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Communication

Unveiling Hidden Allies: In Silico Discovery of Prophages in Tenacibaculum Species

by
Carolina Ramírez
and
Jaime Romero
*
Laboratorio de Biotecnología de Alimentos, Instituto de Nutrición y Tecnología de los Alimentos (INTA), Universidad de Chile, El Líbano 5524, Santiago 7830489, Chile
*
Author to whom correspondence should be addressed.
Antibiotics 2024, 13(12), 1184; https://doi.org/10.3390/antibiotics13121184
Submission received: 30 October 2024 / Revised: 20 November 2024 / Accepted: 23 November 2024 / Published: 5 December 2024
(This article belongs to the Special Issue Antibiotics and Antimicrobial Resistance in Aquaculture: Alternative and Complementary Treatment Methods)
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Abstract

:
Tenacibaculosis, caused by Tenacibaculum species, is a significant disease in aquaculture, leading to high mortality and economic losses. Antibiotic treatment raises concerns about resistance, making phage therapy an interesting alternative. Analyzing phage traces in Tenacibaculum genomes is crucial for developing these bacteriophage-based strategies. Methods: We assessed the presence of prophages in 212 Tenacibaculum genomes/assemblies available in the NCBI repository, comprising several species and global locations, using the PHASTEST program. Then, we focused on those regions classified as intact, evaluating the most common phages found using VICTOR. The protein of interest discovered in the prophages was evaluated using the ProtParam, DeepTMHMM, InterPro, and Phyre2 tools. In addition, we evaluated the presence of antiphage defense systems in those genomes with intact prophages using the DefenseFinder tool. Results: We identified 25 phage elements in 24 out of the 212 Tenacibaculum genomes/assemblies analyzed, with 11% of the assemblies containing phage elements. These were concentrated in T. maritimum and T. mesophilum, which harbored 10 and 7 prophage regions, respectively. Of the identified elements, six were classified as intact, including four in T. maritimum, with the most common phages belonging to the Pippivirus and Siphoviridae families. Bioinformatic analysis showed that the putative endolysin is a stable protein of 432 amino acids and 49.8 kDa, with three transmembrane helices and a CHAP domain, structurally similar to the CHAP lytic domain of S. aureus bacteriophage K. Conclusions: Key prophage elements in Tenacibaculum, especially in T. maritimum, show promise for phage therapy against tenacibaculosis, supporting sustainable, antibiotic-free treatments in aquaculture.

