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Page 8 of 12 Jones et al. Microbiome Res Rep 2024;3:24 https://dx.doi.org/10.20517/mrr.2023.78

Analysis of various novel PBGCs identified from Streptococcus macedonicus
[50]
A study by Hilt et al. found Streptococcus to be a prevalent genus within the healthy female urobiome .
[51]
Streptococcus species produce many well-characterised bacteriocins such as salivaricin, streptolysin, and
[52]
mutacin , but none to date have been associated with the urobiome. Streptococcus macedonicus has
previously been linked to bacteriocin production (macedocin) in dairy fermentations . S. macedonicus
[53]
[30]
UMB0733 was collected by catheter from a participant with no urinary symptoms or diseases . BAGEL4
analysis on this strain identified multiple PBGCs.
The PBGC shown in Figure 2B was initially identified by BAGEL4 as an ubericin A bacteriocin gene cluster.
Numerous Streptococcus uberis strains, producing multiple bacteriocins such as uberolysin A, ubericin A,
and the lantibiotic, nisin U, have shown in vitro activity against different mastitis-inducing pathogens .
[54]
Further analysis of all three putative bacteriocin hits using BLASTP and EMBI-EBI EMBOSS Needle
identified novel variants of class II bacteriocin peptides. The first bacteriocin hit shared 78% identity with an
uncharacterised bacteriocin isolated from Streptococcus gallolyticus. The ubericin A hit shared 93.5%
identity with a BIp family class II bacteriocin previously characterised by Dawid et al. .
[39]
Finally, the bovicin 225 peptide shared 93.4% identity with a previously characterised class II bacteriocin
with a double-glycine leader peptide isolated from Streptococcus infantarius. Alongside the three core
peptides, two EntA immunity proteins were present on the genome with LanT, HlyD, and two ABC
transporters. With three putative novel bacteriocins and all the accessory genes necessary for production, it
can be inferred that this is a novel variant of class II bacteriocin gene cluster. Figure 2B shows a novel
variant of the bacteriocin nisin U which displayed 75% identity to a gallidermin/nisin family lantibiotic
previously characterised by Christ et al. . With all eleven accessory genes present on the gene cluster
[55]
[56]
[Figure 2B], it can be assumed to be an active variant of nisin U .

Analysis of novel Colicin PBGCs identified from Proteus mirabilis
Proteus mirabilis UMB0315 [Figure 2C] was collected by catheter from a participant with symptoms of an
overactive bladder . BAGEL4 analysis identified a novel colicin bacteriocin gene cluster. Colicins, which
[30]
are usually produced by E. coli, are among the most well-studied bacteriocins and are effective
antimicrobials against other E. coli and Enterobacteriaceae strains [57,58] . The colicin of interest in this study
was a novel variant produced by Proteus mirabilis. To date, it appears that colicins produced by Proteus
have not been extensively characterised, but a crude bacteriocin extract from Proteus mirabilis has been
described for its colicin-like antibiofilm properties .
[59]

The PBGC, shown in Figure 2C, was identified by BAGEL4 as a colicin E2 bacteriocin gene cluster. Further
analysis of the bacteriocin core peptide using BLASTP and EMBI-EBI EMBOSS demonstrated a novel
variant of a colicin E2 peptide sharing 99.5% identity with six amino acid differences at positions 1, 57, 59,
86, 128, and 188. With both immunity proteins also present [Figure 2C], this suggests that this novel colicin
variant is one of the first predicted to be produced by a Proteus species. Colicins have been previously
highlighted for their antimicrobial potential by coating catheters to inhibit colonisation by UTI-causing
pathogenic bacteria . Colicins have numerous favourable properties such as the low concentrations needed
[60]
for antimicrobial activity and also their specificity in killing, making them desirable antimicrobials that
inflict limited collateral damage on the commensal microbiota . Other putative colicin clusters identified
[61]
in this study were found among the following urobiome strains: Citrobacter murliniae (UMB1094),
Escherichia fergusonii (UMB0727, UMB0901, UMB0900, UMB0789), Morganella morganii (UMB1297) and
Pseudomonas aeruginosa (UMB0710). New bacteriocins that have recently obtained GRAS status from the
FDA for their use in food include colicins and colicin-like peptides (salmocins from Salmonella) , further
[58]