Rapid identification of <i>Yersinia ruckeri</i> by PCR amplification of <i>yruI–yruR</i> quorum sensing

Abstract

Yersinia ruckeri possesses a quorum‐sensing system detected by cross‐streaking against the white mutant Chromobacterium voilaceum CV0blu. Quorum sensing, which occurs in a number of Gram‐negative pathogens, is known to control virulence gene expression through cell to cell communication. There are two genes required for quorum sensing which are luxR/luxI homologues and there is an N‐ acylhomoserine lactone (AHL, commonly called autoinducer) synthesized by the product of luxI homologue which interacts with a response regulator (the product of luxR homologue). The Y. ruckeri quorum sensing system, termed yruR/yruI , was cloned from a gene library constructed in pUC18 plasmid vector. Nucleotide sequence analysis of Y.ruckeri yruR and yruI revealed convergent transcription with overlapped 3′ ends and two open reading frames (ORFs) of 247 and 217 amino acids, respectively. Two pairs of synthetic oligonucleotide primers of 24 bases were used in a PCR assay to amplify yruR/yruI genes with short flanking sequences as well as an internal DNA fragment within yruR/yruI genes. DNA fragments of 1900 and 1000 bp were amplified from both sources, lysed Y. ruckeri cells and isolated DNA. Amplified sequences were detected in ethidium‐bromide agarose gels or by Southern blot analysis with an internal 336‐bp fragment as a hybridization probe. Detection of Y. ruckeri by PCR amplification of yruR/yruI genes has great potential for rapid identification of this fish pathogen bacterium as it has proved to be highly specific.