Comparative analysis and mutation effects of fpp2–fpp1 tandem genes encoding proteolytic extracellular enzymes of Flavobacterium psychrophilum

Abstract

Flavobacterium psychrophilum is a very significant fish pathogen that secretes two biochemically characterized extracellular proteolytic enzymes, Fpp1 and Fpp2. The genes encoding these enzymes are organized as an fpp2–fpp1 tandem in the genome of strain F. psychrophilum THC02/90. Analysis of the corresponding encoded proteins showed that they belong to two different protease families. For gene function analysis, new genetic tools were developed in F. psychrophilum by constructing stable isogenic fpp1 and fpp2 mutants via single-crossover homologous recombination. RT-PCR analysis of wild-type and mutant strains suggested that both genes are transcribed as a single mRNA from the promoter located upstream of the fpp2 gene. Phenotypic characterization of the fpp2 mutant showed lack of caseinolytic activity and higher colony spreading compared with the wild-type strain. Both characteristics were recovered in the complemented strain. One objective of this work was to assess the contribution to virulence of these proteolytic enzymes. LD50 experiments using the wild-type strain and mutants showed no significant differences in virulence in a rainbow trout challenge model, suggesting instead a possible nutritional role. The gene disruption procedure developed in this work, together with the knowledge of the complete genome sequence of F. psychrophilum, open new perspectives for the study of gene function in this bacterium.

Chromosome-Encoded Hemolysin, Phospholipase, and Collagenase in Plasmidless Isolates of Photobacterium damselae subsp. damselae Contribute to Virulence for Fish

“…In this regard, proteolytic enzymes of Vibrio anguillarum (29) and of Aeromonas salmonicida (30) are considered true virulence factors since they degrade fish tissues. However, there are examples where proteolytic enzymes do not play a role in virulence (31,32). We found that a single deletion of colP in the AVL49 strain reduced virulence with respect to the parental strain, albeit this reduction was lower than that observed after hlyA ch and plpV mutations (Fig.…”

Construction and validation of a GFP-based vector for promoter expression analysis in the fish pathogen Flavobacterium psychrophilum

“…These putative promoter sequences were amplified by PCR from the following genes: pdhB gene (pyruvate dehydrogenase beta subunit; GenBank ID FP0337) using the prompdhB-F and R oligonucleotides (Supplemental Table 2); the upstream sequence of the fpp2-fpp1 operon (Pérez-Pascual et al, 2011), (GenBank ID FP0232 and FP0231, respectively) with the promfpp2-F and R oligonucleotides (Supplemental Table 2); the intergenic fpp2-fpp1 sequence using the promfpp1-F and R primers (Supplemental Table 2); and the upstream sequence of the gldJ gene (GenBank ID FP1389) using promgldJ-F and R oligonucleotides (Supplemental Table 2). The presence and correct orientation of the inserted sequences was confirmed by enzymatic digestion, PCR and nucleic acid sequencing.…”

“…In spite of the increasing importance of this infection (Cipriano and Holt, 2005), little is known about the factors that govern the physiology and genetics of this pathogen. At present, F. psychrophilum is a bacterium difficult to manipulate genetically (Álvarez et al, 2004, 2006Pérez-Pascual et al, 2011). The main reasons for this are that transformation and conjugation frequencies are very low (Álvarez et al, 2004), growth is difficult and slow (Álvarez and Guijarro, 2007) and recovery of the cells on solid media comes up against the presence of viable non-cultivable cells (Michel et al, 1999).…”

“…Therefore, the knowledge of the virulence factors employed by this microorganism to cause disease is still fairly limited. In the last few years, the development of some genetic techniques (Álvarez et al, 2004;Pérez-Pascual et al, 2011), together with the availability of the complete genome sequence of F. psychrophilum (Duchaud et al, 2007) have opened new possibilities for the study of virulence determinants in this bacterium. Thus, a few factors associated to pathogenesis have been described (Álvarez et al, 2006Bertolini et al, 1994;Crump et al, 2005;Lorenzen and Olesen, 1997;Møller et al, 2003;Nematollahi et al, 2003;Ostland et al, 2000) and putative virulence genes have been identified in the genome analysis of this bacterium (Duchaud et al, 2007).…”

TheyrpABoperon ofYersinia ruckeriencoding two putative U32 peptidases is involved in virulence and induced under microaerobic conditions

“…However, this is not a general rule, since some proteolytic enzymes of bacteria causing disease in fish, such as the AspA of A. salmonicida 9 and Fpp2 of Flavobacterium psychrophilum, are not involved in virulence. 10 Therefore, the implication of this type of enzyme in virulence should be assessed in each particular case.…”