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Published in final edited form as: Virology. 2026 Apr 9;620:110911. doi: 10.1016/j.virol.2026.110911

A novel phage satellite class induces prophage excision in Mycolicibacterium aichiense

Heather L Qian a, Anne N Roman a,1, Sudip Paudel a,2, Grace E Hussey a, Kate B R Carline a, Margaret S Saha a
PMCID: PMC13102486  NIHMSID: NIHMS2166327  PMID: 41980317

Abstract

Phage satellites are mobile genetic elements that parasitize a helper phage to complete their life cycle. While phage satellites are widespread in diverse bacterial hosts, none have yet been isolated in Mycobacteriaceae. Here, we report the first phage satellites isolated and characterized in Mycobacteriaceae, Extracellular Prophage-Inducing Particles (EPIPs). EPIPs induce a helper phage—HerbertWM, a Mycolicibacterium aichiense prophage—upon infection. Genomic sequencing of thirteen isolates revealed a genome size of ~11 kb and ~83.3% nucleotide similarity calculated using Virus Intergenomic Distance Calculator (VIRIDIC). Although gene content and synteny were broadly conserved across the isolates, the EPIPs were categorized into distinct groups based on VIRIDIC similarity scores, gene content, and synteny. Notably, EPIPs lack capsid proteins, tail proteins, and holins, suggesting EPIPs hijack the machinery of HerbertWM to replicate, assemble, and lyse the host. Furthermore, the unique gene content of EPIPs compared to other phage satellites, particularly the presence of a tape measure protein and absence of antirepressors, suggests they are distinct from other phage satellite classes. Transmission electron microscopy of EPIPs reveals that they not only feature smaller capsids compared to HerbertWM, but that they feature highly flexible tails of variable length. Additionally, greater EPIP protein similarity to proteins in bacteria as opposed to free phage proteins suggests a functional or evolutionary relationship may exist. The nucleotide similarity, synteny, and unique gene content of EPIPs suggest EPIPs are a distinct class of phage satellites that induce Mycobacteriaceae prophages.

Graphical Abstract

graphic file with name nihms-2166327-f0001.jpg

1. Introduction

Phage satellites (satellites) are mobile genetic elements that hijack helper phage machinery to complete their life cycle. They can exist extrachromosomally as a plasmid or phagemid, integrated in the bacterial host genome, or as a free infective particle (reviewed in Penadés et al., 2025; Ibarra-Chávez et al., 2021). These interactions create a parasitism-mutualism continuum between the helper phage and satellite; satellites benefit the helper phage by blocking non-helper phage infection and competing mobile genetic element transfer while simultaneously inhibiting helper phage replication. Satellites also confer selective advantages to the host: immunity against non-helper phages, increased host virulence, and transduction of bacterial genes (Penadés et al., 2025; Ibarra-Chávez et al., 2021).

Three classes of satellites—P4-like elements, phage-inducible chromosomal islands (PICIs), and PICI-like elements (PLEs)—have been well characterized. All satellites remodel helper capsids and have DNA sequences recognized by a native or hijacked helper phage terminase (Penadés et al., 2025; Ibarra-Chávez et al., 2021). However, these satellite classes display differences: P4-like elements exhibit mutual induction with the satellite and helper phage inducing the other (Lindqvist et al., 1993); PICIs display significant genetic diversity and independent replication facilitated by a Pri-Rep-ori segment in their genomes (Fillol-Salom et al., 2018; Martínez-Rubio et al., 2017); and PLEs feature a relatively small host range limited to the Vibrio genus, complete inhibition of helper phage replication, and acceleration of host lysis (Boyd and Seed, 2024). In addition to these well characterized examples, discoveries of novel phage satellite classes are growing rapidly. For example, capsid-forming PICIs (cf-PICIs), MiniFlayer, MulchRoom, and Phanie encode capsid proteins; phage-inducible chromosomal minimalist islands (PICMIs) and virion-encapsidated integrative mobile elements (VEIMEs) package concatemeric genomes; MiniFlayer and Phanie attach to their helper phages for simultaneous infection; ɸAH14b and its helper lack tails; and tycheposons encode phylogenetically distinct tyrosine recombinases (deCarvalho et al., 2023; Pourcel et al., 2024; Barcia-Cruz et al., 2024; Eppley et al., 2022; Hackl et al., 2023). Despite this diversity, satellites all share small genomes and rely on helper phage machinery (Penadés et al., 2025; Ibarra-Chávez et al., 2021). Here we report yet another novel class of satellites named “Extracellular Prophage-Inducing Particles” (EPIPs) due to their ability to induce the excision of the HerbertWM helper phage, a Mycolicibacterium aichiense prophage, making EPIPs the first satellites experimentally isolated from Mycobacteriaceae.

