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J Bacteriol. Nov 2006; 188(21): 7405–7415.
Published online Aug 25, 2006. doi:  10.1128/JB.00758-06
PMCID: PMC1636279
Whole-Genome Sequence of Listeria welshimeri Reveals Common Steps in Genome Reduction with Listeria innocua as Compared to Listeria monocytogenes[down-pointing small open triangle]
Torsten Hain,1 Christiane Steinweg,1 Carsten Tobias Kuenne,1 André Billion,1 Rohit Ghai,1 Som Subhra Chatterjee,1 Eugen Domann,1 Uwe Kärst,2 Alexander Goesmann,3 Thomas Bekel,3 Daniela Bartels,3 Olaf Kaiser,3 Folker Meyer,3 Alfred Pühler,4 Bernd Weisshaar,5 Jürgen Wehland,2 Chunguang Liang,6 Thomas Dandekar,6 Robert Lampidis,7 Jürgen Kreft,7 Werner Goebel,7 and Trinad Chakraborty1*
Institute for Medical Microbiology, Justus-Liebig-University, Frankfurter Strasse 107, D-35392 Giessen, Germany,1 Gesellschaft für Biotechnologische Forschung GmbH, Department of Cell Biology, Mascheroder Weg 1, D-38124 Braunschweig, Germany,2 Bioinformatics Resource Facility, Centrum für Biotechnologie, University of Bielefeld, Universitätsstrasse 25, D-33615 Bielefeld, Germany,3 Lehrstuhl für Genetik, University of Bielefeld, Universitätsstrasse 25, D-33615 Bielefeld, Germany,4 Lehrstuhl für Genomforschung, University of Bielefeld, Universitätsstrasse 25, D-33615 Bielefeld, Germany,5 Lehrstuhl für Bioinformatik, University of Würzburg, Am Hubland/Biozentrum, D-97074 Würzburg, Germany,6 Lehrstuhl für Mikrobiologie, University of Würzburg, Am Hubland/Biozentrum, D-97074 Würzburg, Germany7
*Corresponding author. Mailing address: Institute for Medical Microbiology, Justus-Liebig-University, Frankfurter Strasse 107, D-35392 Giessen, Germany. Phone: 49-641 99 41250. Fax: 49-641 99 41259. E-mail: trinad.chakraborty/at/mikrobio.med.uni-giessen.de.
Both authors contributed equally to this work.
Received May 26, 2006; Accepted August 14, 2006.
We present the complete genome sequence of Listeria welshimeri, a nonpathogenic member of the genus Listeria. Listeria welshimeri harbors a circular chromosome of 2,814,130 bp with 2,780 open reading frames. Comparative genomic analysis of chromosomal regions between L. welshimeri, Listeria innocua, and Listeria monocytogenes shows strong overall conservation of synteny, with the exception of the translocation of an FoF1 ATP synthase. The smaller size of the L. welshimeri genome is the result of deletions in all of the genes involved in virulence and of “fitness” genes required for intracellular survival, transcription factors, and LPXTG- and LRR-containing proteins as well as 55 genes involved in carbohydrate transport and metabolism. In total, 482 genes are absent from L. welshimeri relative to L. monocytogenes. Of these, 249 deletions are commonly absent in both L. welshimeri and L. innocua, suggesting similar genome evolutionary paths from an ancestor. We also identified 311 genes specific to L. welshimeri that are absent in the other two species, indicating gene expansion in L. welshimeri, including horizontal gene transfer. The species L. welshimeri appears to have been derived from early evolutionary events and an ancestor more compact than L. monocytogenes that led to the emergence of nonpathogenic Listeria spp.
Listeriae are gram-positive, motile, facultative anaerobic rod-like bacteria that are ubiquitously occurring in nature. Listeriae are members of a group of bacteria with low G+C DNA content that includes species of the genera Bacillus, Clostridium, Enterococcus, Streptococcus, and Staphylococcus. The genus Listeria consists of six different species: Listeria monocytogenes, Listeria ivanovii, Listeria innocua, Listeria welshimeri, Listeria seeligeri, and Listeria grayi. L. monocytogenes and L. ivanovii are the only pathogenic species of the genus. While L. ivanovii is predominantly an animal pathogen, L. monocytogenes can cause infections of both animals and humans. Clinical symptoms of listeriosis in humans are often manifested as meningitis, meningoencephalitis, septicemia, abortion, prenatal infection, and gastroenteritis (39).
