Evolved distal tail protein of skunaviruses facilitates adsorption to exopolysaccharide-encoding lactococci

Bacterial strains and phages

Bacterial strains and phages are listed in Table 1. Lactococcal strains were grown at 30 °C in sterile 11% w/v nonfat dry milk (NFDM) or in M17 broth (Oxoid, UK) supplemented with 0.5% lactose or glucose. Escherichia coli was aerobically propagated in LB broth (BD Difco, USA) at 37 °C. When required, antibiotics were added to the media as follows: erythromycin [Em; 5 µg/mL (lactococci), 150 µg/mL (E. coli)].

The preparation of bacteriophage lysates was performed as previously described^[26]. High titer lysates were passed through a 0.45 µm filter and stored at 4 °C. Phage typing was performed using the multiplex PCR method as described by Labrie and Moineau^[27]. Spot titer assays and plaque assays were performed as previously described^[26] on MRS medium (Oxoid, UK). Briefly, 10-fold serial dilutions in peptone buffer (3M, USA) of phage lysates were prepared. For standard plaque assays, 100 µL of the phage dilution was combined with 150-200 µL of exponentially growing cells (A₆₀₀ ≈ 0.5) and incubated at room temperature for 10 min. The mixture was added to 3 mL of soft agar overlay (0.5% agar w/v) containing 10 mM CaCl₂ and then poured onto a standard MRS agar base plate. For spot assays, exponentially growing cells were added to the soft agar overlay with CaCl₂ and then poured onto a base plate. Once solidified, aliquots of the 10-fold serially diluted phage lysates were spotted onto plates. The Efficiency of Plaquing (EOP) is determined by dividing the phage titer on the test host by the titer on the fully sensitive host strain. Results are averaged from three independent replicates.

Phage adsorption tests were performed as previously described^[28] on MRS medium. Briefly, phage and exponentially growing cells (absorbance at 600 nm = 0.5) were combined and incubated for 15 min at room temperature. The mixture was then centrifuged (RCF = 9,750 × g at 4 °C) and supernatant assayed for residual phage by standard plaque assay on a sensitive host. Percent adsorption is calculated as: (starting phage titer - residual phage titer)/starting phage titer, and then multiplied by 100. Adsorption on the sensitive host serves as the control against which test strains are compared. Results are averaged from three independent replicates.

PCR, DNA preparation, sequencing, and bioinformatic tools

PCRs were performed with GoTaq® Colorless Master Mix (Promega Corp., USA) or Phusion HF Mastermix (Thermo Scientific, USA) according to the manufacturer’s instructions with 35 cycles of denaturation/annealing/extension. Primer sequences and additional thermocycler conditions are listed in Table 2. Amplicons were purified using the Promega Wizard® SV Gel and PCR Clean-Up System (Promega Corp., USA). Primers were synthesized by Integrated DNA Technologies (USA). Phage DNA was isolated from high-titer phage lysate using Invitrogen PureLink Viral RNA/DNA Mini kit (Life Technologies, USA) according to the manufacturer’s instructions. Phage DNA was sequenced using Illumina technology (Illumina, USA). Sanger sequencing was performed by Eurofins Genomics (USA). Sequences were annotated using RAST or PATRIC RASTtk-enabled Genome Annotation Service^[29-31]. Sequences were analyzed using Geneious Prime 2021.0.3 (https://www.geneious.com). Protein analysis was performed using HHpred (Homology detection & structure prediction by HMM-HMM comparison)^[32,33].

Molecular cloning and phage engineering

Development of pG9Dit₆₈₈₇ was performed using NEBuilder® HiFi DNA Assembly (New England Biolabs, USA). PCR amplification of dit was conducted from D6887 phage lysate with primers clone 6073DIT-F and clone 6073DIT-R [Table 2]. A linear amplicon of pGhost9, a vector commonly used for cloning experiments, was created by PCR with primers VF-pG9 and VR-pG9 [Table 2]. Purified Dit amplicon was assembled with purified linear pGhost9 using NEBuilder® HiFi DNA Assembly Master Mix (New England Biolabs, USA) according to the manufacturer’s instructions. Assembly was electroporated into E. coli TG1 RepA⁺ cells according to the Dower method^[34]. Recombinant plasmids were isolated from TG1 RepA⁺ cells using GeneJET Plasmid Miniprep Kit (Thermo Scientific, USA). Purified plasmid was electroporated into lactococci as previously described^[35].

