Garvicins AG1 and AG2: Two Novel Class IId Bacteriocins of Lactococcus garvieae Lg-Granada

Abstract

Lactococcus garvieae causes infectious diseases in animals and is considered an emerging zoonotic pathogen involved in human clinical conditions. In silico analysis of plasmid pLG50 of L. garvieae Lg-Granada, an isolate from a patient with endocarditis, revealed the presence of two gene clusters (orf46-47 and orf48-49), each one encoding a novel putative bacteriocin, i.e., garvicin AG1 (GarAG1; orf46) and garvicin AG2 (GarAG2; orf48), and their corresponding immunity proteins (orf47 and orf49). The chemically synthesised bacteriocins GarAG1 and GarAG2 presented inhibitory activity against pathogenic L. garvieae strains, with AG2 also being active against Listeria monocytogenes, Listeria ivanovii and Enterococcus faecalis. Genetic organisation, amino acid sequences and antimicrobial activities of GarAG1 and GarAG2 indicate that they belong to linear non-pediocin-like one-peptide class IId bacteriocins. Gram-positive bacteria that were sensitive to GarAG2 were also able to ferment mannose, suggesting that this bacteriocin could use the mannose phosphotransferase transport system (Man-PTS) involved in mannose uptake as a receptor in sensitive strains. Intriguingly, GarAG1 and GarAG2 were highly active against their own host, L. garvieae Lg-Granada, which could be envisaged as a new strategy to combat pathogens via their own weapons.

Keywords: antimicrobial peptides; in silico analysis; zoonotic diseases.

Figure 1

Alignment of the amino acid sequences of garvicin AG1 (A) and garvicin AG2 (B) precursor peptides with their known homologs and with class IId bacteriocins that use a mannose-specific phosphotransferase system (Man-PTS) as a receptor (C). The double-glycine motifs (GG or GS) of the leader peptides are highlighted with a grey background, and the black arrow indicates the cleavage site that generates the mature peptide bacteriocins. The sequences were aligned using the Multalin interface page (http://multalin.toulouse.inra.fr/multalin/ accessed on 1 December 2021). Highly conserved residues (consensus level = 90%) are indicated in red, whereas weakly conserved residues (consensus level = 50%) are indicated in blue. Consensus symbols are: !, I or V; $, L or M; %, F or Y; #, N, D, Q, E, B or Z. NCBI reference sequences, UniProtKB/Swiss-Prot or GenBank accession numbers: Garvicin AG1, WP_225667055.1 (Lactococcus garvieae Lg-Granada, plasmid); Garvicin AG2, WP_165719065.1 (Lactococcus garvieae Lg-Granada, plasmid); Garvicin A, CCF71073.1 (Lactococcus garvieae 21881, plasmid); Garvicin Q, AEN79392.1 (Lactococcus garvieae BCC 43578, plasmid); Bacteriocin BacSJ, CAR92206.2 (Lacticaseibacillus paracasei subsp. paracasei BGSJ2-8, plasmid); Acidocin M, partial, BAB86318.1 (Lactobacillus acidophilus TK8912, plasmid); Bovicin 255, AAG29818.1 (Streptococcus sp. LRC 0255, chromosome); Garvicin B, CCF55365.1 (Lactococcus garvieae 21881, plasmid); Garvicin C, CCF55362.1 (Lactococcus garvieae 21881, plasmid); Lactococcin A, P0A313.1 (Lactococcus lactis subsp. cremoris 9B4, plasmid); Lactococcin B, P35518.1 (Lactococcus lactis subsp. cremoris 9B4, plasmid); Lactococcin Z, BAU29928.1 (Lactococcus lactis QU7, chromosome).

Figure 2

Schematic representation of a 3.1-kpb DNA fragment from plasmid pLG50 of L. garvieae Lg-Granada that contains the genes encoding garvicins AG1 (orf46) and AG2 (orf48) and their corresponding immunity proteins, orf47 and orf49, respectively (A); detailed analysis of predicted promoters and Rho-independent terminator (B); and inverted repeats sequences of the IS1216 transposase (C). (A) P1 and P2 are putative promoter sequences, whereas T1 is a putative Rho-independent transcription terminator. IRR and IRL correspond to the right and left inverted repeat sequences flanking the transposase of IS1216, respectively. Resolvase indicates the orf encoding a putative transposon gamma–delta resolvase gene. (B) The putative promoters P1 and P2 were detected with Softberry BProm. The typical −35 and −10 boxes and the ribosome binding sites (RBS) are shown; “Met” in the P1 and P2 sequences indicates the methionine residues of GarAG1 and AG2, respectively. “Stop” in the P2 sequence indicates the termination codon of orf46. ARNold was used to predict the Rho-independent terminator (T1); base pairs of the hairpin are shown in blue boldface, and apical loops are indicated by red italics. The predicted free energy of terminator hairpins (kcal/mol) is given in parentheses. (C) The 22 bp right and left inverted repeats (IRR and IRL, respectively) of IS1216 showed 100% identity in their nucleotide sequence.