Variation in 16S-23S rRNA intergenic spacer regions in Photobacterium damselae: a mosaic-like structure

Abstract

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(Glu(UUC)), tRNA(Lys(UUU)), tRNA(Val(UAC)), and tRNA(Ala(GGC)). Five amplicons contained tRNA(Glu(UUC)) combined with two additional tRNA genes, including tRNA(Lys(UUU)), tRNA(Val(UAC)), or tRNA(Ala(UGC)). Five amplicons contained tRNA(Ile(GAU)) and tRNA(Ala(UGC)). Two amplicons contained tRNA(Glu(UUC)) and tRNA(Ala(UGC)). Two different isoacceptor tRNA(Ala) genes (GGC and UGC anticodons) were found. The five smallest amplicons contained no tRNA genes. The tRNA-gene combinations tRNA(Glu(UUC))-tRNA(Val(UAC))-tRNA(Ala(UGC)) and tRNA(Glu(UUC))-tRNA(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.

FIG. 1.

Alignment of representative ITS sequences of P. damselae subspecies and strains corresponding to group 1 (A), group 2 (B), group 3 (C), and group 4 (D). Nucleotide positions that are conserved are indicated by dots. Gaps that have been included to obtain optimal sequence alignment are indicated by dashes. The different sequence blocks are enclosed in boxes and labeled with numbers, indicating their sequence length. Inverted repeats with palindromic structure are denoted by pairs of inverted arrows and designated as L-1 to L-6. The type of ITS spacer and the strain are indicated on the left. Numbers on the right indicate spacer lengths at different intervals.

FIG. 1.

Alignment of representative ITS sequences of P. damselae subspecies and strains corresponding to group 1 (A), group 2 (B), group 3 (C), and group 4 (D). Nucleotide positions that are conserved are indicated by dots. Gaps that have been included to obtain optimal sequence alignment are indicated by dashes. The different sequence blocks are enclosed in boxes and labeled with numbers, indicating their sequence length. Inverted repeats with palindromic structure are denoted by pairs of inverted arrows and designated as L-1 to L-6. The type of ITS spacer and the strain are indicated on the left. Numbers on the right indicate spacer lengths at different intervals.

FIG. 1.

Alignment of representative ITS sequences of P. damselae subspecies and strains corresponding to group 1 (A), group 2 (B), group 3 (C), and group 4 (D). Nucleotide positions that are conserved are indicated by dots. Gaps that have been included to obtain optimal sequence alignment are indicated by dashes. The different sequence blocks are enclosed in boxes and labeled with numbers, indicating their sequence length. Inverted repeats with palindromic structure are denoted by pairs of inverted arrows and designated as L-1 to L-6. The type of ITS spacer and the strain are indicated on the left. Numbers on the right indicate spacer lengths at different intervals.

FIG. 2.

Mosaic-like structure of ITS from sequence alignments in Fig. 1. Colors indicate discrete sequence blocks that are common between different ITS. Mosaic structures of ITS from group 1 (A), group 2 (B), and groups 3 and 4 (C) are shown. Numbers on the left indicate the sequence lengths of the ITS. Numbers at the bottom indicate the lengths of the discrete sequence blocks.

FIG. 3.

(A) Restriction map of P. damselae rrn operon. (B) Results of hybridization with 16S probe on chromosomal DNA digested with BglII and double-digested with BglII-PstI. (C) Results of hybridization with 16S probe on chromosomal DNA digested with HindIII and double-digested with HindIII-BglII. Lane designations: 1, DI21; 2, ATCC 29690; 3, RG91; 4, ATCC 33539; a, DNA digested with BglII; b, DNA double digested with BglII and PstI; c, DNA digested with HindIII, d, DNA double digested with HindIII-BglII.