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Millen et al. Microbiome Res Rep 2023;2:26 Microbiome Research
DOI: 10.20517/mrr.2023.29
Reports

Original Article Open Access

Evolved distal tail protein of skunaviruses facilitates
adsorption to exopolysaccharide-encoding

lactococci

1
2
1
Anne M. Millen , Damian Magill , Dennis Romero , Laura Simdon 1
1
Health and Biosciences, IFF, Madison, WI 53716, USA.
2
Health and Biosciences, IFF, Dangé-Saint-Romain 86220, France.
Correspondence to: Ms. Anne M. Millen, Health and Biosciences, IFF, Madison, 3329 Agriculture Dr., Madison, WI 53716, USA.
E-mail: Anne.Millen@IFF.com
How to cite this article: Millen AM, Magill D, Romero D, Simdon L. Evolved distal tail protein of skunaviruses facilitates
adsorption to exopolysaccharide-encoding lactococci. Microbiome Res Rep 2023;2:26. https://dx.doi.org/10.20517/mrr.2023.29

Received: 26 Apr 2023 First Decision: 1 Jun 2023 Revised: 10 Jun 2023 Accepted: 29 Jun 2023 Published: 18 Jul 2023

Academic Editor: Douwe van Sinderen Copy Editor: Pei-Yun Wang Production Editor: Pei-Yun Wang

Abstract
Aim: Lactococcal skunaviruses are diverse and problematic in the industrial dairy environment. Host recognition
involves the specific interaction of phage-encoded proteins with saccharidic host cell surface structures.
Lactococcal plasmid pEPS6073 encodes genes required for the biosynthesis of a cell surface-associated
exopolysaccharide (EPS), designated 6073-like. Here, the impact of this EPS on Skunavirus sensitivity was assessed.

Methods: Conjugal transfer of pEPS6073 into two model strains followed by phage plaque assays and adsorption
assays were performed to assess its effect on phage sensitivity. Phage distal tail proteins were analyzed
bioinformatically using HHpred and modeling with AlphaFold. Construction of recombinant phages carrying
evolved Dits was performed by supplying a plasmid-encoded template for homologous recombination.

Results: pEPS6073 confers resistance against a subset of skunaviruses via adsorption inhibition. IFF collection
skunaviruses that infect strains encoding the 6073-like eps gene cluster carry insertions in their distal tail protein-
encoding (dit) genes that result in longer Dit proteins (so-called evolved Dits), which encode carbohydrate-binding
domains. Three skunaviruses with classical Dits (no insertion) were unable to fully infect their hosts following the
conjugal introduction of pEPS6073, showing reductions in both adsorption and efficiency of plaquing. Cloning the
evolved Dit into these phages enabled full infectivity on their host strains, both wild type and transconjugant
+
carrying pEPS6073, with recombinant phages adsorbing slightly better to the EPS host than wild type.

© The Author(s) 2023. Open Access This article is licensed under a Creative Commons Attribution 4.0
International License (https://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, sharing,
adaptation, distribution and reproduction in any medium or format, for any purpose, even commercially, as
long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and
indicate if changes were made.

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