respective copies of three 23S ribosomal RNA genes with primer
combinations of an internal sequence (New100R2) of a 16S ribosomal
RNA gene (upstream of 23S ribosomal gene) and outside sequences
(New061F2, New022F, New041R2) specific to the downstream region
of 23S ribosomal genes (Table 1). The amplified DNA contained the
whole sequence of 23S ribosomal RNA gene, and we determined
nucleotide sequences of the respective copy of 23S ribosomal RNA
genes of both B. bifidum YIT 4001 and YIT 4007. Among 2795 bp of the
23S ribosomal gene, we found that the same nucleotide substitution
(A2090C) was present on all three copies of 23S ribosomal RNA genes
which is A for B. bifidum YIT 4001 and C for B. bifidum YIT 4007
(Table 2). We therefore isolated new spontaneous mutants resistant
to erythromycin at a high concentration from the progenitor strain B.
bifidum YIT 4001. At first we selected mutants resistant to erythromycin
at the concentration higher than 100 μg/mL, but we failed in
the isolation of resistant mutants.We suspected that the resistance to
erythromycin may be correlated with copy numbers of mutated 23S
ribosomal RNA genes, and designed a two step screening to select
mutants resistant to erythromycin. At first, we selected mutants
resistant to erythromycin at a low concentration (0.1 μg/mL) not to