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Article

Pijolavirus UFJF_PfSW6 Infection in Pseudomonas fluorescens Induces a Prophage Belonging to a Novel Genus in Peduoviridae Family

by
Pedro Marcus Pereira Vidigal
1,*,
João Mattos Brum
2,
Maryoris Elisa Soto Lopez
3,
Hilário Cuquetto Mantovani
4 and
Humberto Moreira Hungaro
2,*
1
Núcleo de Análise de Biomoléculas (NuBioMol), Campus da UFV, Universidade Federal de Viçosa (UFV), Viçosa 36570-900, MG, Brazil
2
Departamento de Ciências Farmacêuticas, Faculdade de Farmácia, Universidade Federal de Juiz de Fora (UFJF), Juiz de Fora 36036-900, MG, Brazil
3
Departamento de Ingeniería de Alimentos, Universidad de Córdoba, Montería 230002, COR, Colombia
4
Animal and Dairy Sciences Department, University of Wisconsin-Madison (UW-Madison), Madison, WI 53706-1205, USA
*
Authors to whom correspondence should be addressed.
DNA 2024, 4(4), 519-529; https://doi.org/10.3390/dna4040035
Submission received: 14 October 2024 / Revised: 15 November 2024 / Accepted: 3 December 2024 / Published: 5 December 2024
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Abstract

Background/Objectives: This study explores the genome sequencing data from the infection of Pseudomonas fluorescens UFV 041 by the bacteriophage Pijolavirus UFJF_PfSW6, aiming to identify and characterize prophages induced in the host bacterium during the infection. Methods: Scaffolds from sequencing data were analyzed, and reads were mapped to identify potential prophages using phage-to-host coverage metrics. The putative prophage scaffold was annotated, taxonomically classified, and its integration in the host bacterium was verified by PCR amplification of two target genes. We also tested whether mitomycin treatment could induce the prophage to enter the lytic cycle. Results: The prophage UFJF_PfPro was identified with a high phage-to-host coverage ratio. Its genome is 32,700 bp in length, containing 42 genes, 3 terminators, and 11 promoters, with 98.84% completeness. PCR confirmed its integration into P. fluorescens UFV 041, but mitomycin treatment did not induce the lytic cycle. The UFJF_PfPro genome shares 38.60% similarity with the closest lytic phages in the Phitrevirus genus, below genus and species assignment thresholds. A viral proteomic tree clustered UFJF_PfPro with Phitrevirus in a clade representing the Peduoviridae family. Conclusions: The UFJF_PfPro is a prophage integrated into the P. fluorescens UFV 041 genome, but we were unable to induce it to enter the lytic cycle using mitomycin treatment. The genome of UFJF_PfPro encodes all structural proteins typical of the Caudoviricetes class and shares low genomic similarity with species of the genus Phitrevirus, suggesting that UFJF_PfPro represents a new genus and species within the Peduoviridae family.