In addition to these two pathogens, there are four nonpathogenic species of Listeria, viz., L. innocua, L. welshimeri, L. seeligeri, and L. grayi. Phylogenetic analyses, based on the 16S and 23S rRNA genes and the iap, prs, vclA, vclB, and ldh genes, indicate that L. innocua is highly related to L. monocytogenes. The second group has L. ivanovii, together with L. seeligeri, while L. welshimeri is more distant, exhibiting the deepest branching of this group. L. grayi seems to be very distant from these two groups (34).
L. welshimeri (SLCC5334, CIP8149, and Welshimer V8) was first isolated from decaying plants (33, 40) and is a serovar 6b strain; other serovars (1/2a, 1/2b, 6a, 4c, and 4f) (20, 22) have also been reported for this species. Like other species of Listeria, L. welshimeri bacteria are small (0.5 to 2.0 μm), non-spore-forming, gram-positive rods which are motile below 30°C by means of peritrichous flagella. Growth at low temperatures (4°C) proceeds within 5 days. Results from a CAMP test with Staphylococcus aureus and Rhodococcus equi were negative, and strains of the species also tested negative for oxidase but were positive for catalase activity. Acid production occurs by fermentation of d-xylose and α-methyl-d-mannoside but not from l-rhamnose and d-mannitol (32). These biochemical properties are used to distinguish L. welshimeri from other Listeria species.
The major virulence determinants in Listeria pathogenesis are localized on a chromosomal locus between prs and ldh designated the virulence gene cluster “vgc” (8) or Listeria pathogenicity island 1, “LIPI-1” (39), which is responsible for the intracellular life cycle of the bacterium. All of these virulence genes are missing in L. welshimeri, which suggests that the genus Listeria probably evolved from the loss of the vgc region leading to the generation of nonpathogenic species from a progenitor strain already harboring the virulence genes.
Thus, L. welshimeri strains are nonhemolytic and even a high infecting dose of L. welshimeri (>1 × 108 CFU/ml), which is at least 100,000-fold higher than the 50% lethal dose of L. monocytogenes (1 × 103 CFU/ml), does not kill mice. Primary inoculation with L. welshimeri does not confer protection to subsequent challenge with a lethal dose of L. monocytogenes in mice (22).
Here we report the complete genome sequence and analysis of the type strain of L. welshimeri. We also compared this sequence to the complete genome sequences of three listerial strains, L. monocytogenes 1/2a EGD-e, L. monocytogenes 4b F2365, and L. innocua. The data provide strong evidence for the evolution of nonpathogenic Listeria spp. from an ancestral pathogenic listerial strain and suggest that this species emerged by additional gene loss and the acquisition of novel genes, probably by horizontal gene transfer.
Genome sequencing.
Bacterial strains, plasmids, and primers are listed in Table S1S of the supplemental material. The type strain of Listeria welshimeri (serovar 6b, SLCC5334) was selected for genome sequencing. Two small insert libraries (1.5 to 2 kb and 2 to 3 kb) were constructed using the plasmid pCR4Blunt-TOPO (Invitrogen) for shotgun sequencing. In addition, fosmid (pCCC1FOS; Epicenter) and bacterial artificial chromosome (BAC) (pBeloBAC11; New England Biolabs) libraries were generated for contig ordering and gap closure with large fragments of around 40 kb and 50 kb, respectively. DNA sequencing was performed on the MegaBACE 1000 sequencing system (GE Healthcare) and ABI PRISM 3100 or 3730xl genetic analyzer (Applied Biosystems). Sequence data were analyzed and assembled by using Phred/Phrap/Consed software (14, 15, 18, 19). A total number of 42.146 sequences of the shotgun libraries, 692 BAC sequences, and 1,379 fosmid sequences were assembled by the Phrap software, resulting in ~6.4-fold coverage. Gaps in the assembled genome sequence of L. welshimeri were closed by primer walking on shotgun clones, fosmids, and BACs (421 sequences). To complete the remaining gaps, we determined the order of the contigs of the L. welshimeri genome relative to that of the L. monocytogenes EGD-e genome by using the program NUCmer (10). Specific primers were designed near the ends of neighboring contigs, and PCRs were performed with chromosomal template DNA. Ninety-two sequences were obtained from PCR products that spanned these gaps.