1. Introduction

Tenacibaculum is a genus of Gram-negative bacteria belonging to the Flavobacteriaceae family in the phylum Bacteroidetes, commonly found in marine environments and affecting various aquatic organisms, primarily fish. This bacterium is known for causing skin diseases in fish, such as tenacibaculosis, characterized by skin lesions, ulcers, and tissue necrosis. This pathogen is a significant concern in aquaculture, particularly in the farming of marine fish, as it impacts both fish health and the profitability of production [1].
The genus Tenacibaculum comprises more than 30 distinct species, of which eight have been linked to diseases in fish [2]. Tenacibaculosis, the disease caused by this genus, affects a variety of fish species, including Atlantic salmon (Salmo salar), European seabass (Dicentrarchus labrax), sole (Solea senegalensis), and turbot (Scophthalmus maximus) [3,4,5]. The majority of existing studies on Tenacibaculum species link disease in marine fish to infections caused by Tenacibaculum maritimum [2,3].
Tenacibaculum maritimum was formally described by Suzuki in 2001 [6], following its initial isolation from fish suffering from skin diseases. Prior to this definitive classification, bacteria within this genus were commonly categorized as part of the “Flexibacter group” due to their connection with the genus Flexibacter, which also belongs to the Flavobacteriaceae family. Since then, mortality events linked to Tenacibaculum have been on the rise and spreading worldwide [7]. In 2023, mortality rates among salmon in the grow-out phase in Norway reached unprecedented levels, largely due to disease syndromes like winter ulcer disease and complex gill disease. Winter ulcer disease, primarily caused by Moritella viscosa and Tenacibaculum species, represents the most significant bacterial health and welfare challenge in Norwegian aquaculture. Furthermore, Spilsberg 2022 [8] reported a strong association between the predominant presence of T. finnmarkense and the development of severe cranial lesions in Atlantic salmon smolts that had recently been transferred to the sea. Meanwhile, in Chile, the second-largest producer of salmonids after Norway, the health authority has recently reported that, in terms of infectious mortality in Atlantic salmon, tenacibaculosis ranks second, accounting for 37.9% of cases. Compared to 2022, tenacibaculosis has seen an increase of 9% [9].
Currently, no widely available and effective commercial vaccines exist for controlling Tenacibaculum maritimum, the primary causative agent of tenacibaculosis in marine fish. The only commercial vaccine available is ICTHIOVAC® TM, an inactivated injectable vaccine for tenacibaculosis in turbot [10]. Due to the bacterium’s complexity and the variable conditions in aquaculture, infection prevention and management typically depend on good husbandry practices, environmental controls, and, in some cases, antibiotic treatments.
Bacteriophages have been proposed as antimicrobial agents to combat pathogenic bacteria in various fields, including aquaculture. Research has highlighted numerous examples of phage applications aimed at controlling pathogenic vibrios, which pose significant challenges in aquaculture [11,12,13]. Additionally, the bacteriophage-based product Curtus has been developed specifically to address Yersinia infections in aquaculture settings, offering a targeted and eco-friendly approach to managing this pathogen and improving fish health [14]. Recently, Tsertou [15] reported the isolation and characterization of a bacteriophage against Tenacibaculum larymnensis sp. nov., a novel species within the Tenacibaculum genus, which was isolated from a commercial fish hatchery in Greece.
In this context, this research aims to deepen our understanding of the interactions between Tenacibaculum host bacteria and their corresponding bacteriophages through genomic analysis, with a particular focus on the bacterial defense mechanisms [16]. By elucidating these relationships, the study endeavors to identify novel insights that may contribute to the formulation of effective strategies for the control of these pathogenic bacteria.

2. Results