1. Introduction

Bacteriophages or phages are viruses that infect bacteria and are the most abundant organisms on Earth, found in diverse environments [1]. Although at least four cycles of phage infection have been described [2], they primarily replicate within bacterial cells through two well-characterized modes: the lytic and lysogenic cycles. The lytic cycle involves adhesion, infection, production, and release of new viral particles through the lysis of the host cell. In the lysogenic cycle, the phage genome integrates into the host’s chromosome, becoming a prophage, and is passed on to daughter cells during bacterial replication [3]. The prophage’s state is maintained through regulatory mechanisms that represses genes related to the lytic cycle while allowing the continued expression of certain regulatory and lysogenic conversion genes [4]. Prophages, whether intact or not, can benefit their host by protecting against secondary infections through two major mechanisms: mutual exclusion and superinfection. Mutual exclusion occurs when different phages infect the same host cell, leading to competition between the prophage and lytic phage interfering and preventing one of them replicating [5,6]. Superinfection exclusion occurs when prophages prevent secondary infection by similar phages within the same immunity group by expressing proteins that block their attachment to the host cell [7]. Additionally, the expression of prophage genes can provide competitive advantages to the host through a process known as lysogenic conversion [8]. On the other hand, expressing certain prophage genes represents a metabolic burden in some environments, and switching from the lysogenic to the lytic cycle can be fatal to the host [2,9]. Over successive host generations, prophages can undergo genomic rearrangements such as point mutations, gene deletions, or inactivation, leading to incomplete remnants incapable of switching between lysogenic and lytic cycles [10,11].
In recent years, genome mining and comparative genomics studies have revealed the widespread prevalence of prophages in most bacterial genomes [9,12,13,14]. Prophages can comprise 10–20% of a bacterial genome and often more than one prophage, including intact prophages, is found, a condition known as polylysogeny, [4,15]. However, we only understand a small fraction of the total diversity of prophages from bacterial genomes in terms of genetic content and their role in modulating the host [16].
Although the presence of prophages in bacterial genomes can limit the infection by new phages [17], the induction of intact prophages in the genome or regulation of its gene expression could become an interesting mechanism for microbial inactivation, and modulation of microbial ecology in complex environments [18,19]. Prophage induction can occur spontaneously, but more commonly is triggered by external stressors that damage DNA and activate cellular repair mechanisms, ultimately leading to changes in the repressors of the lytic-lysogenic regulatory genes [20]. Several studies demonstrate that the induction of prophages can be a collateral effect of the use of antimicrobial mechanisms or substances. DNA-damaging agents such as UV light, mitomycin C, and quinolones are frequently used to induce prophages in laboratory conditions [21]. However, some phages cannot be induced under these standard laboratory conditions, suggesting that other mechanisms are involved in prophage induction, particularly in polylysogenic bacteria and complex environments where competition exists [22].
In recent decades, the bioprospecting and application of lytic phages to combat bacteria have increased significantly in various fields, including agriculture, food safety, veterinary practice, and human medicine [23,24,25]. Despite their potential as biocontrol agents, our understanding of the diversity of lytic phages targeting specific bacterial species, such as Pseudomonas fluorescens, remains limited due to the scarcity of characterized isolates and sequenced genomes. Bacteria of the Pseudomonas genus, particularly P. fluorescens, are common contaminants in raw milk, posing significant challenges in dairy production due to their role in product spoilage and biofilm formation [26,27]. Our research group has been exploring several lytic phages able to infect and control P. fluorescens proliferation in dairy industry environments. Among the recent findings, we reported isolating a novel phage species, Pijolavirus UFJF_PfSW6, specifically targeting P. fluorescens, effectively reducing bacterial counts on raw milk [28,29]. Beyond lytic phages, there is a growing interest in characterizing prophage diversity, particularly due to their significant influence on bacterial pathogenesis, resistance to novel phage infections, and microbial ecology [9,16]. However, prophages remain a relatively unexplored topic, particularly within P. fluorescens genomes, representing a ‘viral dark matter’ that warrants further investigation.
In this study, we explore the genome sequencing data of P. UFJF_PfSW6 infecting P. fluorescens UFV 041, investigating the following questions: (i) Are remnants of prophage sequences present in the sequencing data? (ii) Are prophages integrated into the genome of P. fluorescens UFV 041? (iii) Can these prophages be induced? (iv) How conserved are these prophages compared to previously known lytic phages?

2. Materials and Methods

2.1. Pijolavirus UFJF_PfSW6 Infection Sequencing Data and Prophage Screening

We previously isolated the phage UFJF_PfSW6 from dairy industry wastewater and propagated it using Pseudomonas fluorescens UFV 041 as the host bacterium, following the method in [29]. The phage suspension was then subjected to a nuclease mix to remove bacterial DNA and RNA, followed by the precipitation of viral particles. We extracted the phage genomic DNA and sequenced it using the Illumina NovaSeq 6000 platform with a read length of 2 × 150 bp, as detailed in [25]. After quality processing and trimming of the sequencing data, the assembly revealed the scaffold of the lytic phage genome, identifying it as a novel Pijolavirus species. For details on the software and parameters used, see [28].
To further identify putative-induced prophages, we selected the remaining assembled scaffolds, analyzed their features, and mapped the trimmed reads to them using BBMap version 38.76 (https://sourceforge.net/projects/bbmap; accessed on 29 January 2020), looking for high or mid-coverage sequences. The BLASTn tool in BLAST version 2.15.0 (https://blast.ncbi.nlm.nih.gov; accessed on 7 June 2024) aligned the scaffolds to the sequences from Bacteria (taxonomy ID 2) and Viruses (taxonomy ID 10239) available in the NCIB Nucleotide collection (nt) database (https://www.ncbi.nlm.nih.gov/nucleotide; accessed on 7 June 2024), selecting as significant alignments those with a maximum E-value score of 1 × 10−10.

