All bacterial strains and information on phage F341 are listed in Supplementary Table S1. Campylobacter jejuni was standardly grown on base II plates with 5% calf blood (BA) at 37°C under microaerobic conditions (6% O_2, 6% CO₂, 88% H₂N₂ or 6% O_2, 6% CO₂, 88% N₂).

The genome sequences of C. jejuni strain NCTC12658 and Campylobacter phage F341 are deposited in NCBI Genbank under the NZ_CP081480.1 and OQ864999 Accession numbers. The fastq sequencing files of the LJ variants can be found at the European Nucleotide Archive under project Accession number PRJEB61440.

C. jejuni strains of interest were grown under standard growth conditions and harvested into cation adjusted (1 mM CaCl₂, 10 mM MgSO₄). Brain Hearth Infusion broth (Oxoid) (CBHI) and subsequently adjusted to OD₆₀₀ of 0.01 in 10 mL cultures. Phage F341 was added to a 10 mL culture containing C. jejuni strain NCTC12658 to reach a multiplicity of infection (MOI) of 0.001, whereas no phage was added to a NCTC12568 control culture and a culture containing NCTC12658∆motA. All cultures were incubated for 24 h under standard growth conditions. Following incubation, serial dilutions (10⁻⁴–10⁻⁸) of the cultures were made in phosphate buffered saline, and 100 μL of each serial dilution was plated onto BA plates with 5 mL of NZCYM overlay agar with 0.6% agar concentration that had been pre-dried for 45 min under a flow hood. The plates were then incubated under standard growth conditions for 2–3 days until single colonies were visible. Swarming colonies appearing on plates where serial dilutions of the NCTC12658 + phage F341 culture had been plated were re-streaked on regular BA plates, harvested into BHI, and stored at −80°C. Swarming colonies named LJ1–LJ14 were later tested for phage sensitivity and motility as mentioned below.

Motility assays were performed as described previously (Sørensen et al., 2011). In brief, C. jejuni strains of interest were grown at standard conditions for 18–24 h. Cells were then harvested into BHI and adjusted to an OD₆₀₀ of 0.1. One microliter of the suspension was spotted in the center of five Heart infusion broth (HIB) (Difco) 0.25% agar plates that had been pre-dried for 45 min in a flow hood. Plates were incubated under standard growth conditions and growth zones representing motility were measured after 24, 42, and 48 h. Measurements and standard deviations represent the mean counts from two independent experiments.

Phage F341 was propagated on C. jejuni NCTC12658 as described previously using the double-layer agar method (Baldvinsson et al., 2014). Phages from lysed plates were harvested into 5 mL SM buffer (50 mM Tris-HCl, pH-7.5, 100 mM CaCl, 8 mM MgSO₄) and stored at 4°C. Titration and standard plaque assays were performed as previously described using spot assays on bacterial lawns (Sørensen et al., 2021a). Briefly, tenfold serial dilutions of the F341 phage stock were made in SM buffer up to 10⁻⁷, and three aliquots of 10 μL of the undiluted stock (10⁰) and each dilution were spotted on bacterial lawns with the strains of interest. Plates were incubated for 18–24 h at 37°C under standard C. jejuni growth conditions and plaques were counted and the mean plaque forming units per ml (PFU/mL) were calculated. The RBP antibody plaque assay was performed as previously described (Sørensen et al., 2021a). Briefly, anti-F341_RBP serum and pre-serum (serum extracted before immunization) were added at a 1:10 ratio to the phage F341 stock for 1 h at room temperature before serial dilutions were made and spotted on NCTC12658 lawns. All experiments were performed in duplicate and the data presented are the means from two independent experiments.

Phage adsorption assays were performed as previously described (Baldvinsson et al., 2014). Briefly, C. jejuni cultures were washed and adjusted to an OD₆₀₀ of 0.4 in CBHI and inoculated with phage F341 at an MOI of 0.001. Cultures were incubated at 37°C and samples containing free phages were collected at 0, 30, 60, and 90 min and filtered through a 0.22 μm sterile syringe filter (Millipore). Filtered samples were stored at 4°C until enumeration (plaque assay). Free phages were enumerated on C. jejuni NCTC12658 and calculated as the percentage of free phages compared to time point zero for each sample (phage PFU/mL at time zero was accounted as 100%). The data are representative of two independent experiments and represent the mean percentages of free phages and standard deviations thereof.

