# From Demodification to Sequencing: the Genome of Bacteriophage Carin-5

Rémi Sieskind, Sophia Missoury, Frédéric Bonhomme, Pauline Nogaret, Marc Monot, Mikael Skurnik, Anne-Claire Baudoux, Marc Delarue

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Rémi Sieskind, Sophia Missoury, Frédéric Bonhomme, Pauline Nogaret, Marc Monot, et al. From Demodification to Sequencing: the Genome of Bacteriophage Carin-5. 2025. hal-05351923

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# From Demodification to Sequencing: the Genome of Bacteriophage Carin-5

**Rémi SIESKIND**

Institut Pasteur, Université Paris-Cité, CNRS UMR 3528

**Sophia MISSOURY**

Institut Pasteur, Université Paris-Cité, CNRS UMR 3528

**Frédéric BONHOMME**

Institut Pasteur, Université Paris Cité, CNRS UMR 3523

**Pauline NOGARET**

Sorbonne Université, CNRS, Adaptation et Diversité en Milieu Marin

**Marc MONOT**

Institut Pasteur, Université Paris Cité

**Mikael SKURNIK**

University of Helsinki

**Anne-Claire BAUDOUX**

Institut Pasteur, Université Paris Cité, CNRS UMR 3523

**Marc DELARUE**

marc.delarue@pasteur.fr

Institut Pasteur, Université Paris-Cité, CNRS UMR 3528

## Article

### Keywords:

**Posted Date:** July 25th, 2025

**DOI:** https://doi.org/10.21203/rs.3.rs-7091417/v1

**License:** © This work is licensed under a Creative Commons Attribution 4.0 International License.

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**Additional Declarations:** No competing interests reported.
# From Demodification to Sequencing: the Genome of Bacteriophage Carin-5

Rémi SIESKIND<fnref n="1" />, Sophia MISSOURY<fnref n="1" />, Frédéric BONHOMME<fnref n="2" />, Pauline NOGARET<fnref n="3" />, Marc MONOT<fnref n="4" />, Mikael SKURNIK<fnref n="5" />, Anne-Claire BAUDOUX<fnref n="2" />, Marc DELARUE<fnref n="1" />§

**Abstract:** The bacteriophage Carin-5, which infects *Cobetia marina*, presented significant challenges for genome sequencing due to DNA modifications that hindered efficient amplification of its DNA. Initial attempts using an optimized Illumina library preparation protocol yielded only a partial genome. Among the obtained contigs, the gene encoding the replicative DNA polymerase was identified. This gene was subsequently cloned into an expression vector and the encoded DNA polymerase protein was purified. The purified DNA polymerase was used to replicate the Carin-5 genomic DNA *in vitro*, effectively bypassing the modification-induced amplification barriers. This approach enabled the amplification of the entire Carin-5 genomic DNA and the use of a standard NGS library preparation protocol and allowed to obtain the complete sequence of the Carin-5 genome. The DNA modifications were characterized by mass spectrometry, and they showed a clear analogy with the oligosaccharide-hypermodified thymidines of bacteriophage SP-15. The annotated genome of Carin-5 carried a gene block encoding enzymes for nucleotide sugar modifications and glycosyltransferases.

## Introduction

Carin-5 is a recently identified lytic marine podophage that targets the γ-proteobacteria *Cobetia marina* DSMZ 4741. This bacterium is notable for its production of exopolysaccharides, which can either remain attached to the cell as a capsule or be released into the surrounding water as dissolved organic matter [1]. While several *C. marina* phages have been well-characterized, such as Carin-1, which carries a structural polysaccharide depolymerase enzyme (Dpo36) to degrade the host's capsule and facilitate infection, Carin-5 has remained largely unexplored due to significant sequencing challenges [2], [3].

The genomic analysis of Carin-5 has been hindered by sequencing difficulties, likely due to the presence of modified bases in its genetic material. Such modifications - commonly including methylation, glycosylation, and other chemical alterations - interfere with standard sequencing technologies, complicating genome assembly and annotation. Similar challenges were observed in other bacteriophages, such as the *Yersinia ruckeri* phage YerA41, which contains highly modified thymidine [4]. Despite these obstacles, YerA41 was successfully sequenced using RNA sequencing followed by in vitro DNA replication with its own DNA polymerase Gp144, demonstrating a potential strategy for resolving complex phage genomes [4], [5]. A comparable phenomenon has also been reported for Ralstonia phage RP13, a lytic bacteriophage isolated from tomato fields in Phang Nga, Thailand, which possesses highly modified bases that hindered sequencing efforts [6]. RP13 and YerA41 are both bacteriophages with large genomes of ca. 150 kb, however, both have capsid sizes comparable to those of > 200 kb jumbo phages, very likely to accommodate the extra space needed

<footnote n="1">¹ Institut Pasteur, Université Paris-Cité, CNRS UMR 3528, Unit of Architecture and Dynamics of Biological Macromolecules, F-75724 Paris, France</footnote>

<footnote n="2">² Institut Pasteur, Université Paris Cité, CNRS UMR 3523, Unit of Epigenetic Chemical Biology, F-75724 Paris, France</footnote>

