new aspects regarding evolution and virulence of listeria ... · listeria monocytogenes is a...

INFECTION AND IMMUNITY, Feb. 2004, p. 1072–1083 Vol. 72, No. 20019-9567/04/$08.00�0 DOI: 10.1128/IAI.72.2.1072–1083.2004Copyright © 2004, American Society for Microbiology. All Rights Reserved.

New Aspects Regarding Evolution and Virulence of Listeriamonocytogenes Revealed by Comparative Genomics and DNA Arrays

Michel Doumith,1 Christel Cazalet,2 Natalie Simoes,2 Lionel Frangeul,2 Christine Jacquet,1Frank Kunst,2 Paul Martin,1 Pascale Cossart,3 Philippe Glaser,2 and Carmen Buchrieser2*

Laboratoire des Listeria, Centre National de Reference des Listeria, World Health Organization Collaborating Centerfor Foodborne Listeriosis,1 Laboratoire de Genomique des Microorganismes Pathogenes,2 and Unite des

Interactions Bacteries-Cellules,3 Institut Pasteur, 75724 Paris Cedex 15, France

Received 12 September 2003/Returned for modification 20 October 2003/Accepted 22 October 2003

Listeria monocytogenes is a food-borne bacterial pathogen that causes a wide spectrum of diseases, such asmeningitis, septicemia, abortion, and gastroenteritis, in humans and animals. Among the 13 L. monocytogenesserovars described, invasive disease is mostly associated with serovar 4b strains. To investigate the geneticdiversity of L. monocytogenes strains with different virulence potentials, we partially sequenced an epidemicserovar 4b strain and compared it with the complete sequence of the nonepidemic L. monocytogenes EGDeserovar 1/2a strain. We identified an unexpected genetic divergence between the two strains, as about 8% of thesequences were serovar 4b specific. These sequences included seven genes coding for surface proteins, two ofwhich belong to the internalin family, and three genes coding for transcriptional regulators, all of which mightbe important in different steps of the infectious process. Based on the sequence information, we then charac-terized the gene content of 113 Listeria strains by using a newly designed Listeria array containing the “flexible”part of the sequenced Listeria genomes. Hybridization results showed that all of the previously identifiedvirulence factors of L. monocytogenes were present in the 93 L. monocytogenes strains tested. However, distinctpatterns of the presence or absence of other genes were identified among the different L. monocytogenes serovarsand Listeria species. These results allow new insights into the evolution of L. monocytogenes, suggesting thatearly divergence of the ancestral L. monocytogenes serovar 1/2c strains from the serovar 1/2b strains led to twomajor phylogenetic lineages, one of them including the serogroup 4 strains, which branched off the serovar 1/2bancestral lineage, leading (mostly by gene loss) to the species Listeria innocua. The identification of 30 L.monocytogenes-specific and several serovar-specific marker genes, such as three L. monocytogenes serovar4b-specific surface protein-coding genes, should prove powerful for the rapid tracing of listeriosis outbreaks,but it also represents a fundamental basis for the functional study of virulence differences between L.monocytogenes strains.

The current revolution in microbial investigations due to thesequencing of complete genomes has revealed insights into thegenetic structures of a number of bacterial species. However, ithas become evident that the genome sequence of one strain isnot entirely representative of other members of the species andthat the broad spectrum of physiological and virulence prop-erties of bacterial pathogens mirrors the existence of differentsubsets of genes enabling different lifestyles. Besides whole-genome comparisons (1, 27), micro- and macroarray tech-niques are extensively used to study inter- and intraspeciesdiversity (16, 26). For example, microarrays and related tech-niques have been employed to study the genetic differencesamong members of the Mycobacterium tuberculosis complex (2,7), Helicobacter pylori strains (30), Staphylococcus aureusstrains (17), and Vibrio cholerae strains (14) and between Yer-sinia pestis and Yersinia pseudotuberculosis (23). The resultsunderscore the fact that although considerable diversity ispresent among different bacterial isolates of the same species,clonal expansion of highly virulent subpopulations of a bacte-rial pathogen may exist.

Listeria monocytogenes, an intracellular pathogen, is thecausative agent of serious epidemic and sporadic food-bornelisteriosis. The clinical features of listeriosis include meningitis,meningoencephalitis, septicemia, abortion, perinatal infec-tions, and gastroenteritis (34). Although rare when comparedto other food-borne diseases, a significant feature of listeriosisis the high lethality rate (about 30%), which makes L. mono-cytogenes an important human pathogen. L. monocytogenes hasthe capacity to adapt and survive under extreme conditions,allowing it to ubiquitously exist in the environment and tosurvive and proliferate under conditions that exist within thefood chain.

However, not all strains of L. monocytogenes seem to beequally capable of causing disease in humans. Isolates from 4(1/2a, 1/2c, 1/2b, and 4b) of the 13 serovars identified withinthis species are responsible for over 98% of reported humanlisteriosis cases (24). Furthermore, all major food-borne out-breaks of listeriosis, as well as the majority of sporadic cases,have been caused by serovar 4b strains, suggesting that strainsof this serovar may possess unique virulence properties. Anumber of different typing and population genetic studies sug-gested that different genetic divisions or lineages, which cor-relate with serovars, exist within the species L. monocytogenes(3, 6, 20, 28). Hereafter we will designate serovar 1/2a, 1/2c,and 3c strains as lineage I strains; serovar 4b, 1/2b, and 3b

* Corresponding author. Mailing address: Laboratoire de Genom-ique des Microorganismes Pathogenes, Institut Pasteur, 28 rue du Dr.Roux, 75724 Paris Cedex 15, France. Phone: (33-1)-45-68-83-72. Fax:(33-1)-45-68-87-86. E-mail: [email protected].

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strains as lineage II strains; and serovar 4a and 4c strains aslineage III strains. Genetic analyses using multilocus sequencetyping of virulence-associated genes, restriction fragmentlength polymorphism analysis, and ribotyping suggested thatepidemic strains are mostly found in lineage II and that spo-radic strains are found in lineages I and II, while lineage IIIstrains are extremely rare and are mostly animal pathogens(25, 36). However, these methods are unable to further char-acterize the genetic basis for this observed variability.

Recently, the complete genome sequences of L. monocyto-genes strain EGDe and Listeria innocua strain CLIP 11262were determined (19). A comparison of these sequences re-vealed 10.5 and 14% specific sequences for each strain, respec-tively (19). The L. monocytogenes strain that was sequenced isfrom serovar 1/2a and belongs to lineage I. Therefore, it wasimportant to investigate differences in gene content betweenlineage I and lineage II isolates and/or between different se-rovars.

