Variation in 16S-23S rRNA Intergenic Spacer Regions in Photobacterium damselae: a Mosaic-Like Structure

Phenotypically, Photobacterium damselae subsp. piscicida and P. damselae subsp. damselae are easily distinguished. However, their 16S rRNA gene sequences are identical, and attempts to discriminate these two subspecies by molecular tools are hampered by their high level of DNA-DNA similarity. The 16S-23S rRNA internal transcribed spacers (ITS) were sequenced in two strains of Photobacterium damselae subsp. piscicida and two strains of P. damselae subsp. damselae to determine the level of molecular diversity in this DNA region. A total of 17 different ITS variants, ranging from 803 to 296 bp were found, some of which were subspecies or strain specific. The largest ITS contained four tRNA genes (tDNAs) coding for tRNA[superscript Glu(UUC)], tRNA[superscript Lys(UUU)], tRNA[superscript Val(UAC)], and tRNA[superscript Ala(GGC)]. Five amplicons contained tRNA[superscript Glu(UUC)] combined with two additional tRNA genes, including tRNA[superscript Lys(UUU)], tRNA[superscript Val(UAC)], or tRNA[superscript Ala(UGC)]. Five amplicons contained tRNA[superscript Ile(GAU)] and tRNA[superscript Ala(UGC)]. Two amplicons contained tRNA[superscript Glu(UUC)] and tRNA[superscript Ala(UGC)]. Two different isoacceptor tRNA[superscript Ala] genes (GGC and UGC anticodons) were found. The five smallest amplicons contained no tRNA genes. The tRNA-gene combinations tRNA[superscript Glu(UUC)]-tRNA[superscript Val(UAC)]-tRNA[superscript Ala(UGC)] and tRNA[superscript Glu(UUC)]-tRNA[superscript Ala(UGC)] have not been previously reported in bacterial ITS regions. The number of copies of the ribosomal operon (rrn) in the P. damselae chromosome ranged from at least 9 to 12. For ITS variants coexisting in two strains of different subspecies or in strains of the same subspecies, nucleotide substitution percentages ranged from 0 to 2%. The main source of variation between ITS variants was due to different combinations of DNA sequence blocks, constituting a mosaic-like structure.