<footnote n="3">³ Sorbonne Université, CNRS, Adaptation et Diversité en Milieu Marin, AD2M, F-29680 Roscoff, France</footnote>

<footnote n="4">⁴ Institut Pasteur, Université Paris Cité, Plate-forme Technologique Biomics, F-75015 Paris, France</footnote>

<footnote n="5">⁵ Department of Bacteriology and Immunology, Human Microbiome Research Program, Faculty of Medicine, University of Helsinki, Helsinki, Finland</footnote>

<footnote>§ corresponding author: marc.delarue@pasteur.fr</footnote>
by the heavily modified genomic DNA. These findings suggest that phages with heavily modified
 genomes are underrepresented in current databases, limiting our understanding of their biological
 functions and evolutionary significance. Addressing the sequencing challenges of Carin-5 could
 provide valuable insights into its genetic composition, infection mechanisms, and ecological roles,
 contributing to a broader understanding of phage diversity. Here, we describe how the sequencing
 challenges of Carin-5 parallelled those of the phages YerA41 and RP13 and discuss strategies to
 accurately determine its genome sequence. We also carried out LC-MS analysis of nucleosides of the
 phage genome and found that it carries a highly modified thymidine base, with a 88 Da linker, a
 phosphate group and an oligosaccharide. This pattern is highly reminiscent of the modification of
 phage SP-15 [7], to which the DNA polymerase of Carin-5 is clearly closely related, together with
 RP13, YerA41 and a dozen of other phages that we propose to cluster in a new family called SP15-
 like in genus *Thornevirus* [17].

**Materials and Methods**

1. Bacterial host culture

The marine Gamma Proteobacterium *C. marina* DSMZ 4741 was cryo-conserved at -80°C in a 17% v/v glycerol solution. Cultures were reactivated on a solid autoclaved ZoBell medium (5 g peptone, 1 yeast extract, 15 g agar diluted in 1 L of 0.2 µm filtered seawater). One day prior to the virus production, a single colony was picked from the solid medium and transferred into 5 ml autoclaved ZoBell Broth (5 g Peptone, 1 g Yeast extract in 1 L of 0.2 µm filtered seawater). The culture was incubated overnight at 25°C under vigorous agitation (180 rpm) on a rotary shaker.

2. Viral culture

Bacteriophage Carin-5 that infects *C. marina* DSMZ 4741 was isolated by plaque purification assays from coastal Atlantic marine waters [3]. Stock solution of the viral culture was kept at 4°C in the dark. Prior to the production for genome analysis, the infectivity of the viral suspension was assessed by spot test. Therefore, an aliquot the stock viral suspension was serially diluted in SM buffer (100 mM NaCl, 8 mM MgSO₄×7H₂O, 50 mM Tris-HCl, pH 7.5 in MilliQ water), and a 5 µL drop of each dilution was spotted on a host lawn obtained by plating a 1:4 mixture of host culture in molten agar (ZoBell Broth completed with 0.6% wt/vol noble agar) onto a Zobell agar plate (5 g Peptone, 1 g Yeast extract, 15 g Agar in 1 L of 0.2 µm filtered seawater). The plate was incubated overnight at room temperature, and translucent spots were enumerated to compute the titer of the viral suspension.

3. Bacteriophage production and purification

A volume of 2000 mL of host culture growing in ZoBell medium was infected at a multiplicity of infection on 0.1 based on the spot assay conducted previously. The mixture was incubated at room temperature under low agitation (60 rpm) on a rotary shaker for 20 h. The infection was stopped by centrifugation at 7000 × g for 30 min (Eppendorf 5810R) to remove intact bacteria and large bacterial debris. The supernatant was filtered through 0.2 mm PES filters (Filtration unit Stericup GP Millipore) to collect the virions. The viral suspension was concentrated by tangential filtration using a Vivaflow 200 cartridge (Sartorius Stedim) made of polyethylsulfone (PES) with a 30 kDa cutoff. The suspension was concentrated down to a volume of 50 mL. The 50 mL-sample was further concentrated using a Vivaspin20 centrifugal concentrators (Sartorius Stedim) made of PES with a cutoff 30 kDa. Repeated runs of centrifugation (6000 × g, 15 min) were performed until the sample reached a final volume of 1 to 2 mL. The concentrator was then incubated overnight at 4°C in order to maximize virus recovery. The viral concentrate was collected and purified on a linear 10 to 40% sucrose gradient by ultracentrifugation using a SW41Ti rotor at 134,400 × g for 30 min, 4°C (Optima XPN-80, Beckman). The band was extracted with a sterile syringe (1 mL) and washed 3 times with
15 mL of SM buffer using a 30 kDa PES Vivaspin column to remove traces of sucrose. The purified viral suspension was also kept overnight at 4°C to improve virus recovery.

## 4. Viral genome extraction

Viral DNA was extracted from the purified bacteriophage suspension using the DNeasy Blood & Tissue Kit (Qiagen) following the manufacturer's recommendations except for the proteinase K incubation time which was prolonged to 60 min (instead of 10 min). The DNA was quantified using a Qubit-4 fluorometer (Invitrogen).