In order to address questions regarding epidemiological andevolutionary relationships between pathogenic and nonpatho-genic Listeria species and to define characteristics of particu-larly successful clonal pathovariants for causing disease, wepartially sequenced an epidemic isolate of L. monocytogenesserovar 4b. Based on the comparison of the three Listeriasequences, we constructed specific arrays that were used tocharacterize 113 Listeria strains. The correlation of genomic,phylogenetic, and epidemiological properties of the strains al-lowed us to identify lineage-specific marker genes and to pro-pose new evolutionary relationships. The results open newavenues for the development of rapid typing tools as well as forfunctional analysis of species- and serovar-specific genes tounderstand their roles in pathogenicity.

MATERIALS AND METHODS

Bacterial strains. The Listeria strains used in this study were selected from theculture collection of the National Reference Center for Listeria, Institut Pasteur,Paris, France, and from the production environment of different food plants. Thestrains were selected to represent all serovars of L. monocytogenes as well asdisease- and non-disease-related isolates. A total of 93 L. monocytogenes strainsfrom 12 different serovars isolated from humans (sporadic and epidemic cases),foods, animals, and the environment, as well as 20 representative strains of thedifferent species of the genus Listeria (eight L. innocua strains, six Listeriaivanovii strains, two Listeria welshimeri strains, two Listeria seeligeri strains, andtwo Listeria grayi strains), were studied (Table 1). This set included eight epi-demic strains from five outbreaks, 15 isolates from sporadic cases, and 19 strainsfrom food plants. Strains were routinely grown overnight at 37°C without agita-tion in TPB broth (GIBCO).

Sequencing, assembly, and sequence analysis. Cloning, sequencing, assembly,and annotation of L. monocytogenes CLIP 80489 were done as described byGlaser et al. (19). Essentially, 13,234 shotgun sequences were assembled into1,020 contigs. For annotation, the program CAAT-box (18) was used.

Probe selection, primer design, PCR amplification, and array construction.The L. monocytogenes EGDe-specific probes spotted onto the Listeria arraycorresponded to the 270 genes defined previously as being specific for L. mono-cytogenes EGDe with respect to L. innocua CLIP 11262 (19). As some genes weretoo small to allow amplification of a PCR product of optimal size, the final arraycontained 262 EGDe genes. The 94 probes that were specific for L. innocuaCLIP 11262 relative to L. monocytogenes EGDe were also selected from thepreviously defined list of 149 genes specific for L. innocua CLIP 11262 (19). Forgenomic regions containing several genes, only representatives were chosen forthe array. In order to identify L. monocytogenes CLIP 80459-specific sequences,we used the program Cross-match (http:/bozeman.mbt.washington.edu/). Withthis approach, 141 sequence fragments, ranging from 33 to 3,025 bp, wereidentified as missing from both L. monocytogenes EGDe and L. innocua CLIP11262. For probe design, only fragments longer than 1 kb were taken into

account, allowing us finally to select 53 sequences that were specific for L.monocytogenes CLIP 80459. Primers were designed by use of a modified versionof Primer 3 software (CAAT-box [18]) to amplify a specific fragment of 300 to600 bp for each gene (melting temperatures were 55 to 65°C) (Eurogentec).Amplification reactions were performed in a 100-�l reaction volume containing10 to 20 ng of chromosomal DNA. The concentration and size of each PCRproduct were verified on agarose gels. For array preparation, nylon membranes(Qfilter; Genetix) were soaked in TE solution (10 mM Tris [pH 7], 1 mM EDTA[pH 7.6]). Spot blots of PCR products and controls were performed by a Qpixrobot (Genetix). Following spot deposition, membranes were fixed for 15 min in0.5 M NaOH–1.5 M NaCl, washed briefly in distilled water, and stored wet at�20°C until use.

Hybridization. Genomic DNA was extracted by using a Qiagen DNeasy kitand was radiolabeled by using a random priming DNA labeling kit (Roche).Labeling was performed with 500 ng of genomic DNA and 50 �Ci of 33P-labeleddCTP (Amersham). Labeled DNA was purified through Sephadex G-50 (Roche)or Qiaquick (Qiagen) minicolumns. High-density arrays were wetted in 2� SSC(1� SSC is 0.15 M NaCl plus 0.015 M sodium citrate) and prehybridized for 1 hin 10 ml of a solution containing 5� SSPE (1� SSPE is 0.18 M NaCl, 10 mMNaH2PO4, and 1 mM EDTA [pH 7.7]), 4% sodium dodecyl sulfate, 1� Den-hardt’s solution (50� Denhardt’s solution is 1% Ficoll, 1% polyvinylpyrrolidone,and 1% bovine serum albumin), and 1 mg of denatured salmon sperm DNA.Hybridization was performed overnight at 60°C. Membranes were washed twiceat room temperature and twice at 60°C in 0.5% SSPE–0.2% sodium dodecylsulfate. Arrays were then sealed in polypropylene bags and exposed to a phos-phorimager screen (Molecular Dynamics) for 24 h.

Verification of the specificity and quality of the macroarray. Fifteen percent ofall PCR products were randomly chosen and sequenced. All 64 sequences cor-responded to the expected PCR products. The membrane was then hybridizedwith chromosomal DNAs isolated from the three Listeria strains used to amplifythe probes (L. monocytogenes EGDe, L. innocua CLIP 11262, and L. monocy-togenes CLIP 80459) to test the quality and the correct spotting of probes.

Data analysis. For scanning, a model 445SI phosphorimager (Molecular Dy-namics) was used. ArrayVision software (Imaging Research) was used for quan-tification of hybridization intensities and for normalization of data. For eachspot, the hybridization intensity value was normalized by dividing it by theaverage of all significant intensity values on each filter (see supplemental tablesat http://www.pasteur.fr/recherche/unites/gmp/sitegmp/gmp�projects.html). Forratio calculation, a reference array, which was built by combining the averagenormalized data from three replicate hybridizations of the genomic DNAs of L.monocytogenes EGDe, L. innocua CLIP 11262, and L. monocytogenes CLIP80459 to the corresponding spots on the array (http://www.pasteur.fr/recherche/unites/gmp/sitegmp/gmp�projects.html), was used. In order to define the cutoffratio for the presence of a gene, we analyzed the results for L. monocytogenesEGDe genes hybridized with L. innocua chromosomal DNA. The threshold forthe presence of a gene was defined as �0.3. This corresponds to a DNA similarityof �80%, which was verified by sequence comparisons of these genes for bothgenomes. The data were then converted into binary scores (for ratios of �0.3, agene was scored as present [1], and for ratios of �0.3, a gene was scored as absent[0]) (http://www.pasteur.fr/recherche/unites/gmp/sitegmp/gmp�projects.html).The binary data were analyzed by hierarchical clustering with the programJ-Express (13), by neighbor joining with the program MVSP 3.1 (Kovach Com-puting Services), and by intensive expert-based data mining with Excel spread-sheets.

