DNA sequence heterogeneity in the three copies of the long 16S–23S rDNA spacer of Enterococcus faecalis isolates

Abstract

The possibility of intragenic heterogeneity between copies of the long intergenic (1 63-235 rDNA) spacer region (LISR) was investigated by specific amplification of this region from 21 Enterococcus faecalis isolates. Three copies of the LlSR (rrnA, B and C) were demonstrated by hybridization of the LlSR to genomic DNA cleaved with I-Ceul and Smal. When the LISR amplicon was digested with Tsp5091, two known nucleotide substitutions were detected, one 4 nt upstream from the 5' end of the tRNAaIa gene (allele rrnB has the TspSOSl site and rrnA and Cdo not) and the other 22 nt downstream from the 3' end of the tRNAa'* gene (rmC has the Tsp5091 site). Sequence differences at these sites were detected at the allelic level (alleles rrnA, B and C) and different combinations of these alleles were designated lsp Types. Using densitometry to analyse bands from electrophoresis gels, the intra-isolate ratios of the separate alleles (rmA:rmB:rmC) were determined in each Tsp Type: I (0:3:0), II (l:2:U), 111 (2:O:I)' IV (3:0:0)# V (2:1:0) and VI (1:l:l). Sequence variation between the three copies of the LlSR was confirmed by the detection of at least five other intra-isolate nucleotide substitutions using heteroduplex analysis by conformation-sensitive gel electrophoresis (CSGE) that were not detected by Tsp5091 cleavage. Perpendicular denaturing gradient gel electrophoresis was capable of resolving homoduplexes; six to seven out of a possible nine curves were obtained in some isolates. In the isolate where seven curves were obtained one or more further nucleotide substitutions, not detected by TspSO9l cleavage or CSGE, were detected. On the basis of LlSR sequence heterogeneity, isolates were categorized into homogeneous (only one allele sequence present) and heterogeneous (two or three allele sequences present). The transition between homogeneous and heterogeneous LlSRs may be useful in studying evolutionary mechanisms between E. faecalis isolates.

The Mosaic Nature of Intergenic 16S-23S rRNA Spacer Regions Suggests rRNA Operon Copy Number Variation in Clostridium difficile Strains

“…These sequence data suggest that intraspecific diversity in the ISRs in C. difficile is much greater than that reported for any other bacterial species (4,5,12,15,17,20,27,30,40,50,51). Many authors have compared ISRs between different species and strains of a single bacterial species (15,17,33,44,46).…”

“…In some cases the ISR diversity between species of a genus is lower than the diversity seen between the strains of C. difficile examined here. Sequence differences between ISRs of the same size most likely arise from nucleotide substitution events (4,5,25,30). Also, some ISRs were of similar length (e.g., rrnE and rrnF of S. aureus) but had sequence blocks with different nucleotide sequences, probably because of recombination events (25,27,28).…”

Analysis of the 16S–23S rRNA Gene Internal Transcribed Spacer Region in Klebsiella Species

“…Sequence and length polymorphisms found in the ITS are increasingly being used as tools for bacterial species and subspecies identification (17,27,29,37), typing (25,37), and evolutionary studies (1,12,33). Gürtler et al reported that ITS sequence analysis is complementary to the 16S rRNA gene for phylogenetic analysis (13). A PCR method based on ITS sequences has been developed for the detection and identification of K. pneumoniae subsp.…”

Bacillus anthracis Diverges from Related Clades of the Bacillus cereus Group in 16S-23S Ribosomal DNA Intergenic Transcribed Spacers Containing tRNA Genes

“…The 16S-23S rDNA intergenic transcribed spacers (ITS) are the most variable regions of the ribosomal operon, and, apart from interoperonic nucleotide substitutions, insertions, and deletions, such ITS can be differentiated, given the presence of the different numbers and types of tRNA genes (9,10,31,49). Since the ITS have fewer functional constraints than the adjacent ribosomal genes, which undergo concerted evolution (17)(18)(19), their sequences can contain traces of ribosomal operon rearrangements and species-specific or even strain-specific traits that are useful for strain typing.…”