Evolutionary Aspects

Based on in vitro data, all Listeria species are regarded as normally noncompetent [71]. It was therefore totally unexpected to find genes in L. monocytogenes and L. innocua coding for putative DNA uptake systems, homologous to B. subtilis competence genes [2, 71]. The uptake apparatus may either not be functional any more, or its regulation or the signals that induce competence may differ from those of B. subtilis and the inducing conditions have not yet been met during laboratory culture. Nevertheless, the possibility of gene transfer by transformation could well explain the genomic differences between the two species L. monocytogenes and L. innocua. These differences are mainly found in blocks dispersed around the chromosome, resulting in a mosaic genome structure. Furthermore, the collinearity identified for the L. monocytogenes and L. innocua chromosomes also extends to numerous regions of the B. subtilis chromosome [2]. The hundreds of insertions found in the three chromosomes are best explained by multiple independent transformation events followed by DNA integration at various sites on the chromosomes.

The origin of the known virulence genes in Listeria is still unclear. The large family of internalins seems to have evolved in Listeria after the initial combination of the LPXTG membrane anchor motif with a LRR motif (both of which are of unknown origin) to form a protointernalin. This then (probably) duplicated several times and evolved further by recombinations and point mutations [2]. Even among the completely sequenced L. monocytogenes strains there are significant differences in the numbers and types of internalins, which indicates an unusually high rate of mutation in these genes [3]. The virulence gene cluster was obviously either acquired a long time ago or evolved in Listeria. Looking at the individual genes present in the virulence gene cluster, homologues of the listeriolysin gene [24], the metalloprotease gene [38], and the two phospholipase genes [21, 34] are present in many related species but never found in a cluster as in L. monocytogenes. The origin of the actA gene is totally obscure since no bacterial gene with significant homology (besides the actA genes present in L. ivanovii and L. seeligeri [72, 73]) has yet been isolated. It has been speculated, however, that the actA gene may be of eukaryotic origin since parts of ActA show some homology to eukaryotic cytoskeletal proteins [74]. At present, fully functional virulence clusters are found in L. monocytogenes and in the animal pathogen L. ivanovii, and a similar cluster with additional genes is present in the nonpathogenic species L. seeligeri [73-76] (Fig. 16.2]. Interestingly, one of the ORFs with unknown function at the right border of the cluster shows some weak homology to genes of Listeria phages [73, 74]. This might point to phage transduction events involved in the early evolution of this gene cluster.

The analysis of the sequences of the virulence gene cluster and the flanking regions in the different Listeria species (Fig. 16.2) indicates that the virulence gene cluster was present in the common ancestor of L. monocytogenes, L. innocua, L. ivanovii, L. seeligeri, and L. welshimeri. It is present at exactly the same chromosomal

Prfa Listeria

Fig. 16.2 The virulence gene cluster locus in the six species of the genus Listeria. The cluster is flanked by the housekeeping genes (black boxes) prs, vclB (lmo0209), and Idh in all six species. Genes controlled by PrfA are shown as boxes with black arrowheads. vclA (lmo0208) is present in all species except L. grayi. vclP is present in L. welshimeri, L. seeli-geri, and L. ivanovii. vclZ (lmo0207) is present in L. monocytogenes and L. innocua. vclYand vclX are inverted in Lseeligeri. Species-specific genes (medium gray) not under PrfA control include vclJ, vclFI, vclGI, vclG2, vclF2 of L. grayi, and vclCof L. seeligeri. Homologous genes are represented by boxes of the same color. Reprinted with permission from Refs. [73, 75]. (This figure also appears with the color plates.)

Fig. 16.2 The virulence gene cluster locus in the six species of the genus Listeria. The cluster is flanked by the housekeeping genes (black boxes) prs, vclB (lmo0209), and Idh in all six species. Genes controlled by PrfA are shown as boxes with black arrowheads. vclA (lmo0208) is present in all species except L. grayi. vclP is present in L. welshimeri, L. seeli-geri, and L. ivanovii. vclZ (lmo0207) is present in L. monocytogenes and L. innocua. vclYand vclX are inverted in Lseeligeri. Species-specific genes (medium gray) not under PrfA control include vclJ, vclFI, vclGI, vclG2, vclF2 of L. grayi, and vclCof L. seeligeri. Homologous genes are represented by boxes of the same color. Reprinted with permission from Refs. [73, 75]. (This figure also appears with the color plates.)

position in all hemolytic isolates. The pathogenic capability associated with the virulence gene cluster has been lost in two separate events in L. innocua and L. Welshmen [73, 74, 77]. The two independent deletion events in the two species are indicated by the presence of short DNA sequences believed to originally belong to the virulence gene cluster [73, 77]. The more distantly related species L. grayi obviously never harbored the virulence gene cluster [73]. The availability of the complete genome sequences of at least one strain of each species of the genus Listeria in the near future and their comparison will certainly further enlarge our understanding of the evolution of the genus Listeria.

The recent sequencing projects with additional members of the species L. monocytogenes [3, 70] also shed light on the evolutionary history of this species comprising 13 different serovars, out of which only 4 (Sv 1/2a, 1/2c, 1/2b, and 4b) account for 98% of reported human listeriosis cases [4]. In the pregenomic era, different genetic methods, including multilocus sequence typing, restriction fragment length polymorphism, and ribotyping, suggested that L. monocytogenes can be subdivided into either two or three lineages, with most of the epidemic strains found in only one lineage [4, 70, 78]. The construction of a DNA macroarray representing those genes which are unique to L. innocua and the L. monocytogenes Sv1/2a EGD-e strain and the Sv4b strain CLIP 80489 allowed the comparison of more than 100 Listeria strains by genomic hybridization [70]. This study only partially confirmed the previous classification of L. monocytogenes strains; the evolutionary scheme derived from the genome comparison groups the L. monocytogenes isolates into two main lineages which can be further subdivided. Most interestingly, L. innocua could be grouped together with the L. monocytogenes Sv4 strains, and it is now evident that this species once derived from an ancestor of the L. monocytogenes Sv4 strains by successive gene loss including loss of the virulence gene cluster.

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