RESULTS

Genome diversity among an epidemic L. monocytogenesstrain, L. monocytogenes EGDe, and L. innocua and macroar-ray conception. The partial sequence of the epidemic L. mono-cytogenes serovar 4b strain CLIP 80459 (lineage II) was assem-bled into 1,020 contigs that encompassed 2.3 Mb of thegenome (estimated complete genome, 2.9 Mb) and was com-pared to the genome sequence of L. monocytogenes EGDe(serovar 1/2a; lineage I). About 8% of the CLIP 80459 se-quences were missing from EGDe. Thus, the genetic diversitybetween the two L. monocytogenes isolates seems to be close tothat between L. monocytogenes EGDe and L. innocua (10.5%),which belong to two different species. One hundred forty-oneserovar 4b-specific fragments, ranging from 33 to 3,025 bp,

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TABLE 1. Strains used in the DNA-DNA macroarray hybridization analysis

Species Straina Originb Serovar Year isolated Countryd

L. monocytogenes CLIP 61673 Human, sporadic 7 1995 FranceCLIP 74917 * 7CLIP 42636/T23 Human, epidemic 1/2a UKCLIP 46664/T20 Food 1/2a USACLIP 46682/T56 Human, sporadic 1/2a USACLIP 42663/T77 Human, sporadic 1/2a USACLIP 46661/T14 Human, sporadic 1/2a USACLIP 42651/T53 Animal, sporadic 1/2a UKCLIP 9429 Human, sporadic 1/2a 1988 FranceCLIP 74902/ATCC31152 * 1/2aCLIP 61523 Food 1/2a 1994 FranceCLIP 61678 Food 1/2a FranceCLIP 61634 Human, sporadic 1/2a 1995 FranceCLIP 61839 Food 1/2a FranceCLIP 61868 Human, sporadic 1/2a 1995 FranceCLIP 87880 Food 1/2a 1992 FranceCLIP 87881 Food, environment 1/2a 1993 FranceCLIP 87884 Food, environment 1/2a 1992 FranceCLIP 87890 Food 1/2a 1992 FranceCLIP 69566 Environmental 1/2a FranceCLIP 80559 Environmental 1/2aCLIP 60368 Environmental 1/2a 1994 FranceCLIP 14843 Environmental 1/2a FranceCLIP 89461 Environmental 1/2a DenmarkCLIP 89458 Food 1/2a DenmarkCLIP 87908 1/2aCLIP EGD 1/2aCLIP 46686/T64 Human, sporadic 1/2b USACLIP 42655/T61 Food 1/2b USACLIP 989 Human, sporadic 1/2b 1981 USACLIP 3559 Human, sporadic 1/2b ArgentinaCLIP 87878 Food, environmental 1/2b 1993 FranceCLIP 87879 Food 1/2b 1992 FranceCLIP 87888 Food 1/2b 1992 FranceCLIP 87897 Food, environmental 1/2b 1999 FranceCLIP 87909 Food 1/2b 1999 FranceCLIP 87910 Food, environmental 1/2b 1993 FranceCLIP 70086 Environmental 1/2b 1995 ArgentinaCLIP 70848 Environmental 1/2b 1996 SpainCLIP 76278 Environmental 1/2b 1998 FranceCLIP 14842 Environmental 1/2b FranceCLIP 89460 Environmental 1/2b DenmarkCLIP 46694/T80 Human, sporadic 1/2c UKCLIP 42653/T57 Human, sporadic 1/2c UKCLIP 42972/T8 Human, sporadic 1/2c UKCLIP 2710 Human, sporadic 1/2c Czech RepublicCLIP 11550 Food 1/2c FranceCLIP 87891 Food, environmental 1/2cCLIP 87892 Food, environmental 1/2cCLIP 87893 Food, environmental 1/2cCLIP LO028 1/2cCLIP 86436 Food 3a 2000 FranceCLIP 74905/ATCC19113 * 3aCLIP 8053 Environmental 3a 1987 FinlandCLIP 74906 * 3bCLIP 87900 Food, environmental 3bCLIP 3558 Animal 3b ArgentinaCLIP 11962 Food 3c FranceCLIP 85412 Food 3c 2000 FranceCLIP 74907 * 3cCLIP 74908/ATCC19114 * 4aCLIP 78025 4a GermanyCLIP 71988 4aCLIP 73722 Human, sporadic 4b 1997 FranceCLIP 46684/T60 Environmental 4b SwitzerlandCLIP 46679/T50 Food, epidemic 4b CanadaCLIP 42635/T21 Human, epidemic 4b Switzerland

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were identified. An analysis of these fragments identified par-tial or complete genes with homologies to genes encoding cellproteins, e.g., 7 surface proteins with an LPXTG motif, 6transport proteins, 3 transcriptional regulators, 1 phosphotrans-ferase system (PTS) components and 26 unknown proteins (seesupplemental material at http://www.pasteur.fr/recherche/unites/gmp/sitegmp/biodiversitylist.html), suggestingthat epi-demic L. monocytogenes strains differ substantially in gene con-tent from the L. monocytogenes EGDe strain.

To extend the comparison from the three sequenced strainsto a large collection of strains, we designed high-density mem-branes that were mainly focused on genes specific for eachsequenced Listeria strain. This approach was chosen to in-

crease the discriminatory power of the array. The membranecontained 409 probes, including 262 that were specific for L.monocytogenes EGDe relative to L. innocua CLIP 11262 andall its virulence genes, 94 that were specific for L. innocuaCLIP 11262 relative to L. monocytogenes EGDe, and 53 thatwere specific for L. monocytogenes CLIP 80459. This mem-brane was used to analyze 113 Listeria isolates. Probes andprimary hybridization results are available online as supple-mental material (http://www.pasteur.fr/recherche/unites/gmp/sitegmp/biodiversitylist.html).