Automatic annotation of the genome sequence of L. welshimeri was performed using GenDB 2.0 (25). Subsequently, all orthologs between the L. welshimeri genome and the genomes of L. monocytogenes EGD-e, L. innocua CLIP 11262 (17), and L. monocytogenes F2365 (29) were predicted and the L. monocytogenes F2365 annotation was adapted over for the corresponding orthologs (see below) of the L. welshimeri genome. The only exception was a discrepancy between the annotation of L. monocytogenes F2365 and the automatic annotation of GenDB 2.0 in case the latter one was identical to the annotations of L. monocytogenes EGD-e and L. innocua. Finally, all genes were manually inspected and annotated using GenDB 2.0.
For comparative genome analyses, orthologs were calculated using BLASTP. Two genes are considered an orthologous pair if they feature (i) a BLASTP E value of <0.001, (ii) protein identity of >50%, and (iii) both coverages (alignment to protein 1 and alignment to protein 2) between 75 to 125%. Two genes were considered a unidirectional orthologous pair if they were orthologs and the query gene was the best BLASTP hit in the compared genome. Two genes were considered a bidirectional ortholog pair if they were orthologs and reciprocal best BLASTP hits when comparing two genomes. Cluster analysis was performed by merging all pairs that share one protein (pair AB plus BC results in cluster ABC), which leads to a soft protein family-like grouping. The program GECO (C. T. Kuenne) for comparative genome visualization and the computational pipeline Augur (A. Billion) for surface-associated protein prediction and protein classification are unpublished software.
Nucleotide sequence accession number.
The genome sequence of L. welshimeri serovar 6b (SLCC5334) reported here has been deposited in the EMBL database under accession number AM263198.
General and specific features of the genome.
The overall features of L. welshimeri and the recently sequenced genomes of L. monocytogenes and L. innocua (17) are given in Table Table1.1. The circular genome of the type strain of L. welshimeri (SLCC5334) is 2,814,130 bp in length (Fig. (Fig.1A).1A). In comparison with the genome sizes of L. monocytogenes and L. innocua, L. welshimeri has the smallest genome of the genus Listeria. As with the genomes of the other listerial strains, L. welshimeri has a low G+C content, 36.4%, which is slightly lower than those of L. monocytogenes (38.0%) and L. innocua (37.4%). Its origin of replication and terminus, as determined by G/C skew analysis, are located ~1,400 kb apart in positions similar to those observed for L. monocytogenes and L. innocua. The genome of L. welshimeri contains six complete copies of rRNA operons (16S-23S-5S); two are located on the right replichore and four are located on the left one (Fig. (Fig.1A).1A). We detected 66 tRNA genes, a number similar to that for L. innocua but one less than that for L. monocytogenes.
TABLE 1.
TABLE 1.
Characteristics of L. welshimeri, L. innocua, and L. monocytogenes
FIG. 1.
FIG. 1.
(A) Circular representation of the L. welshimeri genome. The first circle represents the scale in kilobases starting with the origin of replication at position 0. The second circle shows the distribution of CDS (gray) in the leading and lagging strand. (more ...)
The protein coding sequences (CDS) represent 88.7% of the genome and are organized into 2,780 CDS. The average length of predicted CDS was 299 amino acids. The majority of CDS (79.3%) are organized on the leading strand in the direction of DNA replication, which is similar to those of the genomes of L. monocytogenes (78.7%) and L. innocua (80%). Six pseudogenes were detected, which is a number similar to those in L. monocytogenes (9 pseudogenes) and L. innocua (13 pseudogenes). Following gene prediction and annotation, we assigned functions for 2,186 proteins (79%) in L. welshimeri and 594 proteins (21%) were similar to hypothetical and conserved hypothetical proteins. A total of 1,492 out of 2,780 CDS showed similarities to proteins from other low-G+C-content Firmicutes species, like Bacillus subtilis 168 (1,317 orthologous genes) and S. aureus N315 (1,100 orthologous genes), when a cutoff for bidirectional ortholog pair calculation of >30% protein identity and 75 to 125% coverage was used.
