Isolation and analysis of a protease gene with an ABC transport system in the fish pathogen Yersinia ruckeri: insertional mutagenesis and involvement in virulence

Área de Microbiologia, Departamento de Biologia Funcional, Facultad de Medicina, Instituto Universitario de Biotecnologia de Asturias, Universidad de Oviedo, 33006 Oviedo, Spain 1 SERIDA, Laboratorio de Sanidad Animal de Jove, 33299 Gijon, Spain 2 Author for correspondence: J. A. Guijarro. Tel: +34 985 104 218. Fax: +34 985 103 148. e-mail: jaga{at}sauron.quimica.uniovi.es Yersinia ruckeri is a Gram-negative pathogen that causes enteric redmouth disease in salmonids. A gene from Y. ruckeri encoding an extracellular protease termed yrp1 ( Y ersinia r uckeri p rotease 1 ) was cloned from a Sau 3AI library constructed in pUC19 and analysed in gelatin-supplemented medium. The nucleotide sequence of the yrp1 gene indicated an ORF encoding a protein of 477 aa. On the basis of the high degree of homology in the amino acid sequence as well as its conservative motifs, this protein was included within the serralysin metalloendopeptidase subfamily (EC 3.4.24.12). The yrp1 N-terminal sequence showed a 14 aa propeptide followed by a 10 aa sequence identical to the one deduced previously from the 47 kDa purified protease. Additional results demonstrated that the yrp1 gene encodes the 47 kDa protein. In contrast to other Yersinia species, the yrp1 protease is secreted by a type I Gram-negative bacterial ABC exporter protein secretion system composed of three genes termed yrpD , yrpE and yrpF , and a protease inhibitor inh . The development of genetic methods for this species has allowed the exploration of the organization and the putative role of the Yrp1 genetic locus. Thus, site-directed insertion mutations into the yrp1 and the yrpE genes were constructed by the integration of the mobilizable suicide vector pIVET8 containing internal portions of both coding sequences. Complementation studies of those mutants with different loci indicated that they are organized as a single operon. The mutant strains lacked protease activity as well as the Yrp1 protein and, although physiologically similar to the parental strain when growing on nutrient broth medium, they were attenuated in virulence when bacteria were injected intraperitoneally into rainbow trout ( Oncorhynchus mykiss ). This is the first report of defined mutations in Y. ruckeri to show the implication of a factor such as an extracellular protease in pathogenesis. Keywords: enteric redmouth disease, pathogenicity, type I secretion system a The GenBank accession numbers for the sequences reported in this paper are AJ3