2.2. Prophage Genome Annotation and Taxonomic Assignment

The Prokka version 1.14.6 [30] was applied to predict the genes of the putative-prophage scaffold using the following parameters: kingdom: viruses, gcode: 11, and cdsrnaolap. Next, the BLASTp tool of BLAST was used to align the proteins encoded by the predicted genes to the sequences from Bacteria (taxonomy ID 2) and Viruses (taxonomy ID 10239) available in the NCBI Non-redundant protein database (https://www.ncbi.nlm.nih.gov/protein/; accessed on 7 June 2024). A maximum E-value score of 1 × 10−10 was considered to select the significant alignments. In addition, the completeness of the putative-prophage scaffold was assessed with CheckV version 1.0.3 [31] using the default parameters.
To characterize the prophage regulatory elements, we scanned the scaffold using PhagePromoter (https://galaxy.bio.di.uminho.pt; accessed on 7 June 2024) [32], targeting the Podoviridae family, the Pseudomonas host, and the phage type temperate.
The ARNold [33] (http://rssf.i2bc.paris-saclay.fr/toolbox/arnold, accessed on 31 July 2024) and FindTerm version 2.8.1 [34] tools allowed the identification of Rho-independent terminators by selecting a maximum free energy threshold value of −11 kcal/mol for stem-loop regions.
The taxonomic assignment of the putative prophage followed the criteria proposed by the members of the Bacterial Viruses Subcommittee of the International Committee on Taxonomy of Viruses (ICTV) [35], which suggests a minimum genomic similarity of 70% for the genus assignment and 95% for species. We compared the prophage with the genome sequences that significantly aligned to it and with the reference genomes of bacteriophages from the Caudoviricetes class (taxonomy ID 2731619), filtering the ones that infect bacteria from Pseudomonas genus, available in the NCBI RefSeq database using the VIRIDIC tool version 1.1 [36] to calculate the genomic similarity scores. We also used this set of Pseudomonas phages’ reference genomes to generate a viral proteomic tree using the ViPTree web server (https://www.genome.jp/viptree/, accessed on 22 August 2024) [37]. Then, we assessed the synteny and modular organization of the prophage genome, aligning it with the most similar genomes using Clinker version 0.0.23 [38].

2.3. Validation of the Prophage Integration into Host Bacterium

To confirm the integration of the prophage scaffold into the genome of P. fluorescens UFV 041, we carried out a polymerase chain reaction (PCR) to target and amplify two genes that encode the integrase (gene g04) and phage tail protein (gene g35). Two sets of primers, Int-F (5′-GCGAAATGAAGGGGTTTGGT-3′) and Int-R (5′-TGACTAGGTGATTCGGCAGG-3′), as well as Tail-F (5′-TCCTGGCGGTTTTCACTACT-3′) and Tail-R (5′-GCCATAGCCTTCAAGTGTCG-3′), were designed using Primer3web version 4.1.0 (https://primer3.ut.ee/; accessed on 7 June 2024) with default parameters. Additionally, the primers’ specificity was assessed against the genomes of Bacteria and Viruses taxa available in the NCBI Nucleotide database using the PrimerBLAST tool (https://www.ncbi.nlm.nih.gov/tools/primer-blast/; accessed on 7 June 2024).
To perform the PCR amplification, P. fluorescens UFV 041 was cultivated overnight in Tryptic Soy Broth (TSB) at 30 °C. Genomic DNA extraction used the Wizard® Genomic DNA Purification Kit following the manufacturer’s instructions (Promega Corporation; Madison, WI, USA). The PCR procedure amplified each gene in 25 µL reactions containing the following components: 2.5 µL of 10× PCR buffer (Sinapse Inc.; São Paulo, Brazil), 2 mM MgCl2, 0.8 mM dNTPs (Uniscience; São Paulo, Brazil), 0.4 µM of each primer, 1 unit of Taq DNA polymerase (Sinapse Inc.; São Paulo, Brazil), and 2 µL of extracted DNA. The amplification used the following thermal cycling conditions: an initial denaturation step at 95 °C for 5 min, followed by 30 cycles of denaturation at 94 °C for 60 s, annealing at 53.9 °C for 60 s, and extension at 72 °C for 120 s. A final extension step at 72 °C for 5 min was also performed. The electrophoresis separated aliquots of 2 µL of the PCR products in a 1.5% agarose gel. The gel was run for 60 min at 100 volts, stained with ethidium bromide solution (0.5 µg/mL) for 30 min, destained in water for one h, and visualized using a UV gel documentation system (Bio-Rad Gel Doc™ XR+; Bio-Rad Laboratories, Hercules, CA, USA). We used a 100-bp DNA ladder (Ludwig Biotecnologia; Alvorada, Brazil) as a molecular weight marker for the PCR products. We also used the Pseudomonas aeruginosa ATCC 25619 as a negative control in the PCR reactions for both genes.