We identified 25 phage elements in 24 out of the 212 Tenacibaculum genomes/assemblies analyzed, representing approximately 11% of the total assemblies. These elements were predominantly found in two species, T. maritimum and T. mesophilum, which harbored 10 and 7 prophage regions, respectively (Table 1). Notably, this distribution is not due to a larger number of strains analyzed for these species; 40 assemblies of T. maritimum and 10 of T. mesophilum were examined. In contrast, T. finnmarkense, the most represented species in the dataset with 129 assemblies, revealed only a single assembly containing phage-related elements.
Based on the classification provided by PHASTEST, of the 25 identified phage elements, 6 were categorized as intact, 8 as questionable, and 11 as incomplete. Notably, four of the intact phages are found in T. maritimum. Among the intact prophage regions, the most frequently identified phages include two associated with Flavobacterium—vB_FspM_pippi8-1 (NC_048830) and vB_FspM_lotta8-1 (NC_048829)—and two associated with Cellulophaga—phi19:1 (NC_021799) and phi39:1 (NC_021804). The former two belong to the family Pippivirus, whereas the latter two are classified under Siphoviridae.
For the phylogenetic analysis of the most common phages from intact regions and the previously described Tenacibaculum phages (PTm1 and PTm5), the OPTSIL clustering results (Figure 1) show that these phages cluster differently. The prophages found in this study form distinct groups from those described in earlier research. Specifically, three clusters are observed at the family level and four clusters at the subfamily, genus, and species levels. Additionally, the previously described Tenacibaculum phages have notably longer sequences compared to those identified in this study.
A total of 101 phage-associated CDS were identified among the 6 intact prophages (Supplementary Table S1). These were grouped according to their description in the BLAST hit (Figure 2), which highlighted that most of the proteins present in the different Tenacibaculum strains correspond to structural or hypothetical proteins (ranging from 14 to 39%). The coding sequences identified belong to phages of the class Caudoviricetes, except for one, which is unclassified (Supplementary Table S1). Therefore, it is possible to note several tail-associated proteins (tail tube, tail protein, and phage-related minor tail protein, among others). Other relevant components were also found, such as terminase and endolysin, which are involved in the phage DNA packaging and infection-related process, respectively (Figure 2).
A putative endolysin emerged as a protein of interest in this analysis. It was present in two of the six strains with intact classified prophages, T. maritimum strain FC and NBRC 15946. According to the BLAST hit, this protein showed homology with an endolysin described in Streptomyces phage TG1 (NC_018853). ProParam analyses showed that the putative endolysin, a protein of 432 amino acids in length, has a molecular weight of 49.8 kDa and an instability index of 34.38, which classifies the protein as stable. In addition, transmembrane helix prediction showed three TMhelix sites in the putative endolysin, positioned between amino acids 12–29, 48–66, and 100–109. Moreover, the results of the InterPro analysis showed the presence of the CHAP (cysteine, histidine-dependent amidohydrolases/peptidases) domain at amino acid positions 158–246 of the putative endolysin (Supplementary Figure S1). Structural prediction and homology modeling of the putative endolysin using the Phyre2 server showed that the CHAP domain had similar folding topology to that of the CHAP lytic domain of endolysin k from Staphylococcus aureus bacteriophage k, with 98.37% confidence but with a low percentage identity of 19% (Supplementary Figure S2).
In addition, we evaluated the antiphage systems present in Tenacibaculum genomes that had regions with prophages classified as intact. The results showed an average of 10 defense systems per strain. The systems present in the six genomes were the restriction-modification (R-M) and Mokosh systems, whereas CRISP-Cas was only present in the four genomes of T. maritimum (Figure 3). The results also highlighted that, in general, the R-M system was present in greater proportions in the genomes of the strains (Supplementary Table S2).

