Draft genome sequence of Enterococcus lactis SP-50a, isolated from artisanal cheese, with potential to produce antimicrobial agents and biopolymers

ABSTRACT

Artisanal cheeses are a source of bacteria with biotechnological potential. The genome size of Enterococcus lactis SP-50a was 2.71 Mb with 2,637 genes. Analysis identified genes for bacteriocin and peptidoglycan hydrolase synthesis, associated with antimicrobial activity. Additionally, the fabG gene was detected, indicating a possible polyhydroxyalkanoate production.

ANNOUNCEMENT

Enterococcus lactis could provide a dual biotechnological solution for reducing antimicrobial resistance (1) and plastic contamination (2). This genus synthesizes antimicrobial agents, including bacteriocins (3) and peptidoglycan hydrolases (4), and potentially harbors the metabolic routes for biopolymer biosynthesis (5, 6).

The E. lactis SP-50a strain was isolated from cheese samples from local farms in the municipality of La Joya, Veracruz (19°36′36″ N, 97°01′32″ W), previously reported (7). The bacteria were grown in De Man, Rogosa and Sharpe (MRS) medium at 37°C for 12 h without agitation (OD_600nm: 0.771 ± 0.02). The initial analysis of the bacteria showed 99.87% similarity to Enterococcus faecium DSM 20477 (ID: 99.87%, NR_114742). However, 16S sequencing is insufficient to distinguish between E. faecium and E. lactis (8). Whole-genome sequencing was performed for definitive species identification and for biotechnological applications. Genomic DNA was extracted using the Allprep Bacterial/DNA/RNA/Protein Kit (QIAGEN, catalog number 47054). DNA purity was confirmed via spectroscopy by BioTek Epoch 2 microplate spectrophotometer (A₂₆₀/A₂₈₀: 1.97; concentration: 219 ng/µL). DNA integrity was verified by 1% agarose gel electrophoresis to confirm the presence of high-molecular-weight genomic material. Library preparation was performed using the Rapid Sequencing Kit v14 (SQK-RAD114) following the manufacturer’s instructions. Briefly, 100–150 ng of genomic DNA was adjusted to a volume of 10 µL with nuclease-free water. Enzymatic fragmentation and adapter attachment were achieved through a transposase-based method using the fragmentation mix. The reaction was incubated at 30°C for 2 min, then heated to 80°C for 2 min to inactivate the enzyme and immediately cooled on ice. Finally, 1 µL of rapid adapter was added to the fragmented DNA and incubated at room temperature for 5 min. Basecalling was performed using Dorado V1.4 (https://github.com/nanoporetech/dorado) with the dna_r10.4.1_e8.2_400bps_sup@v4.3.0 model. Reads were filtered to retain those with a minimum Q score of 10 and a minimum length of 200 bp. The de novo genome was assembled using Flye v2.9.4 (9) and polished with Medaka v2.2.1 (https://github.com/nanoporetech/medaka). Both tools were run using default parameters unless otherwise specified. The assembly of E. lactis SP-50a resulted in a draft genome of 2,715,204 base pairs across two contigs: a 2.54 Mb bacterial chromosome (coverage: 153×) and a 172 kb plasmid (coverage: 151×). The genome was annotated using the NCBI Prokaryotic Genome Annotation Pipeline (PGAP) v.6.10. Genome assembly and annotation metrics are shown in Table 1. Taxonomic identity was confirmed using FastANI v1.34 tools (10), showing an average nucleotide identity of 99.26% against E. lactis strain SU-B46 (GCA_030490365.1). Several genes were identified as bacteriocin immunity protein (MHN6393358.1) and the class II bacteriocin (MHN6393359.1), involved in bacteriocin biosynthesis. Additionally, a lysozyme family protein (MHN6395638.1) and a glucosaminidase domain-containing protein (MHN6394094.1) have been identified, both of which belong to the peptidoglycan hydrolase family (11, 12). The fabG gene (MHN6393495.1), which can function as a phaB for polyhydroxybutyrate biosynthesis, was also identified (13). Preliminary analyses supporting the antimicrobial activity and PHB production of the E. faecium SP-50a strain are shown in Fig. 1.

TABLE 1.

Assembly and genome annotation statistics for Enterococcus lactis SP-50a^a

Parameters	Values
Assembly statistics
Median read quality	22.5
Median read length (bp)	2611
Total base pairs (Mb)	418.96
Total reads	98,646
Number of contigs in assembly	2
N50 (Kb)	7.294
Genome metrics
Genome size (bp)	2,715,204
G + C %	38
CDS (total)	2,547
CDSs (with protein)	2,417
CDSs (without protein)	130
Genes (total)	2,637
rRNA	6 (5s), 6 (16s), 6 (23s)
tRNA	68
Checkm completeness (%)	99.06
CheckM contamination (%)	0.94
SRA accession number	SRR37701720
GenBank accession number	JBWDIX000000000.1
BioProject	PRJNA224116
BioSample	SAMN56532058

^a The assembly statistics were obtained using NanoPlot v 1.46.2 (14) and annotated using the NCBI Prokaryotic Genome Annotation Pipeline (PGAP) V. 6.10.

Fig 1.

Preliminary testing of the antimicrobial activity assays, peptidoglycan hydrolase, and PHA production by the Enterococcus lactis SP-50a strain. (A) Inhibition zone of crude bacteriocin extract against Listeria innocua AST 062; (B) inhibition zone of peptidoglycan hydrolase against Klebsiella pneumoniae ATCC 10031; (C) colonies of Enterococcus lactis SP-50a and E. coli BL 21 stained with Sudan Black; (D) micrograph of Enterococcus lactis (SP-50a) stained with Sudan Black using an optical microscope at 100× (Leica Microsystems, Wetzlar, Germany): the green arrow points to a PHA-accumulating cell shown in blue-black, and the black arrow points to a non-PHA-accumulating cell showing the characteristic rose color.

DATA AVAILABILITY

The Whole Genome Shotgun project has been deposited at DDBJ/ENA/GenBank under the accession number JBWDIX000000000.1. The raw sequencing data generated in this study have been deposited in the NCBI Sequence Read Archive database under accession number SRR37701720.