FSL J1-208, a virulent uncommon phylogenetic lineage IV Listeria monocytogenes strain with a small chromosome size and a putative virulence plasmid carrying internalin-like genes

Abstract

The bacterial genus Listeria contains both saprotrophic and facultative pathogenic species. A small genome size has been suggested to be associated with the loss of pathogenic potential of L. welshimeri and L. seeligeri. In this paper we present data on the genome of L. monocytogenes strain FSL J1-208, a representative of phylogenetic lineage IV. Although this strain was isolated from a clinical case in a caprine host and has no decreased invasiveness in human intestinal epithelial cells, our analyses show that this strain has one of the smallest Listeria chromosomes reported to date (2.78 Mb). The chromosome contains 2,772 protein-coding genes, including well-characterized virulence-associated genes, such as inlA, inlB, and inlC and the full prfA gene cluster. The small genome size is mainly caused by the absence of prophages in the genome of L. monocytogenes FSL J1-208, and further analyses showed that the total size of prophage-related regions is highly correlated to chromosome size in the genus Listeria. L. monocytogenes FSL J1-208 carries a unique type of plasmid of approximately 80 kbp that does not carry genes annotated as being involved in resistance to antibiotics or heavy metals. The accessory genes in this plasmid belong to the internalin family, a family of virulence-associated proteins, and therefore this is the first report of a potential virulence plasmid in the genus Listeria.

Fig 1

Scatter plot of chromosome sizes of various Listeria strains versus total sizes of prophage regions. Values on the x axis indicate the total chromosome sizes (in bp), and values on the y axis indicate the total sizes of prophage regions. Abbreviations: L.m., Listeria monocytogenes; L.i., Listeria innocua CLIP11262; L.w., L. welshimeri SLCC5334; L.s., L. seeligeri SLCC3954.

Fig 2

Phylogenetic classification of pLMIV based on sequence information for repA. The tree was constructed using a maximum likelihood optimization criterion. Values on the branches are bootstrap values based on 100 maximum likelihood bootstrap replicates. Abbreviations: Lactob., Lactobacillus; Lactoc., Lactococcus; Pedioc., Pediococcus; Staph., Staphylococcus; Strep., Streptococcus. The compositions of plasmids in Listeria groups 1 and 2 have been described by Kuenne et al. (23).

Fig 3

(A) Map of pLMIV. Arrows, ORFs encoding proteins with similarity to proteins found in other Gram-positive bacteria; bars, hypothetical proteins without sequence similarity to currently known proteins. Green arrows, proteins with BLAST similarity to genes associated with plasmid replication and conjugation; red arrows, internalins or internalin-related proteins; blue arrows, transposases or integrases. Asterisks, proteins with >20% similarity to proteins found in plasmid pMG1 of Enterococcus faecium; dots, proteins with >20% similarity to proteins found in plasmid pCP13 of Clostridium perfringens. (Inner circle) Histogram of the GC skew. Yellow bars, relative overrepresentation of GC; purple bars, relative underrepresentation of GC. (B) Overview of conserved domains/repeats found in the four plasmid-borne internalins and the one internalin-like protein. Numbers within the regions represent the number of repeats; numbers behind the bars represent the total lengths of the given genes (in amino acid residues).

Fig 4

Phylogenetic placement of the pLMIV internalin genes. Shown is a maximum likelihood tree of internalin genes found in selected L. monocytogenes strains and internalin genes found in L. monocytogenes FSL J1-208. Values on the branches are approximate likelihood ratio test values. The L. monocytogenes strains are abbreviated as follows: lmo, strain EGD-e; IIIA, strain HCC23; IIIC, strain FSL F2-208; FI, strain F2365; Lm4b, strain CLIP80459; IV, strain FSL J1-208. Internalins marked with an asterisk are found in FSL J1-208, and internalins marked with a P and an asterisk are found on the plasmid of FSL J1-208.

Fig 5

Invasion efficiencies in Caco-2 cells of L. monocytogenes strains 10403S, FSL B2-294 (FSL J1-208 strain cured of the plasmid pLMIV), and FSL J1-208. Invasion efficiency (the number of recovered cells per number of cells used for inoculation) was normalized to the invasion efficiency obtained for L. monocytogenes 10403S of the same biological replicate (hence, 10403S shows an invasion efficiency of 100% without an error bar in this figure). Three independent invasion assays (consisting of three technical replicates each) were performed for each strain tested.

Fig 6

Maximum likelihood tree based on the concatenated sequences of the MLST scheme described by den Bakker et al. (10). Values on the branches are bootstrap values based on 100 maximum likelihood bootstrap replicates. The intraspecific phylogenies of L. welshimeri, L. seeligeri, L. innocua, and L. marthii, the subspecies of L. ivanovii, and lineages I and II of L. monocytogenes have been collapsed to triangles. Open circles indicate the presence of a pLMIV-like plasmid (as determined by PCR of the origin of replication of the plasmid and inlP3), and the arrow indicates the presence of a putative pLMIV-like integrated conjugative element (as determined by PCR of P60 and cnaB).