|Phillips, Robert - USDA-FSIS|
Submitted to: Systematic and Applied Microbiology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: November 19, 2008
Publication Date: February 11, 2009
Citation: Meinersmann, R.J., Phillips, R.W., Ladely, S.R. 2009. Inter- and intra-genomic heterogeneity of the intervening sequence in the 23S ribosomal RNA gene of Campylobacter jejuni and Campylobacter coli. Systematic and Applied Microbiology. 32(2):91-100. Interpretive Summary: Campylobacter are one of the most common bacterial causes of diarrheal disease in human. As with other bacteria, when researchers wish to track the spread of this organism analyses are performed to assign a type that hopefully coincides with the family tree of the organism. Transmission of the Campylobacter has proven to be very difficult to trace because the characteristics that are used to identify specific types are mixed between types at a rate that causes efforts to reconstruct the history of a type to be confused by the mixtures. It is commonly thought that the genes for ribosomes are unlikely to become mixed because of the essential nature of the gene. With that idea we set out to see if the ribosome genes can be used to trace Campylobacter and we decided to start with a convenient marker in the ribosome genes. This marker is a fragment found in some strains, not in others, and is known as the intervening sequence (IVS). As it happens, every Campylobacter has three copies of the ribosome genes and it is expected that all three will be identical. However, we found that a substantial proportion of strains had differences in the presence of the IVS in the copies of their ribosome genes. We devised a method to separately determine the DNA sequence of each copy of the gene in several of these strains. This allowed us to confirm that there were differences between copies of the gene and these differences could only have been created by the mixing of these genes between different strains. Thus, it turns out that the ribosome is not stable in Campylobacter and it can not be expected to give a more accurate history than any other method used to characterize the organism.
Technical Abstract: An intervening sequence (IVS) can be present or absent in the 23S rRNA of Campylobacter jejuni and C. coli. As part of a survey, we used a polymerase chain reaction (PCR) assay to detect the presence of the IVS in 43 isolates of C. coli and 82 isolates of C. jejuni. An IVS was present in 40 (93%) of the C. coli and only 34 (41%) of the C. jejuni isolates. Twelve of the C. coli isolates and 7 of the C. jejuni isolates resulted in two PCR products, indicating heterogeneity in the presence of the 23S rRNA IVS. Fourteen of the isolates with two products were evaluated by pulse-field gel electrophoresis; thirteen different patterns were observed and only in one case was the total band size substantially greater than the expected 1.7 Mb, possibly indicating a mixed culture. Southern blot analyses demonstrated the expected three rRNA operons in all tested isolates. Nested PCR reactions with operon-specific primers followed by primers for the IVS confirmed that the strains of interest contained either one or two operons carrying the IVS and the remaining operon(s) did not. Sequence analysis of the IVS and flanking regions of the 23 S rRNA genes did not discriminate C. jejuni and C. coli as distinct populations. These results indicate horizontal transfer of 23S rRNA genes or portions of the gene between C. jejuni and C. coli. Also, data showing sequence polymorphisms between the three 23S rRNA loci outside of the IVS region suggest that the isolates with intragenomic heterogeneity appear to be members of clones that have an ancient defect in gene conversion mechanisms needed for concerted evolution of the ribosomal operons.