## 5. Illumina sequencing

Genomic libraries were prepared using the TruSeq™ DNA PCR-Free kit and IDT for Illumina TruSeq DNA UDI indexes. Genomic DNA was fragmented into ~350 bp fragments using a Covaris E220 focused ultrasonicator. After end repair, index-containing adapters were ligated. The libraries were sequenced on an Illumina MiSeq system using a Nano v2 flow cell with a 2 × 151 bp paired-end read protocol, including index reads. The sequencing reaction was performed with a final library concentration of 8 pM in a 95:5 mixture of the libraries and PhiX control (20 pM). For libraries requiring pre-amplification, the prepared libraries underwent a 14-cycle amplification step using P5 and P7 Illumina primers. Sequencing was conducted on an Illumina MiSeq system using a Nano v2 flow cell with a 2 × 151 bp paired-end read protocol, excluding index reads. These libraries were sequenced at a final concentration of 30 pM as a single sample.

## 6. Genome reconstruction

The reads were first mapped on the *C. marina* genome using Bowtie2 (v2.3.5.1) [8] to remove host DNA contamination from the read genomic library. The unaligned reads were kept for further genome assembly. The reads were assembled using Spades (v3.15.0) [9], the obtained contigs were visualized with SnapGene (v8.0.2) and the predicted gene product sequences were submitted to BlastP for initial function assignment.

## 7. DNA polymerase production and purification

The DNA polymerase gene was codon optimized for *E. coli* bacterial expression using the Thermo Geneart online service and ordered from Genscript cloned into pRSF-duet backbone. The plasmid was transformed into *E. coli* NEB10β cloning strain (New England Biolabs) and plated on LB agar plate supplemented with kanamycin (50 mg/L). Bacteria from a single colony were grown in LB supplemented with kanamycin overnight at 37°C, the plasmid DNA was isolated with a miniprep kit and sequenced by Sanger sequencing to check the integrity of the DNA polymerase encoding gene. The purified plasmid was transformed into the *E. coli* BL21 *star* (DE3) expression strain (Thermo) and plated on LB agar plate supplemented with kanamycin (50 mg/L). A few clones were picked and cultured in LB medium with kanamycin at 37°C until the OD600 reached 0.6. IPTG was added (0.5 mM) and the cultures were incubated at 20°C overnight on a multitron shaker incubator (Infors HT) set at 110 rpm.

The bacteria were harvested by centrifuging the cultures at 3,600 × g, 4°C for 30 min. The pellets were transferred to a clean 50 mL tube and mixed with 3 volumes of Lysis buffer (50 mM Tris-HCl, pH 8.0, 300 mM NaCl, 10 mM MgCl2, 1 µL of Benzonase nuclease / 50 mL of solution (Millipore), 1 tablet of Pierce protease inhibitor / 100 mL of solution (ThermoScientific)). The cell lysates were obtained using a cell disruptor (CellD) and centrifuged at 20,000 g, 4°C for 30 min. The protein was purified at 4°C by affinity (HisTrap, Cytiva), cation-exchange (Heparin, Cytiva) and size exclusion (Superdex 200 Increase, Cytiva) chromatography steps using the buffers described in **Table S1**.
## 8. Non-modified neosynthesized genomic DNA production and amplification

The genome amplification by the purified DNA polymerase was performed by mixing the purified genomic DNA (5-10 ng/µL), with the reaction buffer (20 mM Tris-HCl pH 8.0, 50 mM NaCl, 5 mM MgCl₂, 10 mM DTT), 1 mM of dNTPs mix (New England Biolabs) and 100 ng/mL of DNA polymerase and by incubating the mixture at 25°C overnight.

PCR of fragments of the genome were amplified using the LongAmp Taq DNA polymerase (New England Biolabs) following the manufacturer's protocol with the primers presented in Table S2.

## 9. Phage genome annotation and comparison

The circular reconstructed genome was annotated using Prokka (v1.14.5) [10]. To confirm the presence and the specificity of tRNA inside the genome, the online version of tRNAscan-SE (v2.0.12) [11] was used. Knowing the close relatedness between the Carin-5 DNA polymerase and the YerA41 DNA polymerase, the proteome comparison service of the BV-BRC (v3.46.3) [12] was used to compare genes two-by-two and improve the initial automatic annotation. HHpred tool was used to search functional details to selected gene products [13].

## 10. Nucleoside analysis

Nucleoside Digestion Mix (New England Biolabs, Ipswich, MA, cat#M0649S) was used to digest Carin-5 genomic material into nucleosides. Briefly, 50 µl reactions were prepared with 2 µl of the enzyme mix, 5 µl of the 10× nucleoside digestion mix buffer and 43 µl of Carin-5 genomic material. Samples were incubated overnight at 37°C and stored at 4°C. The LC-MS chromatograms of Carin-5 genomic samples digested with the Nucleoside Digestion Mix were recorded with a Agilent Technologies 1260 Infinity Binary LC and Agilent 6100 Series Quadrupole LC/MS Systems with Phenomemex 150 × 4.6 SynergiTM 4 µm Fusion-RP 80 Å analytical column. The nucleoside samples were eluted for 2.5 min with 1% MeCN and 99% formic acid (0.1%) buffer in mQ water, and then increasing MeCN volume linearly to 10% at 25 min. Tandem mass spectroscopy (LC-MS/MS) was recorded with Thermo Scientific Q Exactive Hybrid Quadrupole-Orbitrap Mass Spectrometer using HPLC conditions mentioned above.

