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Despite the recent advances in sequencing technologies, the complete assembly of multi-chromosome genomes of the Vibrionaceae , often containing several plasmids, remains challenging. Using a combination of Oxford Nanopore MinION long reads and short Illumina reads, we fully sequenced, closed and curated the genomes of two strains of a primary aquatic pathogen Photobacterium damselae subsp. piscicida isolated in Australia. These are also the first genome sequences of P. damselae subsp. piscicida isolated in Oceania and, to our knowledge, in the Southern hemisphere. We also investigated the phylogenetic relationships between Australian and overseas isolates, revealing that Australian P. damselae subsp. piscicida are more closely related to the Asian and American strains rather than to the European ones. We investigated the mobilome and present new evidence showing that a host specialization process and progressive adaptive evolution to fish are ongoing in P. damselae subsp. piscicida , and are largely mediated by transposable elements, predominantly in chromosome 2, and by plasmids. Finally, we identified two novel potential virulence determinants in P. damselae subsp. piscicida – a chorismate mutase gene, which is ubiquitously retained and co-localized with the AIP56 apoptogenic toxin-encoding gene on the pPHDP10 plasmid, and transfer-messenger RNA gene ssrA located on the main chromosome, homologous to a critical-to-virulence determinant in Yersinia pseudotuberculosis . Our study describes, to our knowledge, the only fully closed and manually curated genomes of P. damselae subsp. piscicida available to date, offering new insights into this important fish pathogen and its evolution.

Allelic exchange mutagenesis that relies on RecA-mediated homologous recombination up- and downstream from the targeted gene is a generalizable method of site-specific bacterial gene knock-out and knock-in. However, generation of a mutagenic DNA construct (alternative allele flanked by regions surrounding the gene target) and subsequent mutant selection are laborious procedures. Here we demonstrate allelic exchange knock-out facilitated by Gibson Assembly in Streptococcus iniae. Gibson Assembly allows rapid construction of a large allelic exchange cassette simultaneous with cloning, as well as rapid reconstruction of complete recombinant vector sequence when required. Additionally, we show that during two-step mutant selection, absence of recombination at one of the homologous regions (single cross-over) might be rapidly detected by colony PCR of meroploid clones and resolved by extension/shifting of corresponding sequence in DNA construct. The combination of Gibson Assembly for mutagenic DNA construction/redesign with colony PCR screening of meroploids to detect recombination at both sides of the exchange target may significantly accelerate generation of chromosomal mutants in a wide range of bacterial taxa.

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Aquaculture produces more than 50% of fish for human consumption and, in spite of major improvements since the adoption of injectable vaccines in the 1990s, bacterial diseases still account for considerable losses, particularly in tropical and warm temperate species. Streptococcosis, caused predominantly by Streptococcus iniae and S. agalactiae, manifests as a generalised septicaemia and meningitis followed by rapid mortality. Vaccination against streptococcal infections is difficult as a result of multiple, poorly defined serotypes and consequent vaccine escape (reinfection of previously vaccinated animals). However, genomics applied to reverse vaccinology is providing novel insights into diversity among these aquatic pathogens and is identifying cross-serotype targets that may be exploited for new generation streptococcal vaccines for aquaculture.Aquaculture reached a significant milestone in 2013 as global production for food use overtook beef production for the first time and now accounts for more than 50% of the global seafood supply 1 .Aquaculture has wrestled with social license throughout its rapid growth in developed economies, including Australia, through the