Genomic DNA from phage F341 was isolated as described previously (Sørensen et al., 2021a). The DNA concentration was estimated visually based on 0.7%–1% agarose gel electrophoresis as Campylobacter phages contain modified DNA (Crippen et al., 2019) that cannot be measured correctly by fluorometric quantification. Chromosomal DNA from C. jejuni NCTC12658 was isolated using Maxwell 16 Tissue DNA extraction kit (Promega) (Illumina sequencing) and the MagAttract HMW DNA kit (Qiagen) (SMRT sequencing, Pacific Biosciences) according to the manufacturer’s instructions. Chromosomal DNA from the LJ variants was isolated using Maxwell 16 Tissue DNA extraction kit (Promega) according to the manufacturer’s instructions.

Genome sequencing of C. jejuni NCTC12658 was done on the Illumina platform (Illumina Hiseq and Miseq) and on a PacBio RS II device (Pacific Biosciences, Menlo Park, CA, United States) using P6/C4 chemistry using one SMRT cell. The complete de novo genome was assembled using SMRT Analysis version 2.3 and the HGAP3 algorithm. Average coverage was 634-fold. The genome was annotated using the NCBI Prokaryotic Genome Annotation Pipeline (PGAP). Genome sequencing of phage F341 resistant NCTC12658 variants LJ1, LJ6, LJ8, LJ9, LJ10, LJ11, and LJ13 was performed using the Illumina platform (Illumina Miseq). Sequencing of LJ4 was not performed due to failed library preparation. Basic variant detection analysis was performed using CLC genomics workbench version 21.0.3 using default settings. Reads mapping to the NCTC12658 reference genome were as follows for the phage F341 resistant variants; LJ1: 384.052, LJ6: 674.826, LJ8: 656.167, LJ9: 535.977, LJ10: 565.008, LJ11: 537.104 and LJ13: 460.315 with coverage of detected SNP’s ranging from 10-fold to 132-fold. De novo assembly of the phage F341 resistant variants was performed using CLC genomic workbench version 21.0.3 with a total number of contigs generated: 144 (LJ1), 157 (LJ6), 141 (LJ8), 173 (LJ9), 113 (LJ10), 151 (LJ11) and 39 (LJ13).

The phage F341 genome was sequenced on a PacBio RS II device (Pacific Biosciences, Menlo Park, CA, United States) using P6/C4 chemistry using one SMRT cell. Sequencing produced 30,591 reads with a mean read length of 25,853. The complete de novo genome was assembled using SMRT Analysis version 2.3 and the HGAP3 algorithm. The average coverage of the phage F341 genome was 6,006-fold. The phage genome was annotated using CPT Galaxy and WebApollo platforms (Afgan et al., 2018). Comparative genomics of Fletchervirus phages was performed in CLC main workbench version 20.0.4 using the whole genome alignment 20.1 plugin tool with default settings. Upper comparison gradient: ANI (average nucleotide identity), lower comparison: AP (alignment percentage). Detailed in silico analysis of phage F341 proteins was performed using InterPro (Mitchell et al., 2019), BLASTp (Johnson et al., 2008), HHpred (Zimmermann et al., 2018), Colab AlphaFold2 (Mirdita et al., 2022) and Dali server (Holm, 2022). Proteins are visualized using PyMOL (Schrödinger, 2015).

Pulsed field gel electrophoresis (PFGE) analyses of C. jejuni NCTC12658 and LJ variants were performed according to Ribot et al. (2001) using two restriction enzymes, SmaI and KpnI (New England Biolabs), separately.

The deletion of 05515-05525 in NCTC12658 was constructed by replacing most of the genes (the first 287 bp of 05525 and the last 36 bp of 05515 are maintained) with a kanamycin (kan) cassette by homologous recombination. A vector was constructed by fusing three PCR amplicons as described below using In-fusion cloning (Takara) and used for homologous recombination. Two PCR fragments (746 bp and 829 bp) flanking the region upstream and downstream of the 05515-05525 deletion were amplified from C. jejuni NCTC12658 using the primers LOS_F_up and LOS_R_up and LOS_F_down and LOS_R_down, respectively. The kan gene (1,398 bp) was amplified from pBCα3 (Bijlsma et al., 1999) using the primers Kan + LOS_F and Kan + LOS_R. The kan gene was then inserted between the upstream and downstream 05515-05525 flanking PCR fragments and simultaneously inserted into the pGEM-7Zf vector (Promega) in the BamHI and XhoI sites using the In-Fusion kit according to the manufacturer’s instructions. The resulting plasmid pMP810 was transformed into Escherichia coli StellarTM Competent Cells (Takara) according to manufacturer’s instructions and verified by sequencing. Purified pMP802 plasmid was electroporated into C. jejuni NCTC12658 promoting homologous recombination of the flanking 05515-05525 regions to create NCTC12658Δ05515-05525. The mutant was verified by PCR using the LOS_control_F and LOS_control_R primers and sequencing. Plasmids and primers are listed in Supplementary Table S1.

