The 165-235 and 235-55 rRNA spacer DNA regions (spacer regions 1 and 2, respectively) from Enterococcus faecalis, Enterococcus faecium, Enterncoccus hirae, Enterococcus durans and Enterncoccus mundtii were amplified by PCR. Their nucleotide sequences were established and a secondary structure model showing the interaction between the two spacer regions was built. Whereas lactococci and Streptococcus sensu stricto are characterized by a single type of spacer region 1 , the enterococci show a high degree of variability in this region; thus the spacer regions 1 with and without tRNAAia were characterized. However, as shown for lactococci and Streptococcus sensu stricto, the tRNAAia gene does not encode the 3'4erminal CCA trinucleotide. A putative antitermination signal is found downstream from the tRNAAia gene. Based on comparison with Lactococcus lactis and Streptococcus thermophilus, a doublestranded processing stem is proposed. In €. hirae, one of the three different types of spacer region 1 contains no tRNAAia, but displays a 107 nt insertion that forms a long stem-loop structure. A similar insertion (1 15 nt in length) was found in E. faecium and base compensatory mutations preserve the ability to form the long stem-loop structure. Such insertions may correspond to mobile intervening sequences, as found in the 235 rRNA coding sequences of some Gram-negative bacteria. The spacer regions 1 and 2 from the three subgroups of streptococci were compared, and except for the tRNAAia gene and the double-stranded processing sites, little similarity was found, which opens large possibilities for future development of DNA-based typing methods.

The 16S-23S intergenic spacer region (spacer region 1) of Streptococcus salivarius, S. thermophilus, and Lactococcus lactis subsp. cremoris and the 23S-5S intergenic spacer region (spacer region 2) of S. salivarius and L. lactis subsp. cremoris were sequenced and compared with the spacer regions 1 and 2 of other streptococci. A high degree of intraspecific conservation was observed for S. thermophilus and L. lactis, and very similar sequences were found for S. salivarius and S. thermophilus. Whereas spacer region 1 is highly conserved in the genus Streptococcus sensu-stricto, only the tRNA gene and the rRNA processing stems are highly conserved in the three genera: Streptococcus sensu-stricto, Lactococcus, and Enterococcus. The presence of a unique tRNA(Ala) gene without the 3' terminal CCA sequence seems to be a general feature of the streptococci spacer region 1. A secondary structure model was built to show the interaction between the spacer regions 1 and 2 of S. thermophilus and S. salivarius. The rapid evolution of spacer region 1 in streptococci is in part due to insertions and deletions of small RNA stem/loop structures.

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