SEQUENCE DIVERSITY IN THE 16S-23S INTERGENIC SPACER REGION (ISR) OF THE RIBOSOMAL-RNA OPERONS IN REPRESENTATIVES OF THE ESCHERICHIA-COLI ECOR COLLECTION

The ribosomal. RNA multigene family in Escherichia coli comprises seve n rrn operons of similar, but not identical, sequence. Four operons (r rnC, B, G, and E) contain genes in the 16S-23S intergenic spacer regio n (ISR) for tRNA(Glu-2) and three (rrnA, D, and H) contain genes for t RNA(Ile-1) and tRNA(Ala-1B). TO increase our understanding of their mo lecular evolution, we have determined the ISR sequence of the seven op erons in a set of 12 strains from the ECOR collection. Each operon was specifically amplified using polymerase chain reaction primers design ed from genes or open reading frames located upstream of the 16S rRNA genes in E. coli K12. With a single exception (ECOR 40), ISRs containi ng one or two tRNA genes were found at the same respective loci as tho se of strain K12. Intercistronic heterogeneity already found in K12 wa s representative of most variation among the strains studied and the l ocation of polymorphic sites was the same. Dispersed nucleotide substi tutions were very few but 21 variable sites were found grouped in a st em-loop, although the secondary structure was conserved. Some regions were found in which a stretch of nucleotides was substituted in block by one alternative, apparently unrelated, sequence (as illustrated by the known putative insertion of rsl in K12). Except for substitutions of different sizes and insertions/deletions found in the ISR, the patt ern of nucleotide variation is very similar to that found for the 16S rRNA gene in E. coli. Strains K12 and ECOR 40 showed the highest inter cistronic heterogeneity. Most strains showed a strong tendency to homo genization, Concerted evolution could explain the notorious conservati on of this region that is supposed to have low functional restrictions .