As a control, the genome of phage T4 was analyzed by the same procedure and the results confirmed the presence of a majority of glycosylated 5hmdC instead of dC.

## Results

**First sequencing and partial assembly.** Purified genomic DNA of Carin-5 was submitted to the Institut Pasteur Biomics sequencing platform for an Illumina NGS service. Following fragmentation and library preparation, initial quality control indicated that the library did not meet internal quality standards, resulting in a very low number of reads. To address this issue, the library was re-prepared with an additional pre-amplification step to increase the quantity of DNA deposited on the sequencing flowcell. This adjustment led to a typical yield of sequencing reads. Attempts at direct genome assembly using SPAdes did not produce the expected single contig corresponding to the target genome. Instead, multiple large contigs were obtained, some reaching up to 960,000 bp in length with coverage values ranging from 20 to 30. BLASTn analysis revealed that the first 16 contigs matched perfectly with the *C. marina* genome, whereas the 17th contig, approximately 39,000 bp in length and with a coverage of 131, showed no significant similarity to any known genome in the databases. Additionally, a few smaller contigs exhibited sequence similarity to *Chitinophaga* species. To eliminate contaminating reads contributing to contigs associated with *Chitinophaga* and *C. marina*, the sequencing reads were first mapped against the complete genomes of these species using Bowtie2. This analysis revealed that 6% of reads aligned with *Chitinophaga* and 80% with *C. marina*. Only the unmapped reads were retained for a second round of SPAdes-based genome assembly. From this filtered read
set, approximately ten contigs exceeding 6,000 bp in length and a coverage of 50 or more were obtained. While the longest contig (~39,000 bp) still showed perfect alignment with the *C. marina* genome, all remaining contigs exhibited no significant similarity to known genomes in BLASTn queries.

**DNA polymerase discovery.** Each contig was then thoroughly annotated, and all predicted open reading frames (ORFs) encoding proteins larger than 200 amino acid residues were translated and systematically submitted to BLASTp searches. Several large proteins were identified as being involved in nucleic acid metabolism. Notably, one protein shared 43.2% identity (97% query coverage, E-value = 0) with the DNA polymerase of YerA41, a bacteriophage that infects *Yersinia ruckeri* and exhibits sequencing challenges similar to those encountered with Carin-5. The Carin-5 gene encoding the DNA polymerase was codon-optimized for expression in *Escherichia coli*, synthesized by GenScript, and cloned into a modified pRSF1-Duet expression vector containing an N-terminal 14-histidine tag and a TEV protease cleavage site. The recombinant protein was subsequently expressed in *E. coli* and purified using Fast Protein Liquid Chromatography (FPLC).

**PCR after treatment.** To confirm the presence of modifications in the genomic DNA that hinder replication by exogenous polymerases, primers were designed within the contig containing the DNA polymerase gene to assess PCR amplification of fragments ranging from 500 to 3,000 bp. Several commercial PCR kits incorporating different polymerases were tested, but none successfully amplified even the smallest fragment (500 bp). The recombinant polymerase was then used to treat the genomic DNA overnight at room temperature in the presence of dNTPs. The treated sample was subsequently subjected to the same PCR protocols. Following this treatment, both Q5 and LongAmp Taq polymerases were able to amplify fragments up to 3,000 bp (Figure 1).

**Treatment and second sequencing.** Building on this result and following a protocol similar to that applied to the YerA41 genome [5], the treated Carin-5 genome was further purified using phenol-chloroform extraction and ethanol precipitation before being resubmitted to the Institut Pasteur Biomics sequencing platform. This time, following fragmentation, the library passed all quality control checks without the need for preamplification, and the sequencing run yielded a standard number of reads. Mapping of the obtained reads revealed no alignment with *C. marina* and *Chitinophaga* sp., confirming the absence of contaminant sequences. The unaligned reads were then used for SPAdes-based genome assembly. The primary contig obtained was 154,676 bp in length with a coverage of 352, while all remaining contigs were approximately 200 bp long with coverages below 1. The 154,676 bp contig, exhibiting a GC content of 37%, was considered to represent the complete genome of the Carin-5 bacteriophage. Furthermore, the previously identified smaller contigs (from the untreated genome) were successfully aligned to this reference sequence. Notably, the initial genome assembly—consisting of 10 disjoint contigs longer than 2,000 bp—had already reconstructed 87% of the complete genome, despite a significantly lower coverage.