To isolate recombinant phages that have exchanged the wild-type dit for dit₆₈₈₇, phages were propagated on their respective lactococcal host containing pG9Dit₆₈₈₇. With sufficient DNA homology between the respective 5’ and 3’ ends of the wild-type dits and dit₆₈₈₇, recombination between the phages and pG9Dit₆₈₈₇ would occur via double crossover [Supplementary Figure 1]. Selection of recombinant phages was performed by plating the propagations on the respective host + pEPS6073.

Molecular modeling

Molecular modeling of Dit proteins was conducted using the AlphaFold package in monomer mode and employing the fully compiled database of Protein Data Bank (PDB) structures^[36]. Predictions were scored based on the pLDDT criteria in order to choose the top-ranked model. Putative carbohydrate-binding domains were predicted using an in-house software package. Structural alignments were carried out using the TM-align algorithm^[37]. Visualization of models and preparation of publication-grade figures was conducted within Pymol^[38].

Statistical analysis

One-tailed t-tests were conducted for the comparison of adsorption data across recombinant and native phages on the relevant strains. This was carried out using the SciPy package within Python.

pEPS6073 confers resistance against skunaviruses in model lactococci

Lactococcal plasmid pEPS6073 encodes a previously characterized cell surface-associated exopolysaccharide (EPS) correlated to sensitivity to a subgroup of P335 phages^[18]. Because plasmid-encoded EPS has been shown to provide phage resistance in lactococci by reducing phage adsorption^[14], L. cremoris LM2345 and L. lactis 1403S transconjugants containing pEPS6073 (2345-EPS and 1403S-EPS) were tested against homologous skunaviruses. Plaque assays, which measure the level of phages present that are able to lyse the cell, showed pEPS6073 provided a high level of resistance to each phage as evidenced by a low efficiency of plaquing (EOP) [Table 3]. Adsorption assays, which measure the level of phages that are able to attach to the cells, showed a reduction in adsorption of each phage to the EPS⁺ transconjugant compared to the fully sensitive parent strain [Table 3].

Skunaviruses that infect 6073-like EPS⁺ strains contain insertions in Dit

The IFF collection includes skunaviruses that infect strains, including L. cremoris DGCC6073, that encode the 6073-like eps gene cluster. To determine why these phages are able to infect EPS⁺ hosts, while pEPS6073 provides a level of resistance against p2, bIL170, and P008NC (a derivative of P008 received from North Carolina State University that shares 97% nucleotide identity with P008), whole genome sequencing was performed on five skunaviruses that infect DGCC6073 and two that infect L. cremoris DGCC6071, a strain that encodes the EpsM variant of the 6073-like eps gene cluster. Examination of the genes encoding the phage distal tail proteins (Dits) found that the skunaviruses infecting DGCC6073 and DGCC6071 contain evolved Dits, which harbor insertions encoding carbohydrate-binding domains. The Dits of the phages infecting DGCC6073 (D4839, D6869, D6875, D6887, and D6888) share > 97% deduced amino acid identity and contain inserts of 168 aa [Supplementary Table 1 and Figure 1]. This Dit insert is consistent with group 14, based on our previous analysis of IFF collection skunaviruses that identified 16 distinct groups of Dit insertions^[4]. The Dits of the phages infecting DGCC6071 (D4842 and D5604) share 100% deduced amino acid identity and contain inserts of 319 aa [Supplementary Table 1 and Figure 1], which are consistent with group 7. HHpred analyses of these Dit insertions result in top domain hit 5E7T_B; minor structural protein 5; bacteriophages; Lactococcus lactis. Minor structural protein 5 is identified as ORF 52/BppA in lactococcal phage TUC2009 (accession number NC_002703), a component of the tripod baseplate structure with a putative carbohydrate-binding domain and is involved in receptor binding^[39,40]. We observed the presence of sequences in the public domain similar to our group 7 classified Dits, with an average identity of 55%. Through HHpred analysis, the proteins from these phages (62601, 62605, and CHPC958) were also found to be in possession of the 5E7T_B domain.

