Staying below the Radar: Unraveling a New Family of Ubiquitous "Cryptic" Non-Tailed Temperate Vibriophages and Implications for Their Bacterial Hosts

Abstract

Bacteriophages are the most abundant biological entities in the oceans and play key roles in bacterial activity, diversity and evolution. While extensive research has been conducted on the role of tailed viruses (Class: Caudoviricetes), very little is known about the distribution and functions of the non-tailed viruses (Class: Tectiliviricetes). The recent discovery of the lytic Autolykiviridae family demonstrated the potential importance of this structural lineage, emphasizing the need for further exploration of the role of this group of marine viruses. Here, we report the novel family of temperate phages under the class of Tectiliviricetes, which we propose to name "Asemoviridae" with phage NO16 as a main representative. These phages are widely distributed across geographical regions and isolation sources and found inside the genomes of at least 30 species of Vibrio, in addition to the original V. anguillarum isolation host. Genomic analysis identified dif-like sites, suggesting that NO16 prophages recombine with the bacterial genome based on the XerCD site-specific recombination mechanism. The interactions between the NO16 phage and its V. anguillarum host were linked to cell density and phage-host ratio. High cell density and low phage predation levels were shown to favor the temperate over the lytic lifestyle for NO16 viruses, and their spontaneous induction rate was highly variable between different V. anguillarum lysogenic strains. NO16 prophages coexist with the V. anguillarum host in a mutualistic interaction by rendering fitness properties to the host, such as increased virulence and biofilm formation through lysogenic conversion, likely contributing to their global distribution.

Keywords: Asemoviridae; NO16; Vibrio; dif sites; integration; lysogenic conversion; non-tailed phages; spontaneous induction; ubiquitous presence.

Figure 1

(A) Successful infection and consequent cell lysis of V. anguillarum strain A023 by phage NO16 under TEM, (B) Morphology of the NO16 non-tailed virions under TEM, (C) Genomic map of bacteriophage NO16 (GenBank accession no. MH730557) illustrating the 23-gene content of the novel phage. The viral core genes, DJR MCP and ATPase, are slightly shaded while the attributed functions of all 23 genes are listed on the side. (D) Dendrogram generated by GRAViTy (Database selection: DB-B: Baltimore Group Ib—Prokaryotic and archaeal dsDNA viruses). The major viral families are color coded and the phylogenetic clade, which includes the novel phage NO16, is highlighted and magnified in order to illustrate its single monophyletic taxon with only one member, marked with an asterisk *.

Figure 2

(Left): Successful HMM hits of DJR and ATPase and DJR, ATPase and SAH in 42 and 30 different Vibrio species (including the unidentified Vibrio sp. group), respectively, other than V. anguillarum. The asterisk indicates the species where NO16 phages were detected using only the DJR and ATPase core genes. (Right): Genomic organization of representative strains of the species where NO16 prophages were detected. The synteny of NO16-like prophage genomes are vertically illustrated, while the key genes (DJR MCP, ATPase and SAH) are consistently present and highlighted (blue: SAH, brown: DJR MCP, gray: ATPase).

Figure 3

Fifteen different Vibrio species with more than one strain. The percentage in the colored piece of each pie chart corresponds to the NO16-containing sequences and geographic location is painted on the map. Information on isolation sources (marine, human, seafood, terrestrial animals or non-available) are also reported in the legend.

Figure 4

(A). Progress of lysogenization and resistance development over 72 h where potential responses between free-swimming and aggregate-forming subpopulations were assessed, (B). Number of lysogenized CFUs in the NO16-infected V. anguillarum strain A023 cultures, (C). Gradual decrease in free viruses over the 72 h experiment, (D). EOP percentages of the 306 NO16-sensitive bacterial clones over the 72 h experiment, in both free-swimming and aggregate-forming subpopulations. The values are means ± standard deviation of the three replicates.

Figure 5

(A) Relative integrated NO16 prophage genomes (prophage per bacteria genomes) among different MOI (0.01, 0.1, 10) according to the quantitative assessment of the prophage–host integration sites, (B) Abundance of free phages 48 h post infection at different MOI (0.1, 1, 10) and cell densities (addition of 6 h, 12 h and 24 h host’s spent supernatant) conditions, (C) Expression levels of NO16-related genes at the early stage of infection (9 h) under different MOI (0.01, 10). The values are means ± standard deviation of the three replicates. (a, b, c, d: different groups of statistical significance, p-value < 0.05; * statistical significance, p-value < 0.05).

Figure 6

(A) Spontaneous induction of NO16 phages from 10 different V. anguillarum strains with considerable variations over 72 h, (B) Seven different lysogenized A023 clones that form two different groups based on their spontaneous induction rates over 72 h: the upper box corresponds to the high whereas the lower box to the low induction rate clones, (C) Spontaneous induction of lysogenized A023 clone #20 remains unaffected by different cell density levels (addition of 6 h, 12 h and 24 h host’s spent supernatant), (D) Spontaneous induction of lysogenized A023 clone cl15 is inhibited by increased cell density (addition of 6 h, 12 h and 24 h host’s spent supernatant). The values are means ± standard deviation of the three replicates.

Figure 7

(A) Biofilm formation of lysogenized (#20, #28, #40, cl5, cl13, cl15, cl26) and non-lysogenized (#8, #9, #10, #11, #16, #17, cl23), but exposed to NO16, A023 clones after 48 h. A023 was used as control. (B) Biofilm formation of A023 under low (MOI: 0.01) and high (MOI: 10) phage predation, as well as in the presence of host’s spent supernatant (A023 sn) and supernatant from bacteria–phage lysis event (p-f sn). A023 was used as control. (C) In vivo mortality rates of S. aurata larvae infected by A023 lysogens (cl13 and cl15), wild type (A023) and non-infected (bacteria-free) condition as negative control, over a 7-day period. Letters a and b indicate statistically significant groups (Dunn’s method versus control). The values are means ± standard deviation of the three replicates.