2.4. Assessment of the Prophage Induction and Lytic Conversion

To verify whether the prophage detected in the bacterial genome could be induced and retain infectivity, we employed a methodology adapted from Ambroa et al. [39]. Briefly, P. fluorescens UFV 041 was grown overnight in TSB broth at 30 °C and diluted in broth TSB (1:10). Then, the culture was grown at 30 °C with 100 rpm shaking until reaching a DO600 of 0.5 before being treated with mitomycin C (MMC) at a concentration of 10 µg/mL. After incubation at 30 °C and 100 rpm shaking for 6 h, the lysate was centrifuged at 5000× g for 10 min. The supernatant was filtered through a 0.22 µm filter (Millipore, Burlington, MA, USA). The drop-on-lawn technique [40] determined the presence of inducible phage in the filtrate using P. fluorescens UFV 041 as the host bacterium. Briefly, 500 µL of P. fluorescens UFV 041, previously grown overnight in TSB at 30 °C, was mixed with 5 mL of soft agar (TSB with 0.7% agar) and poured onto Petri dishes containing Tryptic Soy Agar (TSA). After solidification, 20 µL of filtered supernatant containing the induced prophage was spotted on the soft agar surface. After inoculum absorption, the plates were incubated at 30 °C for 24 h and examined for lysis plaques.

3. Results

3.1. Unveiling a Novel Prophage of the Peduoviridae Family

The sequencing data analysis of Pijolavirus UFJF_PfSW6 infecting P. fluorescens UFV 041 revealed one scaffold sequence from a putative prophage. While the P. UFJF_PfSW6 genome of 38,758 bp showed a high sequence coverage of 28,048, after mapping the trimmed reads, the remaining 355 scaffolds, primarily remnants from the host bacterium, ranged in size from 128 to 41,069 bp, and showed an average coverage of 5-fold. Notably, one scaffold with 32,700 bp stood out with a 628-fold coverage (Supplementary Materials, Table S1), corresponding to a phage-to-host bacteria sequencing coverage ratio of 125. This scaffold, the only one showing features of bacteriophage genomes, was named UFJF_PfPro and selected for further comparison.
The UFJF_PfPro scaffold corresponds to a double-strand DNA genome showing a GC context of 58.3%, containing 42 genes, 3 terminators, and 11 promoters (7 host- and 4 phage-promoters) (Figure 1 and Tables S2, S5 and S6). The terminators are positioned at the ends of their respective genome strands, with one on the positive strand and two on the negative strand, while the promoters are located at both ends of the genome strands. Among the proteins encoded by the predicted genes, 27 significantly aligned with other proteins and had their function assigned. In contrast, we annotated 18 proteins as hypothetical proteins as they did not align with viral or bacterial proteins. In addition, CheckV classified the UFJF_PfPro scaffold as a high-quality viral genome with a completeness of 98.84%.
Similarity searches for the UFJF_PfPro against the Viruses taxa genomes in the NCBI nucleotide database revealed significant alignments with low query coverage (14%) and sequence identity (76.03%) with two bacteriophages of the Phitrevirus genus from the Peduoviridae family: phages phi3 (Genbank accession NC_030940) and PaBSM-2607-JFK (accession OQ849765) (Table S3). In contrast, UFJF_PfPro showed a higher query coverage (average of 63%) and identity (average of 86.28%) when compared with Bacteria genomes from P. fluorescens and other Pseudomonas species. The genome of Pseudomonas sp. WCS374 (accession CP007638.1) exhibited a genomic region with the highest value for the coverage × identity product (55.72%) (Table S3).
The taxonomy analysis using the VIRIDC software compared the UFJF_PfPro scaffold with these genomes and the reference genomes of Caudoviricetes (Table S7) to calculate pairwise genomic similarity scores. All calculated scores are below the threshold recommended for genus (70%) and species (95%) assignments. The known taxon with the highest scores was the Phitrevirus genus, with an average genomic similarity score of 38.60%. In addition, UFJF_PfPro shares a genomic similarity score of 49.78% with the putative prophage genomic region of Pseudomonas sp. WCS374. The synteny analysis showed that the UFJF_PfPro prophage has a modular organization typical for bacteriophages (Figure 1), sharing it with the genomes mentioned above. Despite the lower similarity among the genomes, 27 proteins, mainly those with structural functions, share an average amino acid sequence similarity of 76.82% (Table S8).
The viral proteomic tree of Pseudomonas phages of the Caudoviricetes class also clustered the UFJF_PfPro prophage into a monophyletic clade containing reference genomes of the Peduoviridae family and from of the Phitrevirus and Citexvirus genera (Figure 2).

