Isolation and characterization of the first phage infecting ecologically important marine bacteria Erythrobacter

Abstract

Background: Erythrobacter comprises a widespread and ecologically significant genus of marine bacteria. However, no phage infecting Erythrobacter spp. has been reported to date. This study describes the isolation and characterization of phage vB_EliS-R6L from Erythrobacter.

Methods: Standard virus enrichment and double-layer agar methods were used to isolate and characterize the phage. Morphology was observed by transmission electron microscopy, and a one-step growth curve assay was performed. The phage genome was sequenced using the Illumina Miseq platform and annotated using standard bioinformatics tools. Phylogenetic analyses were performed based on the deduced amino acid sequences of terminase, endolysin, portal protein, and major capsid protein, and genome recruitment analysis was conducted using Jiulong River Estuary Virome, Pacific Ocean Virome and Global Ocean Survey databases.

Results: A novel phage, vB_EliS-R6L, from coastal waters of Xiamen, China, was isolated and found to infect the marine bacterium Erythrobacter litoralis DSM 8509. Morphological observation and genome analysis revealed that phage vB_EliS-R6L is a siphovirus with a 65.7-kb genome that encodes 108 putative gene products. The phage exhibits growth at a wide range of temperature and pH conditions. Genes encoding five methylase-related proteins were found in the genome, and recognition site predictions suggested its resistance to restriction-modification host systems. Genomic comparisons and phylogenetic analyses indicate that phage vB_EliS-R6L is distinct from other known phages. Metagenomic recruitment analysis revealed that vB_EliS-R6L-like phages are widespread in marine environments, with likely distribution in coastal waters.

Conclusions: Isolation of the first Erythrobacter phage (vB_EliS-R6L) will contribute to our understanding of host-phage interactions, the ecology of marine Erythrobacter and viral metagenome annotation efforts.

Keywords: Complete genome sequence; Erythrobacter; Marine; Siphovirus.

Fig. 1

Plaque (a) and TEM (b) images of Erythrobacter litoralis DSM 8509 phage vB_EliS-R6L particles. The scale bar (b) equals 100 nm

Fig. 2

One-step growth curve analysis of Erythrobacter litoralis phage vB_EliS-R6L. (a) the Plaque forming Unints (PFUs) of the phage and (b) the optical density (OD₆₀₀) of Erythrobacter litoralis DSM 8509. Open circles (a), chloroform-treated samples; closed circles (a), non-chloroform-treated samples. Open circles (b), without phage-inoculated samples; closed circles (b), phage-inoculated samples. OD, optical density

Fig. 3

Stability of Erythrobacter litoralis phage vB_EliS-R6L under various stress conditions. (a) pH stability and (b) temperature stability. PFU, Plaque Forming Unit. Error bars show standard deviations among triplicate samples

Fig. 4

Phylogenetic relationships of four genes of vB_EliS-R6L-like phages. The neighbor-joining trees were based on the ClustalW alignment of amino acid sequences by MEGA 6.06. The bootstrap values were based on 200 replicates. (a) terminase; (b) endolysin; (c) portal protein; (d) major capsid protein

Fig. 5

Relative abundance of vB_EliS-R6L -like phage genes in the metagenomes. (a) Heatmap of the normalized relative abundance of vB_EliS-R6L ORFs identified in the Jiulong River Estuary, Xiamen, China (JRE), Pacific Ocean Virome (POV) and Global Ocean Survey (GOS). (b) Normalized relative abundance of ORF 32, 49, 54 and 100 in the metagenomes