**The genome is circularly permuted.** A duplicate sequencing run was carried out, and the analysis was repeated from the beginning. As observed in the first library, no reads aligned with *C. marina* or *Chitinophaga* sp., and the SPAdes assembly once again produced a single contig of 154,676 bp. Alignment of the two assemblies revealed a shift of 27,246 bp between the linear sequences, with perfect sequence identity except for the first 55 bases of each contig. Further analysis indicated that the first 55 bases of the initial assembly were repeated at the end of the sequence, whereas in the second assembly, this sequence appeared only once but was absent from the beginning. The observed shift between assemblies from two independent sequencing runs strongly suggested that the genome was circularly permuted. We also run the PhageTerm software [14] and no termini was found in line with a circular genome. Additionally, the presence of the 55-bp repeat at both ends supported the conclusion that the actual genome length was 154,621 bp, after accounting for the removal of one redundant 55-bp sequence prior to circularization. The complete genome sequence has been
submitted to GenBank (https://www.ncbi.nlm.nih.gov/genbank/) and is available under the accession
number PV155638.1.

**BLASTn** A BLASTn search using BLAST+ (v2.10.0) [15] against the GenBank bacteriophage database (gbphg), with an E-value cutoff of 10⁻³, revealed that 25.5% of the Carin-5 genome exhibited a high degree of identity with known phages in the database. After excluding matches corresponding to the two closest related phages—*Yersinia* phage vB_Yru_GN1 (GenBank accession number LC779065.1) and *Yersinia* phage YerA41 (GenBank accession number MW570730.1)—only 1.5% of the Carin-5 genome showed similarity to elements within the remaining database entries. These findings suggest that Carin-5 belongs to a new family within the class *Caudoviricetes*. The homology between Carin-5 and YerA41 genomes is illustrated by a dot plot in **Figure 2**. The position of Carin-5 in the phylogenetic proteomic tree is illustrated in **Figure 3A**, while a phylogenetic tree restricted to phage genomes containing a DNA polymerase highly similar to the one of YerA41, in particular those containing a N-terminal UDG domain [17], is shown in **Figure 3B**.

**Prokka, BLASTp and BV-BRC.** The Carin-5 genome was annotated using Prokka, a rapid prokar-
yotic genome annotation tool [10]. This workflow detects coding sequences (CDS), rRNAs, tRNAs,
signal leader peptides, and non-coding RNAs (ncRNAs) using gold-standard bioinformatics tools.
Gene annotation is performed progressively through comparison with large databases, with genes
remaining classified as “hypothetical proteins” in the absence of significant matches. Prokka identi-
fied 222 genes and four tRNAs (specific for arginine, methionine, leucine, and glycine). Functional
annotations were assigned to 47 genes (21%). The presence of only four tRNAs was further confirmed
using the online tRNAscan-SE tool [11]. Functional predictions based on homology searches were
performed using GHOSTX against the GenomeNet nr-aa database via the ViPTree webserver [16].
Among the 222 genes, 112 (50.5%) exhibited significant similarity with database sequences, with E-
values below 10⁻³. Notably, 71 genes (32%) had their closest homologs in *Yersinia* phage YerA41
(GenBank accession number MW570730.1), 44 genes (20%) showed similarity to hypothetical or
uncharacterized proteins, and 21 genes (9.5%) were homologous to unspecified structural proteins.
These homology-based predictions were further validated through systematic BLASTp searches us-
ing BLAST+ (v2.10.0) [15] against the NCBI RefSeq database, with an E-value cutoff of 10⁻³. A total
of 95 genes (43%) had significant matches in RefSeq (Supplementary Table S3), among which 50
genes (22.5%) were predicted to have known functions. Given the high proportion of matches with
YerA41 proteins, the online BV-BRC proteome comparison tool was employed to compare the pro-
teomes of Carin-5 and YerA41 [12]. This analysis identified homologs for 89 genes (40%) within the
YerA41 genome, of which 29 genes (13%) had predicted functions and 24 genes (11%) were anno-
tated as structural proteins (Figure 2 and Supplementary Table S3). This comprehensive annotation
process enabled the identification of several DNA replication enzymes, including a family A DNA
polymerase (gene #181), a DNA polymerase X (gene #164), a DNA ligase (gene #196), DNA hel-
icases (genes #108, #214, #215), and DNA topoisomerases (genes #190, #223, #224). Additionally,
genes potentially involved in the formation of highly modified nucleotide bases were identified, such
as thymidylate synthetases (genes #049, #169). The position of the main genes and the GC-biases are
illustrated in Figure 4.

The initial observation of the ability of Carin-5 to carry an efficient EPS depolymerase activity
prompted us to search for the possible gene encoding the enzyme. The gene #152 (and #153 and
#154) was predicted to encode a tail spike protein (1844 aa residues) with a pectin lyase-like fold or
glycosyl hydrolase family 28-related protein, and the gene #156 also a tailspike glycosidase protein
(2186 aa, also glycosyl hydrolase family 28-related protein) with a pectin lyase fold. The gene # 159
in turn was predicted to encode Predicted Peptidoglycan domain/Chitosanase, subunit A/Glycosyl
hydrolase (N-acetyl muramidase), a glycoside hydrolase family 108-related protein.
The sample of Carin-5 DNA was treated with a mix of nucleases (NEB) and purified using phenol-chloroform treatment followed by ethanol precipitation to be analyzed by LC-MS/MS. A deficit in dT vs dA was observed, implying the presence of modified T of **MW 1073 Da**, whose percentage was estimated at about **75%**.

