Intraspecific diversity of the 23S rRNA gene and the spacer region downstream in Escherichia coli

Abstract

The molecular microevolution of the 23S rRNA gene (rrl) plus the spacer downstream has been studied by sequencing of different operons from some representative strains of the Escherichia coli ECOR collection. The rrl gene was fully sequenced in six strains showing a total of 67 polymorphic sites, a level of variation per nucleotide similar to that found for the 16S rRNA gene (rrs) in a previous study. The size of the gene was highly conserved (2902 to 2905 nucleotides). Most polymorphic sites were clustered in five secondary-structure helices. Those regions in a large number of operons were sequenced, and several variations were found. Sequences of the same helix from two different strains were often widely divergent, and no intermediate forms existed. Intercistronic variability was detected, although it seemed to be lower than for the rrs gene. The presence of two characteristic sequences was determined by PCR analysis throughout all of the strains of the ECOR collection, and some correlations with the multilocus enzyme electrophoresis clusters were detected. The mode of variation of the rrl gene seems to be quite similar to that of the rrs gene. Homogenization of the gene families and transfer of sequences from different clonal lines could explain this pattern of variation detected; perhaps these factors are more relevant to evolution than single mutation. The spacer region between the 23S and 5S rRNA genes exhibited a highly polymorphic region, particularly at the 3' end.

FIG. 1

Variable positions in the rrl genes of E. coli K-12 and other strains used in this study. Periods indicate the same nucleotide composition as the rrlA sequence of E. coli K-12 (Escherichia coli Database Collection). The numbers at the top indicate positions in the sequences reported by Brosius et al. (3). Insertion of nucleotides G and C between positions 545 and 546 is indicated by the arrow.

FIG. 2

Secondary structures of variable regions in the rrl genes from E. coli strains. The numbers indicate positions reported by Brosius et al. (3). Conserved nucleotides are in boldface. In parentheses are the nucleotides that have been determined in the rrl genes of the following strains: helix 25, (U)¹ in rrlH of ECOR 40, (U)² in rrlB, rrlC, rrlE, and rrlG of ECOR 35, and a (U)³ insertion in rrlB, rrlC, and rrlD of ECOR 24 and rrlG of A8190; helix 45, (G)¹ in rrlB, rrlG, and rrlH of ECOR 10 and rrlB, rrlC, and rrlD of ECOR 24, (U)² in rrlC of ECOR 24; and (G)³ in rrlA and rrlC of A8190; helix 63, (A)¹ in rrlC of ECOR 40 and (U)² in rrlB and rrlC of ECOR 10 and rrlB, rrlC, and rrlD of ECOR 24; helix 79, (G)¹ in rrlA, rrlC, rrlD, and rrlE of ECOR 52 and (G)² and (G)³ in rrlB and rrlC of ECOR 10 and rrlA, rrlC, and rrlG of A8190; helix 98, (A)¹ in rrlC of A8190 and (G)² in rrlA and rrlE of ECOR 52.

FIG. 3

Distribution of structures I, II, and III of helix 25 and structures I and II of helix 63 among the ECOR strains as revealed by a PCR test using sequence-specific probes. The MLEE relationships shown in the tree are those described by Herzer et al. (16) modified by the addition of E. coli K-12 and A8190.

FIG. 4

Alignment of rrl-rrf ITS sequences determined for ECOR strains and A8190 and for rrnA of E. coli K-12. (B) Alignment of rrl-rrf ITSs of E. coli K-12, ECOR 40, and S. typhi. Mosaic similarities are underlined. Periods indicate the same nucleotide composition.