3.2. The UFJF_PfPro Is Integrated into P. fluorescens UFV 041 Genome

We assessed the integration of the UFJF_PfPro prophage into its likely host bacterium by amplifying PCR products of two target genes: the integrase (gene g04) and the phage tail protein (gene g35). The PCR successfully amplified both genes and confirmed their integration into the P. fluorescens UFV 041 genome (Figure 3). Amplicons with the expected sizes were obtained for the designed primers: 488 bp for the gene g04 and 253 bp for the gene g35. Importantly, we did not obtain PCR products for P. aeruginosa ATCC 25619 used as the negative control.

3.3. Mitomycin Treatment Did Not Induce the UFJF_PfPro on Its Host Bacterium

We attempted to induce the UFJF_PfPro prophage by treating the P. fluorescens UFV 041 host with mitomycin C during its logarithmic growth phase. However, no lysis plaques were observed on the bacterial lawn from lysates, indicating that the prophage was either not induced by this treatment or was unable to infect the host cell.

4. Discussion

Raw sequencing data from lytic bacteriophages are valuable for identifying induced prophages, based on the assumption that, in addition to lytic phages, they will outnumber the host bacterium sequence counts [41]. Estimating phage-to-host ratios provides a method to calculate a prophage’s lytic potential by assessing the sequence abundances or coverages of prophages and the bacterial genomes in which they are integrated [41,42]. Ratios greater than 10 can reliably identify induced prophages [41,42]. Applying these principles to our study, we analyzed the sequencing data of Pijolavirus UFJF_PfSW6 in P. fluorescens UFV 041 [28]. Our analysis revealed scaffolds with low sequence coverage that are remnants of the host bacterium. However, one scaffold, named UFJF_PfPro, showed a high coverage corresponding to a phage-to-host ratio of 125 (Table S1), which led us to speculate that UFJF_PfPro is a prophage integrated into P. fluorescens UFV 041 induced by Pijolavirus UFJF_PfSW6 infection. This induction of UFJF_PfPro suggests a mechanism distinct from mutual exclusion, in which lytic infection by the Pijolavirus UFJF_PfSW6 partially activated UFJF_PfPro, resulting in the production of genomic copies of both the lytic phage and the prophage. A similar phenomenon of prophage induction following secondary infection has also been observed in Vibrio cholerae [43].
The UFJF_PfPro sequence has a gene repertoire showing high completeness regarding the expected genes for the bacteriophage genomes. It includes a gene encoding an integrase (gene g04) at the beginning of its sequence, as is typical for many prophages. However, identifying one integrase gene alone cannot confirm a genomic element as a prophage [4]. The sequence also contains a set of genes encoding structural proteins for the virion assembly that correspond to those expected for the typical head-tail architecture of bacteriophages of the Caudovirecetes class [44]. Additionally, the UFJF_PfPro has two lytic enzymes for the bacterium host’s lysis and the release of viral particles. Most of these genes are likely under regulation by the predicted phage and host promoters and Rho-independent terminators, reflecting the expected genetic and regulatory elements necessary to produce viable viral particles upon induction of the lytic cycle. This reinforces the hypothesis that UFJF_PfPro is a prophage.
We confirmed the integration of UFJF_PfPro into the genome of P. fluorescens UFV 041 genome by amplifying the integrase gene (g04) and one of the tail fiber protein genes (g35) via PCR. We chose these two genes as evidence of the recombination event of the UFJF_PfPro into the host genome and as markers of one of its structural components responsible for host recognition during infection. To test if UFJF_PfPro is an inducible prophage, we stressed the bacterium host with mitomycin, which is a DNA-damaging agent that induces bacterial SOS responses and triggers the lysogenic-to-lytic transition in bacteriophages. This process typically results in prophages excising from the host genome to produce viral particles [22]. However, our treatment did not induce the lytic cycle of UFJF_PfPro, nor did it produce viral particles capable of infecting P. fluorescens UFV 041.
Upon confirming that UFJF_PfPro is a prophage, we compared its genome to other bacteriophage isolates and putative prophages integrated into bacterial genomes. The sequence of UFJF_PfPro shares low genomic similarity with other isolates of Phitrevirus from the Peduoviridae family and putative prophages of Pseudomonas sp. genomes, falling below the reference thresholds for bacteriophage genus and species assignment. Despite the low genomic similarity, the synteny analysis showed that UFJF_PfPro shares a modular organization with these genomes and a higher sequence similarity for the structural proteins, as expected for bacteriophages infecting hosts from the same genus. The viral proteomic tree of Pseudomonas phages of the Caudoviricetes class corroborated the comparative genomic analysis and allowed the assignment of UFJFPfPro to the Peduoriviridae family. Therefore, the UFJF_PfPro represents a new genus and species within the known taxa of the Peduoviridae family.
Prophage elements have been reported in other Pseudomonas fluorescens strains, such as P. fluorescens DS206 [45] and P. fluorescens Pf-5 [46]. However, this study is the first to detect an inducible prophage in P. fluorescens triggered by the infection of another lytic bacteriophage, Pijolavirus UFJF_PfSW6, employing a comprehensive sequencing data analysis. By thoroughly taxonomically classifying the prophage UFJF_PfPro, we expanded the diversity of bacteriophage infecting P. fluorescens and opened new perspectives for exploring prophage diversity, aiming to target their potential biotechnological applications.