The thymidine modification was explored by MS-MS experiments (**m/z 1074 in positive mode**) that suggested the presence of the following groups substituting the thymidine moiety of MW 242 Da: (1) one 88 Da linker, (2) two 162 Da hexose residues, (3) one 203 Da *N*-acetylamine hexose residue, (4) one unknown 216 Da residue.

In addition, the MS-MS in negative mode (**m/z 1072**) indicated the presence of a 80 Da phosphate residue (HPO₃⁻) and a 154 Da ion C₃H₇O₅P, possibly containing the phosphate itself, leaving an unknown 74 Da anion (**Figures S1-S3**).

The genomic DNA of YerA41 was also re-analyzed by the same LC-MS method and the results of MS/MS in the positive mode confirmed what was observed earlier in [5]. In particular, i) the presence of hypermodified dT was observed, with an estimated percentage of **70%**, as well as ii) the presence of two peaks at m/z 918 and 1102, both with a common fragment of m/z 346, implying a linker of MW 104 Da (346=242+104). The difference of the 1102 and 918 peaks confirmed the presence of an unknown 184 Da residue (close to glucose of 180 Da), and the total mass of the modified thymidine suggested that the residue is present up to four to five times (glycosylation). In addition, the LC-MS performed in the negative mode detected the presence of a phosphate moiety in both the 918 and 1102 Da fragments.

Taking into consideration that the Blastp search of both the YerA41 and Carin-5 DNA polymerases showed similarity to the SP-15 DNA polymerase [17] (**Figure 5A**), prompted us to compare new mass spectrometry data on the thymidine modifications to what is known on the hypermodification of the SP-15 DNA [7] : it contains a dihydroxypentyl linker on the thymidine ring, with one (4') of the hydroxyl groups directly glycosylated and the other (5') hydroxyl group is phosphorylated and then glucuronated (**Figure 5B**).

This immediately suggests a situation for both Carin-5 and YerA41 with T-modifications that are similar to that of SP-15, containing a linker of 104 or 88 Da, respectively (note that MW of di-hydroxypentane is 104 Da while that of hydroxypentane is 87 Da), phosphorylated on the hydroxyl group, and then further glycosylated/substituted by glucose(s) or *N*-acetyl-glucosamine(s).

Regarding the observed nucleotide modifications, the #174 - #179 genes encode proteins associated with sugar modifications and glycosyltransferases (**Suppl. Figure S4**), located analogously to the YerA41 genome neighboring the DNAP encoding gene (#181). The #174 gene product is a glycosyl-transferase with similarity to YerA41 putative glycosyl (KDO) transferase. While the #175 gene product is annotated as CTP:phosphoglutamine cytidylyltransferase in HHpred it shows with >99% probability similarity to glucose-1-phosphate thymidylyltransferase, the enzyme that generates the NDP-activated sugar donor for the glycosyltransferases. Very likely the product dTDP-glucose functions as the starting point for the further sugar modifications.

The #177 gene product is annotated as a polysaccharide deacetylase and shows in HHpred search with >95% probability similarity to *N*-acetyl-D-glucosamine *N*-deacetylase. The #178 gene product, even though annotated as a hypothetical protein shows also with 70-80% probability local similarity to glycosyltransferases in HHpred. The *N*-terminal half of #179 gene product shows similarity to *N*-acetylhexosaminyltransferases in HHPred search with 96.7 % probability. In conclusion, these genes are likely involved in the building of the thymidylate modifying oligosaccharide, however, details of the functions and the structure remain to be solved.

**Discussion**
The first bulky modifications of pyrimidines in bacteriophages were reported in the early 1970s [18], [19]. Given the structural constraints of most DNA polymerase active sites, their inability to replicate such modified DNA is readily understood because of a steric hindrance. In the era of metagenomics— where thousands of genomes can be reconstructed simultaneously from environmental samples—it is increasingly likely that a significant fraction of microbial genomes are chemically modified and thus inaccessible to conventional polymerase-based sequencing technologies. Current Oxford Nanopore Technologies (ONT) nanopore designs have been optimized for native, unmodified DNA. While some efforts have been made to adapt base-calling algorithms to detect small DNA modifications, such as methylation [20], these subtle changes do not impede DNA passage through the nanopore. In contrast, bulky chemical modifications are likely to physically block translocation through the pore, thus halting the sequencing process altogether. This limitation was confirmed in the case of the highly modified *Carin-5* genome, which resisted ONT sequencing. Although bulky modifications severely impair DNA replication, the low DNA yield during Illumina library preparation prompted us to attempt a preamplification step—a strategy that has previously improved library quality in our laboratory. While this did increase the library quality, downstream bioinformatic analysis revealed that the majority of sequencing reads aligned with *C. marina* and, to a lesser extent, *Chitinophaga* species. Trace amounts of host DNA or bacterial contaminants are commonly present in phage DNA preparations. In this instance, the preamplification preferentially amplified the contaminant DNA rather than the phage genome, which remained resistant to standard enzymatic processing. This result highlights the non-trivial challenges associated with manipulating highly modified, non-amplifiable phage DNA and the broader difficulties of exploring this "biological dark matter," the true extent of which remains largely unknown. In this context, the ability of an endogenous phage-encoded DNA polymerase to replicate its own genome is of particular significance. To demonstrate this activity, we incubated purified *Carin-5* genomic DNA with the recombinant DNA polymerase overnight at 25°C in the presence of dNTPs. Subsequent PCR testing showed that amplification was only possible following this treatment. This result indicates that while the native, modified genome resists amplification, the newly in vitro synthesized strands—produced by the endogenous polymerase—lack the obstructive modifications and are thus accessible to standard PCR enzymes. Following this enzymatic pretreatment, Illumina library preparation proceeded successfully. Sequencing yielded high coverage and depth, allowing for complete, accurate, and reproducible genome reconstruction.

