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Page 10 of 17 Wottrich et al. Microbiome Res Rep 2024;3:27 https://dx.doi.org/10.20517/mrr.2023.42

Identification of Ltp-like protein
In addition to genome annotation, bioinformatics was used to characterize gene(s) of unknown function
that are putatively involved in phage-mediated SIR. Previous research has shown that the Ltp protein in
Streptococcus thermophilus and Lactococcus lactis phages TP-J34 and TP-778L confers SIR via an
electrostatic interaction between Ltp and the tape measure protein C-terminus, resulting in a stalling of the
ejection complex and prevention of infection [22,23] . The BLASTp for Ltp of TP-J34 and TP-778L yielded a hit
with a protein of unknown function (gp41) in C. acnes phage Lauchelly, as well as in Propionibacterium
[33]
freudenreichii phages PFR1 and PFR2 [Table 5] .

InterPro was used to compare signatures between LtpTP-J34, LtpTP-778L, and gp41 of Lauchelly and
[44]
Aquarius . There was a remarkably similar signature profile between gp41 and Ltp, most noticeably the
conservation of a long non-cytoplasmic domain at a similar locus and relative length within the sequences,
as well as several signal peptide signatures. A roughly 90 amino acid-long region of disorder was also
predicted in Ltp and gp41 by InterPro and RaptorX, corresponding to the region after the end of the signal
peptide at its C-terminal region and the beginning of the region within the non-cytoplasmic domain with
which the first HTH domain of Ltp begins [44,47] . MEME was employed to search for putative motifs within
these non-cytoplasmic domains, considering a hallmark of Ltp family proteins is the presence of two repeat
HTH domains. The output for TP-J34 Ltp and TP-778L Ltp compared to gp41 from sixteen C. acnes phages
demonstrated two motifs of similar size with low P-values overlapping the non-cytoplasmic domain [22,45] .
Lastly, Mega7 was used to identify charge conserved residues within the putative active site domains by
conducting a protein alignment of TP-778L and TP-J34 Ltp proteins with several C. acnes phage gp41
proteins . The output indicated the presence of several charge conserved residues that may fit with the
[46]
model of the Ltp-Tape Measure Protein (TMP) interaction according to Bebeacua et al. (2013), although
further study is required to make definitive claims about active roles for any specific residue(s) . The
[22]
combined findings of InterPro, MEME, and RaptorX are visually depicted in Figure 5, which correspond to
the outputs for Ltp of TP-J34 and gp41 of Aquarius, respectively.

Protein structure prediction with AlphaFold2 (AF2) indicates that the N-terminal residues of Aquarius gp41
(1-50) do not adopt a high-confidence structure. This correlates with the disorder prediction by InterPro
and RaptorX [Figure 5B and Figure 6A]. The gp41 N-terminal region is also predicted to contain a signal
peptide and transmembrane motif that targets gp41 to the cell membrane [Figure 5B and Figure 6A]. Gp41
residues 52-84 adopt a confidently predicted α-helical element that connects the N-terminal transmembrane
sequence to a confidently predicted C-terminal element that is predominantly β-conformation (95-179) but
includes a single α-helix (94-112) [Figure 5A and Figure 6B]. Secondary structure predicted by AF2 agrees
with PsiPRED and Spider predictions implemented through the Max Plank Institute (MPI), Quick2D server
(not shown). A structural homology search using DALI shows that the Aquarius gp41 C-terminal motif (95-
179) adopts a structure similar to extracellular matrix proteins cystatin/latexin and VirB8-like proteins of
type IV secretion systems with Z scores of 8.9 and 7.0 indicating a high likelihood of structural similarity,
respectively - gp41 aligns with mouse cystatin with a RMSD of 2.14 Å [Figure 6C]. Notably, cystatins have
been shown to assemble into nonpathological amyloid matrices that are thought to be involved in cell
maturation and protection [53,54] . Additionally, Vir8B is an essential structural component of type IV secretion
[55]
systems with a periplasmic motif (res 86-226) involved in the assembly of these multiprotein complexes .
It is possible that gp41 facilitates SIE by assembling a protective matrix around the pseudolysogen,
protecting them from infection or by directly interacting with and inhibiting the activity of proteins
required for secondary phage infection. Gp41 surface electrostatics were calculated with the Adaptive
Poisson-Boltzmann Solver (APBS) which shows a patch of negatively charged surface analogous to that
observed for the TP-J34 Ltp [Figure 6D]. Thus, it may be that gp41 is involved in SIE through interactions
with phage TMP, similar to the proposed mechanism described for S. thermophilus phage TP-J34 Ltp.