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Page 6 of 14 Millen et al. Microbiome Res Rep 2023;2:26 https://dx.doi.org/10.20517/mrr.2023.29

RESULTS
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 . Because plasmid-encoded EPS has been
[18]
[14]
shown to provide phage resistance in lactococci by reducing phage adsorption , 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 . The Dits of the phages infecting DGCC6071 (D4842 and D5604) share 100% deduced amino
[4]
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.

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
[18]
eps gene cluster: typical (as described on pEPS6073) or the closely related EpsM variant . We previously
described the EpsM variant as it resides on pEPS7158 , and an alignment of the typical vs. EpsM variant
[18]
eps clusters as they reside on respective plasmids pEPS6073 and pEPS7158 extracted from Millen et al.,
2022 can be found in Figure 2. The EpsM variant can be distinguished from the typical 6073-like cluster
[18]
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 . Since GTFs are responsible for linking the sugars of the EPS, and each GTF
[18]
may have a different substrate specificity , the differences in GTF content between the 6073-like strain and
[41]
its variant likely affect the composition of the EPS.