2. Materials and methods

2.1. EPIP isolation

EPIPs were isolated from soil samples obtained from geographically diverse locations within a five mile radius of Jamestown, Virginia over a two-year period using standard procedures for phage isolation. As previously described (Carline et al., 2023), after inoculation of 5 mL of soil with 25 mL Middlebrook 7H9 media, 3 mL AD supplement, and 1 mL liquid M. aichiense NCTC 10820 (ATCC 27280) culture, samples were incubated at 37°C with shaking at 250 rpm for three days. Following centrifugation at 3,400 rpm for 15 minutes, the culture was filtered through a 0.22 μm PES filter. The resulting lysate was incubated with M. aichiense for 20 minutes at room temperature, then the infected culture was plated using Middlebrook 2X 7H9 top agar on Luria broth plates (15 g/L agar, tryptone 10 g/L, yeast extract 5 g/L, NaCl 10 g/L) and incubated for two days. Resulting plaques were purified through two additional rounds of plating before lysates were collected by flooding webbed-lysis plates with 5 mL phage buffer (1 mM CaCl2, 10mM Tris, 10 mM MgSO4, 0.4% w/v NaCl) and incubating the flooded plates for hours at 20°C. Collected lysates were filtered using a 0.22 μm PES filter.

2.2. Transmission Electron Microscopy

Three EPIPs, ChrisB, Bernie, and Alyssa1, were imaged using transmission electron microscopy (TEM). Bernie lysate was collected by flooding a webbed-lysis plate, while ChrisB and Alyssa1 lysates were collected by extracting spots resulting from a spot assay and placing each plug in 500 μL phage buffer. All lysates were stored at 4°C for a minimum of 14 hours before being used to prepare a TEM grid. Following a standard protocol for TEM grid preparation (Carline et al., 2023), 4 mL lysate was filtered using a 0.22 μm PES filter into two 2 mL tubes. Following centrifugation at 13.3k rpm at 4°C for 1 hour, supernatant was decanted and pellets were resuspended in 50 μL phage buffer. After incubating at 4°C for 1 hour, the two 50 μL volumes were combined. TEM grids were prepared by placing 10 μL lysate onto carbon-formvar-coated copper grids (Ted Pella), and EPIPs were allowed to attach for 7 minutes. The grid was rinsed with 10 μL nuclease free water twice before being stained with 10 μL 1% uranyl acetate for 2 minutes. Samples were imaged using a JEM-2100F field electron microscope ranging from 6,000x to 80,000x magnification.

2.3. DNA extraction

To maximize DNA recovery, polyethylene glycol (PEG) precipitation was performed (Paithankar and Prasad, 1991). Chloroform was added to an infected culture of M. aichiense cells to a final volume of 1% then incubated for 90 minutes at 37°C. A sample of this culture was then centrifuged at 3,500 rpm for 5 minutes and was followed by filtration with a 0.22 μm PES filter. To 95 mL filtered lysate, NaCl was added to 1M followed by 10% volume PEG 8000, and the sample was incubated overnight at 4°C with stirring. The sample was then centrifuged at 4°C for 10 minutes at 5,500 g and supernatant was removed. The remaining pellet was resuspended in 5 mL phage buffer.