Based on its phenotype and phylogeny, Carin-5, along with phages YerA41 and RP13, appears to represent a distinct group of bacteriophages with highly modified DNA. Despite infecting different bacterial hosts, these phages share a common trait: extensive DNA modifications that not only complicate sequencing efforts but also serve as a defense mechanism against host-encoded restriction-modification (RM) and CRISPR-Cas systems. This reflects the ongoing evolutionary arms race between bacteriophages and their bacterial hosts, where phages continuously develop strategies to evade host defenses, while bacteria, in turn, evolve mechanisms to counteract phage infections. While other bacteriophages, such as T4 and P1, are also known to harbor modified bases—like glucosylated hydroxymethylcytosine in T4—these modifications have not been reported to hinder sequencing to the same extent as those observed in YerA41, RP13, and Carin-5. The extreme difficulty in sequencing these phages suggests the presence of more complex or extensive modifications that interfere with standard sequencing technologies, including Sanger and next-generation sequencing (NGS).

To overcome these challenges, *in vitro* expression of phage-encoded DNA polymerases, as successfully demonstrated with YerA41 [5], could facilitate the amplification of highly modified DNA, enabling more efficient and complete genome assembly.

The presence of a phosphate group is a new feature that is present not only in SP-15, but also in YerA41 and Carin-5. While it is certainly sticking out in solution in the major groove of the DNA and surrounded by counter ions, its presence in the DNA binding site of the DNAP, close to the DNA
phosphodiester backbone, may have necessitated the evolution of a new subfamily of DNA polymerases.

We note that Weigele et al. [21] deciphered the biosynthesis pathway of various hypermodified thymidines, all involving the post-replication modification of 5hmdU, essentially through the addition of an amino-acid, followed by its decarboxylation.

The biosynthesis of 5hmdU itself is usually the result of the transformation of dUMP by a variant of thymidylate synthase, while dUMP can be the product of dCTP deaminase followed by dUTP diphosphatase, or dCMP deaminase. We looked for the presence of these genes in the genomes of Carin-5, YerA41 and SP15 and other phage genomes whose DNA polymerase is similar to the one of YerA41 [17]: PhiLo, RP13, IME392, RHph-TM30, LiS04. The results are summarized in **Table 1**. Carin-5 is the only one which contains both a dCTP deaminase and a dCMP deaminase.

In summary, we propose to refer to this family of phages containing this type of DNA modification as SP15-like. While our study does not decipher the complete biosynthesis pathway of the modified thymidine, it narrows down the search to genes common to this new family. Other members of the family are currently under study in our lab, while monthly database searches will likely indicate more members as time goes by.
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**Statements and Declarations**

**Funding**

MD, SM and RS acknowledges financial support CNRS (PEPR MoleculArxiv Grant No. ANR-22-PEXM-0002). MD's laboratory is also supported by I. Pasteur and CNRS. MM's Biomics Platform, C2RT, Institut Pasteur, Paris, France, is supported by France Génomique (ANR-10-INBS-09) and IBISA.

**Competing interests**

The authors declare no competing interests

**Author contributions**

PN & ACB extracted and purified the phage Carin-5 genomic DNA.

MS purified phage YerA41 genomic DNA and helped extensively with the writing of the manuscript.

FB performed and analyzed the mass spectrometry experiments.

MM supervised the phage sequencing experiments.

RS & SM reconstructed the genome, produced and purified the DNA polymerase and performed
functional tests.

MD, SM & RS designed the study.

MD supervised the entire study.

All the authors reviewed the manuscript.

**Data availability**

The genome of Carin-5 bacteriophage is available online under the Genbank accession number
PV155638.1.

**Ethics approval**

This research involves no human or animal subjects

**Supplementary materials**

This article refers to supplementary figures and tables gathered in the supplementary information file.

**Acknowledgments**

We thank Laurence Ma of the BIOMICS sequencing facility at the Institut Pasteur for carrying out
libraries preparation and Illumina sequencing.