We observed that the synteny between L. welshimeri, L. monocytogenes, and L. innocua is highly conserved (see Fig. S1S in the supplemental material), indicating that the overall organization of these genomes is stable. Strong conservation of the genome organization has also been observed with other closely related members of low-G+C bacteria, such as Bacillus and Staphylococcus (5). The chromosome of L. welshimeri contains a single putative prophage, located at around 1.2 Mb (lwe1196-lwe1257), with strong homology to a prophage in L. innocua located 2.6 Mb from its origin of replication (see Fig. S1S in the supplemental material). Interestingly, this prophage of L. welshimeri is inserted within the region between the tRNAArg and ydeI genes compared to L. monocytogenes EGD-e and L. innocua. At the same chromosomal location in L. ivanovii, the species-specific Listeria pathogenicity island 2 (12) is flanked by the tRNAArg and ydeI genes, suggesting that this region is an evolutionary “hot spot” of genome evolution for Listeria spp., which is effected by transduction processes of bacteriophages.
Two regions that interrupt the synteny are located at 0.5 Mb and 0.8 Mb of the L. welshimeri chromosome (see Fig. S1S in the supplemental material). These specific regions harbor insertions of several genes for L. welshimeri. A small cluster of genes encoding the FoF1-ATP synthase (lwe0418-lwe0428) (see Fig. Fig.1S1S in the supplemental material) is translocated with respect to L. monocytogenes and L. innocua. The chromosome is devoid of mobile genetic elements and harbors no plasmid.
Genome reduction through gene loss in L. welshimeri.
Comparative genomic analyses of L. welshimeri with L. innocua and L. monocytogenes allowed us to identify different classes of deleted genes in L. welshimeri and L. innocua. The first class includes genes that are commonly absent in both L. innocua and L. welshimeri relative to L. monocytogenes, while the second and the third classes comprise genes which are specifically absent in L. innocua and L. welshimeri, respectively. A total of 545 genes are absent in all three classes. A total of 249 genes are absent in both L. innocua and L. welshimeri: 63 are specific deletions for L. innocua, and 233 comprise deletions specific for L. welshimeri (Fig. (Fig.1B).1B). The majority of the deleted genes are organized in gene clusters, 71 in all, whereas just 49 represent deletions of individual genes. To confirm that the deletions are specific for the species L. welshimeri, we used PCR to examine them by using other serovar strains of L. welshimeri (Fig. (Fig.2;2; see Table S1S in the supplemental material). For all of the regions tested, we obtained PCR products of identical sizes for all strains. This result indicates that these deletions are common and stable within the species and that the development of the species L. welshimeri seems to have been a clonal event.
FIG. 2.
FIG. 2.
Confirmation of deleted gene regions in L. welshimeri compared to those in L. monocytogenes EGD-e using PCR. Lanes: M, 1 kb Plus DNA ladder (Gibco); 1, L. welshimeri SLCC5877 (1/2b); 2, L. welshimeri SLCC5828 (6b); 3, L. welshimeri SLCC6199 (6a); 4, (more ...)
The localization of the deleted regions on the circular chromosomal map of L. monocytogenes (Fig. (Fig.1B)1B) indicates that the missing genes of L. innocua and L. welshimeri originate largely at the same loci within the chromosomes. Indeed, in many cases, the deletion that is observed in the L. innocua genome has been extended to include neighboring genes for the same locus in the L. welshimeri genome (Fig. (Fig.3).3). An inspection of the distribution of these deletions in the chromosome of L. monocytogenes revealed that the majority of genes (~50%) that are absent are located in the first third of the genome on the right replichore. We also detected larger deletions in the regions around 120 kb, 1.133 Mb, and 2.36 Mb that resulted in the loss of several genes, including those coding for monocin production (lm0113-lmo0129), a Tn916-like transposon (lmo1097-lmo1114), and a putative prophage (lmo2271-lmo2332), that are present in L. monocytogenes.
FIG. 3.
FIG. 3.
Analysis of six chromosomal regions of L. monocytogenes EGD-e in comparison to those of L. innocua, a close relative of L. monocytogenes EGD-e, and L. welshimeri showing the reductive evolution of Listeria spp. With increasing distance from the common (more ...)
