Page 38 - Read Online

Millen et al. Microbiome Res Rep 2023;2:26 https://dx.doi.org/10.20517/mrr.2023.29 Page 11 of 14

sought to understand why the 6073-like EPS may provide resistance to some, but not all, skunaviruses.

+
Available genomes of IFF collection skunaviruses that infect 6073-like EPS strains were compared to those
+
of bIL170, P008NC, and p2. Noticeably, phages that infect EPS strains had insertions in their distal tail
proteins, consistent with an evolved Dit. Furthermore, the specific insertion could be correlated to the
subtype of the 6073-like eps cluster encoded by the host, typical vs. EpsM variant. This was notable as Dit is
[9]
a component of the Skunavirus baseplate, which is involved in phage-host interactions . Classical and long
(evolved) Dits have been previously described in skunaviruses, with internal insertions present in the
[4,8]
evolved Dits found to contain carbohydrate-binding modules (CBMs) . Likewise, the Dit inserts identified
in this study were also predicted to contain CBMs, which is consistent with the evolved Dits potentially
interacting with an EPS. Notably, HHpred analyses identified that the Dit insertions of the phages that
infect hosts encoding the typical 6073-like eps gene cluster harbor a single 5E7T_B CBM, while Dit
insertions of phages that infect hosts encoding the EpsM variant eps gene cluster contain two 5E7T_B
domains. Modeling of representative Dits using AlphaFold further predicted two carbohydrate-binding
domains in the D2929 Dit insert, with the proximal domain containing two contiguous regions involved in
carbohydrate binding and the distal domain containing only one. In contrast, the Dit insert of D6887 lacked
the distal domain and was predicted to encode only one carbohydrate-binding motif in the proximal
domain. These differences likely account for the correlation observed between Dit insert and host EPS
subtype. We previously showed that despite the differences in GTF content between the 6073-like EPS
subtypes, strains encoding either EPS subtype adsorbed a specific subset of P335 group phages at high
[18]
efficiency . In contrast, this study would suggest that the differences between the EPS produced by the
6073-like eps gene cluster subtypes affect Skunavirus adsorption.

The evolved Dit encoded by D6887 was cloned into phages bIL170, P008NC, and p2. Recombinant phages
were tested on their respective isogenic EPS wild-type and pEPS6073 transconjugant hosts. While wild-
-
+
type phages were inhibited on their pEPS6073 host, displaying hazy plaques and/or reduced EOPs, all
+
recombinant phages formed clear plaques on both wild-type and pEPS6073 hosts, although plaque size was
+
+
slightly reduced on EPS strains. Recombinant phages were found to plaque with comparable efficiencies on
-
both their EPS and EPS hosts, indicating that the Dit insertion is indeed responsible for overcoming
+
pEPS6073-mediated phage resistance. Furthermore, this demonstrated that the acquisition of the evolved
Dit results in an expansion of the host range.
Because the pEPS6073-mediated phage resistance was shown to result, at least in part, from phage
adsorption inhibition, adsorption assays were performed using recombinant phages. These assays found
that the recombinant phages were able to adsorb at high efficiency to their EPS isogenic host, confirming
+
that the Dit insertion enabled the phages to overcome the putative EPS-mediated adsorption inhibition.
Adsorption assays were also used to determine if the Dit insertion facilitates phage adsorption rather than
just enables the phages to circumvent the putative EPS barrier. Assays found that the adsorption of the
recombinant phages to the EPS host was reduced compared to the adsorption of the wild-type phages,
-
indicating that the recombinant Dit is less suited to interact with the wild-type, EPS host receptors. Assays
-
also found that the recombinant phages adsorb to the EPS host with higher efficiency than the EPS
-
+
isogenic host, indicating that the Dit insertion may facilitate adsorption to the putative EPS receptor.
However, this is somewhat inconsistent with the adsorption assay results of the recombinant phages on the
non-permissive host, which showed only a slight increase in adsorption to the EPS strain vs the EPS
+
-
isogenic strain. The low efficiency of recombinant phage adsorption to the EPS non-permissive host could
+
be due to incompatibilities of the strains’ cell wall polysaccharides (CWPS), which serve as a primary
receptor for skunaviruses ; LM2345 encodes a type C.1 CWPS, while 1403S encodes a type B. This shows
[7,8]