A 1.439 bp fragment including F341_rbp and the downstream F341_034 chaperone plus an additional TAA stop codon was synthetically produced and inserted into the NdeI and XhoI cloning sites of pET28a + producing the F341_RBP protein with an N-terminal his-tag used for purification of the protein.

The F341_RBP was expressed and purified as previously described using E. coli BL21-CodonPlus (DE3)-RIL competent cells (Agilent Technologies) and 0.5 mM IPTG (isopropyl β-D-1-thiogalactopyranoside) induction (Sørensen et al., 2021a). The F341_RBP was purified using Ni²⁺-NTA-affinity chromatography (His GraviTrap, GE Healthcare) according to the manufacturer’s instructions and exchanged into PBS buffer using 10 kDA Amicon^® Ultra-15 Centrifugal filter units. The purified protein was used to raise polyclonal rabbit antibodies generated by Davids Biotechnologie GmbH (Germany). Anti-F341_RBP serum (anti-RBP serum) was purified on a protein A column and stored at −20°C until use. The binding ability of purified anti-RBP serum to phage F341 was confirmed by standard Western blotting.

Immunogold electron microscopy was performed as described previously (Sørensen et al., 2021a). Staining was performed as previously reported with 2% (w/v) uranyl acetate on freshly prepared carbon films (Sørensen et al., 2015). Grids were analyzed in a Tecnai 10 transmission electron microscope (TEM) (FEI Thermo Fisher, Eindhoven, The Netherlands) at an acceleration voltage of 80 kV. Micrographs were taken with a MegaView G2 charge-coupled device camera (EMSIS, Muenster, Germany).

Data in the graphs and figures are representative of at least two independent experiments and show mean values with error bars that represent the standard deviations as reported in the figure legends.

To investigate phage resistance in C. jejuni against flagellotropic phages when motility is maintained, we developed a soft agar assay that would allow us to screen for motile phage resistant variants in vitro. We exposed C. jejuni NCTC12658 to phage F341 in liquid culture for 24 h followed by plating serial dilutions on double layered soft agar (SA) plates where swarming/motility of single colonies could be observed (Figure 1A). To evaluate motility levels, we also plated the C. jejuni NCTC12658 wildtype (motile) and an NCTC12658∆motA mutant (non-motile) as controls. As expected, many colonies of the NCTC12658 wildtype exhibited swarming behavior, while no swarming was observed for the NCTC12658∆motA colonies (Figure 1A). Most colonies originating from NCTC12658 exposed to phage F341 demonstrated no swarming behavior, consistent with previous findings that loss of motility rapidly occurs when C. jejuni is exposed to flagellotropic phages in vitro. Still, some motile colonies could be observed (Figure 1A), suggesting that phage F341 resistance without loss of motility can also develop in vitro. To verify that the swarming colonies exposed to phage F341 indeed were fully motile and resistant to phage F341, we isolated 14 swarming colonies from the SA plate (named LJ1 to LJ14) and investigated phage F341 sensitivity by plaque formation and assessed motility in soft agar. Six of the LJ colonies were still highly motile but had not gained phage resistance, as F341 plaque formation was comparable to the NCTC12658 wildtype (data not shown). However, eight colonies (LJ1, LJ4, LJ6, LJ8, LJ9, LJ10, LJ11, and LJ13) demonstrated complete resistance to phage F341 as no lysis nor plaque formation was observed, while still exhibiting motility at levels comparable to the NCTC12658 wildtype (Figure 1B). To determine if phage F341 resistance was associated with lack of phage binding, we investigated whether F341 could adsorb to the eight motile phage resistant LJ strains (Figure 1C). Our data showed that for all eight LJ strains, phage adsorption was prevented suggesting changes in surface structures such as alteration or masking of the receptor recognized by phage F341. Thus, our soft agar assay allowed us to isolate motile C. jejuni NCTC12658 variants in vitro exhibiting complete resistance towards flagellotropic phage F341.