Figure 1. Amino acid alignment of representative Dits. Deduced amino acid alignment of the classical Dit from phage p2 and the evolved Dits from representative phages that infect hosts encoding a 6073-like eps gene cluster. Phages that infect strains encoding the typical 6073-like EPS vs. EpsM variant are indicated. In the consensus identity bar, green indicates identity, yellow indicates polymorphism, and red indicates low identity. Dit: distal tail protein; EPS: distal tail protein; EpsM: 6073-like variant.

Additional sequenced IFF collection phages that encode Dits sharing high nucleotide identity to those present in phages infecting DGCC6073 or DGCC6071 were identified (representative translated Dits shown in Figure 1). Each of these phages was found to infect a host that encodes a 6073-like eps gene cluster. Furthermore, the specific Dit insertion was found to correlate to the subtype of the host-encoded 6073-like eps gene cluster: typical (as described on pEPS6073) or the closely related EpsM variant^[18]. We previously described the EpsM variant as it resides on pEPS7158^[18], and an alignment of the typical vs. EpsM variant eps clusters as they reside on respective plasmids pEPS6073 and pEPS7158 extracted from Millen et al., 2022^[18] can be found in Figure 2. The EpsM variant can be distinguished from the typical 6073-like cluster by differences in a set of two glycosyltransferase (GTF) genes found outside of the contiguous eps operon. pEPS6073 contains GTF genes pEPS6073_22 and pEPS6073_24, whereas pEPS7158 contains GTF genes annotated as epsM and epsN^[18]. Since GTFs are responsible for linking the sugars of the EPS, and each GTF may have a different substrate specificity^[41], the differences in GTF content between the 6073-like strain and its variant likely affect the composition of the EPS.

Figure 2. Alignment of typical and EpsM variant eps gene clusters as they reside on pEPS6073 and pEPS7158, respectively (Excerpted from Millen et al., 2022^[18]). Numbers appearing over gene depictions correspond to locus tags as annotated in GenBank (accession numbers OP323065-OP323068). Genes are colored based on their putative function, including EPS assembly, EPS modulation, glycosyltransferase, transposase, or others. The putative polymerase (wzy), flippase (wzx), and attachment (lytR) genes are annotated as such. Gray bars are used to indicate sequence identity. EPS: Distal tail protein; EpsM: 6073-like variant.

