Draft Genome Sequence of the Virulent Strain 01-B526 of the Fish Pathogen Aeromonas salmonicida

Abstract

Aeromonas salmonicida is an important fish pathogen, mainly of salmonids. This bacterium causes a disease named furunculosis, which is particularly detrimental for the aquaculture industry. Here, we present the draft genome sequence of A. salmonicida 01-B526, a strain isolated from a brook trout that is more virulent than A. salmonicida reference strain A449, for which a genome sequence is available.

GENOME ANNOUNCEMENT

The Gram-negative bacterium Aeromonas salmonicida subsp. salmonicida is the causative agent of furunculosis, especially in salmonids (11, 17). The complete genome sequence of A. salmonicida reference strain A449 has already been published (16). Unfortunately, this strain was not virulent when facing the alternative host model Dictyostelium discoideum amoeba, probably due to the absence of proteolytic activity (6). In this context, it appeared important to obtain the genomic sequence of a true virulent strain of A. salmonicida, such as 01-B526, a strain isolated from an infected brook trout in the province of Quebec (Canada) displaying high virulence against both fish and amoeba (6, 7).

The total genomic DNA of A. salmonicida 01-B526 was extracted using the DNeasy blood and tissue kit (Qiagen, Streetsville, ON, Canada). Whole-genome 3-kb paired-end DNA sequencing of A. salmonicida 01-B526 was performed using the Roche/454 pyrosequencing method on the Genome Sequencer FLX system with titanium chemistry at the Plateforme d'Analyse Génomique of the Institut de Biologie Intégrative et des Systèmes (IBIS; Université Laval). In total, 214,046,091 bases were analyzed using the de novo assembler module (gsAssembler) of Newbler version 2.5.3 (454 Life Sciences). A total of 1,108 contigs were produced (173 contigs were larger than 500 bases, and 935 contigs ranged from 100 to 499 bases). Paired-end data (average pair distance of 2.38 kb) joined 135 large contigs into 31 scaffolds. A reference assembly of the same data set of A. salmonicida A449 (GenBank accession numbers CP000646.1, CP000645.1, and CP000644.1) using the gsMapper module of Newbler version 2.5.3 produced 108 contigs. This high number of contigs despite an average coverage of more than 40× is due to the presence of a high number of repeated elements and especially insertion sequences (IS) in the A. salmonicida genome (16). Manual gap closing is under way to complement de novo and reference assemblies.

The ongoing assembly of the data set shows that the A. salmonicida 01-B526 chromosome has 4.75 Mb compared to 4.70 Mb for A. salmonicida A449 (16). 01-B526 holds a large plasmid, pAsa5, of 155 kb (16). This strain also possesses 3 small plasmids, pAsa1, pAsa2, and pAsa3, of 5,424 bases, 5,247 bases, and 5,616 bases, respectively (5). The genome structure analysis of 01-B526 was further investigated with PCR and EcoRI restriction profiles (5). Restriction analyses suggested the additional presence of the pAsal1 plasmid (5) in A. salmonicida 01-B526 (data not shown). This plasmid has been missed in sequence analysis for two reasons: the very high homology of a big part of this plasmid with pAsa3, and the presence of one IS also found elsewhere in the genome. These kinds of elements cannot be easily managed by next-generation assemblers and most often require manual intervention (1). The total genome assembly has a mean G+C content of 58.5%. The annotation of the sequences was made by the NCBI Prokaryotic Genomes Automatic Annotation Pipeline (PGAAP) (2–4, 8–10, 12–15).

In the future, a deep comparative analysis of the genome sequences of A449 and 01-B526 might allow the identification of new virulence factors used by A. salmonicida to infect fish. The fact that the 01-B526 chromosome is 50 kb bigger than the one of A449 is of particular interest.

Nucleotide sequence accession number.

The nucleotide sequence for the draft genome sequence was deposited in DDBJ/EMBL/GenBank under accession number AGVO00000000.