To extract DNA (Russell and Sambrook, 2006), 12.5 μL 1M MgCl2, 14 μL nuclease mix (4 μL 200 U/mL DNAsel and 10 μL 10 mg/mL RNase A) was added to the PEG-treated lysate. The tube was vortexed to mix then incubated at room temperature for 30 minutes. Then, 40 μL 0.5 M EDTA, 5 μL 10 mg/mL Proteinase K, and 50 μL 10% SDS were added, and the tube was vortexed before incubating at 55°C for 1 hour, vortexing twice during this period at 20 minute intervals. Samples were divided into 500 μL volumes, and DNA was extracted using 500 μL PCI (25 phenol : 24 chloroform : 1 isoamyl alcohol). The tube was inverted to mix, then centrifuged at 13,000 rpm for 5 minutes. After transferring the upper phase to a new tube, 500 μL CI (24 chloroform : 1 isoamyl alcohol) was added, inverted several times to mix, and centrifuged at 13,000 rpm for 5 minutes. The upper phase was transferred to a new tube, and 50 μL 3M sodium acetate and 1 mL ice cold 100% ethanol were added to precipitate the DNA. Following incubation on ice for 7 minutes and centrifugation at 13,000 rpm for 10 minutes, supernatant was decanted and the pellet was washed with 500 μL ice cold 70% ethanol. Samples were centrifuged again at 13,000 rpm for 10 minutes, and supernatant was discarded. Pellets were air dried for 20 minutes before being resuspended in 50 μL nuclease free water. DNA was quantified using a Nanodrop spectrophotometer and visualized using agarose gel electrophoresis.

2.4. Genome sequencing and assembly

Thirteen isolates were selected for DNA sequencing and analysis. EPIP and helper phage genomes were sequenced using an Illumina MiSeq sequencer and reads were assembled using Newbler v2.9. The final assembly was determined using Consed v29.0 (Gordon et al., 1998) at the Pittsburgh Bacteriophage Institute or using CLC Genomics Workbench v22.0 (QIAGEN), and all assembled genomes were deposited in GenBank (GenBank: MN224566 (HerbertWM), OR387110, OR387111, OR387112, OR387113, OR387114, OR387115, OR387116, OR387117, OR387118, OR387119, OR387120, OR387121, PX657209). The starts of the circular EPIP genomes were aligned using CLC Genomics Workbench v22.0 (QIAGEN). Additionally, Bacterial Whole Genome sequencing was performed by Plasmidsaurus on M. aichiense cultures using Oxford Nanopore Technology.

2.5. Genome annotation and comparative genomics

Genome annotations were determined using multiple metrics including DNA Master v5.23.6 (Pope and Jacobs-Sera, 2018), Glimmer v3.02 (Delcher et al., 2007), GeneMark v2.5 (Besemer and Borodovsky, 2005), BLAST (Altschul et al., 1990), and HHPred (Söding et al., 2005). EPIPs were categorized into groups defined by a Virus Intergenomic Distance Calculator (VIRIDIC) similarity score greater than 95%—the VIRIDIC species threshold (Moraru et al., 2020), identical protein function, and synteny, with each group comprising a distinct branch on the genome-based phylogenetic tree generated with Clustal Omega Multiple Sequence Alignment (Sievers et al., 2011). EPIP protein structure alignment was also used as a metric for genome annotations. One EPIP from each group and the two ungrouped EPIPs were selected for protein structure analysis where EPIP protein structures were predicted using AlphaFold 3 (Abramson et al., 2024), and predicted structures were aligned with proteins in Protein Data Bank (Burley et al., 2021) using Foldseek (vanKempen et al., 2023) and DALI (Holm et al., 2022). Alignment quality was evaluated using overall fold similarity, percent coverage, and the context of the EPIP protein within the EPIP genome. For Foldseek results, overall fold similarity was determined using the overlay function in ChimeraX v1.10.1 (Meng et al., 2023), while for DALI results, overall fold similarity was determined using side-by-side comparison.

Four EPIPs (one from each group and the two ungrouped EPIPs) were selected for further analysis: WillG, JSForest2, Bernie, and Alyssa1. Following genome annotations, BLASTp (Altschul et al., 1990) was used to align all 83 EPIP proteins with proteins in NCBI ClusteredNR database. As a control, 32 A2 phage proteins were aligned with proteins in ClusteredNR as well. For each gene, the top five BLASTp results were recorded and the proportion of bacterial results was calculated by dividing the number of bacterial results by the total number of recorded results. A Mann-Whitney U test was performed, revealing that EPIP proteins had a higher proportion of bacterial results than A2 phages that were used as a “control” for proteins present in free phages. Additionally, homology of EPIPs to HerbertWM was analyzed by aligning all thirteen EPIP genomes to the HerbertWM genome using MegaBLAST (Altschul et al., 1990).

