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Page 12 of 14 Millen et al. Microbiome Res Rep 2023;2:26 https://dx.doi.org/10.20517/mrr.2023.29

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that while Dit helps facilitate adsorption to EPS strains, other phage and host factors are likely required for
efficient adsorption, including the primary receptor/antireceptor (host CWPS/phage-encoded receptor-
binding protein [43,44] ). CBMs have also been found to decorate various phage structural components in
[42]
addition to Dit, including the neck passage structures and major tail proteins of skunaviruses . Together,
these CBMs were proposed to contribute to phage adsorption by allowing more flexibility in binding
[42]
orientation or by increasing host binding affinity due to their avidity .
This study demonstrates that, like other previously described plasmid-encoded EPS, pEPS6073 reduces
adsorption and provides resistance against a subset of skunaviruses. Coupled with our previous study that
implicates the same plasmid-encoded EPS in P335 phage sensitivity , this work shows the acquisition of
[18]
EPS can alter a strain’s spectrum of phage sensitivity, providing resistance to some phages but acquiring
sensitivity to others. This study found that skunaviruses that were inhibited by pEPS6073 encode classical
Dits, while skunaviruses capable of infecting 6073-like EPS hosts contain insertions in their Dits that
+
encode carbohydrate-binding domains. Such domains were previously shown to be involved in the host
attachment of skunaviruses, likely recognizing a component of the CWPS . This study indicates that a
[8]
component of EPS is likely recognized by the evoDits encoded by strains that produce a 6073-like EPS, as
recombinant phages with Dit show increased adsorption to strains encoding the EPS. However, the EPS
6887
is not required for the adsorption of these phages, nor is the EPS alone sufficient to achieve a high level of
phage adsorption, indicating additional phage/host factors are required for successful host attachment. This
study increases our understanding of phage-host interactions regarding lactococcal EPS, which can be
applied to the development of improved dairy starter cultures with increased phage robustness.

DECLARATIONS
Acknowledgments
The authors would like to thank the Moineau lab at Université Laval, Stuart Huntley, Philippe Horvath, and
Sabine Schermann for their bioinformatic support. We would also like to thank Christophe Fremaux and
Carsten Hansen for the helpful discussion.

Authors’ contributions
Made substantial contributions to the conception and design of the study, performed experiments, and
performed data analysis and interpretation: Millen AM, Magill D
Provided administrative, technical, and material support: Romero D, Simdon L

Availability of data and materials
Representative evolved Dit sequences presented in this study have been deposited in the NCBI GenBank
repository and can be found at: https://www.ncbi.nlm.nih.gov/genbank/; OR271589-OR271593.

Financial support and sponsorship
None.

Conflicts of interest
The authors are all employees of International Flavors and Fragrances (IFF), a company that produces and
markets cultures for industrial dairy applications.

Ethical approval and consent to participate
Not applicable.