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

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 approximately 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 gamma-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.

Characterization of culturable bacterial flora in yolk sac larvae of Atlantic halibut (Hippoglossus hippoglossus L.) with

“…The profile of normally occurring, culturable, heterotrophic bacteria may serve as an indicator of the health status of halibut larvae and a stable community may give protection against pathogens such as Vibrio species (Bolinches & Egidius, 1987;Bergh et al, 1994;Bergh, 1995). The bacterial community of halibut larvae has also been monitored using a combination of molecular techniques like PCR-RFLP of 16S rRNA genes and partial 16S rDNA gene analysis (Jensen et al, 2002;Verner-Jeffreys et al, 2003). In order to compare the endogenous flora present in larvae with jaw gaping syndrome, the culture method was employed, and representative colonies differentiated by morphological criteria were characterized using the RFPL-PCR and sequencing of the 16S rRNA genes.…”

“…The restriction fragments were resolved on a 2% agarose/TAE gel and visualized using ethidium bromide under UV illumination. A molecular weight ladder was included in each run (100-bp ladder, Invitrogen) (Jensen et al, 2002). The partial sequences of 16S ribosomal RNA gene from 33 isolates are available in the GenBank with the following access numbers: JQ861982 to JQ862014.…”

Isolation and characterization of culturable bacteria from tropical coastal waters

“…La secuenciación completa del gen 16S rDNA (~1500 bp) para estas cepas también confirmó su similitud. La divergencia entre especies (Urakawa et al 1999, Jensen et al 2002 probablemente causó la inconsistencia de uno o dos patrones de restricción. Tal divergencia también se observó para las cepas KK9 y PD1F, las cuales presentaron diferentes RFLP de 16S rDNA pero fueron identificadas con un valor de similitud de 99% con Alteromonas sp.…”

Immunohistochemistry of great scallop Pecten maximus larvae experimentally challenged with pathogenic bacteria

“…Full sequencing of the 16S rDNA and Restriction Fragment Length Polymorphism (RFLP) for genotyping 16S rRNA are 2 methods for characterising unknown cultured bacteria. The latter was used by Jensen et al (2002) to characterise bacteria cultured from halibut Hippoglossus hippoglossus fry. LT 13 has been isolated from the microalgae Chaetoceros calcitrans, which is used as feed for scallop larvae (Torkildsen et al 2005).…”