High-quality genome assembly of Listeria monocytogenes Lm208 (CNL895805): a domestic strain able to grow faster at low temperature when exogenous unsaturated fatty acids are present

TL;DR

Scientists sequenced and analyzed the genome of a Listeria monocytogenes strain that grows faster at low temperatures when unsaturated fatty acids are present.

Contribution

The study provides a high-quality genome assembly and investigates genes related to fatty-acid metabolism in a cold-adapted Listeria strain.

Findings

The genome of Lm208 was sequenced and annotated with a focus on fatty-acid metabolism genes.

The strain shows enhanced growth at low temperatures when exogenous unsaturated fatty acids are present.

Genes related to fatty-acid metabolism were analyzed using sequence alignment methods.

Abstract

The genome of Listeria monocytogenes Lm208 strain has been sequenced and assembled. The draft genome has been automatically annotated. Considering its specific ability to grow faster at low temperatures in the presence of exogenous fatty acids, the annotation of genes related to fatty-acid metabolism was investigated using local and multiple sequence alignments.

Tables

TABLE 1. Genome metrics of L. monocytogenes Lm208 and comparison to domestic reference strains EGD-e, EGD, ScottA, LO258, and 10403S

Strain	Genomic elementa	RefSeq	ANI (%)	Contig no.	Length(Mb)	G + Ccontent (%)	No. ofCDS	No. of tRNA	No. of rRNAs	Source	CC
Lm208	Chr	GCF_048381475.1	100	12	2.87	37.9	2,824	49	4	Sheep’s brain	7
EGD-e	Chr	GCF_000196035.1	98.94	1	2.94	38.0	2,867	67	18	Derivative of EGD	9
EGD	Chr	GCF_000582845.1	99.93	1	2.91	38.0	2,818	67	18	Guinea pig tissue	7
10403S	Chr	GCF_000168695.2	99.99	1	2.90	38.0	2,802	67	6	Human skin lesions	7
ScottA	Chr	GCF_000212455.1	94.94	5	3.03	38	2,934	67	6	Listeriosis outbreak	7
LO28	Chr	GCF_021378215.1	98.90	1	2.98	38	2,911	67	6	Pregnant carrier	9

Full text

ANNOUNCEMENT

Listeria monocytogenes is a foodborne pathogen responsible for listeriosis. Lmo208 (CNL895805, serotype 1/2a), isolated in 1992 from a sheep’s brain, can grow faster at low temperature in the presence of exogenous unsaturated fatty acids (FA) (1, 2). Genomic DNA was extracted and purified from a 16-h culture in Tryptic Soy Broth (TSB) using the Wizard Genomic DNA Purification Kit (Promega, France). DNA sequencing was performed at the “Institut du Cerveau et de la Moëlle” (Paris, France). After quantification, DNA-seq libraries were prepared with the Nextera XT DNA Flex Library kit (Illumina, San Diego, USA). Whole Genome Sequencing (WGS) was performed on the NextSeq 500 system using a paired-end read length of 2 × 150 bp with the Illumina High Output kits. Raw reads (1,217,365 sequences; 151 bp) were analyzed using tools of the Galaxy portal (https://galaxy.migale.inrae.fr/). Trimming was performed with the following parameters: Initial IlluminaClip, Leading 3, Trailing 3, Sliding Window 4–20, and Minlen 125 bp (Trimmomatic v39.0) (3). The quality of 421,351 trimmed reads was checked with fastQC (v0.11.9) (4). De novo assembly was performed using SPAdes (v3.15.4) and followed by quality control (Quast v5.2.0) (5, 6). After ordering by progressiveMauve based on the genomic sequence of L. monocytogenes EGD-e (26 February 2015) (7), the size of the genome was 2,871,758 bp made of 12 contigs with a GC content of 37.9%, N50 value of 1,458,283 bp, L50 of 1, and an average coverage of 128×. Automatic annotation was performed with Prokka (v1.14.6) using the proteome of L. monocytogenes EGD-e as reference (UP000000817) (8).

2,824 predicted protein-coding genes were detected with 49 tRNAs and 4 rRNAs. 2,686 Coding Sequences (CDS) were assigned to a gene name based on the locus tag of L. monocytogenes EGD-e. Fifty CDS were identified using ListiWiki, ListeriOmics, or NBCI’s database (9, 10). After enrichment, the remaining 120 CDS encoded hypothetical proteins. Lm208 does not harbour antibiotic resistance genes nor plasmids (Staramr v0.7.2.1) (11). Two phage regions were identified with PHASTEST (https://phastest.ca/): the Listeria phage A118 (NC_003216) prophage in the comK integration site and a partial Streptococcus phage T12 (NC_028700) interrupting the locus lmo208_02875 (12–14). Contigs No. 3, 13, and 15 displayed repeated consensus regions of CRISPR and their associated caspase proteins (CRISPRCasFinder; https://crisprcas.i2bc.paris-saclay.fr/) (15). Clonal complex (CC) and lineage were defined in silico using the BIGSdb database (http://bigsdb.pasteur.fr/listeria), which revealed that Lm208 belongs to lineage II and CC7, clones rarely related to food isolates (16). Lm208 highlights high similarity with the four reference strains with a maximum of average nucleotide identity (ANI) at 99.99% with L. monocytogenes 10403S and a minimum of ANI at 94.94% with L. monocytogenes ScottA (FastANI (v1.3) (Table 1; 17, 18 ).

After automatic annotation, the annotation of Fak and Fab proteins, related to FA metabolism, was deeply investigated based on literature and local or multiple sequence alignments (1). One FakA and two FakB proteins were identified by BLASTp using the well-described Staphylococcus aureus FakAB system (Q2FHL2.1; ABD20605.1; ABD22554.1) as query sequences (18, 19). Eleven Fab proteins were found by Prokka or by NCBI’s BLASTp database and multiple sequence alignment (ClustalOmega v1.2.4) with 114 Fab sequences from Streptococcaceae and Bacillaceae (20). Phenotypic investigation is needed to identify the genetic marker related to its specific ability to grow faster in the presence of unsaturated FA.