Strain diversity and overall gene distribution. Based on themacroarray hybridization data predicting the presence or ab-sence of the studied genes, we built bifurcating trees illustrat-

TABLE 1—Continued

Species Straina Originb Serovar Year isolated Countryd

CLIP 42652/T55 Food, epidemic 4b SwitzerlandCLIP 42639/T29 Human, epidemic 4b USACLIP 42646/T43 Food, epidemic 4b USACLIP 4598 Food 4b SwitzerlandCLIP 2698 Human 4b Czech RepublicCLIP 8715 Human 4b 1988 SwitzerlandCLIP 74910/ATCC 19115 * 4bCLIP 22573 Human, epidemic 4b 1992 FranceCLIP 27993 Food 4b 1992 FranceCLIP 87889 Food, environmental 4bCLIP 87896 Food, environmental 4bCLIP 87911 Food 4bCLIP 87915 Food 4bCLIP 87917 Food 4bCLIP 72783 Environmental 4b 1996 SwitzerlandCLIP 60383 Environmental 4b 1994 FranceCLIP 87691 Environmental 4b PortugalCLIP 80459 Human, epidemic 4b 1999 FranceCLIP 74911/ATCC19116 * 4cCLIP 86309 Food 4c FranceCLIP 81065 Animal 4c 2000 SwitzerlandCLIP 74912/ATCC19117 * 4dCLIP 79619 Animal 4d 1999 SwitzerlandCLIP 74913/ATCC19118 * 4eCLIP 78003 4e GermanyCLIP 79455 4e

L. innocua CLIP 71990 6bCLIP 71989 6bCLIP 74915/ATCC33090 * 6aCLIP 74916/ATCC33091 * 6bCLIP 88566 Food 6a FranceCLIP 88307 Food 6b 2001 FranceCLIP 86490 Food NDc MoroccoCLIP 11262 Food 6a 1988 Morocco

L. ivanovii/ivanovii CLIP 74914 / ATCC19119 * 5CLIP 88111 Food 5 2001 FranceCLIP 86784 Food 5 FranceCLIP87478 / PAM55 5

L. ivanovii/londoniensis CLIP 6645 5 SwitzerlandCLIP 12065 Animal 5 Belgium

L. seeligeri CLIP 73021 / ATCC35967 * 1/2bCLIP 86579 1/2b

L. welshimeri CLIP 87073 Environmental 4c FranceCLIP 87973 6a France

L. grayi CLIP 73019 -L. grayi subsp. murrayi CLIP 12515 -

a CLIP, strain number from Listeria Culture Collection of the National Reference Center for Listeria, Institut Pasteur; ATCC, strain number from American TypeCulture Collection; T, numbers according to the strain set of the International WHO Multicenter Typing Study.

b *, reference strain for serotyping.c ND, not done.d UK, United Kingdom; USA, United States.



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ing possible phylogenetic relationships between the differentListeria species by using the neighbor-joining method. Impor-tant points of gene conservation within each species of thegenus Listeria and within distinct groups of L. monocytogenesstrains were identified. The analysis grouped all strains withoutexception according to their species. Thus, the Listeria arrayallows accurate species identification, although the probeswere defined from only two Listeria species. Each species wasdefined by a combination of genes that were specificallypresent or absent.

For the species L. monocytogenes, we identified 30 markergenes, including 18 that were present in all 93 L. monocyto-genes strains tested (Table 2, group I) and absent from all otherisolates of the remaining Listeria species and 12 that werepresent in all L. monocytogenes strains except the five serovar4a and 4c isolates tested (Table 2, group II). Because serovar4a and 4c strains are very rare and do not cause typical humanlisteriosis (35), thereafter these strains were not considered inthe analysis of species-specific marker genes, but they will bediscussed separately below. The 30 markers comprised thewell-known virulence genes (plcA, plcB, and actA), 7 surface

protein-coding genes (inlA, inlB, inlH, inlE, lmo0333, lmo0835,and lmo2821), 1 soluble internalin-coding gene (lmo0549), 3genes for transcriptional regulators, and 11 genes for proteinsof unknown function. For the species L. innocua, we identifiedfour markers (lin0739, lin0803, lin2741, and lin2918) that wereconsistently present in all L. innocua strains tested. However,29 of the 94 L. innocua genes spotted onto the membrane weredetected only in L. innocua isolates, suggesting them to bespecies specific.

Since the macroarray did not contain probes specific for theother Listeria species, no specific markers for these speciescould be defined. However, orthologs of about a third of theEGDe genes and about a quarter of the CLIP 80459 or L.innocua genes were identified in at least one of the L. ivanovii,L. seeligeri, or L. welshimeri strains. L. grayi was found to be themost distantly related species. From the 409 probes, only 12 (6from EGDe, 3 from L. innocua, and 3 from strain CLIP 80459)hybridized with DNA from L. grayi. These probes corre-sponded to genes present in all Listeria species, such aslmo1136, coding for an LPXTG protein, and genes coding forproteins of unknown function.

Subgrouping within the species L. monocytogenes. In addi-tion to the neighbor-joining method, hierarchical clustering(J-Express) was used for the identification of specific geneclusters. Analysis of the 93 L. monocytogenes strains definedthree lineages (I, II, and III) and distinguished two subdivi-sions within each lineage (Fig. 1). For each lineage and sub-group, specific markers were identified.

Nineteen genes were associated specifically with lineage I(Table 3, group A). Twelve of these genes clustered in tworegions (lmo0734-lmo0739 and lmo1968-lmo1974) coding forproteins putatively involved in sugar metabolism. Further-more, a two-component regulatory system (lmo1060 andlmo1061), an ABC transporter complex (lmo1062 andlmo1063), and a gene coding for a surface protein containingan LPXTG motif (lmo0171) were lineage I specific. Surpris-ingly, the bvr locus (bvrABC) (4) was present only in isolates oflineage I and in the two serovar 4c strains. Eight genes allowedfor the subdivision of lineage I. They were present in lineageI.2 (serovars 1/2c and 3c) but were generally absent from lin-eage I.1 (serovars 1/2a and 3a) (Table 3, group B).

Five of the 53 serovar 4b-specific genes were markers forlineage II (Table 3, group C). Two code for transcriptionalregulators, and three code for surface proteins containing anLPXTG anchor. Because serovar 4b strains are mainly respon-sible for human listeriosis, it is particular interesting to identifymarkers for serovar 4b strains or for the subgroups 4b, 4d, and4e (lineage II.1). One such specific marker was ORF0799,coding for an unknown protein. ORF2372 (encoding a putativeteichoic acid protein precursor C) and ORF2110 (encoding aputative secreted protein) were present only in serovar 4b andin two and four, respectively, of the six L. ivanovii strains.Furthermore, 35 of the 53 serovar 4b genes spotted on themembrane were conserved in all 4b strains, suggesting theirimplication in characteristic features of serovar 4b strains.

