INFECTION AND IMMUNITY,0019-9567/99/$04.0010

Nov. 1999, p. 6119–6129 Vol. 67, No. 11

Copyright © 1999, American Society for Microbiology. All Rights Reserved.

Identification of Regions of the Chromosome of Neisseria meningitidisand Neisseria gonorrhoeae Which Are Specific

to the Pathogenic Neisseria SpeciesAGNES PERRIN, XAVIER NASSIF,* AND COLIN TINSLEY

Laboratoire de Microbiologie, INSERM U411, Faculte de MedecineNecker-Enfants Malades, 75015 Paris, France

Received 7 May 1999/Returned for modification 21 June 1999/Accepted 26 July 1999

Neisseria meningitidis and Neisseria gonorrhoeae give rise to dramatically different diseases. Their interactionswith the host, however, do share common characteristics: they are both human pathogens which do not survivein the environment and which colonize and invade mucosa at their port of entry. It is therefore likely that theyhave common properties that might not be found in nonpathogenic bacteria belonging to the same geneticallyrelated group, such as Neisseria lactamica. Their common properties may be determined by chromosomal re-gions found only in the pathogenic Neisseria species. To address this issue, we used a previously describedtechnique (C. R. Tinsley and X. Nassif, Proc. Natl. Acad. Sci. USA 93:11109–11114, 1996) to identify sequencesof DNA specific for pathogenic neisseriae and not found in N. lactamica. Sequences present in N. lactamica werephysically subtracted from the N. meningitidis Z2491 sequence and also from the N. gonorrhoeae FA1090 se-quence. The clones obtained from each subtraction were tested by Southern blotting for their reactivity with thethree species, and only those which reacted with both N. meningitidis and N. gonorrhoeae (i.e., not specific to ei-ther one of the pathogens) were further investigated. In a first step, these clones were mapped onto the chromo-somes of both N. meningitidis and N. gonorrhoeae. The majority of the clones were arranged in clusters extend-ing up to 10 kb, suggesting the presence of chromosomal regions common to N. meningitidis and N. gonorrhoeaewhich distinguish these pathogens from the commensal N. lactamica. The sequences surrounding these cloneswere determined from the N. meningitidis genome-sequencing project. Several clones corresponded to previ-ously described factors required for colonization and survival at the port of entry, such as immunoglobulin Aprotease and PilC. Others were homologous to virulence-associated proteins in other bacteria, demonstrat-ing that the subtractive clones are capable of pinpointing chromosomal regions shared by N. meningitidis andN. gonorrhoeae which are involved in common aspects of the host interaction of both pathogens.

Neisseria meningitidis and Neisseria gonorrhoeae are twohuman pathogens which belong to the same genospecies.Furthermore, phylogenetic analyses by rRNA similarities andDNA-DNA hybridizations have placed N. meningitidis, N. gon-orrhoeae, N. lactamica, and N. cinerea in a subgroup with par-ticularly close interspecies relatedness (19, 27, 39). Althoughthese bacteria are closely related, they express very differentpathogenicities. N. lactamica and N. cinerea are nonpatho-genic. N. meningitidis colonizes the nasopharynx, from where itmay spread into the bloodstream before crossing the blood-brain barrier to induce meningitis. N. gonorrhoeae colonizesand invades the epithelium of the genitourinary tract and maycause a localized inflammatory process or an ascending infec-tion leading to salpingitis. However, even though N. meningi-tidis and N. gonorrhoeae give rise to two very different diseases,they both have to colonize and cross an epithelium at their portof entry. This is consistent with the fact that in addition tohaving specific virulence factors, they have common virulenceattributes such as pili, immunoglobulin A (IgA) proteases, andclass 5 outer membrane proteins. However other as yet un-identified proteins, some of which are specific for the patho-genic Neisseria species and are not found in N. lactamica, aremost probably involved in this common step of interaction ofthese bacterial pathogens with their host.

While differences in pathogenic potential may theoretically

result from differential expression or subtly differing proteins,the situation is more generally found to involve the possessionof pathogen-specific sequences. Attributes of bacterial viru-lence are often grouped in islands and frequently are passedhorizontally between more or less closely related species (22).Representational difference analysis (33, 44) provides a quickmeans of cloning DNA corresponding to such species-specificsequences, by direct physical subtraction of the chromosomalDNA of a closely related, avirulent strain from the chromo-somal DNA of the pathogen. Thus large islands of DNA whichmay encode N. meningitidis-specific virulence factors which arenot present in N. gonorrhoeae have recently been identified. Toidentify the chromosomal regions that are common to patho-genic Neisseria species and are responsible for the colonizationand survival at the port of entry, we first subtracted from N.meningitidis those sequences which were also present in thecommensal N. lactamica and then performed a similar exper-iment subtracting the N. lactamica sequences from the chro-mosome of N. gonorrhoeae. The results of these experimentsconfirmed that both pathogens have common sequences whichare absent from the nonpathogenic N. lactamica and identifyputative virulence factors involved in survival and dissemina-tion from the port of entry.