2.6. Analysis of intergenic regions

Intergenic regions were operationally defined as any region outside of the start codon and stop codon of a protein-coding gene, with sets defined as intergenic regions between proteins of the same function across EPIPs. Within each set, pairwise similarity of intergenic regions was evaluated using a VIRIDIC score. Then, to determine whether similarity scores were higher for intergenic regions within groups than between groups, pairwise comparisons of similarity scores were performed using Mann-Whitney U tests. Then, a global Fisher p-value was calculated with a similarity threshold of 95 to combine all Mann-Whitney U scores.

To investigate the conservation of intergenic regions across groups, intergenic regions were then marked as positioned between genes transcribed in opposite directions or between genes transcribed in the same direction. A subset of similarity scores was created by selecting pairs of intergenic similarity scores across different groups. Within this subset, a Mann-Whitney U test was used to compare the median similarity of regions between genes transcribed in opposite directions versus regions between genes transcribed in the same direction.

3. Results and Discussion

3.1. EPIP annotation

Assembled reads for all samples revealed two circular contigs. One, 100% identical across all samples, was identified as the cluster A2 prophage, HerbertWM. A second 11-kb contig, the EPIP, varied among samples, showing sequence similarities ranging from 58.1% to 99.8% determined using VIRIDIC (Fig. 1E). Stoichiometric ratios between EPIP and HerbertWM reads varied between samples from an EPIP-to-HerbertWM ratio of 1:1 to 725:1.

Fig. 1. EPIP morphology and categorization.

Fig. 1.

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(A) ChrisB at 40000x magnification (mean capsid diameter 29.8 ± 1.7 nm, N = 10; mean tail length 86.4 ± 8.2 nm, N = 10). (B) Alyssa1 at 10,000x magnification (mean capsid diameter 51.2 ± 1.2 nm, N = 3; mean tail length 384.5 ± 119.4 nm, N = 3). (C) Bernie at 40,000x magnification (mean capsid diameter 32.0 ± 4.1 nm, N = 4; mean tail length 283.8 ± 42.4 nm, N = 3). One particle was excluded from tail length measurements due to likely tail breakage. (D) The HerbertWM helper phage, a Mycolicibacterium aichiense prophage, at 40,000x magnification. (E) Genome similarity generated by VIRIDIC (mean percent within groups 99.76 ± 0.01, Group 1 vs Group 2 73.75 ± 0.2, Alyssa1 vs all other EPIPs 58.20 ± 1.4, Bernie vs all other EPIPs 79.85 ± 2.2). (F) Phylogenetic tree of EPIPs generated by Clustal Omega Multiple Sequence Alignment. Each branch endpoint represents a single EPIP, and each branch represents a group.

All EPIPs encode a terminase small subunit, head-to-tail adaptor, head-to-tail stopper, serine integrase, helix-turn-helix DNA binding domain, scaffolding protein, major capsid protein, tape measure protein, and four conserved hypothetical proteins. However, all EPIPs lack genes critical for phage function such as capsid proteins, tail proteins, and holins, suggesting EPIPs parasitize HerbertWM to replicate, assemble, and lyse the host (Fig. 2A).

Fig. 2. EPIP gene content.

Fig. 2.

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(A) Alignment of EPIP proteins, with proteins of the same color being homologous. A previously described, a homology threshold was defined by ≥ 70% amino acid coverage and ≥ 30% identity (Burgetz et al., 2007; Rost et al., 1999). (B) EPIP proteins common to other phage satellite classes. The column of each dot represents the EPIP protein, and the row represents the phage satellite class that contains a similar protein.

* 8% of VEIMEs encode terminases, and tycheposons typically encode tyrosine integrases.

