Intercistronic heterogeneity of the 16S-23S rRNA spacer region among Pseudomonas strains isolated from subterranean seeds of hog peanut (Amphicarpa bracteata)

Environmental Health Program (Biodiversity), Agriculture and Agri-Food Canada, Ottawa, ON K1A 0C6, Canada Intercistronic heterogeneity of the 16S–23S rRNA internal transcribed spacer regions (ITS1) was investigated in 29 strains of fluorescent pseudomonads isolated from subterranean seeds of Amphicarpa bracteata (hog peanut). PCR amplification of the ITS1 region generated one or two products from the strains. Sequence analysis of the amplified fragments revealed an ITS1 fragment of about 517 bp that contained genes for tRNA Ile and tRNA Ala in all 29 strains; an additional smaller ITS1 of 279 bp without tRNA features was detected in 15 of the strains. The length difference appeared to be due to deletions of several nucleotide blocks between the 70 bp and 359 bp positions of the alignment. The end of the deletions in the variant ITS1 type coincided with the start of antiterminator box A, which is homologous to box A of other bacteria. Phylogenetic analyses using the neighbour-joining algorithm revealed two major phylogenetic clusters, one for each of the ITS1 types. Using a single specific primer set and the DNA-intercalating dye SYBR Green I for real-time PCR and melting-curve analysis produced highly informative curves with one or two recognizable melting peaks that readily distinguished between the two ITS1 types in pure cultures. The assay was used to confirm the presence of the variant ITS1 type in the Pseudomonas community in total DNA from root-zone soil and seed coats of hog peanut. Heterogeneity of the ITS1 region between species has potential for studying molecular systematics and population genetics of the genus Pseudomonas , but the presence of non-identical rRNA operons within a genome may pose problems. Correspondence J. T. Tambong tambongj{at}agr.gc.ca Abbreviations: ITS, internal transcribed spacer The GenBank/EMBL/DDBJ accession numbers for the sequences of isolates obtained in this study are FJ670594 –FJ670637. Two supplementary figures are available with the online version of this paper.