Dits from phages D970, D1113, and D6067, which all infect L. cremoris DGCC8692 (encoding a typical 6073-like eps gene cluster), are ~99% identical at the deduced amino acid level and share 91%-94% pairwise amino acid identity with the Dits of the phages infecting DGCC6073 [Supplementary Table 1 and Figure 1]. The Dits of phages D753, D4006 and D4842, each of which infects a different L. cremoris strain that harbors the EpsM variant 6073-like eps gene cluster (DGCC6871, DGCC7193, or DGCC6071, respectively), share > 99% deduced amino acid identity [Supplementary Table 1]; however, the Dit of D2929, also infecting an L. cremoris strain encoding the EpsM variant EPS (DGCC7168), shares only ~87% deduced amino acid identity with the Dit of the other three phages [Supplementary Table 1 and Figure 1]. Notably, Dits of the phages that infect strains encoding a typical 6073-like EPS share only 58%-62% pairwise amino acid identity with those of the phages that infect strains encoding the EpsM variant EPS. Furthermore, HHpred analyses found that the Dit insertions of the phages infecting hosts that encode the EpsM variant eps gene cluster contain two 5E7T_B domains compared to the single domain encoded by the insertions in the Dits belonging to phages that infect hosts harboring the typical 6073-like eps gene cluster. Representative evolved Dit from D2929 was modeled and compared with the classical Dit from M7361^[4] as well as with D6887 [Figure 3]. A clear conservation of the core region from the classical Dit compared to the evolved counterparts can be observed, with the inserts existing as a distinct region connected to the core region via a series of flexible loops. This highlights a modular evolution within the protein, further exemplified when comparing D6887 with D2929, the latter containing two distinctly visible domains within the insert region. Both domains within the D2929 insert region were predicted to contain motifs involved in carbohydrate binding, supporting the HHpred results. The proximal domain was predicted to contain two contiguous regions located at residues 188-259 and 280-321 involved in carbohydrate binding, whereas the distal domain was predicted to contain one such motif across the amino acid range 391-432. With respect to D6887, the absence of the D2929 distal domain is immediately apparent in the structure. Alignments of the proximal domain revealed a 92% structural similarity; however, it was observed that in the case of D6887, only one carbohydrate-binding motif was predicted, corresponding to the one located in the range 188-259 in D2929. The presence of BppA-like domains within the Dits of some phages also represented a point of interest, as the accessory base plate protein (BppA) has been shown to contain CBMs involved in phage binding to the host^[8,40,42]. Therefore, a model was constructed of the Dit from phage D4006, predicted to contain this domain, and was compared to the solved structure for TUC2009 (5E7T_B) [Figure 3D]. Indeed, it was observed that there exists a structural similarity between the proximal portion of the insert in the D4006 Dit and 5E7T_B.

Figure 3. Molecular modeling and comparison of select classical and evolved Dit proteins. Structural alignment of the AlphaFold predicted Dit models for phages D2929 (green) and M7361 (blue), respectively (image A). The region corresponding to the insert present in D2929 is shown on the left-hand side of the figure, with the locations of the three predicted carbohydrate-binding regions (two of them corresponding to the proximal domain and one corresponding to the distal domain) highlighted in different colors. Structural alignment of the D2929 (green) and D6887 (blue) Dit models is provided in panel B on the right-hand side of the figure with an alignment corresponding only to the insert regions provided in panel C at the bottom. The predicted carbohydrate-binding motif in D6887 is highlighted in yellow. Panel D at the bottom right shows a structural alignment of the D4006 Dit protein predicted to possess BppA domains aligned with the minor structural protein 5 (BppA) from the TUC2009 solved baseplate structure (PDB: 5E7T). It can be clearly seen in the proximal portion of the insert in D4006 that there is a structural similarity with BppA. BppA: accessory base plate protein; Dit: distal tail protein; PDB: protein data bank.

