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

Abstract

Clostridium difficile is a major spore-forming environmental pathogen that causes serious health problems in patients undergoing antibiotic therapy. Consequently, reliable and sensitive methods for typing individual strains are required for epidemiological and environmental studies. Ribotyping is generally considered the best method, but it fails to account for sequence diversity which might exist in intergenic 16S-23S rRNA spacer regions (ISRs) within and among strains of this organism. Therefore, this study was undertaken to compare the sequence of each individual ISR in five strains of C. difficile to explore the extent of this diversity and see whether such information might provide the basis for more sensitive and discriminatory strain typing methods. After targeted PCR amplification, cloning, and sequencing, the diversity of the ISRs was used as a measure of rRNA operon copy number. In C. difficile strains 630, ATCC 43593, A, and B, 11, 11, 7, and 8 ISR length variants, respectively, were found (containing different combinations of sequence groups [i to xiii]), suggesting 11, 11, 7, and 8 rrn copies in the respective strains. Many ISRs of the same length differed markedly in their sequences, and some of these were restricted in occurrence to a single strain. Most of these ISRs did not contain any tRNA genes, and only single copies of the tRNA Ala gene were found in those that did. The presence of ISR sequence groups (i to xiii) varied between strains, with some found in one, two, three, four, or all five strains. We conclude that the intergenic 16S-23S rRNA spacer regions showed a high degree of diversity, not only among the rrn operons in different strains and different rrn copies in a single strain but also among ISRs of the same length. It appears that C. difficile ISRs vary more at the inter-and intragenic levels than those of other species as determined by empirical comparison of sequences. The precise characterization of these sequences has demonstrated a high level of mosaic sequence block rearrangements that are present or absent in multiple strain-variable rrn copies within and between five different strains of C. difficile.

Maternal oral origin of Fusobacterium nucleatum in adverse pregnancy outcomes as determined using the 16S–23S rRNA gene intergenic transcribed spacer region

“…In general, these studies demonstrated a markedly higher heterogeneity of this region compared with the 16S rRNA gene to differentiate closely related taxa. The mosaic pattern of the ITS described for other genera/species has revealed that the presence or absence of one or more regions (also known as block types) were the reason for the length differences (Sadeghifard et al, 2006). Similar patterns were observed in this study for all the F. nucleatum subspecies, where the operon's variability was based mainly on the presence or absence of these block types.…”

“…Using the ITS region, several studies have investigated species/subspecies and phylogenetic relationships from a range of important clinical isolates including the genus Fusobacterium (Christensen et al, 1999;Narayanan et al, 2001;Conrads et al, 2002;Sadeghifard et al, 2006). In general, these studies demonstrated a markedly higher heterogeneity of this region compared with the 16S rRNA gene to differentiate closely related taxa.…”

“…Nevertheless, a disadvantage of using the 16S rRNA component is the lack of sufficient resolution to enable a precise differentiation between strains of the same species. The 16S-23S rRNA gene intergenic transcribed spacer (ITS) region is increasingly being used for the analysis of phylogenetic relationships between species and subspecies of the same genus (Tyrrell et al, 1997;Sadeghifard et al, 2006), due to a higher number of variable sites compared with the more conserved 16S rRNA gene (Söller et al, 2000). The heterogeneity of the ITS region in terms of both copy number and length has raised the possibility of including this fragment as part of the methodology for bacterial identification and typing purposes (Gürtler & Stanisich, 1996;Popa et al, 2009).…”

An oligarchic microbial assemblage in the anoxic bottom waters of a volcanic subglacial lake

“…Rather, their over-representation in our libraries may be a consequence of extraction/PCR bias or a high number of rRNA operons per cell. Some members of the Clostridium (in the order Clostridiales along with Acetobacterium) have X10 rRNA operons (Rainey et al, 1996;Brü ggemann et al, 2003;Acinas et al, 2004;Sadeghifard et al, 2006).…”

Unusual intragenomic and interspecific variability of Geobacillus 16S-23S rRNA internal transcribed spacers

“…In many organisms, intragenomic variability consists of only a few substitutions over the entire ITS length, and results from the presence or absence of tRNA genes. However, intragenomic variability (mosaic-like structure) has been also detected in the bacterial ITS [2][3][4][5][6]. To date, mosaic-like structures have been detected in the non-thermophilic bacteria Salmonella enterica, Haemophilus parainfluenzae, Staphylococcus aureus, Vibrio cholerae, Vibrio mimicus, Vibrio parahaemolyticus, Myxobacterium spp., Acinetobacter baylyi, Photobacterium damselae and Clostridium difficile [2,3,5,[7][8][9].…”