Listeria monocytogenes serotype 7 lacks glycosidic constituents in wall teichoic acids. Here, we present the complete genome sequence of L. monocytogenes serotype 7 strain FSL R9-0915 and an analysis of genes known to affect L. monocytogenes antigenicity. This strain was used as a control strain in Listeria phage host-range analyses.
ABSTRACT
Listeria monocytogenes serotype 7 lacks glycosidic constituents in wall teichoic acids. Here, we present the complete genome sequence of L. monocytogenes serotype 7 strain FSL R9-0915 and an analysis of genes known to affect L. monocytogenes antigenicity. This strain is used as a control strain in Listeria phage host range analyses.
ANNOUNCEMENT
Listeria monocytogenes is a Gram-positive foodborne bacterial pathogen that causes the potentially fatal illness listeriosis (1). L. monocytogenes strains are serotyped based on somatic and flagellar antigens (2). Of the 13 established serotypes, serotype 7 is one of the least commonly studied. Here, we present the complete genome sequence of L. monocytogenes strain FSL R9-0915 (alternative identifiers [IDs] are KC983, KC1716, NTCC 1627, and H3293). FSL R9-0915 was originally isolated from an unknown source on 17 December 1969 by Jeanette Donker-Voet from the Netherlands. This strain was maintained as part of a serotyping collection as a representative serotype 7 strain (3) and was provided to the Centers for Disease Control and Prevention (CDC) on 6 March 1997 upon request by Robert Weaver. This strain is resistant to all wild-type Listeria phages surveyed; thus, it is a useful control strain for Listeria phage host range studies (4, 5).
FSL R9-0915 was grown overnight in brain heart infusion broth (at 37°C, with shaking); genomic DNA was extracted using the Qiagen DNeasy minikit (Hilden, Germany), per the manufacturer’s instructions. For Illumina sequencing, the library was prepared using a Nextera XT kit and sequenced with a NextSeq 550 instrument (150-bp paired-end reads; 3,474,670 total reads were generated; average length of 137.31 bp). For Nanopore sequencing, the library was prepared using an Oxford Nanopore rapid barcoding kit (SQK-RBK0004) and sequenced with a MinION instrument (130,765 total reads were generated; average read length of 5,446.79 bp). FastQC v0.11.7 (6) was used to determine read quality, and Trimmomatic v0.35 (7) was used to trim Illumina reads (parameters were ILLUMINACLIP:NexteraPE-PE.fa:2:30:10 LEADING:3 TRAILING:3 SLIDINGWINDOW:4:15 MINLEN:36). A hybrid assembly was constructed with Unicycler v0.4.8-beta (8) (with pilon polishing), which automatically removes overlaps and circularizes and reorients the assembly to begin at the dnaA gene. QUAST v4.6.3 (9) was used to determine assembly statistics (e.g., length and G+C content), and BBMap v38.08 (10) and SAMtools v1.8 (11) were used to map reads to the assembly and determine average read coverage. The assembly was annotated with the NCBI Prokaryotic Genome Annotation Pipeline (PGAP; v4.13) (12). Default parameters were used for all software unless otherwise specified.
The complete genome assembly of FSL R9-0915 consists of two contigs as follows: one chromosome (2,946,104 bp; G+C content of 38.01%; 153.1× Illumina read coverage) and one plasmid (50,101 bp; G+C content of 35.48%; 253.0× Illumina read coverage). The PGAP annotation identified 2,977 genes, including 2,888 coding DNA sequences (CDSs) and 89 RNA genes (18 rRNAs, 67 tRNAs, and 4 noncoding RNAs [ncRNAs]). A single-nucleotide frameshift deletion (Table 1) was identified in the FSL R9-0915 rmlB homolog (a dTDP-d-glucose-4,6-dehydratase; nucleotide position 1147464) compared with serotype 1/2a strain 10403S (GenBank accession number NC_017544.1); this would result in a truncated protein. These findings support studies that suggest that serotype 7 strains evolved from a serotype 1/2 genetic background (13) by accumulating mutations in genes (e.g., rmlB) that could disrupt glycosylation of wall teichoic acids (WTAs), such as loss of rhamnose in WTAs (14, 15). Additionally, previous wheat germ agglutination assays confirm that FSL R9-0915 lacks N-acetylglucosamine in its WTAs (4). Although no identified mutations are clearly responsible for the lack of N-acetylglucosamine in WTAs, several amino acid changes were found in relevant genes (Table 1).