5. Conclusions

Our data confirmed the presence of the UFFJF_PfPro prophage in the sequencing data of the Pijolavirus UFJF_PfSW6 infecting Pseudomonas fluorescens the UFV 041. The UFFJF_PfPro prophage is integrated into the genome of P. fluorescens, but we were unable to induce the lytic cycle using mitomycin. UFFJF_PfPro contains all the genes encoding structural proteins characteristic of the head-tail morphology of the Caudoviricetes class. It shares low genomic similarity with bacteriophage species of the genus Phitrevirus from the Peduoviridae family, indicating that UFJF_PfPro represents a new genus and species taxa within this family.

Supplementary Materials

The following supporting information can be downloaded at https://www.mdpi.com/article/10.3390/dna4040035/s1, Table S1: Coverage metrics of the scaffolds from sequencing data of Pijolavirus UFJF_PfSW6 infecting Pseudomonas fluorescens UFV 041. Table S2: Annotation of the P. fluorescens UFJF_PfPro genome. Table S3: BLASTN search results for Viral and Bacterial genomes from the Nucleotide Database (nt) similar to the UFJF_PfPro genome. Table S4: BLASTP search results for Viral and Bacterial proteins from the Non-Redundant Protein Database (nr) similar to UFJF_PfPro proteins. Table S5: Identification of Rho-Independent Terminators in the UFJF_PfPro genome. Table S6: Identification of Phage and Host promoters in the UFJF_PfPro genome. Table S7: Completeness of the UFJF_PfPro genome as assessed by CheckV software. Table S8: Intergenomic similarities between the UFJF_PfPro genome and reference genomes for Pseudomonas phages of Caudoviricetes class (taxonomy ID 2731619) available in the NCBI Reference Sequence (RefSeq) database (https://www.ncbi.nlm.nih.gov/refseq). The genomes were aligned pairwise using ViPTree and VIRIDIC. Table S9: Pairwise protein similarities between UFJF_PfPro and its closest related bacteriophages.

