Use of PCR–RFLP for genotyping 16S rRNA and characterizing bacteria cultured from halibut fry

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Abstract
Small subunit ribosomal genes were explored using PCR–RFLP to facilitate the characterization of bacteria cultured from reared fry of the Atlantic halibut (Hippoglossus hippoglossus). Concern has been expressed about pathogen invasion in larvae lacking a counteracting normal flora that may aid the immune system in producing robust noninfected individuals. In this study, pure cultured representatives of normal flora that were previously found to be antagonistic towards a pathogenic Vibrio sp. were subjected to a whole cell PCR protocol amplifying ~1500 bp of 16S rDNA. Amplified DNA was digested by AluI, BstUI, CfoI, and RsaI, to generate restriction profiles. Before the isolates were characterized, a survey was performed to test the discriminative efficiency of the RFLP. Efficient detection of polymorphism and the resolution of species and subspecies were achieved. Using the RFLP on 103 isolates generated as many as 22 genotypes. Based on the restriction profiles, a taxonomic tree incorporating 19 reference strains was constructed. Partial sequencing found this tree to be dominated by γ-Proteobacteria in clusters of Vibrio-, Pseudomonas-, and Alteromonas-affiliated species. Only nine isolates fell outside these genera, including the three isolates Shewanella alga, Deleya marina, and Marinomonas protea. These species have not previously been reported as halibut flora. The most frequently isolated genotype resembled Vibrio salmonicida.Key words: halibut, Vibrio, RFLP, 16S rDNA, phylogeny.