Corrigendum: Characterization and Genomic Analysis of a Novel Pseudomonas Phage vB_PsaP_M1, Representing a New Viral family, Psaeviridae

Yantao Liang ^1* Linyi Ren ¹ Yundan Liu ¹ Baohong Liu ^2* Andrew Mcminn ³ Kaiyang Zheng ¹ Ziyue Wang ¹ Hongmin Wang ¹ Hongbing Shao ¹ Yeong Yik Sung ³ Wen Jye Mok ³ Li Lian Wong ³ Min Wang ¹

• ¹ Ocean University of China, Qingdao, China
• ² Qilu Hospital, Shandong University, Jinan, Shandong Province, China
• ³ University of Tasmania, Hobart, Tasmania, Australia

In the published article, there was an error in the legend for The colored rings represent the host groups (outer ring) and virus families (inner ring). These trees are calculated by BIONJ according to the genome distance matrix and take the midpoint as the root. below each genome indicates sequence similarities between the genomes, with different colors representing the levels of similarity. Fig. 5. Shared gene heat map of vB_PsaP_M1 and vB_PsaP_M1 like virus in rectangular phylogenetic tree. In the right half, color coding allows rapid visualization of phage genome clustering based on intergenic similarity: the closer the relationship between genomes, the darker the color. These numbers represent the similarity values of each genome pair. In the left half, the three indicator values of each genome pair are expressed in order from top to bottom: aligned partial genome 1 (for the genome found in this row), genome length ratio (for two genomes in this pair), and aligned partial genome 2 (for the genome found in this column). Darker colors emphasize lower values, indicating that there is only a small number of genome aligned genome pairs (orange to white gradient), or there is a large difference in the length of the two genomes (black to white gradient). As the distance between phages increases, the aligned genomic fragments are expected to decrease. as published. We recently realized that due to our oversight, we mistakenly named the newly isolated phage M1 as vB_PsaP_M1 in our article. After receiving a reminder, we now recognize that, in order to better reflect the morphology and host specificity of the phage, M1 should be correctly named vB_PaeM_M1. We sincerely apologize for this error and would like to request a correction in the article. Please note that this naming correction does not affect any of the study's results or conclusions, only the phage's designation was incorrect. We are very sorry for this. The corrected legend appears below. The colored rings represent the host groups (outer ring) and virus families (inner ring). These trees are calculated by BIONJ according to the genome distance matrix and take the midpoint as the root. PAGE \* Arabic \* MERGEFORMAT 3 Fig. 5. Shared gene heat map of vB_PaeM_M1 and vB_PaeM_M1 like virus in rectangular phylogenetic tree. In the right half, color coding allows rapid visualization of phage genome clustering based on intergenic similarity: the closer the relationship between genomes, the darker the color. These numbers represent the similarity values of each genome pair. In the left half, the three indicator values of each genome pair are expressed in order from top to bottom: aligned partial genome 1 (for the genome found in this row), genome length ratio (for two genomes in this pair), and aligned partial genome 2 (for the genome found in this column). Darker colors emphasize lower values, indicating that there is only a small number of genome aligned genome pairs (orange to white gradient), or there is a large difference in the length of the two genomes (black to white gradient). As the distance between phages increases, the aligned genomic fragments are expected to decrease. The authors apologize for this error and state that this does not change the scientific conclusions of the article in any way. The original article has been updated.In the published article, there was an error in FIGURE 4,FIGURE 5,FIGURE 6,FIGURE 7 as published. We recently realized that due to our oversight, we mistakenly named the newly isolated phage M1 as vB_PsaP_M1 in our article. After receiving a reminder, we now recognize that, in order to better reflect the morphology and host specificity of the phage, M1 should be correctly named vB_PaeM_M1. We sincerely apologize for this error and would like to request a correction in the article. Please note that this naming correction does not affect any of the study's results or conclusions, only the phage's designation was incorrect. We are very sorry for this. The corrected FIGURE 4,FIGURE 5,FIGURE 6,FIGURE 7 and its caption appear below.FIGURE 4 FIGURE 5 FIGURE 6 FIGURE 7The authors apologize for this error and state that this does not