TABLE 1.
Amino acid differences between genes involved in wall teichoic acid biosynthesis for L. monocytogenes strains FSL R9-0915 and 10403S
| Gene homolog identifier for strain: |
Feature | No. of amino acid changes | Residue | Amino acid change by strain |
Predicted effecta | ||
|---|---|---|---|---|---|---|---|
| 10403S | EGD-e | ||||||
| 10403S | FSL R9-0915 | ||||||
| LMRG_00541 | lmo1079 | YHFO family protein | 5 | 422 | Ile | Thr | RNS |
| 684 | Gly | Asp | RNS | ||||
| 697 | Val | Ile | CNS | ||||
| 909 | Leu | Arg | RNS | ||||
| 948 | Lys | Arg | CNS | ||||
| LMRG_00542 | lmo1080 | rmlT | 8 | 40 | Ser | Ala | RNS |
| 49 | Pro | Ser | RNS | ||||
| 393 | Ser | Ala | RNS | ||||
| 427 | Asp | Asn | RNS | ||||
| 456 | Asn | Asp | RNS | ||||
| 461 | Asp | Glu | CNS | ||||
| 498 | Ile | Val | CNS | ||||
| 544 | Val | Ile | CNS | ||||
| LMRG_00543 | lmo1081 | rmlA | 0 | ||||
| LMRG_00544 | lmo1082 | rmlC | 1 | 38 | Ala | Val | CNS |
| LMRG_00545 | lmo1083 | rmlB | 2 | 24 | His | Tyr | RNS |
| 54 | Val | Ile | CNS | ||||
| 55 | Truncated protein | ||||||
| LMRG_00546 | lmo1084 | rmlD | 2 | 28 | Thr | Asn | CNS |
| 73 | Asn | Asp | RNS | ||||
| LMRG_01697 | lmo2550 | Glycosyltransferase | 1 | 300 | Pro | Leu | CNS |
| LMRG_01698 | lmo2549 | Putative flippase | 0 | ||||
| LMRG_01692 | lmo2555 | Glycosyltransferase | 1 | 195 | Asp | Asn | RNS |
| LMRG_01693 | lmo2554 | Glycosyltransferase | 10 | 129 | Glu | Ala | RNS |
| 150 | Thr | Gly | RNS | ||||
| 157 | Glu | Ala | RNS | ||||
| 160 | Gly | Glu | RNS | ||||
| 222 | Asn | Ser | CNS | ||||
| 225 | Asn | Lys | RNS | ||||
| 237 | Thr | Ser | CNS | ||||
| 264 | Ser | Cys | CNS | ||||
| 314 | Ala | Ser | RNS | ||||
| 332 | Gly | Asp | RNS | ||||
| LMRG_01694 | lmo2553 | Flippase-like domain- containing protein | 5 | 48 | Leu | Ser | RNS |
| 190 | Ile | Val | CNS | ||||
| 338 | Gln | Lys | RNS | ||||
| 350 | Thr | Ala | RNS | ||||
| 352 | Ala | Val | CNS | ||||
| LMRG_02769 | lmo1695 | mprF | 5 | 164 | Asp | Asn | RNS |
| 555 | Glu | Asn | RNS | ||||
| 668 | Asp | Glu | CNS | ||||
| 714 | Ile | Val | CNS | ||||
| 726 | Asp | Glu | CNS | ||||
RNS, radical nonsynonymous mutation; CNS, conservative nonsynonymous mutation; nonsynonymous substitutions were classified based on amino acid charge and polarity (16).
Data availability.
The sequencing data and assembly for FSL R9-0915 are located under BioProject number PRJNA664209 (BioSample SAMN16231355; raw reads SRR12695183 and SRR12695179; annotated assembly CP062124 and CP062125).
ACKNOWLEDGMENT
This work was supported by startup funds provided by The University of Tennessee Institute of Agriculture to T.G.D.
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Associated Data
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Data Availability Statement
The sequencing data and assembly for FSL R9-0915 are located under BioProject number PRJNA664209 (BioSample SAMN16231355; raw reads SRR12695183 and SRR12695179; annotated assembly CP062124 and CP062125).