Author Contributions

Conceptualization, P.M.P.V. and H.M.H.; methodology, P.M.P.V., J.M.B. and H.M.H.; software, P.M.P.V. and H.C.M.; validation, J.M.B. and H.M.H.; formal analysis, P.M.P.V. and H.M.H.; investigation, all authors; resources, P.M.P.V. and H.M.H.; data curation, P.M.P.V.; writing—original draft preparation, P.M.P.V. and H.M.H.; writing—review and editing, M.E.S.L., H.C.M. and H.M.H.; visualization, P.M.P.V.; supervision, H.C.M. and H.M.H.; project administration, P.M.P.V. and H.M.H.; funding acquisition, P.M.P.V. and H.M.H. All authors have read and agreed to the published version of the manuscript.

Funding

This research was funded by Fundação de Amparo à Pesquisa do Estado de Minas Gerais (FAPEMIG), grant number APQ-00146-22, and Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq), grant number 200773/2024-0.

Institutional Review Board Statement

Not applicable.

Informed Consent Statement

Not applicable.

Data Availability Statement

The complete genome sequence of the Pseudomonas fluorescens prophage UFFJF_PfPro was deposited on DDBJ/EMBL/GenBank under accession number PQ412991.

Acknowledgments

We thank the Núcleo de Análise de Biomoléculas (NuBioMol) of the Universidade Federal de Viçosa (UFV) and the Department of Animal and Dairy Sciences of the University of Wisconsin-Madison for providing the facilities for data analysis. We also thank the Collection of Reference Bacteria on Health Surveillance of the Oswaldo Cruz Foundation (FIOCRUZ/CBRVS) for providing the bacterial strains used in this work.

Conflicts of Interest

The authors declare no conflicts of interest.