To identify genetic changes that could account for phage F341 resistance, we sequenced the genomes of seven phage resistant NCTC12658 variants (LJ1, LJ6, LJ8, LJ9, LJ10, LJ11, and LJ13) with Illumina sequencing. We also genome sequenced the NCTC12658 wildtype strain using both Illumina and Pacific Bioscience (PacBio) SMRT sequencing to fully assemble the genome. We then used the complete genome of NCTC12658 and the sequencing reads of the seven phage resistant variants to detect single nucleotide polymorphisms (SNPs) and larger gene insertions or deletions (Table 1; Supplementary Tables S2, S3). To identify potential genomic rearrangements we also performed de novo assembly and pulsed field gel electrophoresis (PFGE), but no rearrangements were observed (Supplementary Figure S1).

As the genome of LJ9 was not fully covered by the sequencing we could not identify genetic changes associated with phage F341 resistance in this variant. However, our analysis identified a limited number of SNPs (≤14) and two gene deletions in the remaining phage resistant NCTC12658 variants (Supplementary Results; Table 1; Supplementary Tables S2, S3). Most SNPs were associated with insertions or deletions (indels) in homopolymeric G (polyG) or A (polyA) tracts causing phase variable gene expression in C. jejuni. Such indels are expected within a given C. jejuni population due to the stochastic nature of slipped strand mispairing of such regions during DNA replication (van der Woude, 2011; Bayliss et al., 2012). Thus, some SNPs may not be due to phage F341 exposure but could be a random mutation event originating from picking single LJ colonies. Also, different homopolymeric tract lengths were in some cases observed between the two sequencing methods for the NCTC12658 wildtype. Hence, when gene expression was found to be both turned on and off in the wildtype NCTC12658 strain sensitive to phage F341, or when different homopolymeric tract lengths were observed that did not lead to differences in the gene expression state, the SNP was not considered relevant. In addition, our experimental data showed that phage resistance was associated with lack of phage adsorption, indicating genetic changes associated with surface structures. Genetic changes not considered relevant for phage F341 resistance development are presented in Supplementary Results and Supplementary Tables S2, S3.

Notably, four out of the seven LJ strains (LJ1, LJ6, LJ8, and LJ11) contain SNPs in genes encoding sugar transferases modifying the LOS structure of C. jejuni NCTC12658 (Table 1). NCTC12658 contains the same LOS locus as C. jejuni NCTC11168 producing an inner LOS core containing ketodeoxytonic acid, heptose, phosphoethanolamine and glucose, and an outer LOS core containing N-acetylgalactoseamine, N-acetylneuraminic acid (sialic acid), glucose and galactose (Figure 2A) (Karlyshev et al., 2005b). LJ6, LJ8, and LJ11 all contain polyA tract variations that either switch off gene expression or lead to the formation of truncated protein products of K5A08_05515, ctsIII, and K5A08_05525, respectively. These genes are responsible for modifying the outer LOS core with galactose and sialic acid, and the inner LOS core with glucose residues suggesting that these LJ strains contain diverse LOS structures compared to the NCTC12658 wildtype (Table 1; Figure 2A). As polymeric tracts found at the gene end in C. jejuni have been proposed to influence transcription and/or translation of the downstream gene (Kim et al., 2012), the polyA tract variation of cstIII in LJ8 may instead affect the downstream neuB1 involved in sialic acid biosynthesis (Table 1). A single nucleotide deletion was observed also in the K5A08_05515 gene in LJ1 introducing an early stop and a truncated protein product. Furthermore, large gaps were found in the LOS loci covering the K5A08_05520 and K5A08_05525 gene regions in LJ10 and LJ13 (Table 1; Supplementary Table S3). The deletion of these two genes in the LOS locus was furthermore confirmed by the de novo assembly of LJ13 (data not shown). K5A08_05520 has been proposed to link the outer LOS core to the inner LOS core. Thus, six out of the seven phage F341 resistant NCTC12658 variants show diverse genetic changes that may all result in a different LOS structure as compared to the wildtype NCTC12658 (Figure 2A).

