Rapid identification of Yersinia ruckeri by PCR amplification of yrul-yruRquorum sensing

Yersinia ruckeri possesses a quorum-sensing system detected by cross-streak ing against the white mutant Chromobacterium voilaceum CV0blu. Quorum sensi ng, which occurs in a number of Gramnegative pathogens, is known to control virulence gene expression through cell to cell communication. There are tw o genes required for quorum sensing which are luxR/luxI homologues and ther e is an N-acylhomoserine lactone (AHL, commonly called autoinducer) synthes ized by the product of luxI homologue which interacts with a response regul ator (the product of luxR homologue). The Y. ruckeri quorum sensing system, termed yruR/yruI was cloned from a gene library constructed in pUC18 plasm id vector. Nucleotide sequence analysis of Y. ruckeri yruR and yruI reveale d convergent transcription with overlapped 3' ends and two open reading fra mes (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 yr uR/yruI genes with short flanking sequences as well as an internal DNA frag ment within yruR/yruI genes. DNA fragments of 1900 and 1000 bp were amplifi ed from both sources, lysed Y. ruckeri cells and isolated DNA. Amplified se quences were detected in ethidium-bromide agarose gels or by Southern blot analysis with an internal 336-bp fragment as a hybridization probe. Detecti on of Y. ruckeri by PCR amplification of yruR/yruI genes has great potentia l for rapid identification of this fish pathogen bacterium as it has proved to be highly specific.