The native producer of Garvicin KS (GarKS), previously described as Lactococcus garvieae KS1546, was analysed using a hybrid sequencing strategy to produce a high-quality closed genome assembly. The assembly revealed that the genes encoding the three-peptide, leaderless multipeptide bacteriocin GarKS are located on a plasmid approximately 50 kb in size, designated pKS50. The closed assembly enabled precise mapping of the biosynthetic locus to this plasmid.
Comparative analysis using updated sequence databases indicated that the organism initially reported as Lactococcus garvieae KS1546 is more accurately classified as Lactococcus petauri. The manuscript reports this taxonomic reassignment based on the genomic comparisons, which is relevant for strain identification in future studies and applications.
Within the GarKS biosynthetic locus, the authors identified a gene annotated as a putative transcriptional regulator, gakR. Using heterologous expression experiments, removal of gakR was shown to decrease GarKS production by approximately sixfold. This functional result supports a role for gakR as a positive regulator of bacteriocin biosynthesis in the examined genetic context.
A second gene in the locus was identified as a putative immunity protein, gakI. When gakI was expressed heterologously, recipient cells exhibited markedly reduced susceptibility to GarKS — a decrease of about 250-fold was reported. The expressed immunity determinant also produced varying degrees of cross-immunity to other multipeptide bacteriocins, indicating that gakI can substantially protect cells from GarKS activity and may influence interactions between related bacteriocin systems.
To investigate resistance mechanisms, spontaneous GarKS-resistant mutants were isolated in Lactococcus lactis and subjected to whole-genome sequencing. The work found that observed resistance levels were generally low. Sequencing of these spontaneous mutants allowed the authors to pinpoint genetic changes associated with altered susceptibility.
Analysis of mutations in the GarKS-resistant isolates implicated ythA, a gene encoding a PspC-domain-containing protein, in the GarKS-susceptible phenotype. The PspC domain is associated with a phage stress response pathway; the data link this stress-response protein to the cellular processes that influence sensitivity to GarKS. The report indicates involvement of stress-response mechanisms rather than high-level resistance development under the conditions tested.
To broaden understanding of conserved features and diversity among leaderless multipeptide bacteriocins, the authors performed genome mining across updated sequence databases. This search identified 11 candidate loci predicted to encode new multipeptide leaderless bacteriocins. The identification relied on conserved genomic features characteristic of this bacteriocin class as inferred from the characterised GarKS locus and related systems.
From the 11 candidates identified by genome mining, four candidate multipeptide bacteriocins were obtained synthetically and tested for activity. These synthetic peptides were confirmed to be bioactive and inhibited clinically and food-relevant Gram-positive pathogens, explicitly including Listeria monocytogenes and enterococci. These confirmations demonstrate that genome-guided discovery can yield functional leaderless multipeptide bacteriocins with activity against important pathogens.
The findings consolidate genomic, regulatory and functional data for a representative leaderless multipeptide bacteriocin system. Locating GarKS genes on plasmid pKS50, demonstrating regulatory influence of gakR, characterising immunity via gakI, and implicating ythA in susceptibility all contribute to an improved mechanistic picture. The discovery and synthetic validation of additional candidate bacteriocins expand the known diversity of this compound class and identify leads that inhibit Listeria monocytogenes and enterococci, supporting potential uses in food preservation and medical contexts.
The article is presented as a preprint and has not been peer reviewed; experimental details, broader generalisability, and in vivo applicability were not reported beyond the described in vitro assays, genome analyses and heterologous expression experiments.