For lineage III strains (serovars 4a and 4c), no specific geneswere identified, as our macroarray did not contain represen-tative sequences of this lineage. However, lineage III was char-acterized by the absence of over 37% (96 genes) of the EGDegenes that were spotted on the membrane. Thirteen genes,

TABLE 2. L. monocytogenes-specific marker genes

Group Genename

No. of isolates withindicated genea

Functional categoryL. monocytogenes

serotype

4c(2 strains)

4a(3 strains)

I Lmo0082 2 3 UnknownLmo0083 2 3 RegulationplcA 2 3 VirulenceactA 2 3 VirulenceplcB 2 3 VirulenceLmo206 2 3 UnknowninlA 2 3 Cell surface proteinsinlB 2 3 Cell surface proteinsLmo0440 2 3 UnknownLmo0638 2 3 UnknownLmo0733 2 3 RegulationLmo0833 2 3 RegulationLmo1133 2 3 UnknownLmo1135 2 3 UnknownLmo2732 2 3 UnknownLmo2733 2 3 Transport and binding

proteins andlipoproteins

Lmo2734 2 3 Specific pathwaysLmo2736 2 3 Unknown

II Lmo0017 2 0 Cell wallLmo0094 0 0 UnknowninlH 2 0 Cell surface proteinsinlE 2 0 Cell surface proteinsLmo0333 2 0 Cell surface proteinsLmo0549 2 0 Soluble internalinsLmo0832 0 0 Transposon and ISLmo0834 2 0 UnknownLmo0835 0 0 Cell surface proteinsLmo1441 0 0 Cell wallLmo1451 0 0 UnknownLmo2821 2 0 Cell surface proteins

a For L. monocytogenes serotypes 1/2a, 1/2b, 1/2c, 3a, 3b, 3c, 7, 4b, 4d, and 4e,all 88 strains had all of the marker genes. For L. ivanovii, L. innocua, L.welshimeri, L. seeligeri, and L. grayi, none of the 20 strains had any of the markergenes.



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clustered in eight different chromosomal regions, were specif-ically absent from lineage III strains. They code for surfaceproteins (lmo1666 and lmo0835), the arginine metabolic path-way proteins (lmo0036-lmo0041), and proteins of unknownfunction. Strains of serovar 4a (lineage III.1) were distin-guished from those of serovar 4c (lineage III.2) by the lack ofan additional 20 genes, 7 of which code for cell surface proteins(inlC, inlEHG, lmo0333, lmo0549, and lmo2821). These geneswere also absent from all L. innocua strains tested.

Distribution of known virulence genes. The virulence genecluster of L. monocytogenes comprises prfA, plcA, hly, mpl,actA, and plcB. Since these genes are a prerequisite for thevirulence of L. monocytogenes, differences in virulence amongdifferent isolates could be due to the absence of one or moreof these genes. However, macroarray hybridization showedthat all 93 L. monocytogenes isolates contained this virulencegene cluster. The above-mentioned genes have been reportedalso to be present in L. ivanovii and L. seeligeri. We detectedhybridization signals for the hly, mpl, and prfA genes, whereasthe plcA, actA, and plcB genes were either absent or did notgive a signal due to a high divergence of the correspondinggene orthologs. Indeed, the sequence similarity of plcA, plcB,

and actA from different L. ivanovii and L. seeligeri strains com-pared to L. monocytogenes EGDe does not exceed 60%.

Several other genes of L. monocytogenes have been impli-cated in adhesion and internalization. Among those, the beststudied are inlA and inlB. Our analysis revealed the presence ofthese two genes in all L. monocytogenes strains tested, confirm-ing their species specificity. The uhpT gene (10) and the bshgene (12) were identified in all isolates of the three hemolyticListeria species (L. monocytogenes, L. ivanovii, and L. seeligeri).

High diversity level of surface proteins within the species L.monocytogenes. Fifty-five genes coding for putative surface pro-teins (http://genolist.pasteur.fr/ListiList/) belonging to thethree sequenced Listeria genomes were spotted onto the array.We identified two groups of genes. The first group comprises25 genes specific for the species L. monocytogenes, includinginlAB, the inlGHE cluster, inlF, and a number of surface pro-teins of unknown function (Table 4). Two of the genes forsurface proteins (lmo0171 and lmo2026) are lineage I specificand three (ORF2568, ORF2017a, and ORF0029) are lineage IIspecific. inlG seems to be specifically absent from all lineage IIand serovar 4a strains. None of the serovar 4b surface protein-coding genes was identified in L. monocytogenes serovar 1/2c

FIG. 1. Listeria genetic diversity. Red and black areas denote the presence and absence of genes, respectively. (A) Dendrogram showingestimates of genomic relationships of the 113 strains constructed by hierarchical cluster analysis with the program J-Express. Phylogenetic lineagesand subgroups are indicated. (B) Enlargements representing the groups of lineage-specific genes whose numbers are indicated to the right. I,lineage I (serovars 1/2a, 1/2c, 3a, and 3c); II, lineage II (serovars 4b, 4d, 4e, 1/2b, and 3b); III, lineage III (serovars 4a and 4c); I.1, serovars 1/2aand 3a; I.2, serovars 1/2c and 3c; II.1, serovars 4b, 4d, and 4e; II.2, serovars 1/2b and 3b.



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TABLE 3. L. monocytogenes lineage-specific marker genes

Group Genename

No. of isolates with indicated gene

Functional categoryLineage I Lineage II Lineage III

I.1 (1/2a, 3a)(27 strains)

I.2 (1/2c, 3c)(12 strains)

II.1 (4b, 4d, 4e)(27 strains)

II.2 (1/2b, 3b)(20 strains)

III.1 (4a)(3 strains)

III.2 (4c)(2 strains)

A Lmo0171 27 12 0 0 0 0 Cell surface proteinsLmo0172 27 12 0 0 0 0 Transposon and ISLmo0525 27 12 0 0 0 0 UnknownLmo0734 27 12 0 0 0 0 RegulationLmo0735 27 12 0 0 0 0 Specific pathwaysLmo0736 27 12 0 0 0 0 Specific pathwaysLmo0737 27 12 0 0 0 0 UnknownLmo0738 27 12 0 0 0 0 Transport and binding

proteins and lipoproteinsLmo0739 27 12 0 0 0 0 Specific pathwaysLmo1060 27 12 0 0 0 0 RegulationLmo1061 27 12 0 0 0 0 SensorsLmo1062 27 12 0 0 0 0 Transport and binding

proteins and lipoproteinsLmo1063 27 12 0 0 0 0 Transport and binding

proteins and lipoproteinsLmo1968 27 12 0 0 0 0 Metabolism of amino acidsLmo1969 27 12 0 0 0 0 Specific pathwaysLmo1970 27 12 0 0 0 0 Metabolism of lipidsLmo1971 27 12 0 0 0 0 Transport and binding

