Nucleotide sequences of the 23S rRNA genes fromBordetella pertussis, B.parapertussis, B.bronchisepticaandB.avium, and their implications for phylogenetic analysis

Virtually all members of the gram-negative bacillus genus Bordetella are pathogenic, being responsible for a variety of diseases in both man and animals. B.pertussis causes whooping cough, while B.parapertussis infections result in milder, but still serious, respiratory diseases in human (1). B.bronchiseptica is pathogenic for both dogs and pigs, and B.avium infects birds (2, 3). While B.pertussis, B.parapertussis and B.bronchiseptica are very closely related and are more correctly considered subspecies (4), B.avium is considerably more distant to the group and, as such, constitutes a distinct species classification (5). We have determined the complete nucleotide sequence of the 23S rRNA genes from B.pertussis Tohama, B.parapertussis ATCC 15311, B.bronchiseptica ATCC 19395 and B.avium ATCC 35086. The 23S rRNA genes, including the 5' and 3' flanking regions, were amplified via the polymerase chain reaction (PCR). The resultant amplification products were directly sequenced by die dideoxy termination method. In addition, we performed Southern blot analysis on the genomic DNA of each bacteria, and detected 3 distinct loci for this rRNA in all 4 species (data not shown). We have found the three rRNA gene sequences in closely linked gene sets in the order 16S-23S-5S in the genome of all 4 Bordetella species. Furthermore, we have discovered two putative tRNA genes located in the intergenic spacer region between the 16S and 23S sequences. All of the Bordetella species examined contain the identical nucleotide sequences for the tRNA'k and tRNA^ genes. The 23S rRNA gene of B.pertussis is 2876 bp long with a G + C content of 53 %. These genes from B.parapertussis and B.bronchiseptica are 2877 bp in length, also with a G + C composition of 53 %. On the other hand, the gene from B.avium consists of 2867 bp, with a G + C content of 52.7%. In Table 1 sequence homologies between the Bordetella species and three other purple bacteria are shown. Rhodobacter capsulatus (6) belongs to the alpha subclass, whereas Pseudomonas cepada (7) is from the beta subclass and Pseudomonas aeruginosa (8) falls in the gamma subclass. A phylogenetic tree based on the DIST values (9) is outlined in Figure 1. The high sequence similarity (>99%) between B.pertussis, B.parapertussis and B.bronchiseptica confirms the close relationship of these organisms. In contrast, the lower homology between B.avium and the other three Bordetella members ( > 97%) is in agreement with the results reported by De Ley et al. (6). REFERENCES