change the scientific conclusions of the article in any way. The original article has been updated.Reminder: Figures, tables, and images will be published under a Creative Commons CC-BY licence and permission must be obtained for use of copyrighted material from other sources (including re-published/adapted/modified/partial figures and images from the internet). It is the responsibility of the authors to acquire the licenses, to follow any citation instructions requested by third-party rights holders, and cover any supplementary charges.In the published article, there was an error. We recently realized that due to our oversight, we mistakenly named the newly isolated phage M1 as vB_PsaP_M1 in our article. After receiving a reminder, we now recognize that, in order to better reflect the morphology and host specificity of the phage, M1 should be correctly named vB_PaeM_M1. We sincerely apologize for this error and would like to request a correction in the article. Please note that this naming correction does not affect any of the study's results or conclusion, only the phage's designation was incorrect. We are very sorry for this.A correction has been made to ABSTRACT, Paragraph 1. This sentence previously stated:Pseudomonas phage vB_PsaP_M1, is described, which was isolated from the surface coastal waters of PAGE \* Arabic \* MERGEFORMAT 3 Qingdao, China. vB_PsaP_M1 contains a linear, double-stranded 89,387-bp genome with a GC content of 41.04% and encoding 184 putative open reading frames (ORFs).Phylogenetic analysis of whole genome amino acid sequence and comparative genomic analysis showed that vB_PsaP_M1 has a distant evolutionary relationship with previously isolated viruses and can be grouped into a family-level novel viral cluster (VC_61) with eleven uncultured, assembled viral genomes, named as Psaeviridae. Combined with its ability to infect Pseudomonas and its representation of an unstudied viral family, vB_PsaP_M1 may be an important and novel model system for the study of interactions between viruses and host cells in marine ecosystems.The corrected sentence appears below:Pseudomonas phage vB_PaeM_M1, is described, which was isolated from the surface coastal waters of Qingdao, China. vB_PaeM_M1 contains a linear, double-stranded 89,387-bp genome with a GC content of 41.04% and encoding 184 putative open reading frames (ORFs).Phylogenetic analysis of whole genome amino acid sequence and comparative genomic analysis showed that vB_PaeM_M1 has a distant evolutionary relationship with previously isolated viruses and can be grouped into a family-level novel viral cluster (VC_61) with eleven uncultured, assembled viral genomes, named as Psaeviridae. Combined with its ability to infect Pseudomonas and its representation of an unstudied viral family, vB_PaeM_M1 may be an important and novel model system for the study of interactions between viruses and host cells in marine ecosystems.A correction has been made to Introduction Paragraph 2. This sentence previously stated: In this study a new Pseudomonas phage vB_PsaP_M1 was isolated from coastal waters off Qingdao, China. Through the genome, phylogeny and comparative genome analysis of vB_PsaP_M1, the Pseudomonas phage database was expanded. Due to its unique evolutionary relationship, vB_PsaP_M1 can be combined with eleven uncultured phage contigs identified from metagenomics to form a new family-level virus cluster (VC).The corrected sentence appears below:In this study a new Pseudomonas phage vB_PaeM_M1 was isolated from coastal waters off Qingdao, China. Through the genome, phylogeny and comparative genome analysis of vB_PaeM_M1, the Pseudomonas phage database was expanded. Due to its unique evolutionary relationship, vB_PaeM_M1 can be combined with eleven uncultured phage contigs identified from metagenomics to form a new family-level virus cluster (VC).A correction has been made to Materials and methods, Phylogenetic analysis and comparative genomic analyses, Paragraph 3 This sentence previously stated:Based on the whole genome amino acid sequences of bacteriophage vB_PsaP_M1 and Pseudomonas phages, the proteome tree was generated by ViPTree v3.1 (https://www.genome.jp/viptree) (Nishimura et al., 2017). tBLASTx and ViPTree were used for genome comparison to describe the relationship between bacteriophage vB_PsaP_M1 and its close relatives. All-vs-all BLASTp analysis used orthofinder v2.5.4 to compute the percentage of shared genes between phage vB_PsaP_M1 and all complete Pseudomonas phages genomes in the NCBI RefSeq database (Emms and Kelly, 2019).In order to find homologous phages related to vB_PsaP_M1, each coding sequence of vB_PsaP_M1 was