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Millen et al. Microbiome Res Rep 2023;2:26 https://dx.doi.org/10.20517/mrr.2023.29 Page 9 of 14

Development of recombinant phages
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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 pG9Dit , which includes the entire dit sequence from D6887, was assembled in E. coli
6887
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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 pG9Dit until the culture cleared from
6887
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
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respective pEPS6073 host strains were > 1 × 10 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 bIL170-Dit , and P008NC-Dit confirmed the
6887
6887
6887,
successful integrations of the Dit insertion. Besides the Dit , no differences between the bIL170-Dit
6887
6887
6887
genome and the published bIL170 genome (AF009630) were found. In addition to the Dit , only one SNP
6887
(silent mutation in the 3’ end of the putative neck passage structure CDS) was observed in P008NC-Dit
6887
compared to the sequence of the P008NC phage lysate used for its development. Three additional SNPs
were observed between the p2-Dit genome and the published p2 genome (NC_042024); one intergenic,
6887
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-
Dit isolates using primers p2RPB-F and p2RPB-R. No mutations were identified in the RBP of these
6887
recombinant phage isolates, confirming that RBP mutation is not required for infection on the EPS host.
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Recombinant phages were plaque assayed on their respective hosts +/- pEPS6073 [Table 4]. The EOPs of
P008NC-Dit were roughly equivalent on both isogenic hosts. The EOPs of bIL170-Dit and p2-Dit
6887
6887
6887
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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
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can infect both their EPS , wild-type host and the respective isogenic EPS transconjugant. To determine
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how the exchanged Dit affects phage adsorption, adsorption assays were performed with P008NC-Dit ,
6887
6887
bIL170-Dit , and p2-Dit [Figure 4]. Wild-type phages were only tested on their homologous (fully
6887
6887
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
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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
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alone is not sufficient for efficient adsorption [Figure 4].