proteins and lipoproteinsLmo1973 27 12 0 0 0 0 Transport and binding

proteins and lipoproteinsLmo1974 27 12 0 0 0 0 RegulationbvrC 27 12 0 0 0 2 UnknownbvrB 27 12 0 0 0 2 Transport and binding

proteins and lipoproteins

B Lmo0151 3 12 0 0 0 0 UnknownLmo0466 2 12 0 0 0 0 UnknownLmo0467 2 12 0 0 0 0 UnknownLmo0469 2 12 0 0 0 0 UnknownLmo0470 2 12 0 0 0 0 DNA restrictions and

modificationsLmo0471 2 12 0 0 0 0 UnknownLmo1118 1 12 0 0 0 0 UnknownLmo1119 1 12 0 0 0 0 DNA restrictions and

modifications

C ORF2819 0 0 27 20 0 0 Unknown, similar tohypothetical transcriptionalregulator

ORF3840 0 0 27 20 0 0 Unknown, similar totranscriptional regulator

ORF2568 0 0 27 20 0 0 Unknown, similar to internalinproteins, putativepeptidoglycan-bound protein(LPXTG)



D ORF0799 0 0 27 0 0 0 UnknownORF2372 0 0 27 0 0 0 Unknown, similar to teichoic

acid protein precursor CORF2110 0 0 27 0 0 0 Unknown, putative secreted

protein



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and 3a strains. For L. innocua, we identified two specific sur-face protein-coding genes (lin0739 and lin0803). The secondgroup comprises surface protein-coding genes that are heter-ogeneously distributed among the different Listeria isolatesand species (Table 4).

For corroboration of the hybridization results, six of thesurface protein genes specific for L. monocytogenes (inlA, inlB,inlE, inlG, inlH, and inlF) and two of the surface protein genesfound within several or all Listeria species (lmo0550 andlmo1289) were amplified by PCR from one reference strain for

TABLE 4. Distribution of cell surface proteins

Gene

Presence of gene in indicated groupa

L. monocytogenesL.

innocua(8 strains)

L.ivanovii

(6 strains)

L.seeligeri

(2 strains)

L.welshimeri(2 strains)

L.grayi

(2 strains)

Lineage I Lineage II Lineage III

I.1 (1/2a, 3a)(27 strains)

I.2 (1/2c, 3c)(12 strains)

II.1 (4b, 4d, 4e)(27 strains)

II.2 (1/2b, 3b, 7)(20 strains)

III.1 (4c)(2 strains)

III.2 (4a)(3 strains)

inlA � � � � � � � � � � �inlB � � � � � � � � � � �Lmo2085 � � � � � � � � � � �Lmo1413 � � � � � � � � � � �Lmo0463 �* � � � � � � � � � �Lmo0460 �* � � � � � � � � � �Lmo0550 � � � � � � �* � � � �Lmo0160 � � �* � � � �* � � � �Lmo0333 � � � � � � � � � � �inlE � � � � � � � � � � �inlH � � � � � � � � � � �Lmo2821 � � � � � � � � � � �Lmo2027 �* � �* �* � � � � � � �inlC � � � � � � � � � � �Lmo0835 � � � � � � � � � � �Lmo1666 � � � � � � � � � � �inlF �* � �* �* � � � � � � �Lmo0320 �* � � �* � �* � � � � �Lmo0842 � � �* �* � �* �* �* � � �Lmo0801 �* � �* �* � �* �* � � � �inlG �* � �* � � � � � � � �Lmo1115 �* �* �* �* � �* �* �* � � �Lmo2576 � � � � � � � � � � �Lmo0171 � � � � � � � � � � �Lmo2026 �* � � � � � � � � � �Lmo2178 � � � � � � � �* � � �Lmo1799 � � � � � � �* � � � �Lmo1289 � � � � � � �* �* � � �Lmo1136 � � � � � � � � � � �Lmo0880 � � � � � � �* �* � � �Lmo0514 � � � � � � �* �* � � �Lmo0175 � � � � � � � � � � �Lmo0732 � � �* �* � � �* � � � �Lmo0610 � � � �* � �* � �* � � �Lin0739 � � � � � � � � � � �Lin0803 � � � � � � � � � � �Lin0559 �* � �* �* � � � � � � �Lin2724 � � � � � � � �* � � �Lin1204 � � � �* � � �* � � � �Lin0372 �* �* � � � � � � � � �Lin0415 �* �* �* �* � � � � � � �Lin0665 �* �* � � � � � � � � �Lin0740 �* �* �* �* � �* � � � � �Lin0661 �* �* �* �* � � � � � � �Lin1328 �* �* �* �* � �* � �* � � �ORF2568 � � � � � � � � � � �ORF1761 � � � � � � � � � � �ORF0029 � � � �* � � � � � � �ORF1590 � � � �* � � � � � � �ORF2541 �* � � � � � � � � � �ORF2013 �* � � �* � � � � � � �ORF2017 �* � � � � � �* � � � �

a � or � indicates gene presence or absence in all strains of the corresponding serovar; �* or �* indicates gene presence or absence in at least two-thirds of thestrains of the corresponding serovar; � was used when the gene was present in one of two strains studied. Lmo and Lin numbers correspond to gene names on theListiList web server (http://genolist.pasteur.fr/ListiList/). Serovar and total number of strains studied for each lineage are indicated between brackets. Bold data indicatespecies- or lineage-specific genes.



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each serovar and one for each species. The PCR amplificationsconfirmed the array results (data not shown).

Carbohydrate metabolism and PTSs. The distribution pat-terns of genes for 12 PTS permeases and of 14 genes encodingproteins predicted to be implicated in sugar metabolism anddegradation were similar to that of surface protein-codinggenes; all of these genes were highly conserved in lineage Istrains and most were lacking in the other Listeria species.Except for two PTS genes (lmo2733 and lmo2782) and threecarbohydrate metabolism genes (lmo2143, lmo2735, andlmo2781), all other genes in this group were missing from theL. monocytogenes serovar 4a strains.

Cell wall proteins—two subdivisions within teichoic acidbiosynthesis genes. Despite the fact that the majority of genesgrouped the 93 L. monocytogenes strains according to previ-ously defined lineages which correlate mainly with flagellarantigen combinations (serovars), we identified 13 genes impli-cated in cell wall biosynthesis that divide the L. monocytogenesstrains into two groups according to their somatic antigens(serogroup 4 and serogroups 1/2, 3, and 7). These genes codefor teichoic acid biosynthesis proteins and were detectedwithin the strains of serogroups 1/2, 3, and 7, but were absentfrom strains of serogroup 4. This finding is in agreement withprevious studies that have identified two distinct structuraltypes of teichoic acid within L. monocytogenes, for which thefirst type was found in strains of serogroups 1/2, 3, and 7 andthe second was found in strains of serogroup 4 (15). Thissuggests that these genes may be implicated in the synthesis ofthe specific teichoic acid type. Nine of these genes, locatedwithin a 19-kb region of the L. monocytogenes chromosome(lmo1076-lmo1077, lmo1080 to lmo1084, lmo1088, andlmo1091), were also shared with the L. seeligeri strains, whichare of serogroup 1/2, suggesting that L. seeligeri has a teichoicacid type similar to that of L. monocytogenes serogroup 1/2.