queried against the Integrated Microbial Genome/Virus (IMG/VR, v.3) database (E value, <1e-10; identity, >30; and alignment region covering >50%) (Paez-Espino et al., 2017;Paez-Espino et al., 2019;Roux et al., 2021).In vConTACT analysis, the selected sequence was compared with vB_PsaP_M1 as a group to obtain more accurate results (similar sequences were selected by Diamond, all of which satisfied the following parameters: E value, <1e-5; alignment region covering more than 50% of the shorter sequence; and identity >30%) (Zhan and Chen, 2019).The ANI (average nucleotide identity) value of vB_PsaP_M1 was calculated by VIRIDIC, and the results were visualized by pheatmap (Moraru et al., 2020).The corrected sentence appears below:Based on the whole genome amino acid sequences of bacteriophage vB_PaeM_M1 and Pseudomonas phages, the proteome tree was generated by ViPTree v3.1 (https://www.genome.jp/viptree) (Nishimura et al., 2017). tBLASTx and ViPTree were used for genome comparison to describe the relationship between bacteriophage vB_PaeM_M1 and its close relatives. All-vs-all BLASTp analysis used orthofinder v2.5.4 to compute the percentage of shared genes between phage vB_PaeM_M1 and all complete Pseudomonas phages genomes in the NCBI RefSeq database (Emms and Kelly, 2019).In order to find homologous phages related to vB_PaeM_M1, each coding sequence of vB_PaeM_M1 was queried against the Integrated Microbial Genome/Virus (IMG/VR, v.3) database (E value, <1e-10; identity, >30; and alignment region covering >50%) (Paez-Espino et al., 2017;Paez-Espino et al., 2019;Roux et al., 2021).In vConTACT analysis, the selected sequence was compared with vB_PaeM_M1 as a group to obtain more accurate results (similar sequences were selected by Diamond, all of which satisfied the following parameters: E value, <1e-5; alignment region covering more than 50% of the shorter sequence; and identity >30%) (Zhan and Chen, 2019).The ANI (average nucleotide identity) value of vB_PaeM_M1 was calculated by VIRIDIC, and the results were visualized by pheatmap (Moraru et al., 2020).A correction has been made to RESULTS AND DISCUSSION, Morphology of vB_PsaP_M1, Paragraph 4, This sentence previously stated:A marine phage, named vB_PsaP_M1, that can infect Pseudomonas ZM01 was isolated from a surface seawater sample collected from the coastal waters of Qingdao, Yellow Sea. TEM images show that phage vB_PsaP_M1 has an isometric head (diameter of 90 to 90.9 nm [average ± standard deviation, 55 ± 3 nm]) and a contractible tail (length of 88 to 91 nm [11 ± 3 nm]) (Fig. 1B).The corrected sentence appears below:A marine phage, named vB_PaeM_M1, that can infect Pseudomonas ZM01 was isolated from a surface seawater sample collected from the coastal waters of Qingdao, Yellow Sea. TEM images show that phage vB_PaeM_M1 has an isometric head (diameter of 90 to 90.9 nm [average ± standard deviation, 55 ± 3 nm]) and a contractible tail (length of 88 to 91 nm [11 ± 3 nm]) (Fig. 1B).A correction has been made to RESULTS AND DISCUSSION, Overall genome features, Paragraph 4, This sentence previously stated: vB_PsaP_M1 was a linear, 89,387-bp, double stranded DNA (dsDNA) molecule genome with a GC content of 41.04%.The corrected sentence appears below: vB_PaeM_M1 was a linear, 89,387-bp, double stranded DNA (dsDNA) molecule genome with a GC content of 41.04%.A correction has been made to RESULTS AND DISCUSSION, Overall genome features, Paragraph 5, This sentence previously stated:Most of the genes related to phage packaging are located in the upper reaches of the vB_PsaP_M1 genome.The corrected sentence appears below:Most of the genes related to phage packaging are located in the upper reaches of the vB_PaeM_M1 genome.A correction has been made to RESULTS AND DISCUSSION, Overall genome features, Paragraph 6, This sentence previously stated: By using PhageTerm to determine the end of vB_PsaP_M1, it was found that the packaging process was similar to that of bacteriophage T7, and the cos site was found at the end of the M1 genome (Supplemental Fig S1). Different virus genes have different origins and vB_PsaP_M1 has a special phylogenetic state and evolutionary history.Two AMGs were predicted in the vB_PsaP_M1 genome, i.e. ORF 87 (Phosphate starvation-inducible protein) and ORF 112 (Signal peptide peptidase).The structure related genes are mainly located in the middle reaches of the vB_PsaP_M1 genome.The corrected sentence appears below: By using PhageTerm to determine the end of vB_PaeM_M1, it was found that the packaging process was similar to that of bacteriophage T7, and the cos site was found at the end of the M1 genome (Supplemental Fig S1). Different virus