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Microbiology and Biotechnology Letters

Genome Report(Note)

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Genome Report  |  Genome Report

Microbiol. Biotechnol. Lett. 2024; 52(3): 339-341

https://doi.org/10.48022/mbl.2407.07018

Received: July 30, 2024; Revised: September 19, 2024; Accepted: September 20, 2024

Complete Genome Sequence of Salmonella Typhimurium-Specific Phage vB_SalA_KFSST3 Possessing Antibiofilm Activity

Su-Hyeon Kim, Jaein Choe, and Mi-Kyung Park*

School of Food Science and Biotechnology, and Food and Bio-industry Research Institute, Kyungpook National University, Daegu 41566, Republic of Korea

Correspondence to :
Mi-Kyung Park,  parkmik@knu.ac.kr

Abstract

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In a previous study, Salmonella Typhimurium-specific phage vB_SalA_KFSST3, which possess antibiofilm activity, was isolated and purified from wastewater used in slaughterhouses. This study aimed to perform bioinformatic analyses to investigate the genes associated with its antibiofilm activity. Phage genome consisted of a single chromosome of 156,555 bp with a GC content of 44.8%. Among its 202 open reading frames (ORFs), three tail spike proteins (TSPs; orf141, orf142, orf143) were identified with high confidence. All TSPs were predicted to encode putative depolymerase activities, including two endoglycosidases and one endorhamnosidase. The genome has been deposited in GenBank under the accession number PP_994976.1.

Keywords: Bacteriophage, antibiofilm activity, tail spike protein, depolymerase, whole genome sequencing

Body

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Biofilms, which are bacterial aggregates embedded in a self-secreted matrix of extracellular polymeric substances (EPS), significantly contribute to most bacterial infections by enhancing microbial survival and antibiotic resistance [1]. A promising strategy to combat biofilms involves the use of bacteriophages (phages) with antibio-film activities. Phages are host-specific viruses, which infect and kill target bacteria through a lytic pathway, and control biofilms primarily via depolymerase activity [2]. This enzyme, often encoded within the same open reading frames (ORFs) as the tail spike protein (TSP), degrades the EPS matrix of biofilms [3]. Consequently, TSPs with depolymerase activity play a crucial role in inhibiting biofilm formation, penetrating existing bio-films, or degrading already-formed biofilms. In our previous study, we isolated and purified Salmonella Typhimurium-specific phage vB_SalA_KFSST3 from wastewater used in slaughterhouses in Daegu, Korea [4]. vB_SalA_KFSST3 demonstrated remarkable lytic and antibiofilm activities at 4℃. Especially, it effectively removed almost all of S. Typhimurium biofilms formed on cantaloupe at cold temperature. Therefore, this study performed detailed bioinformatic analyses of TSPs associated with this antibiofilm activity in the genome of vB_SalA_KFSST3.

The genomic DNA of vB_SalA_KFSST3 was extracted using a Phage DNA Isolation Kit (Norgen Biotek Co., Canada), following the manufacturer’s instructions. Subsequently, the purity of phage DNA was assessed using a Qubit 3.0 fluorometer (Thermo Fisher Scientific, USA) and a NanoDrop spectrophotometer (Bio-Rad, USA). Whole-genome sequencing of vB_SalA_KFSST3 was conducted at SANIGEN Co. (Republic of Korea) using Illumina Miseq platform with paired-end reads of 2 × 300 bp size. Error correction and trimming of the generated raw reads were conducted using FastQC version 0.11.5 (https://www.bioinformatics.babraham.ac.uk/project/fastqc/). De novo assembly was performed using SPAdes version 3.15.0 (Illumina Inc., USA) and annotation was carried out using the BLASTP and the Rapid Annotations Systems Technology (RAST) server [5]. Subsequently, hypothetical proteins located near TSPs were compared with other TSPs listed in the National Center for Biotechnology Information (NCBI) database by alignment of their amino acid sequences using SnapGene software (http://www.snapgene.com/). For further bioinformatic analyses of TSPs (orf141, orf142, and orf143), their conserved domains were analyzed using NCBI conserved domains database and HHpred [6]. Their isoelectric point and the theoretical molecular weight were also predicted using the Prot-Param tool of the Expasy server (https://web.expasy.org/protparam/, accessed on 20 July 2024).