As such, EPIPs feature distinct gene content compared to other satellite classes (Fig. 2B). In particular, the presence of a tape measure protein (TMP) is notable, as TMPs are not present in satellites, although a recent preprint proposes that PLE11 encodes a TMP-like protein (Mathur et al., 2025). The TMP may impact EPIP morphology as TEM imaging of three EPIP isolates, ChrisB, Alyssa1, and Bernie, revealed unconventional tail morphology. All EPIP isolates imaged featured highly flexible tails that varied greatly in length. Alyssa1 and Bernie feature tails of longer length (~385 and ~284 nm respectively) than HerbertWM (~134 nm). In contrast, ChrisB features tails of shorter length (~86 nm) than HerbertWM. Additionally, ChrisB, Alyssa1, and Bernie all possess smaller capsids (~28, ~51, and ~32 nm respectively) than HerbertWM (~67 nm), whose capsid diameter and tail length are common to mycobacteriophages (40–80 nm and 135–350 nm respectively) (Hatfull, 2018) (Fig. 1A–D).

Although the imaged EPIP tail lengths do not reflect the 880 amino acid TMP using the conventional conversion of 0.15 nm per amino acid (Katsura and Hendrix, 1984), it is possible there are other mechanisms influencing EPIP tail length. For example, it has been suggested that chaperone binding could alter the structure the TMP, ultimately affecting tail length (Cumby et al., 2014; Mahony et al., 2016). Additionally, TMP repeats have diverse architectures that may impact the conversion between TMP amino acids and tail length (Belcaid et al., 2011). Furthermore, it is possible that the EPIPs use an uncharacterized mechanism of tail assembly that would explain the tail flexibility, width, and variation in length reflected in the TEM images.

It is also notable that BRO domain-containing proteins are largely conserved across all EPIP isolates. Prior work has suggested that in viruses, some BRO domains function as DNA binding domains, and BRO proteins may influence transcription and replication (Zemskov et al., 2000); however, the BRO domain-containing proteins in EPIPs appear truncated, solely consisting of the BRO domain, which may impact their potential to function as transcriptional activators or antirepressors. Furthermore, EPIPs notably lack canonical antirepressors such as Gpε in P4-like elements (Liu et al., 1997), suggesting EPIPs may feature a unique mechanism of prophage induction.

Additionally, experimental evidence has supported the functionality of the EPIP serine integrase. DNA sequencing of M. aichiense cultures revealed that strains with integrated Bernie but without the HerbertWM prophage do exist. However, it is unknown whether HerbertWM is able to induce Bernie similar to how P2-P4-like elements are able to induce each other (Lindqvist et al., 1993).

Notably, when EPIP proteins were aligned against proteins in the ClusteredNR database, the top results consistently and preferentially aligned to proteins found in bacteria rather than free phage. Even though there were some exceptions where one or two free phages were in the top five BLASTp results (n = 7)—namely, the major capsid proteins in WillG, JSForest2, and Alyssa along with gene products 6 and 7 in both JSForest2 and Bernie—the BLASTp results for the majority of the 83 EPIP proteins (n = 69) consisted solely of proteins in bacteria. The remaining EPIP proteins (n = 7) did not have any BLASTp results. This contrasted with proteins from cluster A2 phages which consistently and preferentially aligned to proteins found in other free phage (p = 6.19 × 10−22). Although PHASTEST (Wishart et al., 2023) and DEPhT (Gauthier et al., 2022) could not be used to investigate the presence of prophages in the bacteria EPIPs aligned to because many of the bacteria did not have sequenced reference genomes, the EPIPs likely have a functional or evolutionary relationship to prophages. The high similarity of EPIP proteins with proteins in bacteria, low homology between the EPIPs and HerbertWM, and the ability of EPIPs to induce HerbertWM suggests that EPIPs fit the previously hypothesized origin of phage satellites: satellites likely originated from genetic exchanges with non-helper phages or other mobile genetic elements, and subsequently repurposed acquired genes to hijack a helper phage (Penadés et al., 2025).

3.2. EPIP comparative analysis

Following genome annotations, EPIPs were categorized into two groups where EPIPs share ~99.8% VIRIDIC similarity within each group, and EPIPs share ~73.7% similarity between groups. The ungrouped EPIPs, Alyssa1 and Bernie, respectively share ~58.2% and ~79.9% similarity with all other EPIPs and were included in comparative analyses (Fig. 1E–F). Additionally, all EPIPs lack significant homology with HerbertWM, the helper phage, showing only ~8.5% nucleotide coverage, ~73.1% identity as determined using MegaBLAST.

