Evolutionary dynamics of the accessory genome of Listeria monocytogenes

Abstract

Listeria monocytogenes, a foodborne bacterial pathogen, is comprised of four phylogenetic lineages that vary with regard to their serotypes and distribution among sources. In order to characterize lineage-specific genomic diversity within L. monocytogenes, we sequenced the genomes of eight strains from several lineages and serotypes, and characterized the accessory genome, which was hypothesized to contribute to phenotypic differences across lineages. The eight L. monocytogenes genomes sequenced range in size from 2.85-3.14 Mb, encode 2,822-3,187 genes, and include the first publicly available sequenced representatives of serotypes 1/2c, 3a and 4c. Mapping of the distribution of accessory genes revealed two distinct regions of the L. monocytogenes chromosome: an accessory-rich region in the first 65° adjacent to the origin of replication and a more stable region in the remaining 295°. This pattern of genome organization is distinct from that of related bacteria Staphylococcus aureus and Bacillus cereus. The accessory genome of all lineages is enriched for cell surface-related genes and phosphotransferase systems, and transcriptional regulators, highlighting the selective pressures faced by contemporary strains from their hosts, other microbes, and their environment. Phylogenetic analysis of O-antigen genes and gene clusters predicts that serotype 4 was ancestral in L. monocytogenes and serotype 1/2 associated gene clusters were putatively introduced through horizontal gene transfer in the ancestral population of L. monocytogenes lineage I and II.

Figure 1. L. monocytogenes phylogenetic tree and accessory genome distribution plots.

Plots show the number of accessory genes in between each core gene as ordered in the reference strain EGDe. Insertion sites of prophages (P), integrated conjugative elements (ICE), and Listeria genomic islands (LGI) as detailed in Table 4 are indicated above each accessory genome distribution plot. Vertical dotted lines with a question mark indicate prophages, which are not assembled in a single contiguous piece, but are hypothesized to be present in the location based on presence of the appropriate phage genes in unalignable fraction of the assembly. Plots are colored by lineage: I, red, II, blue, III, green, IV, purple. Serotypes are shown to the right of each plot. The phylogenetic tree is based on a maximum likelihood analysis of the concatenated alignments of 2,086 core genes.

Figure 2. Cumulative distribution of the accessory genome throughout the chromosome in L. monocytogenes (n = 21), Staphylococcus aureus (n = 17) and strains of the Bacillus cereus group (n = 16).

The circular genome position starts at the origin of replication, which is at 0 degrees.

Figure 3. Clade membership plot of individual genes plotted against the genome of L. monocytogenes F2365.

The order of genome rings is listed in the circle center, with F2365 being the outermost ring. The 7 outermost rings represent lineage I (serotype 4b and 1/2b), the next three rings represent lineage III and lineage IV strains (serotype 4a and 4c), and the last 11 rings represent lineage II strains (serotype 1/2a, 1/2c, and 3a). Clade membership of the individual genes is indicated by color; blue indicates lineage II, red indicates lineage I, and gray is unresolved membership. The two O-antigen gene clusters are highlighted in green and yellow. Genes in these clusters found in serotype 1/2b lineage I cluster phylogenetically with orthologs found in lineage II clade.

Figure 4. Synteny and gene-specific phylogenetic history of the two O-antigen specific gene clusters.

The organismal phylogeny of the genus Listeria is shown in the upper panel (A), while the syntenic relationships of the two O-antigen gene clusters between the two major serotype divisions and the phylogenetic tree based on a representative serotype specific gene are shown in the two lower panels (B and C). Genes are colored by their phylogenetic histories: Serotype-specific genes (i.e., genes found only in specific serotypes) are colored green, while genes displaying an organismal phylogeny across the Listera genus are colored blue. Genes which follow a serotype-related phylogeny across Listeria are shown in orange. Values on the branches represent bootstrap values based on 100 bootstrap replicates. The organismal tree is based on a 10 locus multi-locus sequence analysis as described in Den Bakker et al. . The topology of this tree is congruent with a tree based on the MLST scheme used in Ragon et al. .

Figure 5. Phylogenetic reconstruction of serotype evolution in Listeria. Serotype 4 is shown in red while serotype 1/2 is shown in green.

This construction suggests that serotype 1/2 genes were horizontally transferred from L. seeligeri to an ancestor of L. monocytogenes lineages I and II. The origin of the serotype 1/2 cluster is unclear, we hypothesize that this cluster putatively originated in the most recent common ancestor of the L. seeligeri and L. ivanovii clade (as indicated by the dashed line). Serotype 4 genes appear to be largely inherited by vertical descent, except for a lateral transfer of genes from L. welshimeri into some strains of L. monocytogenes lineage III (dotted red line).