Salmonella enterica subsp. enterica serovar Enteritidis (Salmonella Enteritidis) is a major food-borne pathogen. Its cells tightly attach to food contact surfaces, forming biofilms on poultry products, which leads to severe food-borne illnesses (Myszka and Czaczyk, 2011). Salmonella bacteriophages (phages) have attracted global interest as potential biocontrol agents because of their ability to rapidly lyse a broad range of host bacteria (Pelyuntha et al., 2021). Zindervirus (synonyms SP6-like viruses, Sp6likevirus, and Sp6virus) is a genus in the family Autographiviridae, subfamily Molineuxvirinae (Adriaenssens et al., 2020). The genus is characterized by an isometric head and a non-contractile tail consisting of host-recognizing six prominent tail spike proteins (TSPs) (Knecht et al., 2020), and includes phages infecting Escherichia coli, Salmonella spp., and Vibrio spp. (Cao et al., 2021). Here, we report the complete genome sequence of phage vB_SenAt-pSL2, a new member of the genus Zindervirus, infecting Salmonella Enteritidis from Korean broiler chickens.

The phage vB_SenAt-pSL2 was isolated from the water sample collected from the sewage treatment plant in Deajeon using the host strains Salmonella Enteritidis KCTC 82777 obtained from Korean broiler chickens. Phage propagation and amplification were carried out as described previously (Kim et al., 2012), and total genomic DNA was extracted by Macrogen and sequenced on an Illumina HiSeq X-10 platform of coverage 100x by preparing a DNA library using a TruSeq Nano DNA kit (Illumina). The de novo assembly of the filtered reads (1,972,589,259 bp; 13,126,724 reads; N50, 44,304 bp) with 100× coverage was performed using SPAdes (v3.13.0). Annotation of the putative open reading frames (ORFs) was performed with RAST (https://rast.nmpdr.org/rast.cgi), and conserved domain functions were determined using BLASTP (https://blast.ncbi.nlm.nih.gov/Blast.cgi), Pfam (http://pfam.xfam.org/), and HHpred (https://toolkit.tuebingen.mpg.de/tools/hhpred). Phage termini and packaging mechanism were determined using PhageTerm through the Galaxy server (https://cpt.tamu.edu/galaxy-pub). Transmembrane domains were predicted by using TMHMM (http://www.cbs.dtu.dk/services/TMHMM-2.0/), and signal peptides were predicted using SignalP (http://www.cbs.dtu.dk/services/SignalP/). Phylogenetic analysis based on genome sequences was performed using VICTOR (https://ggdc.dsmz.de/victor.php).

Transmission electron microscopy of phage vB_SenAt-pSL2 revealed morphological characteristics similar to those of other members of Autographiviridae, featuring an isometric head and a putative non-contractile tail (Fig. 1A). The vB_SenAt-pSL2 genome was a linear double-stranded DNA 44,304 bp in length with 47.4% G + C content, 54 predicted protein-coding genes, but no tRNA (Table 1), and including a short terminal repeat region (177 bp). The genome was similar to the Salmonella phages UAB_Phi78 (NC_020414.2, 98.4%) and BP12B (KM 366097.1, 96.5%). Phylogenetic analysis using 20 genome sequences belonging to the subfamily Molineuxvirinae (Autographiviridae) clustered vB_SenAt-pSL2 together with other members of Zindervirus, from which is it distinguished by possessing the largest genome in the genus (Fig. 1B).

Table 1

Features	Value
Genome Size (bp)	44,304
G + C content (%)	47.4
Conitg	1
Total genes	54
Protein-coding genes (CDS)	54

Fig. 1. (A) Transmission electron micrograph (TEM) of Salmonella phage vB_SenAt-pSL2. The scale bar is 200 nm. (B) Genome-BLAST Distance Phylogeny (GBDP) tree based on genome sequences of the strain vB_SenAt-pSL2 and phages of six genera belonging to the subfamily Molineuxvirinae. The tree was created in VICTOR, with branch support determined in terms of formula D0 and inferred from 100 pseudo-bootstrap replicates with an average support of 18%.