Similarly, one (lin1073) of two L. innocua genes that areimplicated in teichoic acid biosynthesis was uniquely sharedwith the L. monocytogenes strains of serogroup 4 and L. welshi-meri. This suggests an implication in the specificity of the cellwall type of serogroup 4 strains of L. monocytogenes, which ismore closely related to that of serogroup 6 of L. innocua thanto that of L. monocytogenes serogroup 1/2 strains (15).

Variable genomic regions and analysis of junction se-quences. Several L. monocytogenes EGDe gene clusters weremissing only from L. monocytogenes serovar 4a strains but werepresent in all other L. monocytogenes strains. Two regions wereabsent from L. monocytogenes serovar 4a and L. innocua butwere present in L. ivanovii, L. seeligeri, and L. welshimeri. Theyseemed good candidates for use as evolutionary markers. Tofurther analyze them, we sequenced the junction regions of theputative deletion sites from six isolates of L. innocua and L.monocytogenes serovar 4a.

Analysis of the region lmo2671-lmo2672 revealed the exis-tence of three deletion events, two of which were located in thecoding sequence of lmo2672, resulting in the deletion of twointernal fragments, of 621 and 35 bp. The third deletion was355 bp and was located downstream of the 5� end of the codingsequence of lmo2671. All three junction sequences were iden-tical, containing either an insertion of TTGCATT, an insertionof A, or no insertion (see supplemental material at http://www.pasteur.fr/recherche/unites/gmp/sitegmp/biodiversitylist

.html). From the analysis of the junction sequence of the re-gion of lmo2771 to lmo2773, we obtained the same result for allthe strains studied, with identical junction sites sequenced 38bp downstream of the 3� end of lmo2770. The junction site hadan insertion of the sequence TTATTTAAG replacing geneslmo2771 to lmo2773 (http://www.pasteur.fr/recherche/unites/gmp/sitegmp/biodiversitylist.html).

The third region investigated (lmo1030 to lmo1036) wasabsent from L. innocua and L. monocytogenes serovar 4astrains and was present in L. ivanovii. The analysis of thejunction region identified a minor sequence variation (inser-tion of TCA in L. innocua and of AT in L. monocytogenesserovar 4a) at the deletion site (http://www.pasteur.fr/recherche/unites/gmp/sitegmp/biodiversitylist.html).

Furthermore, the inlGHE cluster, which is missing from L.monocytogenes 4a and all other Listeria sp., was analyzed. Weidentified again an identical sequence for the five strains se-quenced, suggesting that a single deletion event had occurredin a common ancestor of L. monocytogenes serovar 4a and L.innocua (http://www.pasteur.fr/recherche/unites/gmp/sitegmp/biodiversitylist.html).

DISCUSSION

Overall genetic diversity. One of the first criteria by whichthe L. monocytogenes species could be subdivided was thevarying antigenic properties of diverse subpopulations. Ascheme developed by Seeliger and Hohne described 13 sero-vars (32). Based on somatic antigens, L. monocytogenes isolateswere divided mainly into serogroups 1/2 and 4, and based onflagellar antigen combinations, each of these serogroups wassubdivided into serovars 1/2a, -b, and -c and 4b or some otherless common serovars (32). Today, this scheme is still in use,and numerous studies have identified correlations betweencertain phenotypic or genetic features and specific serovars (3,5, 20, 28). The combined analysis of the genome sequences ofL. monocytogenes serovar 1/2a and L. innocua (19), the partialgenome sequence of L. monocytogenes serovar 4b, and themacroarray hybridization of 113 Listeria DNAs undertaken forthis study substantiated this classification at the genomic level.

One of the most striking observations of this study was themagnitude of divergence that exists within the species L. mono-cytogenes. We found that the genetic divergence between lin-eage I and lineage II of L. monocytogenes was nearly equallyimportant (about 8%) as the interspecies difference betweenthe sequenced L. monocytogenes EGDe serovar 1/2a strain andL. innocua (10%). These results are in line with a previousreport (22) which identified 39 specific gene fragments for theepidemic L. monocytogenes strain F.4565 compared to L. mono-cytogenes EGDe by use of a subtractive hybridization method.This is of particular importance since strains of serovar 4bmainly represent epidemic L. monocytogenes strains and areisolated from severe invasive human cases more frequentlythan strains of other serovars, such as serovar 1/2a. Apart fromthe important divergence between the two lineages of L. mono-cytogenes, the macroarray results identified remarkable geno-mic conservation within the major lineages and subgroups (Fig.1) but variations between the different subgroups. These re-sults seem to mirror the evolution within the genus Listeria.



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Specific features. An important gene family in L. monocy-togenes encodes surface proteins (8, 19). The macroarray hy-bridization and the analysis of the partial L. monocytogenesserovar 4b sequence indicated that a group of surface protein-encoding genes which includes all known internalin genes(inlA, inlB, inlG, inlH, inlE, inlC, and inlF) is highly specific forthe species L. monocytogenes. Furthermore, each subgroup ofL. monocytogenes is characterized by a specific set of surfaceproteins. Finally, a third group of surface protein-coding genesis distributed quite heterogeneously among the different Lis-teria species. Interestingly, in the rarely isolated L. monocyto-genes serovar 4a strains, which are mostly animal pathogens, 13of the 25 L. monocytogenes-specific surface proteins, includingall internalins except inlAB, were missing. The lack of theseproteins may be related to the lower disease potential of thesestrains for humans. The fact that different subgroups of L.monocytogenes strains contain different sets of surface proteinsmay also reflect their different potentials to cause disease or tomultiply in different niches. The elucidation of the functions ofthe different surface proteins and the putative strain-specificcharacteristics that they confer will be one of the challengingquestions of the future and may give additional insights intoour understanding of the tropism of L. monocytogenes towarddifferent cell types.