Many of the genes present in L. monocytogenes and absent from L. welshimeri not only are the result of gene loss but also include genes acquired by horizontal gene transfer (HGT) in L. monocytogenes. To examine for genes acquired by HGT in L. monocytogenes, we used the program SIGI (24), which allows the detection of genes of foreign origin, designated as alien genes. Alien genes are often associated with genomic islands, which are assumed to be frequently acquired via horizontal gene transfer. The gene cluster regions of the prophage, transposon, and monocin of L. monocytogenes were identified by SIGI as genes derived from HGT. Results generated by SIGI (Fig. (Fig.1B)1B) also revealed 16 gene clusters, each harboring a minimal number of one alien gene per cluster, as HGT-derived genes. Thus, ~25% of the regions that are absent in L. welshimeri do not represent bona fide deletions emanating from the L. monocytogenes genome but represent genes acquired by HGT in the latter genome (see Table S2S in the supplemental material for a list of genes derived from HGT and designated alien genes in L. monocytogenes). The large number of deletions and the occurrence of a high number of alien genes in the first third of the chromosome suggest that this particular region of the genome is particularly prone to rearrangements. The examination of the L. innocua and L. welshimeri genomes also revealed that the majority of alien genes were located on the same region of the chromosome. We speculate that the presence of specific elements in this region of the chromosome might account for this phenomenon.
Selective reduction of LRR and LPXTG proteins in L. welshimeri.
Two families of surface-associated proteins, i.e., those harboring LRR (leucine-rich repeat) and LPXTG motifs, appear to have been selectively lost in L. welshimeri. For L. monocytogenes, members of these protein families (e.g., internalins A and B) are required for the adhesion and invasion of nonphagocytic cells. In contrast to the large number of internalins and LPXTG motif proteins present in L. monocytogenes (17), we found that 17 and 20 out of the 25 LRR-motif-containing internalins of L. monocytogenes are absent in L. innocua and L. welshimeri, respectively (see Table S3S in the supplemental material). For L. welshimeri, we also observed that all of the currently studied internalin genes (inlABCEFGHIJ) were absent from its genome (see Table S3S in the supplemental material). Thus, although the inlAB locus is absent in L. welshimeri, the region between lmo0430 and lmo0435, corresponding to the region where the L. monocytogenes inlAB operon is located, was highly divergent compared to those in L. innocua and L. welshimeri (Fig. (Fig.4).4). For L. innocua, upstream of the lmo0435 homolog lin0457 that codes for a putative peptidoglycan-bound protein (2,013 amino acids) harboring a LPXTG motif, is a gene (lin0454) coding for another large protein (2,167 amino acids). This protein shares homologies to the cell wall-associated protein precursor of B. subtilis WapA, which is involved in sucrose binding (31). In L. welshimeri, both homologs, lwe0394 and lwe0431, are present in the same region but they are separated by a large insertion of ~48 kb. The genes adjacent to the putative site of insertion, i.e., from lwe0395 to lwe0417, have lower-than-average G + C contents, suggesting that this region was acquired by horizontal gene transfer. Results generated by SIGI for this region suggest that the origin of these genes was affected by HGT (Fig. (Fig.4).4). The latter part of the insertion harbors the FoF1-ATP synthase genes (lwe0418-lwe0428) that are otherwise positioned around 100 kb from oriC in L. monocytogenes and L. innocua. The inserted region is flanked downstream by lwe0429, a gene coding for another very large putative LPXTG-motif-containing protein (2,753 amino acids), with repeats that are probably involved in bacterial adhesion.
FIG. 4.
FIG. 4.
Comparative representation of the chromosomal “hot spot” region between lwe0390 and lwe0431 of L. welshimeri, including cell wall-associated genes, which are marked. Putative regions of horizontal gene transfer and a rearrangement of the (more ...)
An examination of the chromosomal loci harboring genes encoding LPXTG- and LRR-motif-containing proteins in L. monocytogenes revealed that many of these genes have been lost from the apathogenic species L. innocua and L. welshimeri (Fig. (Fig.1B).1B). We noted that the regions corresponding to the LPXTG- and LRR-motif-containing proteins are part of 16 gene clusters and 6 individual genes that have been lost in the L. welshimeri genome (Fig. (Fig.3).3). In several cases, the deleted regions are flanked by pseudogenes (lmo0171, lmo0172, lmo0410, and lmo0473) or transposase genes (lmo0174, lmo0329, lmo0330, and lmo0464). In contrast to the genes encoding these surface protein families, genes coding for other cell wall-associated proteins, such as GW-motif-containing proteins (see Table S4S in the supplemental material), LysM-motif-containing proteins (see Table S5S in the supplemental material), and NLPC/P60-like proteins (see Table S6S in the supplemental material) were roughly constant in the genomes of all three species. The L. welshimeri genome contained the largest number of genes encoding lipoproteins, 69, versus 65 and 59 for L. monocytogenes and L. innocua (Fig. (Fig.5;5; see Table S7S in the supplemental material).
FIG. 5.
FIG. 5.
