rates on urate (limiting Ca2+) increased with increasing urate concentrations to 10 mM urate. At 10 mM Ca2', germination rates reached a maximum at 1 mM urate and remained constant thereafter. Cations (Na+, K+, Li', and Mg2+), purines, purine
more » ... ines, purine analogs, and EDTA inhibited germination at limiting calcium concentrations but not (except for 10 mM adenine) at 10 mM Ca2 . Methyl viologen or formate did not inhibit germination. Germination was not observed in solutions containing xanthine, hypoxanthine, caffeine, theophylline, 6,8-dihydroxypurine, adenine, allopurinol, formate, glycine, or acetate, even though some of the purines are growth substrates. Clostridium cylindrosporum is an obligately purinolytic anaerobe, sporulating efficiently under appropriate conditions (3, 21) and fermenting uric acid (and some purines) to ammonia, carbon dioxide, acetic acid, and formate (3). It was isolated from bird droppings, of which uric acid is an important constituent. Germination of C. cylindrosporum spores has not been described previously; knowledge of uric acid-triggered germination is based on experiments with Bacillus fastidiosus, a strictly aerobic species that grows solely on uric acid (2, 22) . In B. fastidiosus spores, uricase (a key enzyme of uric acid catabolism) was implicated in the triggering of spore germination by uric acid (22) . Absence of early release of spore cortex fragments and metabolic experiments suggested that aerobic spore germination on urate was a metabolic rather than a purely physical or hydrolytic process. A comparison of anaerobic germination on urate with the reported aerobic germination might be useful in assessing the general importance of key metabolic enzymes in uric acid germination. In this paper, we describe conditions that give consistently rapid and complete germination of C. cylindrosporum spores on uric acid. We show that C. cylindrosporum spore germination differed from B. fastidiosus spore germination in important respects: calcium was required, and it apparently acted by modifying the spore-triggering response to urate. Although calcium and other ions are known to influence the triggering of spore germination in other species (1, 6, (12) (13) (14) (15) 19) , previous studies had not indicated a role in initiating spore germination with urate. We also show that a range of purine bases, xanthine dehydrogenase inhibitors, monovalent cations, and EDTA inhibited germination. These inhibitions could be overcome or reversed by increasing the concentration of calcium or urate alone; it was not necessary to increase both calcium and urate concentrations. based on that of Schiefer-Ullrich et al. (23) and contained 10 mM sodium urate, 4 mM K2HPO4, 4 mM Na2HPO4, 140 ,uM MgSO4, 100 nM sodium selenite, 21 FxM ferric sodium EDTA, 29 p.M CaCl2, 20 nM biotin, 44 ,uM thiamine hydrochloride, 20 mM KHCO3, and 0.0001% (wt/vol) resazurin in glass-distilled water. For some experiments, MnCI2 was added to a concentration of 50 ,uM (2). Sodium urate was prepared from the free acid (Sigma Chemical Co., St. Louis, Mo.) plus NaOH and contained equimolar concentrations of sodium and urate. The medium was prepared anaerobically in 160-ml serum bottles under 80% N2-20% CO2 as described previously (1, 24). Chemicals and reagents. Ferric sodium EDTA, imidazole, Tris, dipicolinic acid, and purines were purchased from Sigma. Ultrapure sucrose and ultrapure Tris were purchased from Schwartz/Mann, Cambridge, Mass. Reagents were prepared from reagent-grade chemicals when possible and from glass-distilled water. Glassware for reagents and medium preparation was rinsed with 6 N HCl followed by glass-distilled water before use. Preparation of spores. Cultures for spore preparation were grown in 20-liter carboys containing 10 liters of defined medium prepared as described above. The medium was prepared aerobically without a reducing agent and then plugged with cotton plugs and autoclaved for 1 h. After autoclaving, the carboys were cooled to ambient temperature under a stream of 80% N2-20% CO2 and stoppered with sterile black rubber stoppers. The stoppers were then wired in place, and the headspace was flushed with a nitrogencarbon dioxide mixture through sterile needles inserted through the stoppers. After the headspace had been flushed, sodium bicarbonate and sodium sulfide (reducing agent) were added to final concentrations of 60 and 600 ,uM, respectively, by injection of concentrated, sterile stock solutions through the stoppers. When the medium became colorless because of reduction of the resazurin, it was inoculated with 5 x 108 spores of heat-shocked (70°C for 20 min) C. cylindrosporum and then incubated at 35°C until growth and lysis of sporangia were complete (5 to 7 days). Spores were harvested with a Pellicon cell harvester (Millipore Corp., Bedford, Mass.) or by continuous-flow centrifugation (12,000 x g) with an RC5C centrifuge (Ivan Sorvall, Inc., Norwalk, Conn.). Harvested spores were 1380 on May 7, 2020 by guest Downloaded from
doi:10.1128/aem.55.6.1380-1385.1989 fatcat:zlslk6vdrva4bfxr3nv7jnzc5u