Cloning, Sequencing and Analysis of the 16S-23S rDNA Intergenic Spacers (IGSs) of Two Strains of Vibrio vulnificus

Abstract

According to the conserved sequences flanking the 3′ end of the 16S and the 5′ end of the 23S rDNAs, PCR primers were designed, and the 16S-23S rDNA intergenic spacers (IGSs) of two strains of Vibrio vulnificus were amplified by PCR and cloned into pGEM-T vector. Different clones were selected to be sequenced and the sequences were analyzed with BLAST and the software DNAstar. Analyses of the IGS sequences suggested that the strain ZSU006 contains five types of polymorphic 16S-23S rDNA intergenic spacers, namely, IGS GLAV, IGS GLV, IGS lA, IGS G and IGS A; while the strain CG021 has the same types of IGSs except lacking IGS A. Among these five IGS types, IGS GLAV is the biggest type, including the gene cluster of tRNA Glu - tRNA Lys - tRNA Ala - tRNA Val; IGS GLV includes that of tRNA Glu-tRNA Lys-tRNA Val; IGS AG, tRNA Ala-tRNA Glu; IGS IA, tRNA Ile-tRNA Ala; IGS G, tRNA Glu and IGS A, tRNA Ala. Intraspecies multiple alignment of all the IGS sequences of these two strains with those of V. vulnificus ATCC27562 available at GenBank revealed several highly conserved sequence blocks in the non-coding regions flanking the tRNA genes within all of strains, most notably the first 40 and last 200 nucleotides, which can be targeted to design species-specific PCR primers or detection probes. The structural variations of the 16S-23S rDNA intergenic spacers lay a foundation for developing diagnostic methods for V. vulnificus.