Development of recombinant phages

To determine if Dit insertions are necessary for Skunavirus virulence on 6073-like EPS⁺ strains, three model phages that contain classical Dits (bIL170, P008NC, and p2) were engineered to encode the Dit insertion from D6887. Vector pG9Dit₆₈₈₇, which includes the entire dit sequence from D6887, was assembled in E. coli TG1 RepA⁺, and the constructed plasmid was then introduced into 1403S and LM2345. Phages p2, P008NC, and bIL170 were propagated on their respective host containing pG9Dit₆₈₈₇ until the culture cleared from phage lysis. Phage lysates were then plaqued on their respective hosts containing pEPS6073. Although pEPS6073 provides a level of resistance to the wild-type phages, titers of these phage propagations on their respective pEPS6073⁺ host strains were > 1 × 10⁶ pfu/mL with clear plaque morphology, indicating a high level of phages within each of the respective lysates was not inhibited by pEPS6073. A representative single plaque isolate from each of the three plaque assays was propagated on its respective host containing pEPS6073. PCR analysis of each lysate with primers checkDIT-F and checkDIT-R showed an increase in dit size, indicative of an exchange of the D6887 Dit with the native Dits of phages p2, bIL170, and P008NC. Illumina sequencing of recombinant phages p2-Dit_6887, bIL170-Dit₆₈₈₇, and P008NC-Dit₆₈₈₇ confirmed the successful integrations of the Dit₆₈₈₇ insertion. Besides the Dit₆₈₈₇, no differences between the bIL170-Dit₆₈₈₇ genome and the published bIL170 genome (AF009630) were found. In addition to the Dit₆₈₈₇, only one SNP (silent mutation in the 3’ end of the putative neck passage structure CDS) was observed in P008NC-Dit₆₈₈₇ compared to the sequence of the P008NC phage lysate used for its development. Three additional SNPs were observed between the p2-Dit₆₈₈₇ genome and the published p2 genome (NC_042024); one intergenic, one silent, and one in the receptor-binding protein (RBP). All three loci in the p2 lysate used for the development of the recombinant phage were confirmed to be consistent with the published genome. As the receptor binding protein is the primary phage antireceptor, the RBP was sequenced in three additional p2-Dit₆₈₈₇ isolates using primers p2RPB-F and p2RPB-R. No mutations were identified in the RBP of these recombinant phage isolates, confirming that RBP mutation is not required for infection on the EPS⁺ host.

Recombinant phages were plaque assayed on their respective hosts +/- pEPS6073 [Table 4]. The EOPs of P008NC-Dit₆₈₈₇ were roughly equivalent on both isogenic hosts. The EOPs of bIL170-Dit₆₈₈₇ and p2-Dit₆₈₈₇ were both slightly reduced on the pEPS6073^- host (< 1 log EOP reduction); however, plaque sizes of all recombinant phages were slightly reduced on the pEPS6073⁺ host. This data shows that recombinant phages can infect both their EPS^-, wild-type host and the respective isogenic EPS⁺ transconjugant. To determine how the exchanged Dit₆₈₈₇ affects phage adsorption, adsorption assays were performed with P008NC-Dit₆₈₈₇, bIL170-Dit₆₈₈₇, and p2-Dit₆₈₈₇ [Figure 4]. Wild-type phages were only tested on their homologous (fully infective) hosts, but recombinant phages were also tested on non-homologous hosts to determine if the EPS facilitates adsorption when the evolved Dit is present. Recombinant phages showed higher adsorption to their EPS⁺ host than the EPS^- isogenic host. Additionally, recombinant phage adsorption to the EPS^- host was reduced compared to that of the wild-type phages. Assays of the recombinant phages on the non-homologous host (p2/1403S, bIL170/LM2345, and P008NC/LM2345) did not show a strong increase in adsorption to the EPS⁺ strain compared to the EPS^- isogenic strain, indicating that the presence of the EPS alone is not sufficient for efficient adsorption [Figure 4].

Figure 4. Dit promotes phage adsorption to EPS⁺ strains. (A): Assays on LM2345 and EPS⁺ transconjugant; (B): Assays on 1403S and EPS⁺ transconjugant. Adsorption assays were performed with wild-type and recombinant phages on isogenic EPS +/- strains. Wild-type phages adsorbed strongly to their native host, and the introduction of pEPS6073 decreased their adsorption (P < 0.05). Dit exchange resulted in poorer adsorption to each native host but strong adsorption to the pEPS6073⁺ transconjugant of the native host (P < 0.05). Recombinant phages adsorbed poorly to non-native hosts, although adsorption was slightly increased on the non-native host when pEPS6073 was present (not statistically significant). Statistical comparisons conducted to support the various conclusions have been annotated using connecting lines to explicitly show the pairwise calculations performed. Statistically significant comparisons are marked with asterisks that indicate the level of statistical significance according to the following scale: (* P < 0.05, ** P < 0.01, *** P < 0.001). Comparisons that are not statistically significant are labeled ns. Wild-type phages were not tested against non-native host strains. Average of three independent trials. Dit: Distal tail protein; Error bars: sample standard deviation; EPS: exopolysaccharides.