Five Gram-negative bacterial isolates, recovered from an outbreak that occurred in March 2006 in Huelva, Spain, affecting adult diseased cultured wedge sole [Dicologlossacuneata(Moreau)], were characterized phenotypically and genotypically in order to clarify their taxonomic position. On the basis of 16S rRNA gene sequence analysis, the isolates were included in the genus Pseudomonas , within the Pseudomonas fluorescens -related species group, their closest relatives being the Pseudomonas jessenii and Pseudomonas koreensis subgroups. The highest sequence similarities were recorded with the type strains of Pseudomonas reinekei , P. moorei , P. umsongensis , P. jessenii and P. mohnii (99.4–99.3 % similarity). Sequence analysis of the housekeeping genesgyrBandrpoDclearly differentiated the isolates from currently described Pseudomonas species, the highest sequence similarities recorded to type strains being below 95 % for both genes. Phylogenetic analysis using concatenated sequences of the three genes showed Pseudomonas moraviensis DSM 16007^Tand P. koreensis DSM 16610^Tas the closest reference strains. DNA–DNA hybridization assays with related strains confirmed that these isolates belong to a novel species of the genus Pseudomonas , for which the namePseudomonas baeticasp. nov. is proposed. The type strain is strain a390^T( = CECT 7720^T = LMG 25716^T). The novel species could be easily distinguished from phylogenetically related species by several phenotypic characteristics, including gelatin hydrolysis, acid production from glucose and growth at 6 % NaCl. Virulence assays revealed that the novel species is pathogenic for wedge sole.

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 protease 1) was cloned from a Sau3AI 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.

Yersinia ruckeri , the etiological agent of the enteric red mouth disease (ERM) of salmonids, produces Yrp1, a serralysin metalloprotease involved in pathogenesis. We describe here the hydrolytic and immunogenic properties of Yrp1. The protease was able to hydrolyze different matrix and muscle proteins as laminin, fibrinogen, gelatine, actin, and myosin but not type II and IV collagens. In addition, the Yrp1 protein, when inactivated by heat and used as an immunogen, was able to elicit a strong protection against the development of ERM. The analysis of different Y. ruckeri strains with (Azo ⁺ ) or without (Azo ⁻ ) Yrp1 activity showed that all of them contained the yrp1 operon. By using yrp1 :: lacZ operon fusions, protease production analysis, and complementation studies, it was possible to show that an Azo ⁻ strain was blocked at the transcription level. The transcriptional study of the yrp1 operon under different environmental conditions showed that it was regulated by osmolarity and temperature, without pH influence. Finally, when β-galactosidase activity was used as a probe in vivo, the progression of the disease in the fish could be visualized, and the tropism of the bacterium and affected organs could be defined. This system opens a vast field of study not only with regard to fish disease progression but also in pathogen interactions, temporal gene expression, carrier stages, antibiotic resistance selection, etc.

Pseudomonas baetica is a pathogen described in 2012 from wedge sole ( Dicologoglossa cuneata ). The aims of this study were to 1) determine the influence of temperature on its virulence; and 2) develop specific protocols for rapid diagnosis. Virulence assays carried out by bath using Senegalese sole fry, showed that virulence is strongly influenced by temperature. So, LD50 at 14°C was of 8.5×105 cfu/ml, meanwhile at 20°C no mortalities were recorded. On the other hand, the high mortality rates observed on virulence assays by intraperitoneal injection of 2.5×105 cfu/g suggest that P. baetica may be pathogenic for the five fish species tested (wedge sole, Senegalese sole, Sea bream, European sea bass and meagre). Two PCR protocols, using specific primers targeting the gyrB and rpoD genes, were developed for rapid diagnosis from pure cultures. An additional protocol, using both primer sets, was also optimized for detection from fish tissue samples. Specificity was tested using 81 strains from 66 bacterial species, taxonomically and/or ecologically related; only the P. baetica strains showed the expected DNA amplicons. A specific dot-blot assay using polyclonal antibodies was also developed for differentiation of P. baetica from related species. Altogether, the protocols described here will constitute useful tools for diagnosis and to clarify the relevance of this pathogen.