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

Conclusion: The 6073-like EPS potentially occludes the phage receptor for skunaviruses that encode a classical Dit
protein. Skunaviruses that infect strains encoding the 6073-like EPS harbor evolved Dits, which likely help promote
phage adsorption rather than just allow the phage to circumvent the putative EPS barrier. This work furthers our
knowledge of phage-host interactions in Lactococcus and proposes a role for insertions in the Dit proteins of a
subset of skunaviruses.

Keywords: Lactococcus, Skunavirus, 936 group bacteriophage, exopolysaccharide, evoDit, dairy fermentations

INTRODUCTION
Lactococcus lactis and Lactococcus cremoris are mesophilic lactic acid bacteria used in starter cultures for
dairy fermentations. The long-term utility of highly specialized starter strains is limited by their
vulnerability to bacteriophages, including those belonging to the Skunavirus genus (936 group), which are
[1,2]
commonly found in the industrial environment . Phage infections can cause significant economic loss due
to negative end-product quality issues and failed manufacturing processes. Extensive research into
lactococcal phage-host interactions has identified a range of phage defenses that can be exploited in
industrial strains for improved phage resistance .
[3-5]

Cell wall polysaccharides (CWPS), encoded by the chromosomal cwps operon (also referred to as rgp
operon), decorate the lactococcal cell wall and have been shown to serve as receptors for skunaviruses [4,6-8] .
In the initial step of infection, a phage attaches to the cell by binding to its receptor. This attachment,
known as phage adsorption, is mediated by phage adhesion modules, which include the C-terminus of the
tail tape measure protein, the distal tail protein (Dit), the N-terminal region of the tail-associated lysin (Tal),
the receptor binding protein (RBP), and baseplate proteins (BPPs), if present [9-11] . Dit is a major component
of the Skunavirus baseplate, and two types of Dit proteins have been found to exist in skunaviruses: classical
[8]
and long (evolved) . Internal insertions are present in the evolved Dits (evoDits), with the N‐terminal and
C‐terminal regions aligning with the full length of the classical Dit. These insertions were found to encode
[8]
carbohydrate-binding modules (CBMs) and were shown to promote host-binding . In addition to
Lactococcus, evoDits have been shown to be involved in phage adsorption in lactobacilli and
Bacillus cereus . Analyses showed the Dit-associated CBMs of lactococcal skunaviruses exhibit the same
[12]
[8]
strain specificity as the RBPs and likely recognize a saccharidic component of the CWPS . Indeed, a Dit/
RBP/CWPS correlation could be made across subtypes within the Skunavirus genus . Lactococcal evoDits
[8]
[8]
were previously grouped into four classes . Subsequently, an analysis of IFF (formerly DuPont) collection
skunaviruses identified as many as 16 distinct groups of Dit insertions .
[4]
In addition to CWPS, some lactococci also produce exopolysaccharides (EPS), which may be excreted into
the growth media or tightly associated with the cell surface . EPS-producing strains are valuable for the
[13]
[13]
desirable textures they produce in fermented dairy products . Additionally, plasmid-encoded EPS
biosynthetic capability has been demonstrated to reduce the adsorption of certain phages to the cell,
increasing the phage robustness of the strain [14-16] . However, EPS-mediated phage resistance appears to be
[17]
limited, as many examples of phages infecting EPS-producing lactococcal strains have been reported .
Furthermore, we recently identified two subsets of P335-group phages, each of which differentially adsorbed
[18]
to strains that encode a distinct set of EPS-associated gene clusters . These gene clusters, originally
identified on plasmids pEPS6073 and pEPS7127, were designated 6073-like and 7127-like, with further
designation of a 6073-like variant (EpsM variant). The 6073-like and 7127-like eps gene clusters were each
found to encode genes required for the synthesis of a cell surface-associated EPS, which, we proposed,
functions as a receptor for the respective P335 phages .
[18]