Proteins implicated in sugar transport and metabolism, inparticular PTSs, form another important gene family in Listeriaspecies (19). An analysis of the distribution of these genesagain underscored the genetic divergence of the different sub-groups of L. monocytogenes, as each lineage was characterizedby a specific set of PTS permeases. Most PTS genes present inserovar 1/2a, 1/2c, 3a, and 3c strains were missing from theserovar 4b and 1/2b strains. The finding that the bvrABC locus,a -glucoside-specific PTS system previously described as be-

ing implicated in virulence gene expression (4), was absentfrom all L. monocytogenes strains of lineage II (serovars 4b, 4d,4e, 7, 1/2b, and 3b) was surprising. Because regulation of thePrfA regulon by -glucosides also takes place in lineage IIstrains, it can be assumed that another PTS system fulfills thefunctions of the bvrABC proteins. The finding that one of thePTS permeases identified in the sequence of the L. monocyto-genes serovar 4b strain was present in all strains in which thebvrABC locus was lacking might be consistent with this hypoth-esis.

Marker genes for L. monocytogenes and each lineage. Weidentified 30 markers for the species L. monocytogenes (Table2), as well as markers of each subpopulation within the speciesL. monocytogenes (Table 3). One of our major questions waswhether the pronounced differences in virulence among differ-ent subgroups of strains can be explained by different genecontents. Scanning of our results for the presence or absence ofknown virulence genes (inlAB, prfA, plcA, hly, mpl, actA, plcB,uhpT, and bsh) revealed that they are present in all L. mono-cytogenes strains tested. However, an analysis of the correlationbetween epidemiological data, the origins of the strains, andthe genomic profiles clustered the L. monocytogenes serovar 4bstrains isolated from epidemics and from incriminated foodsources in a group separate from the other environmental,food, and animal isolates (http://www.pasteur.fr/recherche/unites/gmp/sitegmp/biodiversitylist.html). Thus, disease-relat-ed L. monocytogenes isolates seem to be characterized by aparticular combination of genes, and the Listeria array, com-bined with the knowledge of the marker genes identified in thisstudy, should prove to be a powerful tool for identifying thesestrains (M. Doumith, C. Buchrieser, P. Glaser, C. Jacquet, andP. Martin, unpublished data). The oligonucleotides used toamplify marker genes are available online as supplemental

FIG. 2. Evolutionary scheme of the different lineages and serovars of L. monocytogenes. The scheme is derived from a hypothesis based on thepresence and absence of genes and their correlation with antigenic characteristics.



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material (http://www.pasteur.fr/recherche/unites/gmp/sitegmp/biodiversitylist.html).

Evolutionary genomics of Listeria. As shown in Fig. 1, thecombined use of bioinformatics and macroarray results for 113Listeria strains generated a large data set, from which a de-tailed analysis allows us to group strains according to sharedgenetic profiles. In addition to conclusions concerning genet-ics, epidemiology, and the virulence of Listeria strains, thesedata also allow us to hypothesize how the different Listeriaspecies and phylogenetic lineages may have evolved. Severalstudies, using analysis of 16S and 23S rRNA (11, 31), PCR-based DNA fingerprinting techniques (34), or virulence locusand genome comparisons (9, 19, 34), indicated a phylogeneti-cally close relationship between L. monocytogenes and L. in-nocua and suggested that L. innocua lost the virulence locus bydeletion. Most interestingly, we identified several other regionsmissing from L. monocytogenes serovar 4a strains which werealso missing from L. innocua, such as the inlGHE gene cluster.Sequence analysis of the different junction regions identifiedidentical sequences among L. monocytogenes serovar 4a andthe L. innocua strains, suggesting single deletion events. Thepresence of these genes in the other Listeria species suggeststhat they were part of the genome of a common ancestor andthat L. innocua evolved by successive gene loss from an ances-tor of L. monocytogenes serogroup 4 strains. This hypothesis isalso substantiated by the similar teichoic acid structures of L.monocytogenes serogroup 4 and L. innocua strains (15) and bythe structural and functional similarity of the cell wall anchorof the autolysin Ami of L. monocytogenes serogroup 4 and L.innocua, but divergence between Ami of L. monocytogenesserovar 1/2 and that of serovar 4 (E. Milohanic, R. Jonquieres,P. Glaser, P. Dehoux, C. Jacquet, P. Berche, P. Cossart, J.-L.Gaillard, submitted for publication). Further evidence for thisclose relationship also comes from the flagellar antigen struc-tures of L. monocytogenes serogroup 4 and L. innocua, whichare the same for these two groups but are different from thatof serovar 1/2a and 1/2c strains (32). Based on our analysis andthe literature, we suggest an alternative model of evolutionwithin the L. monocytogenes-L. innocua branch. The separa-tion into phylogenetic lineages is based on the divergence ofserovar 1/2c and serovar 1/2b strains from a common ancestor(Fig. 2). Later in its evolution, the serovar 1/2b branch gainedgenes, such as gtcA, which conferred serogroup-specific ex-pression of TA-associated serotype-specific antigens (29)and evolved into serogroup 4 and later on into the speciesL. innocua, mainly by successive gene loss.

Conclusions. Taken together, the results of DNA-DNA hy-bridization of a specific Listeria array containing genes of threedifferent Listeria isolates showed that L. monocytogenes strainsdiffer substantially in gene content. These differences are mostpronounced in surface proteins and proteins for sugar metab-olism, which are most likely to confer traits that provide selec-tive advantages in the environment and the infected host. Ourresults further provide an explanation for why previous studieshave found an association between various characteristics of L.monocytogenes and serovars. We showed that this association isdue to evolutionary differentiation. To date, the microbiolog-ical surveillance of listeriosis, a disease that causes the death ofat least 400 to 500 persons per year in Europe and NorthAmerica, is based on subtyping by serotyping and pulsed-field

gel electrophoresis (21). The precise characterization of L.monocytogenes is essential for following long-term trends insporadic cases as well as for the detection of clusters of casesand epidemics and the identification of their common source.As such, the selective markers for the different subpopulationsare an essential contribution for the construction of rapid,accurate identification and subtyping methods, which shouldbe powerful tools applicable in health institutions and the foodindustry. Finally, the identification of genes that are consis-tently absent or present in epidemic-associated L. monocyto-genes strains now opens the way for mutational and functionalanalysis of these genes in order to decipher the molecular basisfor the increased pathogenic potential of certain L. monocyto-genes strains.

ACKNOWLEDGMENTS

This work received financial support from the European Commis-sion contract REALIS (QLG2-CT-1999-00932) and the Pasteur Insti-tut (PTR 6 and GPH9). M. Doumith holds a postdoctoral positionfrom the Pasteur Institut (PTR 6).

We thank SOREDAB for providing L. monocytogenes strains,Rachel Purcell and Elisabeth Couve for technical assistance, ClaudeParsot for critical reading of the manuscript, and Roland Brosch forfruitful discussions and ideas. P. Cossart is an international scholarfrom the Howards Hughes Medical Institute.

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