3. Discussion

The distribution of prophage elements observed in Tenacibaculum species reveals a selective presence of phage elements, with notable concentrations in T. maritimum and T. mesophilum. This distribution pattern, where prophage elements are concentrated in specific species rather than in the species with the highest representation in the dataset (e.g., T. finnmarkense, which showed minimal prophage presence despite its 129 assemblies), suggests a unique evolutionary or ecological adaptation in T. maritimum and T. mesophilum. This trend is consistent with studies in other bacterial genera, where prophage presence varies significantly between species within the same genus, often influenced by niche-specific factors or host interactions [17]. Similar results were described by [18,19]. In Mycobacterium strains [18], the distribution of prophages demonstrates notable variability across taxa, with most strains within five major groups lacking prophages entirely, in contrast to the M. abscessus-chelonae complex, where approximately 75% of strains contain at least one prophage. This pattern reflects the significant role of ecological and evolutionary pressures in shaping prophage retention and loss within specific bacterial lineages, suggesting that niche specialization or host interactions may influence prophage prevalence in certain taxa more than others [20]. The low prophage prevalence in T. finnmarkense might suggest reduced exposure to phage interactions or differing survival strategies in its ecological niche. Overall, this distribution underlines how prophage content can reflect bacterial adaptation and highlights T. maritimum and T. mesophilum as potential candidates for exploring phage-based control strategies in aquaculture due to their higher prophage load and potential phage susceptibility.
The diversity observed among Tenacibaculum phages, with distinct clusters at the family, subfamily, genus, and species levels, reflects a broad genetic variability that has also been reported in other bacterial genera with complex prophage populations. For instance, Staphylococcus aureus exhibits a wide range of prophages that form unique phylogenetic clusters, with some strains harboring multiple distinct prophages that contribute to genetic and functional diversity across strains, including virulence factor variation [21]. In Mycobacterium, strains in the M. abscessus-chelonae complex show high prophage diversity, with unique clusters across subgroups that support varied adaptations within environmental or host-associated niches [18]. This genetic diversity in Mycobacterium and other bacteria, as observed in Tenacibaculum, indicates that prophage diversity is often driven by ecological adaptation, host-specific interactions, or environmental pressures, which can lead to the formation of distinct phylogenetic groups [17,20,22].
Bacteriophages are abundant in the environment, particularly in seawater, and have been explored as an alternative to chemical antimicrobials in aquaculture [12,13]. Despite their potential, the use of phages in aquaculture poses risks, including the emergence of new virulent bacteria due to lysogenic conversion, where temperate phages can introduce virulence genes into bacterial genomes, as observed in Vibrio cholerae and V. harveyi [23,24]. Additionally, phages can facilitate horizontal gene transfer through transduction, potentially spreading virulence factors to non-pathogenic strains or even to different bacterial species [23,25]. Although lytic phages may exert selective pressure that leads to phage-resistant bacteria, which are often thought to have reduced virulence, evidence suggests that resistance does not always reduce pathogenicity, raising further concerns about the use of phages as antimicrobials [26,27]. However, phage-derived endolysins do not carry these risks, as they lack the capacity for lysogenic conversion or horizontal gene transfer, making them a safer alternative in antimicrobial strategies. Moreover, unlike endolysins, bacteriophages must overcome bacterial defense mechanisms specifically designed to block the phage replication cycle, such as antiphage defense systems (e.g., CRISPR-Cas, restriction-modification systems, and abortive infection systems).
A recent pangenome analysis revealed new bacterial defense systems that challenge phage effectiveness [28]. Understanding these defenses and their limitations is crucial for advancing phage applications [29]. In this study, the most commonly detected antiphage defense mechanisms were the restriction-modification (R-M) and Mokosh systems, which were present across multiple genomes. In contrast, the CRISPR-Cas system was found exclusively in the four genomes of T. maritimum (Figure 3). The restriction-modification (R-M) system uses two enzymes: a restriction endonuclease and a methyltransferase. The methyltransferase protects bacterial DNA by adding methyl groups to specific sites, whereas unmethylated foreign DNA, like phage DNA, is recognized by the restriction enzyme and cut, effectively degrading it [30]. Mokosh systems were recently discovered; unlike other bacterial immune systems, such as CRISPR-Cas, Mokosh does not rely on a memory of past infections but instead offers an immediate defensive response through the degradation of the phage’s genetic material or interference with essential phage processes [31]. Mokosh includes an RNA helicase domain (COG1112 family) and a PLD family nuclease. In type I Mokosh, the RNA helicase and PLD nuclease are encoded by separate genes, with an additional serine–threonine kinase (STK) domain. In type II, these domains are combined within a single gene, with the RNA helicase at the N-terminus and the PLD nuclease at the C-terminus. In T. larymnensis, Mokosh type II, as described by [15], includes the mkoC gene, featuring an RNA helicase at the N-terminus and a PLD nuclease as the effector domain.
