Closely Related Vibrio alginolyticus Strains Encode an Identical Repertoire of Caudovirales-Like Regions and Filamentous Phages

Abstract

Many filamentous vibriophages encode virulence genes that lead to the emergence of pathogenic bacteria. Most genomes of filamentous vibriophages characterized up until today were isolated from human pathogens. Despite genome-based predictions that environmental Vibrios also contain filamentous phages that contribute to bacterial virulence, empirical evidence is scarce. This study aimed to characterize the bacteriophages of a marine pathogen, Vibrio alginolyticus (Kiel-alginolyticus ecotype) and to determine their role in bacterial virulence. To do so, we sequenced the phage-containing supernatant of eight different V. alginolyticus strains, characterized the phages therein and performed infection experiments on juvenile pipefish to assess their contribution to bacterial virulence. We were able to identify two actively replicating filamentous phages. Unique to this study was that all eight bacteria of the Kiel-alginolyticus ecotype have identical bacteriophages, supporting our previously established theory of a clonal expansion of the Kiel-alginolyticus ecotype. We further found that in one of the two filamentous phages, two phage-morphogenesis proteins (Zot and Ace) share high sequence similarity with putative toxins encoded on the Vibrio cholerae phage CTXΦ. The coverage of this filamentous phage correlated positively with virulence (measured in controlled infection experiments on the eukaryotic host), suggesting that this phage contributes to bacterial virulence.

Keywords: Inoviridae; Vibrio virulence; filamentous phages; prophages; vibriophages; zot.

Figure 1

Left: Bayesian phylogenetic tree of all eight Kiel V. alginolyticus isolates (top) and six other fully sequenced V. alginolyticus isolates based on a core-genome alignment. Node-values represent bootstrap values; for additional information on the tree please see [26]. More information, regarding isolation source can be found in Tables S1 and S3. Middle: Whole chromosome alignment with prophage regions in colored boxes/arrows. Actively replicating prophages are marked in red. Blocks of the same color indicate prophage-types: purple, Vibrio prophage VALGΦ1; light blue, Vibrio prophage VALGΦ2; dark green, Vibrio phage VALGΦ6; light green, Vibrio phage VALGΦ8; dark blue, adjacent sequences of Vibrio phage VALGΦ6 and Vibrio phage VALGΦ8; grey, unknown phages. Right: presence (x) of selected vibriophages in each strain.

Figure 2

Synteny plots of Vibrio prophage VALGΦ1 (top), Vibrio prophage VALGΦ2 (bottom). ORFs are color-coded according to predicted function: red, replication; green, assembly; blue, structural proteins; yellow, integration; purple, lysis; orange, accessory genes; grey, hypothetical proteins.

Figure 3

Synteny plots of Vibrio phage VALGΦ6 (second from top) and Vibrio phage VALGΦ8 (third from top) in comparison to VfO4K68 (top) and VF33 (bottom). ORFs are color-coded according to predicted function: red, replication; green, assembly; blue, structural proteins; grey, hypothetical proteins. Sequences with high sequence identity are indicated by dark grey and low identical sequences by light grey. pI-pX correspond to known filamentous phage proteins and putative homologues.

Figure 4

Extra-chromosomal contigs of Vibrio phage VALGΦ8 sequenced from (a) strain K04M1 and (b) strain K05K4 with the 2-replicon containing contig (left) and the 3-replicon containing contig (right). ORF-coding and protein names as in Figure 3.

Figure 5

Mid-rooted phylogenomic GBDP tree inferred from VICTOR using the formula D0. The numbers above branches are GBDP pseudo-bootstrap support values from 100 replications. The OPTSIL clustering yielded twelve species clusters, four genus clusters and one cluster at the family level. Filamentous phages belonging to the same genus are color-coded. Same species are indicated by S1–S13 in brackets behind each phage name. Both filamentous phages, sequenced in the present study are highlighted with bold letters. Accession numbers of all filamentous phages can be found in Table S2.

Figure 6

Bacterial load (CFU/mL) averaged over all pipefish per infected strain (mean ± s.e.) for each sequenced Kiel V. alginolyticus strain as well as PBS (orange). Strains which do not contain Vibrio phage VALGΦ8 are color coded in blue, strains which contain Vibrio phage VALGΦ8 but have a reduced coverage at the Vibrio phage VALGΦ6 locus are colored in bordeaux, and strains which contain Vibrio phage VALGΦ8 but do not have a reduced coverage at the Vibrio phage VALGΦ6 locus are colored in green.