genes have different origins and vB_PaeM_M1 has a special phylogenetic state and evolutionary history.Two AMGs were predicted in the vB_PaeM_M1 genome, i.e. ORF 87 (Phosphate starvation-inducible protein) and ORF 112 (Signal peptide peptidase).The structure related genes are mainly located in the middle reaches of the vB_PaeM_M1 genome.A correction has been made to RESULTS AND DISCUSSION, Phylogeny and comparative genomic analysis of vB_PsaP_M1, Paragraph 6,8,9, This sentence previously stated:In order to study the phylogenetic relationships between vB_PsaP_M1 and other isolated phages, a circular proteome tree was constructed using VipTree based on the whole-genome amino acid sequences PAGE \* Arabic \* MERGEFORMAT 3 of vB_PsaP_M1 and other reference phages (Fig. 3). Thirty-five viruses with the highest similarity to vB_PsaP_M1 were selected to construct a rectangular proteome tree with vB_PsaP_M1 (Fig. 4A) (Nishimura et al., 2017). The results of the phylogenetic analysis show that vB_PsaP_M1 represents a separate cluster far from the other isolated phages. To confirm this finding, six complete phage genome sequences were selected and a comparative genomic analysis was carried out based on tBLASTx with vB_PsaP_M1 (Fig. 4B). Similar to the phylogenetic tree analysis, vB_PsaP_M1 shows a low similarity to other isolated phages and forms a new single clade by itself.Intergenomic comparisons are useful in determining how viruses are related to each other. In order to further find the phylogenetic relationship between vB_PsaP_M1 and other isolated phages, the 35 viruses closest to vB_PsaP_M1 were selected from the phylogenetic tree for nucleotide-based intergenomic analysis (Fig. 5). The heatmap shows that the ratio of shared genes between vB_PsaP_M1 and other isolated phages is very low. When the nucleotide sequence homology of bacteriophages is greater than 70%, ICTV regards them as members of the same genus (Bin Jang et al., 2019). All phylogenetic analyses showed that vB_PsaP_M1 was notably different from other isolated phages and should therefore be classified as a representative of an unknown virus family.Although vB_PsaP_M1 clearly has the characteristics of a new genus/family, it is difficult to base the characteristics of a new genus/family on only one phage. In order to further explore the relationships between vB_PsaP_M1 and other phages, all-vs-all BLASTp were used to search the Integrated Microbial Genome/Virus (IMG/VR, v.3) database. It was found that 11 metagenomic-assembled uncultured viral genomes (UViGs) were similar to vB_PsaP_M1 (Supplemental Data 1). Comparative genomic analysis of vB_PsaP_M1 and vB_PsaP_M1-like viruses showed that metagenomic assembled genome viruses of vB_PsaP_M1-like viruses contained eight core genes. Most of the homologous ORFs throughout these phage genomes are located in the packaging module (portal protein, large subunit terminase and phage DNA packaging) and structural module (Baseplate protein and Structural protein). Based on the shared single white cluster (PC) between genomes, 354 viruses (336 reference sequences of NCBI, vB_PsaP_M1 and 17 UViGs similar to vB_PsaP_M1) were clustered by vConTACT 2.0. The classification of the virus genome is visualized by gephi (Fig. 6A) (Bastian et al., 2009). Using this method, 60 virus clusters were identified in the whole dataset. vB_PsaP_M1 and 17 phages were grouped as a VC (VC_61) from the results of the genome-content-based analysis.To find phylogenetic relationships between gene sequences of vB_PsaP_M1, 50 phages related to vB_PsaP_M1, based on the results of vConTACT, were selected to study their shared gene rate. Based on these results, a heatmap was drawn by orthofinder (Fig. 7A). vB_PsaP_M1 and UViGs contain shared genes and have low association with other viruses, confirming that these phages (VC_61) might be identified as a new family. In addition, the whole-genome phylogeny results show that vB_PsaP_M1 and 11 other environmental viral sequences cluster together (shown in red) and are comparable to identified podovirus genera in ICTV (Fig. 6B). The gene map (Fig. 7B) shows the core genome of VC_61, where ORFs 117 and 127 (shown in green) are conserved genes. ORF 117 is a head tail binding protein, which plays an important role in the packaging of phages found in Pseudomonas phage Zigelbrucke. ORF 127 is a baseplate organization protein, which is a structural protein in phages. It is also a conservative protein with specificity in VC_61. Using BLASTp to search these two conserved proteins in the NR database, it was found that no virus contained both these two proteins at the same time, so the two proteins can be