Comparative analysis of six major surface protein classes for L. monocytogenes EGD-e, L. innocua, and L. welshimeri as predicted by Augur. Paralogs are not included. Orthologs were identified by unidirectional BLASTP analysis.
The gene locus harboring the ami (28) gene (lmo2558), which is involved in the adhesion of L. monocytogenes to the eukaryotic cell, revealed yet another class of difference for surface-bound proteins. Unlike its counterpart in L. monocytogenes, the Ami protein in L. welshimeri is truncated and contains only two GW repeats (R1 and R3), whereas the L. monocytogenes protein harbors four GW-motif-containing modules (R1 to R4). Interestingly, the Ami protein in L. innocua lacks only one GW-containing repeat, R3 (Fig. (Fig.66).
FIG. 6.
FIG. 6.
Representative overview of the ami region in L. monocytogenes EGD-e, L. innocua, and L. welshimeri. GW modules were identified by using the multiple sequence alignment program CLUSTALW 1.8.3. Repeated GW modules are indicated as R1 to R4 (black boxed) (more ...)
Apathogenic species missing several metabolic pathways of L. monocytogenes.
A major difference between L. monocytogenes and L. welshimeri is the absence of 22 gene clusters and 5 individual genes involved in the transport and metabolism of carbohydrates and amino acids (see Fig. S2S in the supplemental material). These include several genes coding for proteins of phosphotransferase system (PTS)-specific enzyme components involving the uptake of β-glucoside (lmo0738, lmo0874-lmo0876, lmo1035, and lmo2787), fructose (lmo2733 and lmo0503), galactiol (lmo0507 and lmo0508), pentitol (lmo1971-lmo1973), rhamnose (lmo2848-lmo2850), mannitol (lmo2797 and lmo2799), and cellobiose (lmo0034 and lmo0914-lmo0916). The bvrABC locus (lm2786-lmo2788) encoding a β-glucoside-specific PTS has been previously shown to mediate virulence gene repression of L. monocytogenes by β-glucosides (4). A further operon (lmo0178-lmo0184) involved in sugar transport and metabolism is also missing in the genome of L. welshimeri. It was previously shown that this operon is negatively regulated by PrfA (26).
Genes involved in nicotinate and nicotinamide metabolism (nadABC, lmo2022-lmo2025) (Fig. (Fig.3)3) or inositol metabolism (iolRABCD, lmo0383-lmo0386) are specifically absent in L. welshimeri, as are genes involved in arginine catabolism (lmo0036-lmo0039) (Fig. (Fig.3),3), amino acid uptake by an ATP-binding cassette (ABC) transporter (lmo2346-lmo2349), and teichoic acid biosynthesis (lmo1077, lmo1080-lmo1085, and lmo1087) (42). Individual genes specifically absent in L. welshimeri included three genes, ispF (lmo0236), ispG (lmo1441), and ispH (lmo1451), that are part of the deoxyxylulose-5-phosphate pathway for isoprenoid biosynthesis and for cell wall biosynthesis for Escherichia coli and B. subtilis (7).
We also observed that gene clusters containing genes conferring bile and acid resistance are absent in L. welshimeri and L. innocua. For L. monocytogenes, the bile salt hydrolase gene (bsh, lmo2067) (13), the bile tolerance locus (btlB, lmo0752-lmo0754) (1), and the bilE system (lmo1421 and lmo1422) (36) have all been implicated in the protection of the bacterium within the gall bladder, a specific niche for the parasite to avoid host defense responses. However, only the bilE system is commonly present in all three species. L. welshimeri also lacks the gene (lmo2387) encoding a member family of CLC chloride channel proteins regulating ion efflux. Homologs of these proteins in eukaryotic cells are important for cell volume regulation, transepithelial transport, intracellular pH regulation, and membrane excitability. A comparative metabolic reconstruction overview between L. monocytogenes EGD-e, L. innocua, and L. welshimeri is given in Fig. Fig.7.7. In addition, two gene clusters, one coding for a twin arginine secretion apparatus, TatAC (lmo0361-lmo0367), and the sigC gene cluster (lmo0421-lmo0423) (41), are lacking in the genome of L. welshimeri. It was previously reported that sigC codes for σC, which is involved in thermal resistance specific for L. monocytogenes strains of phylogenetic lineage II (41). When cultures of L. welshimeri, L. monocytogenes EGD-e, and L. innocua were assessed for survival following temperature upshift at 60°C for 0, 5, 10, and 15 min, only L. welshimeri was significantly impaired in its ability to recover from exposure to this nonpermissive temperature (see Fig. S3S in the supplemental material).
