Soil Giant Phage: Genome and Biological Characteristics of Sinorhizobium Jumbo Phage

Abstract

This paper presents the first in-depth research on the biological and genomic properties of lytic rhizobiophage AP-J-162 isolated from the soils of the mountainous region of Dagestan (North Caucasus), which belongs to the centers of origin of cultivated plants, according to Vavilov N.I. The rhizobiophage host strains are nitrogen-fixing bacteria of the genus Sinorhizobium spp., symbionts of leguminous forage grasses. The phage particles have a myovirus virion structure. The genome of rhizobiophage AP-J-162 is double-stranded DNA of 471.5 kb in length; 711 ORFs are annotated and 41 types of tRNAs are detected. The closest phylogenetic relative of phage AP-J-162 is Agrobacterium phage Atu-ph07, but no rhizobiophages are known. The replicative machinery, capsid, and baseplate proteins of phage AP-J-162 are structurally similar to those of Escherichia phage T4, but there is no similarity between their tail protein subunits. Amino acid sequence analysis shows that 339 of the ORFs encode hypothetical or functionally relevant products, while the remaining 304 ORFs are unique. Additionally, 153 ORFs are similar to those of Atu_ph07, with one-third of the ORFs encoding different enzymes. The biological properties and genomic characteristics of phage AP-J-162 distinguish it as a unique model for exploring phage-microbe interactions with nitrogen-fixing symbiotic microorganisms.

Keywords: MOI; Sinorhizobium spp.; cas4; jumbo rhizobiophage; lytic activity; phage tRNA; transmission electron microscopy.

Figure 1

Morphological and transmission electron microscopy (TEM) characteristics of the rhizobiophage AP-J-162. (a) Plaques formed by the phage (indicated by arrows) on a lawn of S. meliloti Md3/4 on 0.2% or 0.4% semi-solid agar plates; and (b) microphotographs of phage AP-J-162 particles.

Figure 2

Lytic activity of jumbo phage AP-J-162 on a culture of phage-sensitive S. meliloti strain Md3/4 at different MOIs. The control was the growth curve of an uninfected bacterial strain (blue line), while the remaining curves represent the growth of the phage-infected strain at MOIs 0.0003 and 0.001 (orange and green lines, correspondingly).

Figure 3

Genomic characteristics of the jumbo rhizobiophage AP-J-162. (a) Circular physical map of the AP-J-162 genome. The regions marked in blue represent putative clusters of structural proteins; and (b) GC skew diagram (blue lines) and calculated cumulative GC skew (red lines).

Figure 4

Phylogenetic tree of large-genome phages. The scale bar is 0.01 for the nucleotide substitutions per site. A and B clades; A1, A2, B1 and B2 clusters. The phylogenetic tree was constructed by the N-J method according to the criterion of balanced minimal evolution implemented by the FASTME algorithm and its improvement by topological SPR moves obtained by the D6 formula (average bootstrap 91%).