Page 34 - Read Online

Millen et al. Microbiome Res Rep 2023;2:26 https://dx.doi.org/10.20517/mrr.2023.29 Page 7 of 14

Table 3. Introduction of pEPS6073 into lactococcal host strains provides resistance against select skunaviruses
Phage Strain EOP Adsorption (%) Comments
p2 LM2345 1 89.3 ± 2.1 Clear plaques
-6 -6
2345-EPS 3.4 × 10 (± 3.3 × 10 ) 26.0 ± 2.3 Turbid plaques
bIL170 IL1403S 1 97.7 ± 1.2 -
1403S-EPS < 2.9 × 10 -9 43.9 ± 16.6 No plaquing, poor bacterial lawn at highest phage titers
P008NC IL1403S 1 95.0 ± 0.9 -
-9
1403S-EPS < 1.2 × 10 47.7 ± 10.5 No plaquing, poor bacterial lawn/clear at the highest phage titers
Phage plaque and adsorption assays were carried out on model lactococcal strains +/- pEPS6073. When applicable, the EOP is the average of
three independent trials ± sample standard deviation. When no plaques were visible, EOP is < the highest value of three independent trials.
Adsorption is the average of three independent trials ± sample standard deviation. EOP: Efficiency of plaquing.

Figure 1. Amino acid alignment of representative Dits. Deduced amino acid alignment of the classical Dit from phage p2 and the evolved
Dits from representative phages that infect hosts encoding a 6073-like eps gene cluster. Phages that infect strains encoding the typical
6073-like EPS vs. EpsM variant are indicated. In the consensus identity bar, green indicates identity, yellow indicates polymorphism, and
red indicates low identity. Dit: distal tail protein; EPS: distal tail protein; EpsM: 6073-like variant.

Figure 2. Alignment of typical and EpsM variant eps gene clusters as they reside on pEPS6073 and pEPS7158, respectively (Excerpted
[18]
from Millen et al., 2022 ). Numbers appearing over gene depictions correspond to locus tags as annotated in GenBank (accession
numbers OP323065-OP323068). Genes are colored based on their putative function, including EPS assembly, EPS modulation,
glycosyltransferase, transposase, or others. The putative polymerase (wzy), flippase (wzx), and attachment (lytR) genes are annotated
as such. Gray bars are used to indicate sequence identity. EPS: Distal tail protein; EpsM: 6073-like variant.

Dits from phages D970, D1113, and D6067, which all infect L. cremoris DGCC8692 (encoding a typical
6073-like eps gene cluster), are ~99% identical at the deduced amino acid level and share 91%-94% pairwise
amino acid identity with the Dits of the phages infecting DGCC6073 [Supplementary Table 1 and Figure 1].
The Dits of phages D753, D4006 and D4842, each of which infects a different L. cremoris strain that harbors
the EpsM variant 6073-like eps gene cluster (DGCC6871, DGCC7193, or DGCC6071, respectively), share >
99% deduced amino acid identity [Supplementary Table 1]; however, the Dit of D2929, also infecting an L.
cremoris strain encoding the EpsM variant EPS (DGCC7168), shares only ~87% deduced amino acid
identity with the Dit of the other three phages [Supplementary Table 1 and Figure 1]. Notably, Dits of the
phages that infect strains encoding a typical 6073-like EPS share only 58%-62% pairwise amino acid identity
with those of the phages that infect strains encoding the EpsM variant EPS. Furthermore, HHpred analyses
found that the Dit insertions of the phages infecting hosts that encode the EpsM variant eps gene cluster