CRISPR-Cas is another antiphage defense mechanism. It functions as an adaptive immune system by capturing segments of phage genomes, known as spacers, and storing them in a repeat–spacer array within a CRISPR locus. During a later infection, this array is transcribed into CRISPR-RNA (crRNA) [32,33]. These repeat–spacer sequences then guide endonucleases, like Cas9, to locate and cleave matching sequences in the invading phage genome. CRISPR-Cas systems are notably enriched in certain bacterial pathogens, likely as a response to selective pressures in complex environments like the mucosa [33,34]. This immune mechanism may help pathogens evade phages while retaining virulence, particularly when interacting with the host’s mucosal layers. The presence of mucosa appears to drive a diversification of bacterial immune strategies, as pathogens must balance an effective phage defense with the need to avoid modifications that could hinder mucosal colonization. Studies indicate that exposure to mucosal components like mucin can enhance bacterial susceptibility to phages, as seen in Flavobacterium columnare, Aeromonas species, Clostridium difficile, and Escherichia coli [34,35].
Recently, Wu 2024 noted that certain defense systems that are negatively associated in E. coli show synergistic effects and frequently co-occur in other species, indicating that bacterial immune repertoires are shaped more by selective pressures from host-specific phages than by negative interactions between systems. These findings suggest that bacterial defense systems are compatible and often work together, enabling bacteria to develop adaptable strategies against phage attacks.
Endolysins, or lysins, are enzymes produced by bacteriophages during the later stages of their infection cycle. These proteins target and break down the peptidoglycan (PG) in bacterial cell walls, compromising the structural integrity of the cell. This degradation leads to cell death, typically through osmotic lysis, as the weakened cell wall can no longer withstand the internal pressure, causing the bacterial cell to burst [36,37]. Studies on bacteriophages have enhanced our understanding of endolysins’ role in the phage lytic cycle and their potential as antimicrobial agents. In aquaculture, endolysins show promise as an effective alternative for combating bacterial infections [38]. Their mechanism for breaking down the bacterial cell wall reduces the chances of resistance emerging and is regarded as safe for eukaryotic organisms [37].
Our analysis identified an endolysin similar to LysK from Staphylococcus aureus bacteriophage K, including its CHAP domain (cysteine, histidine-dependent amidohydrolase/peptidase domain) [39]. N-acetylmuramoyl-L-alanine amidase is an enzyme that plays a crucial role in breaking down bacterial cell walls. It specifically targets the bond between N-acetylmuramic acid (a component of the peptidoglycan layer in bacterial cell walls) and L-alanine in the peptide chain. By cleaving this bond, the enzyme contributes to the degradation of the peptidoglycan structure, leading to cell wall disruption and, ultimately, bacterial cell lysis [40].
Phage lysins could be very useful in the control of aquaculture pathogens, like vibrios. The enzyme LysVPp1 effectively lysed 9 out of 12 Vibrio parahaemolyticus strains, displaying a broader range of action than its associated phage VPp1, which was active against only three strains [41]. Similarly, LysVpKK5 exhibited potent lytic activity against the VPATCC-17802 strain, even though the original VpKK5 phage could not infect it [42,43]. Furthermore, several vibriophage endolysins have shown lytic activity on sensitized cells of other Vibrio species, including V. mimicus, V. anguillarum, V. harveyi, and V. alginolyticus [44,45]. These observations underscore a general trend: certain endolysins derived from vibriophages can exhibit broader lytic ranges than their corresponding phages, making them valuable tools against multiple strains or species within the Vibrio genus [36].
The specificity of endolysins toward their host bacteria highlights an evolutionary adaptation to degrade cell walls with distinct structural features. Endolysins typically contain specialized cell wall binding domains (CBDs) that recognize particular components, such as the glycine-rich peptidoglycan in Staphylococcus aureus, enabling the targeted lysis of S. aureus and closely related streptococci [46]. Similarly, Vibrio endolysins, such as those derived from LysVPp1, show lytic effects on several Vibrio species [42]. Some phage-derived endolysins, like LysAB2, initially designed to target Acinetobacter baumannii, and Art-175, a modified endolysin effective against Pseudomonas aeruginosa, incorporate cationic peptides that facilitate passage through the outer membrane, demonstrating potential to act on Gram-negative bacteria when adapted properly [47]. These findings suggest that for broader therapeutic applications, engineering strategies must enhance endolysin permeability to the outer membrane in Gram-negative aquaculture pathogens like Vibrio and Tebacibaculum, thus overcoming natural host specificity and expanding their antibacterial potential.

