Characterisation of four strains of thermophilic spirochetes and characterisation of phosphofructokinase from isolate Rt 118.B2

Abstract

Four strains of spirochetes were purified by the selective use of antibiotics. Microscopy revealed the strains have typical spirochete morphology. The strains were obligate anaerobes and all four strains utilised a variety of carbohydrates including polysaccharides -but not amino acids- as energy sources. The optimum pH of the isolates was between 6.5 and 7.0. The cardinal growth temperatures varied between the different strains from 52°C to 59°C. The lowest temperature any of the four strains could grow at was 37°C and the highest was 62°C. Doubling times of the isolates varied from 221 minutes to 333 minutes. The major products of glucose fermentations were acetate and lactic acid, no ethanol was formed. All strains were resistant to rifampicin, naldixic acid, novobiocin and polymyxin B. Determination of the G+C mol% of DNA revealed that the strains have the lowest G+C % of the species. Cell free extracts from the isolates were assayed for pyrophosphate dependent phosphofructokinase (PFK) activity and the results were compared with those from other members of the species. Attempts were made to purify PFK from one of the isolates. The partially purified enzyme was characterised and was found to have an optimum pH of 7 and optimum temperature of 60°C. The thermostability of the enzyme at various temperatures was established. The kinetics of the enzyme were examined and the Kₘ of fructose-6-phosphate, pyrophosphate and tripolyphosphate established. Various polyphosphates were used as phosphoryl donors; this is the first report of polyphosphates being used as a phosphoryl donor for PFK.