Whole genome comparisons of serotype 4b and 1/2a strains of the food-borne pathogen Listeria monocytogenes reveal new insights into the core genome components of this species

Abstract

The genomes of three strains of Listeria monocytogenes that have been associated with food-borne illness in the USA were subjected to whole genome comparative analysis. A total of 51, 97 and 69 strain-specific genes were identified in L.monocytogenes strains F2365 (serotype 4b, cheese isolate), F6854 (serotype 1/2a, frankfurter isolate) and H7858 (serotype 4b, meat isolate), respectively. Eighty-three genes were restricted to serotype 1/2a and 51 to serotype 4b strains. These strain- and serotype-specific genes probably contribute to observed differences in pathogenicity, and the ability of the organisms to survive and grow in their respective environmental niches. The serotype 1/2a-specific genes include an operon that encodes the rhamnose biosynthetic pathway that is associated with teichoic acid biosynthesis, as well as operons for five glycosyl transferases and an adenine-specific DNA methyltransferase. A total of 8603 and 105 050 high quality single nucleotide polymorphisms (SNPs) were found on the draft genome sequences of strain H7858 and strain F6854, respectively, when compared with strain F2365. Whole genome comparative analyses revealed that the L.monocytogenes genomes are essentially syntenic, with the majority of genomic differences consisting of phage insertions, transposable elements and SNPs.

Figure 1

Circular representation of the three sequenced Listeria genomes. Each concentric circle represents genomic data and is numbered from the outermost to the innermost circle. The outermost circle indicates the AscI (black), NotI (red), SfiI (blue) and SrfI (green) restriction map of the closed L.monocytogenes serotype 4b F2365 strain. The second and third circles represent the predicted strain F2365 ORFs on the + and – strands, respectively, colored by role categories: salmon, amino acid biosynthesis; light blue, biosynthesis of cofactors, prosthetic groups and carriers; light green, cell envelope; red, cellular processes; brown, central intermediary metabolism; yellow, DNA metabolism; green, energy metabolism; purple, fatty acid and phospholipid metabolism; pink, protein fate/synthesis; orange, purines, pyrimidines, nucleosides and nucleotides; blue, regulatory functions; gray, transcription; teal, transport and binding proteins; black, hypothetical and conserved hypothetical proteins. The fourth circle shows the GC-skew. The fifth (strain F2365), seventh (strain H7858) and ninth (F6854) circles indicate ORFs involved in virulence: ORFs other than internalins (red), internalins (blue), putative prophage or monocin regions (dark green), transposable elements (gold), CRISPR elements (light blue), strain-specific genes (half-sized light gray ticks) and contig breakpoints (quarter-sized black ticks) relative to the closed F2365 strain. The sixth circle represents the number of SNPs per 5 kb for the H7858 strain, relative to the F2365 strains: blank, no SNPs (or unsequenced region); quarter-sized gold ticks, 1–30 SNPs; half-sized red ticks, 31–50 SNPs; three quarter-sized dark green ticks, 51–80 SNPs; full-sized blue ticks, more than 81 SNPs. The eighth circle represents the number of SNPs per 5 kb for the F6854 strain, relative to the F2365 strain: blank, no SNPs (or unsequenced region); quarter-sized gold ticks, 1–75 SNPs; half-sized red ticks, 76–200 SNPs; three quarter-sized dark green ticks, 201–300 SNPs; full-sized blue ticks, more than 301 SNPs. The tenth circle denotes atypical regions (χ² value). The eleventh circle depicts tRNA (green), rRNA (blue) and sRNA (red) for the F2365 strain.

Figure 2

Putative prophage regions within the genomes of five sequenced Listeria genomes. ORF colors are based on the annotation of the published sequence of L.monocytogenes 1/2a-specific typing phage φA118 (B). All putative prophage ORFs are colored to match φA118 if the protein sequence has a BLASTP P-value cut-off of ≤1 × 10^–5, and a percentage identity ≥30% over 75% or more of the length of the query sequence. If there is no match to φA118 based on the above cut-offs, the ORF is colored black. The prophages are denoted as follows: (A) putative monocin region from L.monocytogenes F2365; (B) L.monocytogenes 1/2a-specific typing phage φA118 (Genbank accession NC_003216); (C) putative A118-like prophage φEGDe.2 from L.monocytogenes EGD-e inserted into comK; (D) putative A118-like prophage φF6854.2 from L.monocytogenes F6854 inserted into comK; (E) putative A118-like prophage φH7858.2 from L.monocytogenes H7854 inserted into comK; (F) putative A118-like prophage φ11262.5 from L.innocua CLIP 11262 inserted into comK; (G) putative prophage φF6854.3 from L.monocytogenes F6854 inserted into tRNA-Thr-4; (H) putative A118-like prophage φ11262.1 from L.innocua CLIP 11262 inserted into tRNA-Lys-4; (I) putative prophage φ11262.3 from L.innocua CLIP 11262 inserted into a previously undocumented gene similar to lmo1263; (J) putative prophage φ11262.4 from L.innocua CLIP 11262; and (K) putative PSA-like prophage φ11262.6 from L.innocua CLIP 11262 inserted into tRNA-Arg-4. Putative promoters (green bent arrows) were found in the Listeria putative prophages using the predicted promoter sequences from φA118 (22) and the EMBOSS program fuzznuc with a mismatch of 1. Putative transcriptional terminators (red lollypops) were found using the TIGR program TransTerm (www.tigr.org/software). Contig gaps (sequence or physical) are represented by vertical red lines.

Figure 3

Comparison of pLM80 from L.monocytogenes H7858 (B) with pLI100 from L.innocua CLIP 11262 (C) and pXO-1 from Bacillus anthracis (A). ORF colors are based on function. Matches between plasmid ORFs were based on BLASTP data where pLM80 was the query and the other two plasmids were in a WU-BLAST-formatted database. Only those ORFs with a percentage identity ≥35% and P-value of ≤1 × 10^–5 were considered significant. Matches were colored based on percentage identity as follows: blue, 35–49% identity; brown, 50–59% identity; gold, 60–79% identity; green, 80–89% identity; red, 90–100% identity.