4. Materials and Methods

A total of 212 genomes/assemblies available in the NCBI repository were included in this study. The genomic information is derived from different sources, including strain type, isolates from aquaculture species, covering different parts of the world (Figure 4). The assemblies comprise strains of the species T. aiptasiae (5), T. aquimarinum (2), T. bernardetii (3), T. dicentrarchi (1), T. discolor (4), T. finnmarkense (129), T. gallaicum (2), T. larymnensis (1), T. lutimaris (2), T. maritimum (40), T. mesophilum (10), T. ovolyticum (4), T. pacificus (1), T. retecalamus (2), T. singaporense (4), and T. todarodis (2). The assemblies included in this study were filtered based on an average nucleotide identity (ANI) equal to or greater than 90%. The accession codes, host, country, and year of isolation for each genomic sequence used are included in Supplementary Table S3.
Genomic sequences were screened for the presence of prophage elements using the PHASTEST (PHAge Search Tool with Enhanced Sequence Translation) web server [48]. For those strains with complete genomes, the associated accession code was entered; in the case of whole genome shotgun (WGS) sequencing, the FASTA files containing the contigs were entered, and the checkbox was ticked to submit a file consisting of multiple separate contigs. PHASTEST classifies prophage regions into three categories according to a score assigned based on a comparison with the NCBI virus database. Depending on the score, the region is assigned to one of three categories: intact (score above 90), questionable (score between 70 and 90), and incomplete (score below 70). The score assignment is based on three methods. In the first one, if the number of a certain phage in the database is greater than or equal to 100% of the total number of CDS in the region, a total score of 150 is assigned; if this is not the case, the following methods are used. In the second method, a comparison is made similarly to the first, but the number of coding sequences (CDS) in the region is reduced by 50%. The phage with the highest likelihood for that region is identified as the primary candidate. Additionally, the percentage of proteins in the region matching the most probable phage is calculated and multiplied by 100 to generate a partial score. This is followed by comparing the length of the region with the most likely phage and multiplying this percentage by 50, which is then added to the partial score to calculate the final score. This approach also incorporates the identification of specific keywords related to phage components (e.g., capsid, head, integrase, plate, tail, fiber, coat, transposase, portal, terminase, protease, or lysin) to enhance the accuracy of phage predictions. Additionally, it is increased by 10 points if the size of the region is more than 30 Kb and by another 10 points if there are at least 40 proteins in the region. Finally, if all phage-related proteins and hypothetical proteins constitute more than 70% of the total proteins in the region, the score is increased by 10. As a final step, the scores from methods 2 and 3 are compared, and the highest score is chosen as the total score for the region.
The complete genomes of the most common phages found in the regions classified as intact prophages were compared with the genomes of the two phages related to Tenacibaculum, PTm5 (AP019525) and PTm1 (AP019524), using VICTOR [49], the Virus Classification and Tree Building Online Resource, freely available online from https://ggdc.dsmz.de/victor.php (accessed on 3 October 2024), a method for the genome-based phylogeny and classification of prokaryotic viruses. All pairwise comparisons of the amino acid sequences were conducted using the Genome BLAST Distance Phylogeny (GBDP) method [50] under settings recommended for prokaryotic viruses.
The amino acid sequence of the putative endolysin found in the intact prophage regions was first evaluated in the ProtParam tool (available at http://web.expasy.org/protparam/, accessed on 4 October 2024) to investigate the primary structure properties [51]. The prediction of transmembrane helices in the protein was evaluated in DeepTMHMM version 1.0 [52] (available at https://services.healthtech.dtu.dk/services/DeepTMHMM-1.0/, accessed on 4 October 2024). The InterPro tool version 102.0 [53] was used for a functional analysis of the putative endolysin. The endolysin sequence was also evaluated in Phyre2 version 2.0 [54] to identify the closest protein templates based on the predicted structure.
Genome assemblies of Tenacibaculum strains that presented prophage regions classified as intact were evaluated in the DefenseFinder tool version 0.1.0 [55] to discover the presence of antiphage defense systems.

5. Conclusions

This study identifies key prophage elements within Tenacibaculum genomes, with a notable concentration in T. maritimum, which may serve as promising candidates for phage therapy in combating tenacibaculosis in aquaculture. The stable, CHAP domain-containing endolysin found in intact prophage regions demonstrates structural compatibility with known lytic domains, suggesting it could effectively target Tenacibaculum cells. These findings support the feasibility of bacteriophage-based treatments as a sustainable alternative to antibiotics, addressing resistance concerns while paving the way for targeted, environmentally friendly disease management strategies in the aquaculture sector. In this context, genomic and metagenomic studies play a crucial role by uncovering deeper insights into phage–pathogen interactions, providing valuable opportunities to identify new control tools and further advance sustainable approaches to combating bacterial diseases.

