Table 1.
PCR and sequencing primers used.
Figure 1.
Minimum spanning tree analysis of 360 L. monocytogenes and four L. innocua strains based on MLST data.
Each circle corresponds to a sequence type (ST). Grey zones surround STs that belong to the same clonal complex (CC; 24 CCs are visible in total). ST numbers are given inside the circles and are enlarged for the central genotypes that define the major CCs (e.g., ST9 defines the central genotype of CC9). The three major lineages are highlighted by polygons. Four L. innocua sequence types are also represented (black circles). The lines between STs indicate inferred phylogenetic relationships and are represented as bold, plain, discontinuous and light discontinuous depending on the number of allelic mismatches between profiles (1, 2, 3 and 4 or more, respectively); note that discontinuous links are only indicative, as alternative links with equal weight may exist. There were no common alleles between the three major lineages, L. innocua, ST161 (CLIP98) and ST157 (CLIP85); they are arbitrarily linked through ST7 by default. Circles and sectors were colored based on serotyping data according to the provided legend; in addition, rare serotypes (3a, 3c, 4d, 4e) are indicated directly on the Figure. Note that for simplicity, the serotype of strains that were serotyped by the PCR method (Table S1) was equated to the most frequent serotype of each PCR group (e.g., 1/2a for PCR group IIA). STs in which truncated forms of InlA were found are indicated by a black triangle, with the position of the premature stop codons given after letter Δ. The ST of reference genome strains is indicated. The positioning of H7858 (ECII) is based on 6 genes only, as gene dat is incomplete. Inset A. Crosslinks corresponding to one or two allelic mismatches are indicated. Note the absence of links among major clonal complexes, indicative of their neat demarcation. Circles were colored by grey levels according to the number of isolates. Inset B. Correlation between the number of allelic mismatches (number of distinct alleles between MLST profiles) and the average number of nucleotide differences at distinct alleles. Note the regular positive trend, which indicates that L. monocytogenes genotypes diverge predominantly by a mutational process [81]. Allelic mismatch values of 7 correspond mostly to inter-lineages comparisons.
Table 2.
Polymorphism of seven housekeeping protein-coding genes among L. monocytogenes isolates.
Table 3.
Comparison of mutation rates (μ) and recombination rates (r) per base.
Figure 2.
Homologous recombination is rare, but distorts phylogenetic reconstruction.
A) Proportions of ancestry in seven housekeeping genes of L. monocytogenes strains from three ancestral populations as inferred by the linkage model of structure. This plot shows one vertical line for each isolate in which the proportions of ancestry from the three sources are color-coded. For example, strain CLIP85 was inferred as having mixed ancestry, with approximately 75% of nucleotides originated from lineage III (blue), whereas 25% of them were inferred to have an origin in lineage II (green). A number of strains from lineage I have a small proportion of nucleotides with ancestry in lineage III, while strains of lineage II had some nucleotides from lineages I (red) or III (blue). The case of CLIP98 is particular, as it was inferred as deriving from lineage II by imports mainly from L. innocua (see text). B) Neighbor-joining phylogenetic analysis of concatenated housekeeping gene sequences, using the Tamura-Nei+G+I model. The three major L. monocytogenes lineages are recognized. Together, they included all strains except CLIP85 and CLIP98. Bootstrap support of lineages is given at corresponding branches. An asterisk (*) marks strains that were recognized by structure to contain a fraction of nucleotides imported from another ancestral population, with corresponding branches colored according to the source of the recombined nucleotides. Recombination events detected independently using with RDP3 are numbered from 1 to 7 (referring to Table S2), and the involved genes are indicated.
Figure 3.
Phylogeny obtained with ClonalFrame and serotype relationships.
A) 50% consensus tree obtained after 100,000 iterations (after 50,000 burn-in) for the 130 distinct Listeria STs. L. monocytogenes appears monophyletic, with three distinct lineages and one strain (CLIP98) considered as a fourth lineage. Note the close association of CLIP85 with other lineage III strains. B) Detailed view of the inferred relationships within lineage I. Note the monophyly of serotype 4b. C) Detailed view of the inferred relationships within lineage II. Note that all strains of serotype 1/2c (CC9) are nested inside the diversity of 1/2a, and that serotype 1/2c seems to have evolved from 1/2a just after the split from strain EDGe. D) Detailed view of the inferred relationships within lineage III. E) Events on the branch that separates CLIP85 (ST157) from the rest of lineage III. Note the high number of nucleotide changes in lhkA (seventh gene), inferred by clonalFrame to correspond to a single recombination event.
Figure 4.
A) Distribution of the polymorphisms along the 2,400 nt of gene inlA in the 33 distinct inlA alleles encountered. The scale above the graph is in amino-acids (AA). InlA functional domains are represented as distinct blocks: signal peptide (SP), alpha-domain linker, leucine-rich repeats (LRR), Ig-like, B-repeat, Pre-anchor (PA) and cell wall anchor (CWA). Vertical bars above these blocks, correspond to synonymous nucleotide polymorphisms; below these blocks, non-synonymous polymorphisms resulting in amino-acid changes. Note that the LRR domain, especially repeats 7 to 15, is highly conserved. Triangles indicate the position of premature stop codons (PMSC) observed in this study and previous reports; black triangles: PMSCs observed in clonal complex CC9; red: PMSCs in other clones; see Table 4 for details. B) Deduced amino-acid polymorphisms in InlA. Lineages in which the inlA alleles were found are indicated on the left (1, 2 or 3). Blocks of amino-acids that are identical to the sequence in reference strain EGDe (allele 8) are color-shaded. Note the mosaic pattern, with blocks of polymorphisms shared between distinct groups of alleles when scrolling along the sequence.
Table 4.
Premature stop codons identified in internalin gene inlA of Listeria monocytogenes isolates.