A DNA probe for the detection and identification of Bacillus sporothermodurans using the 16S-23S rDNA spacer region and phylogenetic analysis of some field isolates of Bacillus which form highly heat resistant spores.

The spacer regions between the 16S and 23S rRNA genes (spacer regions 1) of Bacillus sporothermodurans were PCR-amplified, cloned and sequenced. Six unique spacer sequences in four size classes were recovered from two strains, rrnA (about 190 bp), rrnB (about 303 bp), rrnC (355 bp) and rrnD (554 bp). rrnD contained two tRNA genes which were deciphered as tRNA(ala) and tRNA(ile) separated from each other by 13 nucleotides. The primary structures of the tRNA molecules clearly resembled those found in Bacillus subtilis; the tRNA(ala) genes were identical and the tRNA(ile) genes were 95% similar. The mixed rrnA and rrnB spacers when PCR-amplified from chromosomal DNA were effective as a hybridization probe for identification of B. sporothermodurans strains. However, high background signals with DNA from some other bacilli were encountered. A more discriminating probe was prepared from the cloned rrnB spacer region. Of eight aerobic, endospore-forming bacteria isolated from silage following heat enrichment, one was identified as B. sporothermodurans using the probe and its identity was confirmed from partial 16S rDNA analysis (phylotyping). This indicated that contamination in milk and dairies by B. sporothermodurans could originate from cattle feeds such as silage. Of the other seven silage strains, only two were identified conclusively by phylotyping and three represented probable new species. The latter three strains were subjected to phylogenetic analysis using almost complete 16S rDNA sequences. Branch lengths, bootstrap percentage values, and 16S rDNA similarity to other Bacillus species suggested that these isolates are likely to constitute new species within the genus Bacillus.