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Page 14 of 17 Wottrich et al. Microbiome Res Rep 2024;3:27 https://dx.doi.org/10.20517/mrr.2023.42

infects E. coli, have SIE properties as well, although the primary benefit of this phenomenon is lengthening
[49]
the phage latent period, which produces a larger burst size . Perdoncini Carvalho et al. (2022) describe a
“bottleneck, isolate, amplify, select” (BIAS) mechanism which characterizes SIE as a beneficial trait by
[58]
blocking all but a few viral genome copies from undergoing intracellular replication . This allows for
beneficial mutations to propagate by blocking highly homologous but less fit phage genomes from
replicating, in addition to promoting a larger burst size as previously described [56-58] . Studies involving
mapping of induced gp41 mutations to reduced efficacy of SIE would support this phenomenon, as well as
aid with identifying key residues involved in SIE if gp41 is in fact a key driver. These phage mechanisms
contrast with the direct phage cleavage CRISPR-Cas system that is documented among several strains of
C. acnes and promotes resistance to infection. Utilization of this system, however, presents an option for the
creation of genomes with induced gp41 mutations, as well as the production of gene knockouts [59,60] .

Despite these findings, it remains unconfirmed whether the mechanism of gp41 in Aquarius is governed by
a mechanism paralleling that of Ltp, which is characterized by negatively charged residues on the Ltp
surface interacting with predominantly positively charged surface peptides on TMP during phage DNA
[22]
ejection into its host . It is intriguing that the gp41 non-cytoplasmic C-terminal does contain a negatively
charged surface analogous to that present in Ltp, indicating the potential for functional similarities
[Figure 6D]. It is notable that other phages, such as T5 which infects E. coli, also have SIE proteins that have
been characterized, which promote SIE by other mechanisms such as by inducing a conformational change
upon the formation of an outer membrane receptor protein complex that blocks host receptors allowing for
[18]
phage internalization in superinfection . However, it may be postulated that given Aquarius is a gram-
positive bacteriophage, its structural resemblance to SIE proteins in phages infecting gram-negative bacteria
may be limited. A detailed study aimed at eliciting the crystal structure of gp41 is warranted for direct
comparison with the protein structures of other SIE proteins (including Ltp). However, it is notable that
membrane glycoproteins are notoriously difficult to crystallize using traditional X-ray crystallography
owing to their non-soluble properties. This has been a limiting factor in other studies aimed at determining
the crystal structure of other glycoproteins, although vapor diffusion crystallography is an alternative
technique that has reported greater success . Should gp41 be involved in an SIE mechanism analogous to
[61]
that of Ltp, it may be difficult to isolate.

Ideally, characterization of this protein would be confirmed via studies in which recombineered phage
strains that do not possess gp41 (i.e., protein knockouts) are isolated as described above, and C. acnes strains
are infected with them to observe for the SIE phenotype, including strains of C. acnes preeminently
engineered to contain gp41. However, cloning via electroporation in C. acnes bacteria is currently not
[62]
possible . Alternatively, escape mutant studies may be reperformed on a larger scale, potentially with
concomitant use of a mutagen such as what was done in the study conducted by Leavitt et al. (2023), to try
to induce mutations that localize to gp41 as an alternative to inducing targeted mutants, e.g., with the use of
CRISPR-Cas systems [19,59,60] . Should escape mutant studies in the future or recombineering prove successful
in isolating phage with a mutated gp41 or gp41 knockout, x-ray crystallography of potential target proteins
(including the tape measure protein) would be warranted to solidify understanding of the gene’s mechanism
in C. acnes phages on a bio-molecular level. We do find that predictive artificial intelligence modeling such
as with Alphafold provides some insights into the prospective function of gp41 in SIR, specifically, that
[48]
gp41 structure resembles factors that facilitate protein-protein interactions to assemble large extracellular
matrices or macromolecular complexes. This could indicate a role for gp41 in SIR through modulation of
aspects of phage adsorption or DNA release. A lack of similarity to known phage SIR systems might suggest
that the mechanism for Aquarius-mediated systems is uncharacterized and novel.