Redefining the bacteriophage mv4 site-specific recombination system and the sequence specificity of its attB and core-attP sites.

Through their involvement in the integration and excision of a large number of mobile genetic elements, such as phages and integrative and conjugative elements (ICEs), site-specific recombination systems based on heterobivalent tyrosine recombinases play a major role in genome dynamics and evolution. However, despite hundreds of these systems having been identified in genome databases, very few have been described in detail, with none from phages that infect Bacillota (formerly Firmicutes). In this study, we reanalyzed the recombination module of Lactobacillus delbrueckii subsp. bulgaricus phage mv4, previously considered atypical compared with classical systems. Our results reveal that mv4 integrase is a 369 aa protein with all the structural hallmarks of recombinases from the Tn916 family and that it cooperatively interacts with its recombination sites. Using randomized DNA libraries, NGS sequencing, and other molecular approaches, we show that the 21-bp core-attP and attB sites have structural similarities to classical systems only if considering the nucleotide degeneracy, with two 7-bp inverted regions corresponding to mv4Int core-binding sites surrounding a 7-bp strand-exchange region. We also examined the different compositional constraints in the core-binding regions, which define the sequence space of permissible recombination sites.

Characterization of Tenacibaculum maritimum isolated from diseased salmonids farmed in Chile reveals high serological and genetic heterogeneity.

The diversity of Tenacibaculum maritimum in Chile remains poorly understood, particularly in terms of antigenic and genetic diversity. This information is crucial for the future development of a vaccine against tenacibaculosis and would increase understanding of this important fish pathogen. With this aim, the biochemical, antigenic, and genetic characteristics were analysed for 14 T. maritimum isolates, recovered from diseased Atlantic salmon (Salmo salar) and rainbow trout (Oncorhynchus mykiss) farmed in Chile between 1998 and 2022. Biochemical analysis showed a homogeneity among all the Chilean T. maritimum isolates and all four other strains included for comparison purposes. Serological characterization using dot-blot assaying revealed antigenic heterogeneity with the use of unabsorbed antisera. The majority of isolates showed cross-reactions, identifying three main serological patterns. When the PCR-based serotyping scheme was performed, the existence of antigenic heterogeneity was confirmed. Four Atlantic salmon isolates were 4-0; and most isolates, including the rainbow trout isolate, were 3-1 (n = 9). A turbot (Scophthalmus maximus) isolate was 1-0. Using an existing Multilocus Sequence Typing system, two newly identified sequence types (ST193 and ST198) in the database were detected. ST193 encompassed nine isolates obtained from Atlantic salmon and rainbow trout, while ST198 regrouped four isolates, all retrieved from diseased Atlantic salmon in 2022. These findings highlight significant antigenic and genetic diversity among the Chilean isolates. This information is useful for epizootiology and the selection of suitable candidate strain(s) for vaccine development against tenacibaculosis caused by T. maritimum in Chilean salmon farming.

Genomic characterization of Tenacibaculum maritimum O-antigen gene cluster and development of a multiplex PCR-based serotyping scheme.

Tenacibaculum maritimum is a devastating bacterial pathogen affecting a large variety of marine fish species. It is responsible for significant economic losses in aquaculture farms worldwide. Different typing methods have been proposed to analyse bacterial diversity and population structure. Serological heterogeneity has been observed and up to four different serotypes have been described so far. However, the underlying molecular factors remain unknown. By combining conventional serotyping and genome-wide association study, we identified the genomic loci likely involved in the O-antigen biosynthesis. This finding allowed the development of a robust multiplex PCR-based serotyping scheme able to detect subgroups within each serotype and therefore performs better than conventional serotyping. This scheme was successfully applied to a large number of isolates from worldwide origin and retrieved from a large variety of fish species. No obvious correlations were observed between the mPCR-based serotype and the host species or the geographic origin of the isolates. Strikingly, the distribution of mPCR-based serotypes does not follow the core genome phylogeny. Nevertheless, this simple and cost-effective mPCR-based serotyping method could be useful for different applications such as population structure analysis, disease surveillance, vaccine formulation and efficacy follow-up.

First Isolation of Virulent Tenacibaculum maritimum Isolates from Diseased Orbicular Batfish (Platax orbicularis) Farmed in Tahiti Island.

The orbicular batfish (Platax orbicularis), also called 'Paraha peue' in Tahitian, is the most important marine fish species reared in French Polynesia. Sudden and widespread outbreaks of severe 'white-patch disease' have occurred since 2011 in batfish farms one to three weeks after the transfer of juveniles from bio-secured hatcheries to lagoon cages. With cumulative mortality ranging from 20 to 90%, the sustainability of aquaculture of this species is severely threatened. In this study, we report for the first time the isolation from diseased batfish of several isolates belonging to the species Tenacibaculum maritimum, a major pathogen of many marine fish species. Histopathological analysis, an experimental bath challenge and a field monitoring study showed that T. maritimum is associated with 'white-patch disease'. Moreover, molecular and serological analyses performed on representative isolates revealed some degree of genetic diversity among the isolates, a finding of primary importance for epidemiological studies and the development of management and control strategies such as vaccination.