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Page 10 of 14 Millen et al. Microbiome Res Rep 2023;2:26 https://dx.doi.org/10.20517/mrr.2023.29

Table 4. Recombinant phages show expanded host range
EOP Plaque size (mm)
1403S 1403S-EPS LM2345 2345-EPS 1403S 1403S-EPS LM2345 2345-EPS
P008NC-Dit 1.08 ± 0.19 1 NA NA 1.75 ± 0.17 1.49 ± 0.04 NA NA
6887
bIL170-Dit 6887 0.36 ± 0.11 1 NA NA 1.54 ± 0.10 0.84 ± 0.24 NA NA
p2-Dit 6887 NA NA 0.47 ± 0.46 1 NA NA 0.64 ± 0.21 0.54 ± 0.08
Plaque assays were performed with recombinant phages on their respective hosts +/- pEPS6073. Average of three independent trials. Three
plaques were measured for each assay. Error bars: Sample standard deviation. EOP: efficiency of plaquing. NA: not applicable as the strain is not a
homologous host for the respective phage.

+
+
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.

DISCUSSION
We recently described lactococcal eps gene clusters that are each associated with sensitivity to a subgroup of
P335 phages . One such cluster, designed 6073-like, was originally identified on pEPS6073, and a closely
[18]
related variant (EpsM variant) with differences in a set of two glycosyltransferases (GTFs) found outside of
the contiguous eps operon was subsequently identified . Despite its previous association with sensitivity to
[18]
a P335 group phage subset, pEPS6073 was found to provide resistance to a subset of skunaviruses in model
lactococcal strains. Upon conjugal introduction, pEPS6073 reduced the adsorption of skunaviruses bIL170,
P008NC, and p2 to their model host transconjugants. Therefore, adsorption inhibition, at least in part,
accounted for this phage resistance phenotype. The majority of the 34.4 kb pEPS6073 encodes the eps gene
cluster, mobile elements, and replication functions; however, there is roughly 8 kb of the plasmid that
encodes several hypothetical proteins (data not shown). Therefore, it cannot be ruled out that other genes
encoded on pEPS6073 could play a role in phage resistance. However, our data are consistent with previous
reports of plasmid-encoded EPS providing phage adsorption inhibition and resistance in lactococci [14,16] .
This adsorption inhibition is proposed to result from the cell surface-associated EPS occluding the
saccharidic cell surface receptor required by the phages [14,16] . Our results are aligned with this proposal, as we
have previously shown via electron microscopy that the EPS produced by pEPS6073 is associated with the
cell surface . We found that this putative 6073-like EPS-associated phage resistance does not apply to all
[18]
skunaviruses, as the IFF phage collection contains several that infect strains encoding a 6073-like eps gene
cluster (either typical or EpsM variant), including DGCC6073, the native host of pEPS6073. Therefore, we