Metabiotics Signature through Genome Sequencing and In Vitro Inhibitory Assessment of a Novel Lactococcus lactis Strain UTNCys6-1 Isolated from Amazonian Camu-Camu Fruits

Abstract

Metabiotics are the structural components of probiotic bacteria, functional metabolites, and/or signaling molecules with numerous beneficial properties. A novel Lactococcus lactis strain, UTNCys6-1, was isolated from wild Amazonian camu-camu fruits (Myrciaria dubia), and various functional metabolites with antibacterial capacity were found. The genome size is 2,226,248 base pairs, and it contains 2248 genes, 2191 protein-coding genes (CDSs), 50 tRNAs, 6 rRNAs, 1 16S rRNA, 1 23S rRNA, and 1 tmRNA. The average GC content is 34.88%. In total, 2148 proteins have been mapped to the EggNOG database. The specific annotation consisted of four incomplete prophage regions, one CRISPR-Cas array, six genomic islands (GIs), four insertion sequences (ISs), and four regions of interest (AOI regions) spanning three classes of bacteriocins (enterolysin_A, nisin_Z, and sactipeptides). Based on pangenome analysis, there were 6932 gene clusters, of which 751 (core genes) were commonly observed within the 11 lactococcal strains. Among them, 3883 were sample-specific genes (cloud genes) and 2298 were shell genes, indicating high genetic diversity. A sucrose transporter of the SemiSWEET family (PTS system: phosphoenolpyruvate-dependent transport system) was detected in the genome of UTNCys6-1 but not the other 11 lactococcal strains. In addition, the metabolic profile, antimicrobial susceptibility, and inhibitory activity of both protein-peptide extract (PPE) and exopolysaccharides (EPSs) against several foodborne pathogens were assessed in vitro. Furthermore, UTNCys6-1 was predicted to be a non-human pathogen that was unable to tolerate all tested antibiotics except gentamicin; metabolized several substrates; and lacks virulence factors (VFs), genes related to the production of biogenic amines, and acquired antibiotic resistance genes (ARGs). Overall, this study highlighted the potential of this strain for producing bioactive metabolites (PPE and EPSs) for agri-food and pharmaceutical industry use.

Keywords: Lactococcus lactis; SemiSWEET sucrose transporter; antimicrobials; exopolysaccharides; metabiotics; pangenome.

Figure 1

Circular maps of the UTNCys6-1 predicted with Proksee viewer (https://proksee.ca, accessed on 6 March 2023). The contents are arranged in feature rings (starting with the outermost ring): ring 1: CRISPR elements; ring 2: mobile genetic element (MGE) annotation with Mobile OG DB marking hsdR gene involved in stability/transfer/defense; ring 3: MGE annotation with Alien Hunter predicting horizontal genetic transfer (HGT) events (blue color arrows); ring 4 shows the UTNCys6-1 protein-coding genes (CDSs) with Prokka annotation (magenta color); tRNA, rRNA, and tmRNA are marked; ring 5 displays the GC content plot (black); ring 6 displays G/C skew information in the + strand (green color) and − strand (purple color).

Figure 2

Phylogenetic tree constructed on TYGS (https://tygs.dsmz.de/ accessed on 6 March 2023). Branch lengths are scaled in terms of GBDP (genome BLAST distance phylogeny method) distance; numbers above branches are GBDP pseudo-bootstrap support values from 100 replications. Leaf labels with different colors indicate the following: (1) species cluster; (2) subspecies clusters; (3) genomic G + C content (min 34.82–39.37); (4) δ values (min 0.061–max 0.17); (5) overall genome sequence length (1,950,384–2,758,410 bp); (6) number of proteins (1847–3118). (7) Target strain is marked. (8) Type species are marked.

Figure 3

EggNOG category distribution of functional annotation result.

Figure 4

(A) A pie chart depicting the number of genes in the UTNCys6-1 strain’s core, soft core, shell, or cloud. (B) A comparison of the genetic content of the 11 strains considered. The matrix depicts the typical genes of each strain as well as those that are conserved among them. The genome assembly codes are described in Table S6.

Figure 5

Bacteriocin gene cluster organization comparison between L. lactis UTNCys6-1 and the references L. lactis ATCC19435, L. lactis KF282, and L. lactis subsp. lactis ATCC11474 strains. (A) Enterolysin_A class; (B) nisin_Z class; (C) sactipeptide class; (D) lactococcin class. Genes with determined function: enterolysin_A: ABC: bacteriocin ABC transporter; BmbF: uncharacterized protein MJ0907 (Methanocaldococcus jannaschii ATCC 43067); Nisin_Z: leaderLanBC (gallidermin); LanB: nisin biosynthesis protein NisB (L. lactis subsp. lactis 1360); ABC: nisin transport ATP-binding protein nisT; LanC: nisin biosynthesis protein nisC; orf00024: nisin immunity protein; protease: nisin leader peptide-processing serine protease NisP (L. lactis subsp. lactis 1360); LanR: nisin biosynthesis regulatory protein nisR; LanK: nisin biosynthesis sensor protein nisK; ABC: NisF; orf00032: NisE; orf00033: NisG; EntA_immun: bacteriocin immunity protein; Gly (orf00023): plantaricin biosynthesis protein PlnO; Gly (orf00024): lipopolysaccharide core biosynthesis glycosyltransferase WaaE. Legend: red blocks: immunity and transport; green blocks and green arrows: core peptide; pink blocks: transport and leader cleavage; blue blocks: peptide modifications; yellow blocks: regulation; grey blocks: no function determined; maroon line with circle ends are the predicted terminators.

Figure 6

Estimation of UTNCys6-1 molecular weight. Legend: M: broad-range molecular marker (Promega); Nis: commercial nisin from L. lactis; L.Lac: PPE obtained from L. lactis subsp. lactis ATCC11474; Cys6-1: L. lactis UTNCys6-1.