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Figure 1. Schematic representation of the P. fluorescens prophage UFJF_PfPro compared to similar prophage and bacteriophage genomes. The genome of the UFJF_PfPro prophage (GenBank accession: PQ412991; shown at the top) exhibits a bidirectional organization with 42 genes, represented by arrows, and is organized into 3 primary functional modules: DNA replication and metabolism, structural proteins, and DNA packaging and host lysis, depicted by colored bars in the background. Each arrow represents an individual gene encoding a protein with a predicted function, as detailed in the annotation list above the genome map. Arrow colors and connecting lines between genes indicate putative homologous genes shared among the genomes, with encoded proteins showing more than 40% sequence identity. Black arrows indicate the integrase gene in the prophages. For a detailed summary of pairwise sequence identities and similarities, refer to Table S9. The genomic segment of Pseudomonas sp. WCS374 (CP007638) represents a putative prophage, while the others are lytic bacteriophages of the Phitrevirus genus (OQ849765 and NC_030940), selected for their similarity to the UFJF_PfPro genome. Vertical bars represent the predicted regulatory elements in UFJF_PfPro genome, each colored according to its category, with dots above the bars indicating elements located on the negative strand.
Figure 1. Schematic representation of the P. fluorescens prophage UFJF_PfPro compared to similar prophage and bacteriophage genomes. The genome of the UFJF_PfPro prophage (GenBank accession: PQ412991; shown at the top) exhibits a bidirectional organization with 42 genes, represented by arrows, and is organized into 3 primary functional modules: DNA replication and metabolism, structural proteins, and DNA packaging and host lysis, depicted by colored bars in the background. Each arrow represents an individual gene encoding a protein with a predicted function, as detailed in the annotation list above the genome map. Arrow colors and connecting lines between genes indicate putative homologous genes shared among the genomes, with encoded proteins showing more than 40% sequence identity. Black arrows indicate the integrase gene in the prophages. For a detailed summary of pairwise sequence identities and similarities, refer to Table S9. The genomic segment of Pseudomonas sp. WCS374 (CP007638) represents a putative prophage, while the others are lytic bacteriophages of the Phitrevirus genus (OQ849765 and NC_030940), selected for their similarity to the UFJF_PfPro genome. Vertical bars represent the predicted regulatory elements in UFJF_PfPro genome, each colored according to its category, with dots above the bars indicating elements located on the negative strand.
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Figure 2. Taxonomic classification of P. fluorescens prophage UFJF_PfPro within the Peduoviridae family. The viral proteomic tree (above) includes prophage UFJF_PfPro, the putative prophage of Pseudomonas sp. WCS374, and 298 reference genomes of Pseudomonas phages from the Caudoviricetes class (taxonomy ID 2731619) available in the NCBI Reference Sequence (RefSeq) database (listed in Table S8). The genomes were aligned pairwise to calculate genomic similarity scores using VIRIDIC and ViPtree. The highlighted cluster (above) represents the clade of lytic phages within the Peduoviridae family, which includes UFJF_PfPro. The table lists the similarity scores of the genomes calculated compared to the UFJF_PfPro prophage. A genomic similarity score of 95% is the threshold for species assignment, and 70% is for genus assignment.
Figure 2. Taxonomic classification of P. fluorescens prophage UFJF_PfPro within the Peduoviridae family. The viral proteomic tree (above) includes prophage UFJF_PfPro, the putative prophage of Pseudomonas sp. WCS374, and 298 reference genomes of Pseudomonas phages from the Caudoviricetes class (taxonomy ID 2731619) available in the NCBI Reference Sequence (RefSeq) database (listed in Table S8). The genomes were aligned pairwise to calculate genomic similarity scores using VIRIDIC and ViPtree. The highlighted cluster (above) represents the clade of lytic phages within the Peduoviridae family, which includes UFJF_PfPro. The table lists the similarity scores of the genomes calculated compared to the UFJF_PfPro prophage. A genomic similarity score of 95% is the threshold for species assignment, and 70% is for genus assignment.
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Figure 3. Detection of UFJF_PfPro genes in P. fluorescens UFV 041. The presence of the genes encoding the integrase (g04) and phage tail protein (g35) from the UFJF_PfPro genome was verified by amplifying their segments from bacterial genomic DNA samples using PCR primers designed in this study. Successful amplification of g04 (488 bp; lane 2) and g35 (253 bp; lane 5) was observed for P. fluorescens UFV 041. No amplification product was detected in the negative control using P. aeruginosa ATCC 25619 (lanes 3 and 6), confirming the specificity of the PCR. A molecular weight marker (100 bp DNA ladder with bands from 100 to 1000 bp and two additional bands at 1500 bp and 2080 bp; Ludwig Biotecnologia, Alvorada, Brazil) served as a reference to confirm the size of the amplicons (lanes 1 and 4).
Figure 3. Detection of UFJF_PfPro genes in P. fluorescens UFV 041. The presence of the genes encoding the integrase (g04) and phage tail protein (g35) from the UFJF_PfPro genome was verified by amplifying their segments from bacterial genomic DNA samples using PCR primers designed in this study. Successful amplification of g04 (488 bp; lane 2) and g35 (253 bp; lane 5) was observed for P. fluorescens UFV 041. No amplification product was detected in the negative control using P. aeruginosa ATCC 25619 (lanes 3 and 6), confirming the specificity of the PCR. A molecular weight marker (100 bp DNA ladder with bands from 100 to 1000 bp and two additional bands at 1500 bp and 2080 bp; Ludwig Biotecnologia, Alvorada, Brazil) served as a reference to confirm the size of the amplicons (lanes 1 and 4).
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Vidigal, P.M.P.; Brum, J.M.; Lopez, M.E.S.; Mantovani, H.C.; Hungaro, H.M. Pijolavirus UFJF_PfSW6 Infection in Pseudomonas fluorescens Induces a Prophage Belonging to a Novel Genus in Peduoviridae Family. DNA 2024, 4, 519-529. https://doi.org/10.3390/dna4040035

AMA Style

Vidigal PMP, Brum JM, Lopez MES, Mantovani HC, Hungaro HM. Pijolavirus UFJF_PfSW6 Infection in Pseudomonas fluorescens Induces a Prophage Belonging to a Novel Genus in Peduoviridae Family. DNA. 2024; 4(4):519-529. https://doi.org/10.3390/dna4040035

Chicago/Turabian Style

Vidigal, Pedro Marcus Pereira, João Mattos Brum, Maryoris Elisa Soto Lopez, Hilário Cuquetto Mantovani, and Humberto Moreira Hungaro. 2024. "Pijolavirus UFJF_PfSW6 Infection in Pseudomonas fluorescens Induces a Prophage Belonging to a Novel Genus in Peduoviridae Family" DNA 4, no. 4: 519-529. https://doi.org/10.3390/dna4040035

APA Style

Vidigal, P. M. P., Brum, J. M., Lopez, M. E. S., Mantovani, H. C., & Hungaro, H. M. (2024). Pijolavirus UFJF_PfSW6 Infection in Pseudomonas fluorescens Induces a Prophage Belonging to a Novel Genus in Peduoviridae Family. DNA, 4(4), 519-529. https://doi.org/10.3390/dna4040035

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