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Millen et al. Microbiome Res Rep 2023;2:26 https://dx.doi.org/10.20517/mrr.2023.29 Page 3 of 14

In this study, the work was extended to another problematic phage group, and pEPS6073 was found to
provide resistance against a subset of skunaviruses via adsorption inhibition. The IFF phage collection,
however, contains several skunaviruses that infect hosts known to harbor the 6073-like eps gene cluster,
indicating this resistance does not extend across the entire Skunavirus genus. Here the genetic determinants
enabling phages to infect 6073-like EPS strains were investigated. Skunaviruses that infect hosts encoding
+
the 6073-like eps gene cluster were found to carry insertions in their distal tail proteins, which encode
carbohydrate-binding domains. The relationship between the Dit insertion and EPS host was confirmed
+
through the construction of recombinant phages carrying Dit insertions. This work furthers our knowledge
of phage-host interactions in Lactococcus and proposes a role for insertions in the Dit proteins of certain
skunaviruses.

METHODS
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 . High titer lysates were
[26]
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 . Spot titer assays and plaque assays were performed as
[27]
[26]
previously described 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 (A ≈ 0.5) and incubated at room temperature for
600
10 min. The mixture was added to 3 mL of soft agar overlay (0.5% agar w/v) containing 10 mM CaCl and
2
then poured onto a standard MRS agar base plate. For spot assays, exponentially growing cells were added
to the soft agar overlay with CaCl and then poured onto a base plate. Once solidified, aliquots of the 10-fold
2
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 on MRS medium. Briefly, phage and
[28]
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.

Table 1. Strains, bacteriophages, and plasmids
Biological Relevant characteristics Reference
material
Bacteria
Lactococcus lactis
R
1403S Spontaneous Sm derivative of IL1403, plasmid-free [19]
R R +
1403S-EPS Sm , Em , pEPS6073 [18]
1403S-Dit 6887 1403S + pG9Dit 6887 This study
Lactococcus cremoris