**Conflicts of interest**

The authors declare no conflict of interest.
<table><caption>**Table 1.** Presence of genes necessary to the biosynthesis of 5hmdUMP in the genomes of Carin-5, YerA41 and SP15, as well as other phages whose DNAP shows detectable homology to the ones of Carin-5 and/or YerA41 [17]. The Genbank reference of the genomes as well as their size is indicated.</caption><thead><tr><th>Phage Name<br>GenBank Ref.</th><th>dCMP deaminase</th><th>dCTP deaminase</th><th>Thymidylate syn-<br>thase</th><th>Genome size<br>(kb)</th></tr></thead><tbody><tr><td>Carin-5<br>PV155638.1</td><td>Yes<br>Carin-5_00189</td><td>Yes<br>CLPJFFLI_00063</td><td>Yes<br>Carin-5_00169</td><td>154.6</td></tr><tr><td>B. subtilis SP-15<br>NC_031245.1</td><td>Yes<br>SP15_219</td><td>No</td><td>Yes<br>SP15_009 & 130</td><td>221.9</td></tr><tr><td>YerA41<br>MW570730.1</td><td>No</td><td>Yes<br>YerA41_068</td><td>Yes<br>YerA41_081</td><td>145.5</td></tr><tr><td>T. thermophilus<br>PhiLo<br>MH673673.1</td><td>?</td><td>?</td><td>Yes<br>phiLo_64</td><td>178.5</td></tr><tr><td>Ralstonia RP13<br>LC554890.1</td><td>g21</td><td>No</td><td>g86</td><td>170.9</td></tr><tr><td>Rhizobium<br>RHph TM30<br>NC_070966.1</td><td>Yes<br>PQC16_gp343</td><td>No</td><td>Yes<br>PQC16_gp196</td><td>207.6</td></tr><tr><td>Listeria LIS04<br>OQ999172.1</td><td>Yes<br>WJZ23590.1</td><td>No</td><td>Yes<br>WJZ23437.1</td><td>181.6</td></tr><tr><td>E. coli EP_H11<br>OP688485.1</td><td>No</td><td>Yes<br>WBU87719.1</td><td>Yes<br>WBU87640.1</td><td>111.4</td></tr><tr><td>EcoM_IME-392<br>MH719082</td><td>No</td><td>Yes<br>AXY86133.1</td><td>Yes</td><td>116.5</td></tr></tbody></table>
# Figures
<figure><img src="image_2.png" /><figcaption>**Figure 1.** Agarose electrophoresis gels of PCR on Carin-5. (A) 1: Negative control, LongAmp Taq PCR on untreated genome. 2: LongAmp Taq PCR after overnight treatment with wild-type Carin-5 endogenous DNA polymerase. 3: LongAmp Taq PCR after overnight treatment with exonuclease-inactivated Carin-5 endogenous DNA polymerase. (B) LongAmp Taq PCR performed on 1: 500 bp-2: 1000 bp-, 3: 1500 bp-, 4: 2000 bp-, 5: 2500 bp-, and 6: 3000 bp-long amplicons after overnight treatment with wild-type Carin-5 endogenous DNA polymerase.</figcaption></figure>
<figure><img src="image_3.png" /><figcaption>**Figure 2.** Dot plot for Carin-5 and YerA41 Proteomes comparison. Each dot corresponds to a protein homology detected by the Proteome comparison tool of the online BV-BRC suite [12]. The color represents the percent of sequence identity between the each pair of considered genes.</figcaption></figure>
**Figure 3.** Proteomic trees showing the phylogenetic position of Cobetia phage Carin-5. (A) Tree drawn using the “with references” mode of the ViPTree server (v 4.0), containing the Carin-5 genome, 8 genomes of organisms presenting replicative DNA polymerase related to Carin-5’s one and 939 related genomes (i.e. showing sequence similarities S<sub>G</sub> > 0.02 to at least one of the 9 aforementioned genomes) from the Virus-Host DB. (B) Tree drawn using the “only query” mode of the ViPTree server (v 4.0), containing the Carin-5 genome and 8 genomes of organisms presenting replicative DNA polymerase related to Carin-5’s one.

A

<figure><img src="image_4.png" /></figure>

B

<figure><img src="image_5.png" /></figure>
**Figure 4.** Genome map of the *Cobetia* bacteriophage Carin-5. The predicted genes and their directions are shown with filled arrows. The predicted functions of the gene products are indicated in the label boxes, and the locations of the tRNA-encoding genes are indicated. The DNA polymerase -encoding and other DNA-modification-associated genes are shown in red, and the genes predicted to be involved in thymidine modification are shown in light green. The RNA metabolism genes are shown in turquoise, tRNA-genes, in dark green, dNTP metabolism, in yellow, and the putative phage particle associated proteins encoding genes, in brown.

<figure><img src="image_6.png" /></figure>
**Figure 5.** Related phages and their DNA modifications. A) Phylogenetic tree of DNA polymerases related to Carin-5 DNAp (PolA family), selected to have a length larger than 900 amino-acids and containing the N-terminal UDG-like domain. Phages known to have a hypermodified thymidine in their genome have a red star: RP13 is annotated as carrying a DNA modification on the thymine (Kawasaki et al., 2021), based on conservation of enzymes putatively involved in its biosynthesis (Kropinski et al., 2008). B) DNA modification of SP15, showing the linker, the phosphate, and the sugars R1 and R2 which can be glucose and glucuronic acid.

A.

<figure><img src="image_7.png" /></figure>

B.

<figure><img src="image_8.png" /></figure>
**Supplementary Files**

This is a list of supplementary files associated with this preprint. Click to download.

* Carin5paperSI.pdf