FIG. 7.
FIG. 7.
An integrated view of the metabolic pathway comparison between L. monocytogenes EGD-e, L. innocua, and L. welshimeri. The map illustrates biochemical pathways for main energy productions, such as glycolysis, starch metabolism, pentose phosphate pathway, (more ...)
Insertions confer new adaptive properties for environmental survival of L. welshimeri.
A well-known characteristic phenotype of L. welshimeri is the degradation of d-xylose, a property used to differentiate among Listeria species. The xylose operon in L. welshimeri consists of five genes, including a glycoside hydrolase family protein gene (lwe0241) coding for a putative alpha-xylosidase, a xylose-proton symporter gene (xylP, lwe0242), a xylose isomerase gene (xylA, lwe0243), and a xylulose kinase gene (xylB, lwe0244) controlled by the transcriptional repressor xylR protein (lwe0240) (Fig. (Fig.7).7). Two genes required for sucrose utilization (lwe1676 and lwe0226) and encoding a putative sucrose-specific IIBC PTS component and a sucrose phosphorylase were also detected. We identified two genes coding for a IIABC (lwe0269) and IIBCA (lwe0278) β-glucoside-specific PTS capable of uptake of a broad range of β-glucosides, like cellulose and cellobiose, lichenin, aryl-β-glucosides, and xylans. Thus, the presence of many uptake and utilization systems for energy sources found almost exclusively in plants and decaying vegetation suggest that L. welshimeri has adapted to a saprophytic strategy to survive in its natural environment.
Two specific secreted proteases were detected for L. welshimeri. A trypsin-like serine/cysteine protease is encoded by lwe1890, while lwe2295 was predicted to encode an otherwise unassigned peptidase. This suggests that L. welshimeri can utilize proteins and peptides as a source of amino acids. Indeed, all of the L. welshimeri strains studied exhibit protease activity on skim milk agar plates, whereas L. innocua has no activity (see Fig. S4S in the supplemental material). The L. monocytogenes EGD-e strain was also protease positive, as described previously (11). However, an isogenic mutant lacking the mpl gene and carrying a zinc-dependent metalloprotease lacked protease activity (see Fig. S4S in the supplemental material). Thus, this novel property of L. welshimeri strains that has not been previously described will be useful in distinguishing it from L. innocua in diagnostic tests.
We also detected two genes (lwe0700 and lwe0701) with homologies to the NatAB ABC transport system, which catalyzes ATP-dependent extrusion of electrogenic Na+, which is cytotoxic to the bacterial cell (9).
We identified an element encoding a complex type IC restriction-modification (R-M) system (lwe0477-lwe0481), comprised of a restriction endonuclease (HsdR) gene, a methylase (HsdM) gene, and two other genes, each encoding a separate HsdS subunit that imparts DNA specificity. Interestingly, the HsdS proteins are located on different strands, separated by a putative integrase gene of bacteriophage origin. The element shares homology with similar structures present in L. innocua and Lactococcus lactis (35) and is reminiscent of features present on staphylococcal cassette chromosomes in Staphylococcus spp. (23). For the latter, it has been postulated that, following integration of such elements, stability is maintained by the selfish R-M system. The function of an R-M system is often considered to confer phage resistance, but it has also been proposed that R-M systems are involved in genetic recombination (21) and allow a low rate of horizontal gene transfer (2). In addition, we detected two genes with homologies to transposase genes of the IS3 family (lwe1615 and lwe2186) and two neighboring genes (lwe1616 and lwe2187) that are involved in the transposition of the insertion sequence.
As discussed earlier, L. welshimeri harbors only 8 internalin-like genes (lwe0309, lwe0471, lwe0580, lwe0702, lwe0759, lwe0842, lwe1429 and lwe2343), while there are 25 and 19 genes encoding internalins for L. monocytogenes and L. innocua, respectively. A characteristic feature of internalins is the presence of multiple LRRs at the N-terminal end of the molecule combined with a LPXTG cell wall-anchoring motif (6). The LRR motif of lwe0702 shared homologies to plant-specific LRR motifs, suggesting a possible role in the saprophytic lifestyle concerning growth and adhesion on plant debris. We identified three L. welshimeri-specific genes coding for internalins harboring LRR motifs and an LPXTG cell wall anchor motif (see Tables S3S and S8S in the supplemental material). Two of these genes (lwe0309 and lwe1429) code for proteins with strong homologies to internalin A, which harbors four and seven LRR repeats. For the remaining gene (lwe0842) we identified a single LRR repeat.