The genome of vB_SalA_KFSST3 consisted of a linear dsDNA with size of 156,555 bp and a GC content of 44.8%. Phage genome contained 48 functional ORFs, 154 hypothetical ORFs, and 5 tRNAs. There are no genes encoding to antibiotic resistance, virulence factors, lysogenic properties, and allergenicity. Based on the RAST database, it is confirmed that two TSPs were encoded by orf142 (XCX11015.1) and orf143 (XCX11016.1)(Table 1). However, most phages belonging to the Ackermannviridae family, including vB_SalA_KFSST3 [4], are typically known to encode up to four multiple TSPs [7]. To investigate the possible presence of additional TSPs, a comparative analysis was performed on hypothetical proteins (orf141 and orf144) adjacent to the identified TSPs (orf142 and orf143) with known TSPs from other Agtrevirus phages, such as those of phages SKML39, Ag3, phiEM4, MK-13, and P46FS4. When aligned (Fig. 1A), the amino acid (AA) sequences of five known TSPs (YP007236096.1, YP003358663.1, BBK03771.1, QBJ04332.1, and QIG62070.1) were similar to those of the hypothetical protein (orf141, XCX11014.1), unlike the other one (orf144, XCX11017.1). Specifically, the N-terminal regions of the TSPs were highly conserved (Fig. 1B), with similarity beginning at AA positions 1 to 67 for known five TSPs and 6 to 72 for orf141. Thus, it is supposed that vB_SalA_KFSST3 genome comprises a total of three TSPs, referred to as TSP1 (orf141), TSP2 (orf142), and TSP3 (orf143) (Fig. 2).

Table 1 . In silico genetic characterization of tail spike proteins in phage genome.

GeneSize (aa1))Molecular weight (kDa)Isoelectric pointDomain possessing enzymatic activity (accession)ProbabilityE-value
Tail spike protein (TSP1, orf141)67571.65.1β-helix, endoglycosidase (6NW9)99.951.3e-24
Tail spike protein (TSP2, orf142)70875.35.2β-helix, endorhamnosidase (3RIQA)100.003.6e-80
Tail spike protein (TSP3, orf143)1,109117.44.7β-helix, endoglycosidase (6NW9)99.901.7e-20

1) AA: amino acid.

Figure 1.(A) Alignment of hypothetical proteins (orf141 and orf144) with other related tail spike proteins (TSPs) and (B) detailed view of the similarity regions at the start positions of the TSPs.

Figure 2.The organization and location of tail spike proteins in the genome of vB_SalA_KFSST3.

From the in silico genetic characterization of three TSPs (Table 1), the molecular weights of TSP1, TSP2, and TSP3 were predicted to be 71.6, 75.3, and 117.4 kDa, respectively. Their isoelectric points were similar, with values of 5.1 for TSP1, 5.2 for TSP2, and 4.7 for TSP3. Furthermore, NCBI conserved domain and HHpred analyses predicts the domain organization of the three TSPs of vB_SalA_KFSST3 with high confidence (Table 1). Residues 29−504 of TSP1 and 344−971 of TSP3 matched putative endoglycosidase (6NW9B) from phage CBA120 with a probability of 99.95% (E-value of 1.3e-24) and 99.90% (E-value of 1.7e-20), respectively. In addition, residues 156−706 of TSP2 matched endorhamnosidase (3RIQA) from phage 9NA with a probability of 100% (E-value 3.6e-80). Consistent with previous findings about the antibiofilm activity of vB_SalA_KFSST3 [4], domains possessing depolymerase activity were found in the ORFs of each TSP. The predicted depolymerases, endoglycosidases and endorhamnosidase have been reported to catalyze the hydrolysis of glycosides and rhamnosides, respectively, in polysaccharides [8, 9]. Consequently, these genes could influence the antibiofilm activity of vB_SalA_KFSST3, as these biofilm dispersal enzymes play a crucial role in degrading the exopolysaccharides of biofilm matrices. This study serves as a basis for further investigations into genes related to the antibiofilm activity of phages and contributes to a better understanding of their functionality.

Nucleotide Sequence Accession Number

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The complete genome of vB_SalA_KFSST3 has been deposited in the GenBank database under an accession number PP_994976.1.

Conflict of Interest

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The authors have no financial conflicts of interest to declare.

References

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