As the genome analysis of the phage F341 resistant motile NCTC12658 variants showed several genetic changes in the LOS locus, we speculated if phage F341 could be dependent on LOS as a secondary receptor for successful host infection. Many of the SNPs and gene deletions were found in the K5A08_05515–K5A08_05525 gene region responsible for both modifying and linking the inner and the outer LOS core. We therefore created a C. jejuni NCTC12658Δ05515-05525 (NCTC12658ΔLOS) deletion mutant only producing the inner LOS core lacking modifying glucose residues (Figure 2A). Before conducting further experiments, we confirmed that the NCTC12658ΔLOS mutant was still motile at levels similar to the NCTC12658 wildtype (Supplementary Figure S2). We then investigated phage F341 sensitivity by plaque formation and compared the results to those obtained on the NCTC12658 wildtype and the non-motile NCTC12658ΔmotA mutant. While phage F341 infected the non-motile NCTC12658ΔmotA mutant with a lower efficiency of plating as demonstrated previously (Baldvinsson et al., 2014), no lysis nor plaque formation was observed on the NCTC12658ΔLOS mutant (Figures 2B,C). These results demonstrate that phage F341 is completely dependent on the LOS for successful infection thereby functioning as a secondary receptor in C. jejuni NCTC12658.

To determine the genetic content of Campylobacter phage F341, we performed whole genome sequencing. Sequencing revealed a genome size of 131.68 kb with an average GC content of 26%, and BLASTn searches showed that phage F341 belongs to the Fletchervirus genus. This is consistent with a genome size of approximately 130 kb being characteristic for phages belonging to this genus (Javed et al., 2014). In addition, phage F341 encodes five tRNA’s and a large number (12) of putative homing endonucleases which has also previously been observed in other Fletchervirus phages (Javed et al., 2014). To further investigate the genetic relationship between phage F341 and other Fletchervirus phages, we performed comparative genomics using the genome sequences of 18 Fletchervirus phages in our collection (Sørensen et al., 2021a). Comparative genomics showed 89%–96% average nucleotide identity (alignment percentage: 85%–94%, average: 88.4%) between the F341 genome and the 18 Fletchervirus genome sequences (Figure 3A) with the closest relative being phage F336 (alignment percentage: 92%, average 96%). Thus, phage F341 shows high sequence similarity to other Fletchervirus phages and we were only able to detect five genes (F341_034, F341_035, F341_109, F341_128 and F341_135) that were either only observed in phage F341, or which showed no or very low sequence similarity to genes/translated proteins encoded by other Fletchervirus phages (Table 2; Supplementary Table S4). Further detailed in silico analyses demonstrated that F341_109 and F341_128 show structural similarity to baseplate wedge proteins. F341_109 also demonstrates structural similarity to adaptor proteins forming base plate attachment sites for tail fibers in other phages (Supplementary Table S4). F341_135 is likely encoding fibritin which in E. coli phage T4 forms neck/collar fibers and exhibits chaperone function by promoting the assembly of the long tail fibers and their attachment to the tail base plate (Tao et al., 1997). Interestingly, this gene was not observed at the corresponding position in other Fletchervirus genomes currently available. The remaining two unique F341 genes (F341_034 and F341_035) were located within the receptor binding protein (RBP) region for capsular-dependent Fletchervirus phages and will be discussed in more detail in the following paragraph. Thus, only a few genes diverge in phage F341 compared to other Fletchervirus phages, yet proposed functions suggest differences in tail associated genes involved in host recognition.

Fletchervirus phages are usually dependent on capsular polysaccharides for infection of C. jejuni and encode up to four receptor binding proteins (rbp1–rpb4) (Sørensen et al., 2021a). In these phages, the RBPs are located between a conserved putative recombination endonuclease and putative antiholin (Sørensen et al., 2021a). However, phage F341 encodes a unique 882 bp gene (F341_035, rbp) downstream of the recombination endonuclease, demonstrating no hits to other genes when using BLASTn. When aligning the putative rbp gene in F341 to the rbp region of capsular-dependent Fletchervirus phages, a 26.5% sequence identity to rbp1 (1.338 bp) could however be observed (Figure 3B). Further protein alignment showed that F341_RBP shares some sequence similarity with the N-terminal of RBP1 up to the point of the pectin lyase domain found in these proteins (Supplementary Figure S3). Interestingly, BLASTp demonstrated sequence similarity to the C-terminal of the tail fiber protein H encoded by the cryptic CJIE1 prophage in C. jejuni strain HF5-4A-4 (Figure 3C; Table 2). Complete alignment of the protein sequences of F341_RBP and the tail fiber protein H showed an almost identical sequence in the far C-terminal of these two RBPs (Figure 3C). These results suggest that F341 encodes a novel hybrid RBP with similarity to RBP1 encoded by the lytic capsular-dependent Fletchervirus phages and the RBP (tail fiber protein H) of a CJIE1 prophage.

