Complete Genome Sequence of Serratia Phage 4S Isolated from Wastewater in Ukraine

Aim. To isolate and characterize phage of Serratia marcescens bacteria. Methods. Phylogenetic analysis. Results. The complete genome of Serratia phage 4S represents a 173,061-bp double-stranded DNA (dsDNA) with a GC content of 39.9 %. The Basic Local Alignment Search Tool (BLAST) results indicated that the closest relative to Serratia phage 4S is Serratia phage CBH8 (7 % query coverage, 76 % identity). According to the electron micrograph images Serratia phage 4S belongs to the order Caudovirales and the family Myoviridae . Following a phylogenetic analysis of Serratia phage 4S MCP (Major Capsid Protein), our results showed that its MCP was highly homologous to Acinetobacter and Enterobacter phages and on the contrary distant from the MCP of Klebsiella phages. The phylogenetic analysis of Serratia phage 4S DNA helicase indicated that it was highly homologous to Yersinia and Enterobacter phages, and on the contrary distant from DNA helicase of Klebsiella phages. Conclusions. Serratia phage 4S has a lytic pathway, which means that it can be considered for further investigation as a control agent against bacterial infections caused by Serratia marcescens .


Introduction
Serratia marcescens is a Gram-negative bacterium of environmental origin like soil, water, and plant surfaces and known to be a plant as well as a human pathogen causing opportunistic infections in hospitals [1]. Serratia marcescens is still an underestimated bacterium that causes a range of infections in severely immunocompromised or critically ill patients with keratitis, conjunctivitis, urinary tract in-fections, pneumonia, surgical wound infections, sepsis, bloodstream infection, and menin gitis [2]. Considering that bacterial resistance to antibiotics increases, phages are one of the most promising alternatives that have to be applied [3]. To date, the NCBI database  ISSN 1993-6842 (on-

Materials and Methods
In this study, the Serratia phage 4S was isolated from wastewater in the Bortnychi aeration station (Kyiv, Ukraine). Serratia phage 4S was detected using the bacterium Serratia marcescens isolate IMBG291 [4] as its host (obtained from the Institute of Molecular Biology and Genetics NAS of Ukraine, Kyiv, Ukraine). Host range was not investigated in this study.
To isolate phage a modified protocol of an enrichment procedure involving a double-layer agar method was used [5]. Briefly, a molten 1.4 % (wt/vol) meat peptone agar (MPA) was poured into Petri dishes and incubated at room temperature for 7 min. Then 500 mL of filtered water sample (i.e., filtered through 0.22-µm pores) were mixed with 100 mL of Serratia marcescens IMBG291 cells and added to 2 mL of molten 0.7 % (wt/vol) MPA and poured into a Petri dishes with underlay 1.4 % (wt/vol) MPA. Disposable Petri dishes were incubated overnight at 25 °C. A single plaque was picked with a pipette tip and transferred into Saline buffer (0.5 %), followed by centrifugation and vortexing to release phages from the agar plaque and stored at 5 °C. The morphology of phage 4S was determined using electron microscopy (M.G. Kholodny Institute of Botany of the National Academy of Sciences of Ukraine, Kyiv, Ukraine). Staining was performed with 2 % uranyl acetate on freshly prepared formvar coated copper grids.
Extraction of genomic DNA from pure Serratia phage 4S suspension was carried out using the DNA-sorb-AM nucleic acid extraction kit (Amplisens biotechnologies, Moscow, Russia) and DNA purification was done using Zymo research DNA Clean & Concentrator (Zymo Research Corporation, Irvine, USA) according to the manufacturer's protocol. A whole-genome amplification using random priming was carried out using REPLI-g Single Cell Kit (Qiagen, Venlo, Netherlands) according to the manufacturer's protocol. DNA library preparation and phage genome sequencing were performed using Ion Torrent next-generation sequencing (Latvian Biomedical Research and Study Centre, Riga, Latvia).
The DNA library was prepared using an Ion Plus Fragment Library Kit (Thermo Fischer Scientific, USA). Trimmomatic [6] and BBNorm (https://jgi.doe.gov/data-and-tools/ bbtools/bb-tools-user-guide/installationguide/) were used for reads trimming and filtering to remove adaptor sequences as well as reads less than 30 bp. FastQC (http://www. bioinformatics.babraham.ac.uk/projects/fastqc) was used as the reads quality control tool, with subsequent assembly using SPAdes Genome Assembler v3.13.1 [7]. Serratia phage 4S genome coverage was achieved 1000× (x?), which accounts for high genome coverage. Putative phage coding sequences were identified using Prodigal v1.20 [8], whereas the genome translation of a nucleotide sequence to a protein sequence was performed using DNA Master [9]. BLASTp was used to iden-tify Query Cover (%) and Percent Identity (%) when comparing [the] isolated phages and phages from NCBI, whereas BLASTp was also used for [the] putative ORF functions prediction. The genome map was built using the online tool CGView Server (http://stothard. afns.ualberta.ca/cgview_server/). Phylogenetic trees were constructed using ClustalX alignment and the neighbor-joining method in MegaX.

