Some thermophilic species of Bacillus were characterized using biochemical tests, antibiotic sensitivity, bacteriocin and bacteriophage sensitivity, esterase patterns, DNA hybridization and % G+C content. The three caldo-active strains of bacilli isolated by Heinen (1971) were compared with strains of B. stearothermophilus. Eight of the strains examined showed characteristics which enabled them to be placed in the three main taxonomic groups suggested by Walker & Wolf (1971). Two strains showed characteristics of both groups 1 and 3. Examination of genotype data showed taxonomic groupings which differed from those based on phenotypic characterization.

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I N T R O D U C T I O NThe inhibition of bacteria by closely related species has long been observed (Gratia, 1925), and bacteriocins have been reported from a wide range of genera (see, for example, Hardy, 1975 ;Holland, 1975). Evidence for bacteriocins produced by the thermophile Bacillus stearothermophilus has previously been presented (Shafia, 1966 ;Yule & Barridge, 1976). This paper describes the production and some properties of a bacteriocin produced by B. stearothermophilus RS93. The bacteriocin, termed thermocin 93, inhibited the growth of all thermophilic bacilli examined but had no effect on mesophilic bacilli. METHODSOrganisms. Bacillus stearothermophilus strain RS93 was isolated from the plant hot water supply (65 k 5 "C) at the Microbiological Research Establishment, Porton. The strain was catalase-and oxidase-positive; nitrate and nitrite were not reduced; and starch, casein and gelatin were not digested. The strain was tolerant to NaCl(5 %, w/v) and fermented glycerol, mannitol, glucose, mannose, fructose, sorbitol and trehalose with the production of acid. The optimum growth temperature was between 60 and 65 "C; growth did not occur at 37 or 70 "C.Bacillus caldolyticus Heinen & Heinen 1972, obtained from Dr W. Heinen, University of Nijmegen, The Netherlands, was used to assay the bacteriocin. Other strains of thermophilic bacilli tested were obtained from the following sources: B. caldovelox and B. caldotenax from Dr Heinen, The Netherlands; B. stearothermophilus 4st and B. stearothermophilus 10 from Dr N. Welker, North Western University, Ill., U.S.A.; B. stearothermophilus R S44 isolated from sandy soil from Jean-De Monts, France; B. stearothermophilus 240 isolated from chalky soil in the Salisbury area ; B. stearothermophilus 262 isolated from an Icelandic hot-water spring.Growth media. Liquid medium for the growth of the thermophiles was that described by Sargeant et al. (1971). Mesophiles were grown in peptone broth. Plating medium was Tryptone Soya Bean Agar (TSBA) (Oxoid CM131).Thermocin production. Thermocin 93 was produced by inoculating 400 ml medium in a 2 1 Erlenmeyer flask from a fresh 18 h plate culture of B. stearothermophilus RS93. The flask was shaken at 200 rev. min-I in a Gallenkamp Orbital Incubator at 60 "C for 18 h. Bacteria were removed by centrifuging (13000g, 15 min, 4 "C) and the supernatant was assayed for thermocin activity using B. caldolyticus as indicator.Thermocin assay. TSBA plates (4 mm deep) were seeded with 1 ml of a 6 h broth culture of B. caldolyticus (prepared by inoculating an overnight plate culture into 10 ml broth) and then dried at 37 "C for 1 h. Wells (8 mm diam.) were cut into the plates and 0.1 ml of B. stearothermophilus RS93 culture supernantant was added to each. The plates were incubated at 60 "C for 18 h and then the zone of inhibition around each well was measured.Activity range. The activity range of thermocin 93 was determined using 6 h cultures of each test organism. TSBA plates were seeded with the appropriate organism, dried for 1 h and 8 ...

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