regarded as conserved proteins. In conclusion, all results confirmed that vB_PsaP_M1 and related UViGs are a novel unassigned viral family, while vB_PsaP_M1 is the only isolate in this putative family which is here named Psaeviridae.The corrected sentence appears below:PAGE \* Arabic \* MERGEFORMAT 3In order to study the phylogenetic relationships between vB_PaeM_M1 and other isolated phages, a circular proteome tree was constructed using VipTree based on the whole-genome amino acid sequences of vB_PaeM_M1 and other reference phages (Fig. 3). Thirty-five viruses with the highest similarity to vB_PaeM_M1 were selected to construct a rectangular proteome tree with vB_PaeM_M1 (Fig. 4A) (Nishimura et al., 2017). The results of the phylogenetic analysis show that vB_PaeM_M1 represents a separate cluster far from the other isolated phages. To confirm this finding, six complete phage genome sequences were selected and a comparative genomic analysis was carried out based on tBLASTx with vB_PaeM_M1 (Fig. 4B). Similar to the phylogenetic tree analysis, vB_PaeM_M1 shows a low similarity to other isolated phages and forms a new single clade by itself.Intergenomic comparisons are useful in determining how viruses are related to each other. In order to further find the phylogenetic relationship between vB_PaeM_M1 and other isolated phages, the 35 viruses closest to vB_PaeM_M1 were selected from the phylogenetic tree for nucleotide-based intergenomic analysis (Fig. 5). The heatmap shows that the ratio of shared genes between vB_PaeM_M1 and other isolated phages is very low. When the nucleotide sequence homology of bacteriophages is greater than 70%, ICTV regards them as members of the same genus (Bin Jang et al., 2019). All phylogenetic analyses showed that vB_PaeM_M1 was notably different from other isolated phages and should therefore be classified as a representative of an unknown virus family.Although vB_PaeM_M1 clearly has the characteristics of a new genus/family, it is difficult to base the characteristics of a new genus/family on only one phage. In order to further explore the relationships between vB_PaeM_M1 and other phages, all-vs-all BLASTp were used to search the Integrated Microbial Genome/Virus (IMG/VR, v.3) database. It was found that 11 metagenomic-assembled uncultured viral genomes (UViGs) were similar to vB_PaeM_M1 (Supplemental Data 1). Comparative genomic analysis of vB_PaeM_M1 and vB_PaeM_M1-like viruses showed that metagenomic assembled genome viruses of vB_PaeM_M1-like viruses contained eight core genes. Most of the homologous ORFs throughout these phage genomes are located in the packaging module (portal protein, large subunit terminase and phage DNA packaging) and structural module (Baseplate protein and Structural protein). Based on the shared single white cluster (PC) between genomes, 354 viruses (336 reference sequences of NCBI, vB_PaeM_M1 and 17 UViGs similar to vB_PaeM_M1) were clustered by vConTACT 2.0. The classification of the virus genome is visualized by gephi (Fig. 6A) (Bastian et al., 2009). Using this method, 60 virus clusters were identified in the whole dataset. vB_PaeM_M1 and 17 phages were grouped as a VC (VC_61) from the results of the genome-content-based analysis.To find phylogenetic relationships between gene sequences of vB_PaeM_M1, 50 phages related to vB_PaeM_M1, based on the results of vConTACT, were selected to study their shared gene rate. Based on these results, a heatmap was drawn by orthofinder (Fig. 7A). vB_PaeM_M1 and UViGs contain shared genes and have low association with other viruses, confirming that these phages (VC_61) might be identified as a new family. In addition, the whole-genome phylogeny results show that vB_PaeM_M1 and 11 other environmental viral sequences cluster together (shown in red) and are comparable to identified podovirus genera in ICTV (Fig. 6B). The gene map (Fig. 7B) shows the core genome of VC_61, where ORFs 117 and 127 (shown in green) are conserved genes. ORF 117 is a head tail binding protein, which plays an important role in the packaging of phages found in Pseudomonas phage Zigelbrucke. ORF 127 is a baseplate organization protein, which is a structural protein in phages. It is also a conservative protein with specificity in VC_61. Using BLASTp to search these two conserved proteins in the NR database, it was found that no virus contained both these two proteins at the same time, so the two proteins can be regarded as conserved proteins. In conclusion, all results confirmed that vB_PaeM_M1 and related UViGs are a novel unassigned viral family, while vB_PaeM_M1 is the only isolate in this putative family which is here named Psaeviridae.