Erina Fujiwara-Nagata

Genomic Characterization of Flavobacterium psychrophilum Serotypes and Development of a Multiplex PCR-Based Serotyping Scheme

Flavobacterium psychrophilum is a devastating bacterial pathogen of salmonids reared in freshwater worldwide. So far, serological diversity between isolates has been described but the underlying molecular factors remain unknown. By combining complete genome sequence analysis and the serotyping method proposed by Lorenzen and Olesen (1997) for a set of 34 strains, we identified key molecular determinants of the serotypes. This knowledge allowed us to develop a robust multiplex PCR-based serotyping scheme, which was applied to 244 bacterial isolates. The results revealed a striking association between PCR-serotype and fish host species and illustrate the use of this approach as a simple and cost-effective method for the determination of F. psychrophilum serogroups. PCR-based serotyping could be a useful tool in a range of applications such as disease surveillance, selection of salmonids for bacterial coldwater disease resistance and future vaccine formulation.

From the Flavobacterium genus to the phylum Bacteroidetes: genomic analysis of dnd gene clusters.

Phosphorothioate modification of DNA and the corresponding DNA degradation (Dnd) phenotype that occurs during gel electrophoresis are caused by dnd genes. Although widely distributed among Bacteria and Archaea, dnd genes have been found in only very few, taxonomically unrelated, bacterial species so far. Here, we report the presence of dnd genes and their associated Dnd phenotype in two Flavobacterium species. Comparison with dnd gene clusters previously described led us to report a noncanonical genetic organization and to identify a gene likely encoding a hybrid DndE protein. Hence, we showed that dnd genes are also present in members of the family Flavobacteriaceae, a bacterial group occurring in a variety of habitats with an interesting diversity of lifestyle. Two main types of genomic organization of dnd loci were uncovered probably denoting their spreading in the phylum Bacteroidetes via distinct genetic transfer events.

Genomic diversity and evolution of the fish pathogen Flavobacterium psychrophilum

Flavobacterium psychrophilum, the etiological agent of rainbow trout fry syndrome and bacterial cold-water disease in salmonid fish, is currently one of the main bacterial pathogens hampering the productivity of salmonid farming worldwide. In this study, the genomic diversity of the F. psychrophilum species is analyzed using a set of 41 genomes, including 30 newly sequenced isolates. These were selected on the basis of available MLST data with the two-fold objective of maximizing the coverage of the species diversity and of allowing a focus on the main clonal complex (CC-ST10) infecting farmed rainbow trout (Oncorhynchus mykiss) worldwide. The results reveal a bacterial species harboring a limited genomic diversity both in terms of nucleotide diversity, with ~0.3% nucleotide divergence inside CDSs in pairwise genome comparisons, and in terms of gene repertoire, with the core genome accounting for ~80% of the genes in each genome. The pan-genome seems nevertheless “open” according to the scaling exponent of a power-law fitted on the rate of new gene discovery when genomes are added one-by-one. Recombination is a key component of the evolutionary process of the species as seen in the high level of apparent homoplasy in the core genome. Using a Hidden Markov Model to delineate recombination tracts in pairs of closely related genomes, the average recombination tract length was estimated to ~4.0 Kbp and the typical ratio of the contributions of recombination and mutations to nucleotide-level differentiation (r/m) was estimated to ~13. Within CC-ST10, evolutionary distances computed on non-recombined regions and comparisons between 22 isolates sampled up to 27 years apart suggest a most recent common ancestor in the second half of the nineteenth century in North America with subsequent diversification and transmission of this clonal complex coinciding with the worldwide expansion of rainbow trout farming. With the goal to promote the development of tools for the genetic manipulation of F. psychrophilum, a particular attention was also paid to plasmids. Their extraction and sequencing to completion revealed plasmid diversity that remained hidden to classical plasmid profiling due to size similarities.

Transcriptional regulation of the Na+-NADH:quinone oxidoreductase gene, nqr, in Vibrio anguillarum, a fish pathogen, in the stationary phase

Vibrio anguillarum kills various kinds of fish over a range of salinities from sea water to fresh water, and causes serious damage to aquaculture systems. In this study, the transcriptional regulation of the Na+-NADH:quinone oxidoreductase (Na+-NQR) operon in V. anguillarum from the logarithmic to stationary phases was investigated. Cloning of the Na+-NQR operon revealed a 7 kb nucleotide sequence composed of six open reading frames with amino acid sequence identity of more than 80% with other Vibrio species. Two promoters, nqrP1 and nqrP2, were identified in the region upstream of the nqrA gene using an S1 nuclease assay. The nqrP1 promoter was constitutively activated throughout the logarithmic to stationary phases and possessed-10 (5′-TAGACT-3′) and −35 (5′-ATGGCA-3′) sequences, which were similar to the consensus sequence of Escherichia coli. On the other hand, the nqrP2 promoter was activated only at the stationary phase and had only a −10 (5′-CATACT-3′) and not a −35 sequence. These results suggest that nqrP2, which works specifically in the stationary phase, contributes to starvation-survival in V. anguillarum.