Supplementary Materials

The following supporting information can be downloaded at https://www.mdpi.com/article/10.3390/antibiotics13121184/s1, Figure S1: Results of the InterPro analysis of the putative endolysin; Figure S2: Predicted structure of the putative endolysin using Phyre2; Table S1: Phage-associated CDS; Table S2: Antiphage systems; Table S3: Assembly/genome data.

Author Contributions

Conceptualization, J.R. and C.R.; methodology, J.R. and C.R.; software, C.R.; writing—review and editing, J.R. and C.R.; funding acquisition, J.R. All authors have read and agreed to the published version of the manuscript.

Funding

The authors are grateful for the financial support provided by FONDECYT Regular 1240430; Anillos Regulares de Tecnología ACT240045 and FONDECYT EXPLORACION 2024, from ANID, Chile.

Institutional Review Board Statement

Not applicable.

Informed Consent Statement

Not applicable.

Data Availability Statement

Data available on request.

Conflicts of Interest

The authors declare no conflicts of interest.

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Antibiotics 13 01184 g001
Figure 1. Phylogenetic analysis of the 4 prophages found as the most common in the regions classified as intact in this study and the phages described for Tenacibaculum in previous studies at the amino acid level using VICTOR. The Genome BLAST Distance Phylogeny tree (GBDP) was derived using the D6 formula and yielded an average support of 100%. The OPTSIL clustering produced 3 clusters at family level and 4 clusters at species, genus, and subfamily levels, these clusters are represented by different colors.
Figure 1. Phylogenetic analysis of the 4 prophages found as the most common in the regions classified as intact in this study and the phages described for Tenacibaculum in previous studies at the amino acid level using VICTOR. The Genome BLAST Distance Phylogeny tree (GBDP) was derived using the D6 formula and yielded an average support of 100%. The OPTSIL clustering produced 3 clusters at family level and 4 clusters at species, genus, and subfamily levels, these clusters are represented by different colors.
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Antibiotics 13 01184 g002
Figure 2. Coding sequences identified in prophages classified as intact according to PHASTEST in Tenacibaculum strains. CDS were grouped in line with the BLAST description and presented as a percentage of proteins per strain. The colors contained in the circles on the left side of the protein description indicate the type of protein: phage-like proteins are highlighted in blue, tail proteins in green, head proteins in red, terminase in yellow, endonuclease in light blue; portal proteins in orange; the hypothetical protein in gray; fiber protein in dark gray; exonuclease in light green; integrase in pink, and DNA helicase in light pink.
Figure 2. Coding sequences identified in prophages classified as intact according to PHASTEST in Tenacibaculum strains. CDS were grouped in line with the BLAST description and presented as a percentage of proteins per strain. The colors contained in the circles on the left side of the protein description indicate the type of protein: phage-like proteins are highlighted in blue, tail proteins in green, head proteins in red, terminase in yellow, endonuclease in light blue; portal proteins in orange; the hypothetical protein in gray; fiber protein in dark gray; exonuclease in light green; integrase in pink, and DNA helicase in light pink.
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Antibiotics 13 01184 g003
Figure 3. Defense systems discovered in Tenacibaculum strains that presented prophage regions classified as intact. The colors represent the proportion of defense system types included in the genome.
Figure 3. Defense systems discovered in Tenacibaculum strains that presented prophage regions classified as intact. The colors represent the proportion of defense system types included in the genome.