Of the putative 54 open reading frames (ORFs), 11 ORFs were functionally annotated as nucleotide metabolism-related proteins: DNA-directed RNA polymerase, HNH homing endonucleases, DNA primase/helicase, DNA polymerase, exonuclease, DNA ligase, lysozyme, holin and peptidase. The presence of virion-encoded RNA polymerase is the defining characteristic of Autographiviridae (Adriaenssens et al., 2020), and its presence in vB_SenAt-pSL2 strongly supported the genome-based phylogeny. 13 ORFs were identified as packaging- and structure-related proteins such as collar head-to-tail connector protein, capsid assembly scaffolding protein, major capsid protein, tail tubular proteins, tail fiber proteins, terminase subunits, and TSPs. Tail fiber proteins are critical components for host recognition and phage DNA ejection (Scholl et al., 2002; Gebhart et al., 2017). TSP, also known as depolymerase, specifically degrades cellular polysaccharides involved in biofilm formation by the host bacterial strain (Harper et al., 2014; Knecht et al., 2020). The high nucleotide similarities of the predicted tail fiber proteins (ORF 37, 38, 39, 41, 42, and 53) TSPs (ORF 51 and 52) between vB_SenAt-pSL2 and other broad-host-range Salmonella phages in the genus Zindervirus (Bardina et al., 2012) suggests a wide host range of the phage against Salmonella serotypes. Moreover, a putative lysozyme (ORF 40) and holin (ORF 43) which showed high sequence similarities to other members in the genus Zindervirus were detected in the genome. The remaining ORFs were assigned to hypothetical proteins, four of which were screened for conserved domains by Pfam search, and antibiotic resistance or bacterial virulence genes were not found in the genome of the phage (Supplementary data Table S1).

Based on the genome comparison and its distinct genome size, the newly isolated Salmonella phage vB_SenAt-pSL2 is a new member of the genus Zindervirus. Its genomic information will provide insights into the biodiversity of phages in Autographiviridae.

Nucleotide sequence accession number

Salmonella phage vB_SenAt-pSL2 was deposited in the Korean Collection for Type Cultures (KCTC 4823). The genome of vB_SenAt-pSL2 was deposited in GenBank (accession number MZ573924.3).

Salmonella enterica subsp. enterica serovar Enteritidis는 전 세계적으로 발생되는 주된 식중독 유발 세균으로 양계 산업 전반에 심각한 피해를 초래한다. 본 연구에서는 국내 도계장에서 분리된 Salmonella enterica subsp. enterica serovar Enteritidis를 특이적으로 감염하는 파지 vB_SenAt-pSL2의 전장 유전체를 분석하였다. 분석 결과, vB_SenAt-pSL2 파지는 총 44,304 bp, G + C 함량 47.4%의 유전체를 지니며 54개의 단백질 코딩 유전자로 구성됨을 확인하였다, 또한 전장 유전체 기반 계통수 분석 결과 vB_SenAt-pSL2 파지는 Zindervirus로 분류되었으며, 동일 속 중 가장 큰 유전체를 지니는 것으로 확인되었다. 또한 Zindervirus 내 광숙주역 파지들과의 tail fiber 및 spike 단백질의 높은 상동성에 근거하여, vB_SenAt-pSL2 파지 역시 광숙주역을 지닐 것으로 추정된다. 본 연구 결과는 Autographiviridae 파지의 분류학적 특성 및 다양성 연구의 기반 자료로 활용될 것이다.

This research was supported by the KRIBB Initiative programs and the National Research Foundation of Korea (NRF-2020 R1I1A2068827) funded by the Ministry of Education in the Republic of Korea and development of technology for biomaterialization of marine fisheries by-products of Korea institute of Marine Scinece & Technology Promotion (KIMST) (KIMST-20220128) funded by the Ministry of Oceans and Fisheries.

The authors declare that they have no conflict of interest.