EPIPs do exhibit variation across groups; a scaffolding protein and several hypothetical proteins are not conserved across all groups (Fig. 2A). Additionally, although intergenic regions are well-conserved within groups (100% median VIRIDIC similarity), intergenic regions across groups are significantly less conserved (86.1% median VIRIDIC similarity, p = 2.23 × 10−68). Interestingly, regions between genes transcribed in opposite directions are more conserved across groups than regions between genes transcribed in the same direction (p < 0.05), suggesting conserved regulatory architecture.

4. Conclusion

The striking similarities between EPIPs across groups—synteny, high VIRIDIC similarity, and homology to bacterial proteins—despite EPIPs being isolated over a five mile radius and a two year period suggests EPIPs are a new class of satellites that induce Mycobacteriaceae prophages. The similarity of EPIP proteins with proteins found in bacteria and the ability of EPIPs to induce the HerbertWM prophage suggests that EPIPs share a functional or evolutionary relationship with prophages. While EPIPs share common features with other satellites, such as small genomes and a lack of proteins required for an independent life cycle (Penadés et al., 2025; Ibarra-Chávez et al., 2021), EPIPs display distinct characteristics as well; the distinct EPIP gene content, particularly their encoding of a tape measure protein and lack of canonical antirepressors and complete BRO proteins may suggest unique mechanisms of assembly and prophage excision (Fig. 2B). Taken together, the data presented here suggest EPIPs are a distinct class of Mycobacteriaceae satellites whose novelty invites further investigation of their mechanisms of action.

  • EPIPs are the first phage satellites experimentally isolated from Mycobacteriaceae

  • EPIPS share 58.1%−99.8% sequence identity, with conserved gene content and synteny

  • Despite lacking canonical antirepressors, EPIPs induce a prophage, HerbertWM

  • EPIPs have longer, more varied tails and smaller capsids than prophage HerbertWM

  • Gene content of EPIPs is distinct from that of other phage satellite classes

Acknowledgements

We thank the many high school students from Jamestown High School who participated in the discovery of these satellite phages. We also thank Taiana James, Mitchell K. Doherty, Mahima Shijo, and Rebecca D. Zheleznyak and the many others for assistance with various aspects of EPIP discovery and characterization. We thank the Pittsburgh Bacteriophage Institute for sequencing and assembling HerbertWM and the Bernie EPIP isolate, and the entire Hatfull lab for their ongoing support. We also thank the Roy R. Charles Center for generous financial support to Heather Qian, Anne Roman, Grace Hussey, and Kate Carline.

Funding

This work was supported by funding from the National Institutes of Health (1R15HD114135-01) to MSS.

Glossary

EPIP

Extracellular Prophage-Inducing Particle, a novel class of phage satellites that induces the HerbertWM helper phage

Helper phage

A phage that is parasitized by a phage satellite

HerbertWM

The temperate phage that EPIPs parasitize

Phage satellite

A mobile genetic element that parasitizes a helper phage

TMP

Tape measure protein, a protein that typically influences phage tail length

VIRIDIC

Virus Intergenomic Distance Calculator, a tool for comparing virus genome similarity using pairwise nucleotide comparisons

Footnotes

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Declaration of interests

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

CRediT authorship contribution statement

Heather L. Qian: Data Curation, Formal analysis, Investigation, Methodology, Software, Validation, Visualization, Writing - original drafts, Writing - review and editing. Anne N. Roman: Data Curation, Formal analysis, Methodology, Software, Writing - original drafts, Writing - review and editing. Sudip Paudel: Data Curation, Formal analysis, Methodology, Software, Writing - original drafts, Writing - review and editing. Grace E. Hussey: Formal analysis, Investigation, Methodology, Validation, Writing - original drafts, Writing - review and editing. Kate B. R. Carline: Investigation, Methodology, Validation, Writing - original drafts, Writing - review and editing. Margaret S. Saha: Conceptualization, Data Curation, Formal analysis, Funding acquisition, Investigation, Methodology, Project administration, Resources, Software, Supervision, Validation, Visualization, Writing - original drafts, Writing - review and editing.

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