A novel Alteromonas phage with tail fiber containing six potential iron-binding domains

Abstract

Viruses play a vital role in regulating microbial communities, contributing to biogeochemical cycles of carbon, nitrogen, and essential metals. Alteromonas is widespread and plays an essential role in marine microbial ecology. However, there is limited knowledge about the interactions of Alteromonas and its viruses (alterophages). This study isolated a novel podovirus, vB_AmeP-R22Y (R22Y), which infects Alteromonas marina SW-47 (T). Phylogenetic analysis suggested that R22Y represented a novel viral genus within the Schitoviridae family. R22Y exhibited a broad host range and a relatively large burst size, exerting an important impact on the adaptability and dynamics of host populations. Two auxiliary metabolic genes, encoding Acyl carrier protein and AAA domain-containing protein, were predicted in R22Y, which may potentially assist in host fatty acid metabolism and VB12 biosynthesis, respectively. Remarkably, the prediction of the R22Y tail fiber structure revealed six conserved histidine residues (HxH motifs) that could potentially bind iron ions, suggesting that alterophages may function as organic iron-binding ligands in the marine environment. Our isolation and characterization of R22Y complements the Trojan Horse hypothesis, proposes the possible role of alterophages for marine iron biogeochemical cycling, and provides new insights into phage-host interactions in the iron-limited ocean.IMPORTANCEIron (Fe), as an essential micronutrient, is often a limiting factor for microbial growth in marine ecosystems. The Trojan Horse hypothesis suggests that iron in the phage tail fibers is recognized by the host's siderophore-bound iron receptor, enabling the phage to attach and initiate infection. The potential role of phages as iron-binding ligands has significant implications for oceanic trace metal biogeochemistry. In this study, we isolated a new phage R22Y with the potential to bind iron ions, using Alteromonas, a major siderophore producer, as the host. The tail fiber structure of R22Y exhibits six conserved HxH motifs, suggesting that each phage could potentially bind up to 36 iron ions. R22Y may contribute to colloidal organically complexed dissolved iron in the marine environment. This finding provides further insights into the Trojan Horse hypothesis, suggesting that alterophages may act as natural iron-binding ligands in the marine environment.

Keywords: Alteromonas; bacteriophage; interaction; iron; tail structure.

Fig 1

Biological features of R22Y. (A) Plaques of R22Y formed on the bacterial lawn of A. marina SW-47 (T) after 24 h of incubation. As a reference, the diameter of the culture dish was 90 mm. (B) TEM image of R22Y. Scale bar, 100 nm. (C) One-step growth curve of phage R22Y and each data point is represented as the mean ± SD derived from three independent replicates.

Fig 2

Genomic analysis of phage R22Y. (A) Annotated genome of phage R22Y. The predicted functions of proteins are illustrated by different colors of arrows representing genes. (B) Full-genome comparison with the phage V19. Homologous proteins are connected using blue shadings of varying transparency to indicate similarity between genes.

Fig 3

Genomic and taxonomic analyses of R22Y. (A) Phylogenetic tree of all 20 Alteromonas phages and 422 selected viruses closely related to alterophages. The tree, constructed using VipTree, displays colored rings denoting virus families (inner ring) and host groups (outer ring). (B) The GBDP tree of 30 phage genomes reconstructed by VICTOR with the formula D6. (C) Protein-based networks of R22Y and its closed phages generated by vConTACT2. Each node represents the phage genomic sequences, with distinct colors indicating their respective host taxonomy. The edges connecting pairs represent the similarity scores between genomes based on shared protein. (D) The intergenomic similarity between R22Y and its related phages generated by VIRIDIC.

Fig 4

Interactions between phage and host. (A) AMGs of R22Y are involved in the metabolism of host bacteria. The flowchart illustrates the involvement of the cobS gene carried by R22Y in the synthesis and transport of host VB12. The related genes, as well as their presence in the 175 strains of host bacteria, are depicted in the figure. (B) R22Y lysis profiles of 175 Alteromonas strains recovered from various seawater depths. Sensitive and insensitive strains are highlighted in red and gray, respectively. Genes involved in the VB synthesis pathway and transport are shown in blue and yellow, while cobS genes are denoted by pentagrams. Solid indicates the presence of the gene and hollow indicates the absence of the gene. The internal phylogenetic tree for the genus Alteromonas, comprising 175 strains, was constructed by concatenating amino acid sequences from 120 bacterial ortholog genes, utilizing GTDB-tk v1.3.0.

Fig 5

Structures analysis of putative receptor-binding proteins in vB_AmaP-R22Y (R22Y) by using AlphaFold 2.0. (A) Ribbon diagrams of RBP trimers (residues1-363) are presented. Chains A, B, and C are colored magenta, green, and cyan, respectively. The six iron ions’ structure are shown in orange. (B) Close-up view of the fourth iron ion with coordinated residues in the ion-binding region (black border). (C) Top view of iron ion docking to HxH structure. (D) Top view of R22Y’s RBP-specific binding region (364–676) to the host surface. (E) Close-up view of R22Y’s RBP-specific binding region. (F) Ribbon diagrams of RBP trimers (residues 1–676).

Fig 6

(A) Alignment of the segmental domains of the tail fiber gp12 protein (1–363) of R22Y, long tail fiber gp37 protein (811–1206) of phage T4, gp4 protein of phage V19. Identical residues are indicated with dark blue or light blue shadow. HxH motifs are also labeled with Fe²⁺ on top of the alignment. (B) HMM logos of the HxH motifs that bind iron ions in phages T4 and conserved HxH motifs identified in the putative tail fiber proteins of R22Y and V19 were created using Skylign. (C) Structure of R22Y with six tail fibers binding 36 iron ions (red spheres).

Fig 7

Ribbon diagrams of R22Y as in the tail RBP trimers (tail fiber, residues 1–363, top). In the center, the superposition of R22Y (residues 105–213, orange) and V19 tail fiber (residues 22–116, gray). The tail fibers in the ion-binding site present a conserved domain architecture (RMSD = 1.595). Ribbon diagrams of V19 tail fiber trimers (residues 1–363, bottom).