In both the Fletchervirus phages and the CJIE1 prophages, a putative chaperone for correct RBP folding is found downstream of the RBPs containing a DUF4376 domain in the C-terminal (Zampara et al., 2021; Sørensen et al., 2021a). Also downstream of the putative rbp gene in phage F341, a hypothetical protein encoded by gene F341_034 with a DUF4376 domain in the C-terminal was observed (Table 2). The F341_034 gene shows sequence similarity in the N-terminal region to a putative chaperone located next to of the tail fiber protein H in C. jejuni HF5-4A-4, whereas the C-terminal region shows similarity to the putative chaperone located downstream of the rbp genes in capsular-dependent Fletchervirus phages (Table 2; Supplementary Figure S4). Thus, these results suggest that F341_034 encodes a putative novel hybrid chaperone associated with the correct folding of the F341 RBP.

To experimentally prove the function of the proposed RBP encoded by phage F341, we raised polyclonal antibodies against the purified protein and used these in an antibody plaque assay to test the inhibition of phage infection. Also, labeled antibodies were applied during transmission electron microscopy to determine the location of the putative RBP on the phage particle. From these experiments, we could confirm that F341_035 encodes the RBP of phage F341, as phage F341 infection of its host was completely blocked in the presence of the RBP antibodies, i.e., no lysis nor plaque formation was observed (Figures 4A,B). Antibodies previously raised against RBP1 from capsular-dependent Fletchervirus phage F358 showed no effect on phage F341 infection as expected (Figure 4B). The lack of lysis and plaque formation is similar to what we observed on the NCTC12658 LOS deletion mutant suggesting that the F341 RBP is recognizing and binding to the LOS (Figure 2B). Also, transmission electron microscopy of phage F341 using immunogold labeled RBP antibodies demonstrated binding to the distal tail fibers/spikes (Figure 4C).

To further characterize the hybrid RBP of phage F341, we modeled the protein structure using AlphaFold2 (Mirdita et al., 2022). For comparisons, we also modeled the structures of RBP1 from capsular-dependent Fletchervirus phage F358 and the tail fiber protein H found in the CJIE1 prophage in the C. jejuni HF5-4A-4 genome (Figure 4D). As RBPs often fold into homotrimers, we predicted all three proteins both as monomers and homotrimers (Figure 4D; Supplementary Figure S5). The F358 RBP1 homotrimer demonstrates a typical tailspike protein (TSP) morphology, i.e., short, rigid and stocky, consisting of two distinct regions, an N-terminal head-like domain and a C-terminal comprised of the pectin lysase domain forming parallel ß-helices. A Dali search (Holm, 2022) comparing protein structures in 3D, further demonstrates high structural similarity between the RBP1 pectin lyase domain and the enzymatic domain of the capsule-degrading tail spike protein (TSP) Gp38 from Klebsiella phage KP32 (Squeglia et al., 2020). The CJIE1 tail fiber protein H from C. jejuni HF5-4A-4 demonstrates typical tail fiber (TF) morphology, i.e., long and slim, with an N-terminal globular head domain, a shaft/stem formed primarily of α-helices and a distal C-terminal knob-like structure consisting of a ß-sandwich. The phage F341 RBP also demonstrates TF morphology similar to the CJIE1 tail fiber protein H although the fiber is much shorter. Overall, the F341 RBP structure consists of an N-terminal head domain followed by a short shaft/stem region and a distal C-terminal ß-sandwich knob-like structure. The distal C-terminal demonstrating sequence similarity between F341_RBP and the CJIE1 tail fiber protein H is responsible for forming the ß-sandwich knob-like structure and a short α-helical hinge region connected to the shaft/stem. Thus, these results suggest that the distal C-terminal knob-like region is responsible for the interaction with the surface receptor. To further investigate, if the predicted F341 RBP structure resembles other known structural characterized RBPs, we ran a Dali search (Holm, 2022), but no significant hits were observed (data not shown). However, we would propose that the F341 RBP shows some structural similarity to the short tail fibers encoded by gp12 in E. coli phage T4 and to the tail fibers encoded by E. coli phage T7 (van Raaij et al., 2001; Garcia-Doval and van Raaij, 2012). In conclusion, our results show that even though phage F341 belongs to the Fletchervirus genus it encodes an unique hybrid RBP with a short tail fiber morphology essential for infection of its C. jejuni host.