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Wottrich et al. Microbiome Res Rep 2024;3:27 https://dx.doi.org/10.20517/mrr.2023.42 Page 5 of 17

lysates were spotted on the lawns to test for SIR.

Lysogen patch testing
Isolated C. acnes bacteria displaying SIR phenotypes were assayed for the pseudolysogeny phenotype.
C. acnes ATCC 6919 was plated on A Media hard agar via the soft agar overlay technique and the putative
pseudolysogens were then streaked onto these plates and monitored for spontaneous phage release after
incubation. Negative control plates were prepared for each putative pseudolysogen by streaking the bacterial
samples on plates without ATCC 6919 to ensure the growth of the streaked bacteria. For the stability patch
tests, the same techniques were employed while serially streaking the individual strains onto a lawn of
ATCC 6919 bacteria every three days over the course of approximately six months (approximately 60
passages).

Pseudolysogeny PCR
Pseudolysogeny PCR, as described by Liu et al. (2015), was performed to identify if the isolated lysogens
harbored the Aquarius genome . A master mix was prepared using the forward primer 5’-CCG AAG CCG
[11]
ACC ACA TCA CAC C-3’ and the reverse primer 5’-TCA TCC AAC ACC TGC TGC TGC C-3’. DNA
from uninfected bacteria and DNA-free negative controls were also assayed. All amplicons were run on a
0.8%-0.9% agarose gel for 25 min at 100 V. A Fisher’s exact test was conducted to assess independence
between PCR results and patch test phenotype.

Aquarius gp41 protein bioinformatics
BLASTp was conducted against the Phagesdb.org database using the sequence of ltp from phages TP-J34
and TP-778L [22,23,33] . The EMBL-EBI protein sequence and classification tool InterPro was then used to
analyze the signature profiles of Ltp from phages TP-778L and TP-J34, as well as gp41 of phages Lauchelly
(Genbank Accession number KR337650) and Aquarius . Multiple Em for Motif Elicitation (MEME) was
[44]
employed to search for putative motifs in the non-cytoplasmic domains identified by InterPro and
BLASTp . The protein alignment and phylogeny analysis tool Mega7 was used to identify residues with
[45]
conserved charge within the putative active site domains of the C. acnes Ltp-like proteins by conducting a
protein alignment of LtpTP-778L and LtpTP-J34 with several C. acnes phage gp41 proteins . The web
[46]
portal for protein structure and function prediction RaptorX was used to compare overall predicted
[47]
disorder between gp41 and Ltp . Aquarius gp41 structure prediction was performed with AlphaFold2 [48]
and structural homology was predicted with DALI . Surface electrostatic potentials were calculated with
[49]
[50]
[51]
the Adaptive Poisson-Boltzmann Solver , structural alignment was performed with TM-Align , and
protein structure images were generated using PyMOL (Schrödinger, LLC).
Escape mutant isolation
Isolation of escape mutants was attempted by incubating 10 μL of phage Aquarius lysate or phage ATCC
29399B_C (Genbank Accession JX262225) lysate with bacterial lysogen strains for 30 min, followed by
plating for lawns using the soft agar overlay technique. Lysate dilutions of 10 and 10 were used as
-0
-1
-4
-7
-6
experimental groups, with dilution spots of 10 , 10 , 10 , and 10 on a lawn of ATCC 6919 as controls.
-2
C. acnes phage infection and qPCR
To analyze RNA levels at various stages in the infection cycle, bacterial cultures of C. acnes ATCC 6919 and
an Aquarius pseudolysogen were grown in RCM and diluted to an OD of 0.2 (approximately 1 × 10 CFU/
8
600
mL). Phage-free ATCC 6919, the pseudolysogen, and ATCC 6919 plus Aquarius at a multiplicity of
infection of 10 were incubated at 37 °C for the 90-minute duration of the active infection period. Total RNA
was isolated using a Qiagen RNeasy® Kit (QIAGEN Group, Valencia, CA, USA) and analyzed for purity via
Bioanalyzer. Following the isolation, cDNA was generated from the RNA according to the Bio-Rad