Results and Discussion
Serratia phage 4S produced clear plaques (≈ 2 mm in diameter) on MPA agar inoculated with Serratia marcescens isolate IMBG291. Therefore, in the next step, [the] electron images of phages were obtained. Serratia phage 4S shows an icosahedral head (of about 68 nm in diameter) and long contractile tail (of about 107 nm in length and 18 nm in diameter). Based on this morphology it can be assigned to order Caudovirales and family Myoviridae (Fig. 1).
Subsequently, bacteriophage was sequenced by whole genome sequencing (WGS). The DNA sequence data was further subjected to genetic analysis. The genome of Serratia phage 4S represents a 173,061-bp doublestranded DNA (dsDNA) with the GC content of 39.9 %. Blastn results indicated that the closest relative to Serratia phage 4S is Serratia phage CBH8 (7 % query coverage, 76 % identity). The total number of coding sequences (CDS) of phage 4S was 290. Serratia phage 4S genome contained genes that can be grouped according to its function: structural and genes for replication/recombination/reparation, transcription, translation, nucleotide metabolism, and additional functions. The first group that includes phage structural genes encodes head structure proteins (major capsid protein, prohead protease, and core proteins, scaffold and head completion proteins), whereas the second -tail/neck structure proteins (tail tube and sheath proteins, tail sheath stabilizer, and completion proteins, tail fiber protein, neck proteins, baseplate tail tube initiator, tail tube protein, baseplate wedge, and hub subunits). Hence, the first and second phage gene groups encode proteins that allow the complete recovery of phage head and tail structures. The third group of replication/recombination/repair genes encodes replication proteins (rIIA protein, DNA topoisomerase II subunits, DNA ligase, DNA primase-helicase subunit, DNA helicases and primase, phage clamp loader subunit, sliding clamp protein, DNA polymerase and exonuclease A, DNA end protector protein, and endonucleases) and recombination/repair proteins (recombination endonucleases, repair and single-stranded DNA binding protein), which suggests that phage 4S has its own replication/recombination/repair systems. The fourth group of nucleotide metabolism genes encodes deoxycytidylate deaminase, thymidylate synthase and kinase, and reductases. The fifth group of transcription/translation genes encodes RNA polymerase sigma factor for late transcription, late promoter transcription accessory protein, and the translational repressor protein. Additional genes encode proteins such as lysozyme, which helps phage to lyse the host cell, thus phage virions are released.
Among the all predicted CDS, on the genome of phage 4S, a Major Capsid Protein and a DNA helicase were used for phylogenetic analysis. Serratia phage 4S was submitted to the GenBank database under the accession number MW082584.
Phage genome map was constructed using CGView Server (Fig. 2). Concentric rings display gene information depending on the phage DNA sequence. A zoomed map represents a part of Serratia phage 4S genome, which mostly includes the genes that code for structural proteins such as tail sheath and tail tube proteins, portal vertex protein, prohead core scaffold and protease, major capsid protein, head vertex protein, inhibitor of prohead protease, head outer capsid protein, tape measure protein, baseplate hub assembly catalyst and hub subunits, baseplate wedge and distal hub subunits, baseplate tail tube initiator. A zoomed map also represents genes that code for replication/recombination/repair proteins including homing endonuclease, putative split helicase, RNA polymerase-ADP-ribosyltransferase Alt, recombination, repair and ssDNA binding protein, RNA-DNA and DNA-DNA helicase, and DNA ligase. Hypothetical proteins are indicated as hp.
Phylogenetic trees were constructed based on conserved sequences of Serratia phage 4S. The first phylogenetic tree was constructed using amino acid sequences of the predicted Major Capsid Protein (MCP), which is often the most conserved sequence in phage genomes. According to phylogenetic analysis of Serratia phage 4S MCP, the results revealed that its MCP was highly homologous to Acinetobacter and Enterobacter phages, and on the contrary distant from MCP of Klebsiella phages (Fig. 3).
The second phylogenetic tree was constructed using amino acid sequences of the predicted DNA helicase. The phylogenetic analysis of Serratia phage 4S DNA helicase indicated that it was highly homologous to Yersinia and Enterobacter phages and on the contrary distant from the DNA helicase of Klebsiella phages (Fig. 4).

Conclusions
In conclusion, a newly isolated Serratia phage 4S able to lyse Serratia marcescens bacterium was characterized. According to the electron micrograph results, Serratia phage 4S belongs to the order Caudovirales and the family Myoviridae. The complete genome of Serratia phage 4S represents a 173,061-bp dsDNA with a GC content of 39.9 %. The Blastn (BLASTp?) results indicated that the closest relative to Serratia phage 4S is Serratia phage CBH8 (7 % query coverage, 76 % identity), which means that Serratia phage 4S is new because query coverage is relatively low. According to phylogenetic analysis of Serratia phage 4S MCP, our results showed that its MCP was highly homologous to Acinetobacter and Enterobacter phages and on the contrary distant from MCP of Klebsiella phages. The phylogenetic analysis of Serratia phage 4S DNA helicase indicated that it was highly homologous to Yersinia and Enterobacter phages, and on the contrary distant from DNA helicase of Klebsiella phages. Serratia phage 4S has a lytic pathway, which means that it can be con- sidered for further investigation as a control agent against bacterial infections caused by Serratia marcescens.