Cumulatively, these data suggest that, for L. welshimeri, the lack of genes required for intracellular replication has been compensated for by the acquisition of genes/gene clusters for uptake systems and metabolic pathways to exploit plant-specific cell wall components.
Phylogenomic analysis of listeriae.
We used MAVID for phylogenetic analysis of the genomes of L. monocytogenes, L. innocua, and L. welshimeri (3) and conclude that the “phylogenomic” relationship (Fig. (Fig.8)8) corresponds exactly to phylogenetic analysis based on 16S rRNA genes (38).
FIG. 8.
FIG. 8.
Phylogenomic tree of three listerial species, L. welshimeri, L. monocytogenes EGD-e, and L. innocua, and B. subtilis. The phylogenomic analysis is based on whole-genome sequence data using MAVID (3). The gain (left arrow) and loss (right arrow) of genes (more ...)
By using BLASTP cluster analyses for whole-genome comparison, we determined the core gene set for all three listerial species to comprise 2,254 conserved CDS (see Fig. S5S in the supplemental material). Of these, 819 CDS revealed orthologs in B. subtilis, with 168 identified by using as a cutoff for ortholog pairs the calculation of >50% protein identity and 75 to 125% coverage. In addition, L. monocytogenes and L. innocua share 205 common CDS, whereas L. welshimeri shares only 55 CDS with L. monocytogenes and 31 CDS with L. innocua, respectively. This is in good agreement with data derived from phylogenomic analysis, indicating that L. monocytogenes and L. innocua are more related to each other than to L. welshimeri.
We note that L. welshimeri harbors 289 species-specific genes comprising ~10% of its genome compared to 205 (~7%) specific CDS for L. monocytogenes and 116 (~4%) specific CDS for L. innocua. Comparative analysis of species such as Brucella suis (42 genes) and Brucella melitensis (32 genes) indicates a low number of species-specific genes (30). On the other hand, genome comparison among three Streptococcus species revealed high numbers of species-specific genes: 416 genes for Streptococcus pyogenes, 683 genes for Streptococcus agalactiae, and 836 genes for Streptococcus pneumoniae (37). These comparisons suggest that the pangenomic gene repertoire of the genus Listeria is relatively large and indicative of active horizontal gene transfer processes in evolution.
An interesting observation is the finding of 157 genes commonly absent in the sequenced L. monocytogenes F2365 serotype 4b, L. innocua, and L. welshimeri strains but present in the L. monocytogenes EGD-e serotype 1/2a genome. Pairwise comparisons indicated that a further 53 genes in L. welshimeri/L. monocytogenes F2365 serotype 4b and 23 genes in the combination L. innocua/L. monocytogenes F2365 serotype 4b were commonly absent. This suggests that these genes were deleted in a clonal ancestor preceding the diversification into the different species. Finally, pairwise comparison of the L. monocytogenes EGD-e serotype 1/2a and L. monocytogenes F2365 serotype 4b genomes indicated the absence of 38 genes in the latter genome. Closer inspection indicated that 18 of the 38 genes are truly absent. For the remaining 20 genes, single nucleotide polymorphisms led to the classification of the respective orf as “absent” in the L. monocytogenes F2365 serotype 4b genome. Thus, as has been described for other species, such single nucleotide polymorphisms mark the beginning of genome variation leading to the deletion and evolutionary reduction in the genome. We expect that whole-genome sequencing of strains comprising other species of this genus will provide a rich resource for understanding the source of variation and the evolutionary history of this genus that comprises only six species.
Supplementary Material
[Supplemental material]
Acknowledgments
We thank Alexandra Amend, Claudia Zörb, Nelli and Juri Schklarenko, and Prisca Viehoever for excellent technical assistance, Herbert Hof for generously supplying the L. welshimeri serovars, and Philippe Glaser and Carmen Buchrieser for fruitful discussions.
This work was supported by funds obtained from the BMBF through the Competence Network PathoGenoMik (031U213B) to T.C. and T.H.
Footnotes
[down-pointing small open triangle]Published ahead of print on 25 August 2006.
Supplemental material for this article may be found at http://jb.asm.org/.
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