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Antibiotics 13 01184 g004
Figure 4. Geographical distribution of genomes/assemblies of Tenacibaculum species used in this study. The size of the circles is proportional to the number of assemblies. Genomic information was grouped by country from where the strains were isolated, considering the average latitude and longitude coordinates per country.
Figure 4. Geographical distribution of genomes/assemblies of Tenacibaculum species used in this study. The size of the circles is proportional to the number of assemblies. Genomic information was grouped by country from where the strains were isolated, considering the average latitude and longitude coordinates per country.
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Table 1. Prophages identified in Tenacibaculum species assemblies using PHASTEST.
Table 1. Prophages identified in Tenacibaculum species assemblies using PHASTEST.
Species
(Strain)		RegionRegion
Length		CompletenessScoreTotal
Proteins		Region
Position		Most Common PhageGC%
T. maritimum
(TF4)		150 KbIntact140553079–53,132Cellul_phi19:130.29
T. maritimum
(FC)		115.8 KbIntact1002213,262–29,093Flavob_vB_FspM_pippi8_133.2
T. maritimum
(NBRC 15946)		115.8 KbIntact1002135,460–51,291Flavob_vB_FspM_lotta8_133.22
T. maritimum
(902)		1
2		21.5 Kb
11.5 Kb		Intact
Incomplete		100
50		20
18		12,829–34,424
18,576–40,083		Cellul_phi19:1
Flavob_vB_FspM_lotta8_1		29.99
31.6
T. maritimum (JIP 32/91-4)117.2 KbQuestionable801231,735–48,966Cellul_phi19:130.34
T. maritimum (P4-45)122.2 KbQuestionable9031139–22,399Flavob_vB_FspM_pippi8_134.09
T. maritimum (NVIB-2097)111.3 KbIncomplete50181–11,355Flavob_vB_FspM_lotta8_131.61
T. maritimum (NVIB-2096)111.3 KbIncomplete50181–11,355Flavob_vB_FspM_pippi8_131.61
T. maritimum (DPIF 89/0239-1)111 KbIncomplete501674,959–86,020Cellul_phi46:131.79
T. mesophilum (Mcav3-52)138 KbIntact11045360,314–398,338Cellul_phi39:131.95
T. mesophilum (bac2)112.7 KbQuestionable70131,752,433–1,765,200Strept_BRock33.26
T. mesophilum (DSM 13764)19.3 KbQuestionable7091,339,122–1,348,490Bacill_BCD733.93
T. mesophilum (DSM 13764)19.3 KbQuestionable7092,013,166–2,022,534Strept_BRock33.93
T. mesophilum (546.fasta)19.3 KbIncomplete60931,278–40,645Strept_BRock33.85
T. mesophilum (XPcli2-G)19.3 KbIncomplete60984,611–93,978Bacill_BCD734
T. mesophilum (SSh1-16)19.3 KbIncomplete60931,113–40,481Bacill_BCD734
T. pacificus
(P3-BQ1)		122 KbIntact110312,677,157–2,699,185Flavob_vB_FspM_lotta8_130.2
T. ovolyticum (20-4135-2)19.3 KbQuestionable8093,744,277–3,753,604Bacill_BCD731.89
T. ovolyticum (To-7Br)19.3 KbQuestionable70921,823–31,151Strept_BRock31.79
T. ovolyticum (da5A-8)19.3 KbQuestionable70980,624–89,952Strept_BRock31.76
T. discolor
(LAR25)		17.4 KbIncomplete608256,719–264,196Serrat_phiMAM132.23
T. discolor
(IMLK18)		15.6 KbIncomplete606338,872–344,494Synech_S_CBP330.82
T. finnmarkense (NVIB-3194)116.7 KbIncomplete40223–16,707Cellul_phiSM30.55
T. dicentrarchi
(TD3509T)		19.3 KbIncomplete508853,075–862,413Flavob_vB_FspM_pippi8_133.5
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Ramírez, C.; Romero, J. Unveiling Hidden Allies: In Silico Discovery of Prophages in Tenacibaculum Species. Antibiotics 2024, 13, 1184. https://doi.org/10.3390/antibiotics13121184

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Ramírez C, Romero J. Unveiling Hidden Allies: In Silico Discovery of Prophages in Tenacibaculum Species. Antibiotics. 2024; 13(12):1184. https://doi.org/10.3390/antibiotics13121184

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Ramírez, Carolina, and Jaime Romero. 2024. "Unveiling Hidden Allies: In Silico Discovery of Prophages in Tenacibaculum Species" Antibiotics 13, no. 12: 1184. https://doi.org/10.3390/antibiotics13121184

APA Style

Ramírez, C., & Romero, J. (2024). Unveiling Hidden Allies: In Silico Discovery of Prophages in Tenacibaculum Species. Antibiotics, 13(12), 1184. https://doi.org/10.3390/antibiotics13121184

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