Abstract
Zonula occludens toxin (Zot) is a conserved protein in filamentous vibriophages and has been reported as a putative toxin in Vibrio cholerae. Recently, widespread distribution of zot-encoding prophages was found among marine Vibrio species, including environmental isolates. However, little is known about the dynamics of these prophages beyond V. cholerae. In this study, we characterized and quantified the zot-encoding filamentous phage VAIϕ, spontaneously induced from the fish pathogen V. anguillarum. VAIϕ contained 6117 bp encoding 11 ORFs, including ORF8pVAI, exhibiting 27%–73% amino acid identity to Inovirus Zot-like proteins. A qPCR method revealed an average of four VAIϕ genomes per host genome during host exponential growth phase, and PCR demonstrated dissemination of induced VAIϕ to other V. anguillarum strains through re-integration in non-lysogens. VAIϕ integrated into both chromosomes of V. anguillarum by recombination, causing changes in a putative ORF in the phage genome. Phylogenetic analysis of the V. anguillarum Inoviridae elements revealed mosaic genome structures related to mainly V. cholerae. Altogether, this study contributes to the understanding of Inovirus infection dynamics and mobilization of zot-like genes beyond human pathogenic vibrios, and discusses their potential role in the evolution of the fish pathogen V. anguillarum.
Highlights
The Inovirus genus comprises filamentous phages encapsulating circular (+) ssDNA genomes.In contrast to dsDNA tailed phages, filamentous phages do not lyse their host cell upon propagation, but are secreted through the host membrane in an infection process called chronic cycle [1].In gram-negative hosts, filamentous phages attach to pili that are thought to spontaneously retract resulting in the entry of the phage genome into the host cytoplasm [1,2]
Recent studies predicted the zot-like genes to reside on prophage elements in three V. anguillarum strains isolated from Chile, the UK and the US, respectively [26], and suggested that the loss of a zot-encoding prophage reduced virulence against fish larvae in a pathogenic V. anguillarum [27]. These results suggest that Inovirus-related prophages may spread in V. anguillarum and that inoviruses could contribute to the virulence of this bacterium, as proposed for the related species V. cholerae and V
PF4-zot-prophage+, which had retained the prophage during the initial screening experiment, revealed that 50 out of 50 colonies contained the zot fragment, suggesting that those clones maintaining the prophage following cultivation established a stable association under the given growth conditions
Summary
The Inovirus genus comprises filamentous phages encapsulating circular (+) ssDNA genomes. In gram-negative hosts, filamentous phages attach to pili that are thought to spontaneously retract resulting in the entry of the phage genome into the host cytoplasm [1,2]. Filamentous phage genomes replicate using the bacterial host machinery to synthesize the (−) strand, producing a circular dsDNA phage genome, usually referred to as the replicative form, which serves as a template for rolling circle replication and transcription of phage genes [1]. Filamentous phage genomes are usually 5–20 kb long, organized in replication, structure and assembly modules [1,3,4]. Due to foundational research in the Escherichia coli Ff phages and the V. cholerae phage CTXφ, genome structures of filamentous phages can be compared and their functions predicted [1,4]
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