MATERIALS AND METHODS

Strains, plasmids, and growth conditions. N. meningitidis Z2491 and N. gon-orrhoeae FA1090 were chosen as reference pathogenic Neisseria strains; both arein the process of being sequenced, and both also have many of their importantgenetic markers positioned on published macrorestriction maps (10, 11). Twostrains of N. lactamica, 8064 and 9764, from this laboratory were used to provide

* Corresponding author. Mailing address: INSERM U411, 156 Ruede Vaugirard, 75015 Paris, France. Phone: 33 140615678. Fax: 33140615592. E-mail: nassif@necker.fr.

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DNA for subtraction. Other strains came from the collection of X. Nassif.Neisseria strains were grown on GCB (Difco) agar plates, containing the Kelloggsupplements and ferric nitrate (26), for 14 to 16 h at 37°C in a humid atmospherecontaining 5% CO2.

Molecular genetic techniques. Routine molecular biological techniques werecarried out as recommended (3, 41). DNA sequences were determined by usingan ABI-Prism 370 automated sequencer with the Big Dye primer-sequencing kit.Southern blotting was performed as previously described (7, 44) but omitting thebovine serum albumin from the hybridization buffer. DNA fragments were la-

belled for Southern hybridizations by random-primed incorporation of [a-32P]dCTP. Chromosomal DNA extraction was performed on cells grown in broth orscraped from agar plates. Bacteria from one 7-cm plate or from 10 ml of brothwere suspended in 1 ml of 10 mM Tris-HCl (pH 8.0)–10 mM EDTA–100 mMNaCl containing 2 mg of RNase A. After addition of 50 ml of 20% sodiumdodecyl sulfate and incubation at 65°C for 30 min, the mixtures were digested for2 h at 37°C with proteinase K (100 mg). The solutions were then extracted oncewith an equal volume of phenol (pH 8), twice with phenol-choroform-isopenta-nol (25:24:1), and once with chloroform-isopentanol (24:1). The solution was

FIG. 1. Procedure for representational difference analysis. Sequences specific to the pathogen are represented in grey; those in common with N. lactamica arehatched. DNA from N. meningitidis or from N. gonorrhoeae was digested with frequently cutting restriction endonucleases and ligated to adapter pairs such that onlythe 59 end of each DNA stand was covalently linked to the 24-bp adapter. On denaturing, mixing, and reannealing, only the (pathogen-specific) sequences with anadapter covalently linked were able to rehybridize with their complementary sequence. The fragments of randomly sheared N. lactamica chromosome are generally over10 times as long as the restriction fragments from the pathogen and not only sequester all pathogen fragments having homologies in N. lactamica but also, in the largemajority of cases, prevent the polymerase from synthesizing the complement of the adapter during the filling-in procedure. Hence, these common fragments areeffectively prevented from being amplified, and only the pathogen-specific fragments possessing an adapter at each end can be exponentially amplified.

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overlaid with an equal volume of ethanol and cooled to 0°C, and the DNA wasspooled from the interface by mixing with a glass Pasteur pipette. The fibrousDNA was washed in 70% ethanol, partially dried, and then redissolved in TEbuffer (10 mM Tris-HCl [pH 8], 1 mM EDTA).

Chromosomal DNA was quantified by UV spectrophotometry. For quantifi-cation of fragmented DNA, preparations were diluted into TE buffer containing1 mg of ethidium bromide per ml and appropriate dilutions were performed toproduce 20-ml samples containing 1, 1/3, 1/10, 1/30, and 1/100 ml of the DNApreparation. In parallel, solutions of 200, 100, 50, 20, 10, 5, and 0 ng of standardDNA per 20 ml (HindIII digest of lambda phage) were prepared in the samesolution. Drops were placed on a sheet of polyethylene film, illuminated with UVlight, and photographed. The concentration of the DNA preparation was mea-sured against the scale of luminosities of the lambda DNA standards.

Representational difference analysis. Clones of DNA fragments present in thegenome of N. meningitidis and/or that of N. gonorrhoeae but absent from N.lactamica were prepared essentially as described previously (44) (Fig. 1). Sixbanks were created, three for N. gonorrhoeae and three for N. meningitidis.Briefly, 20 mg of DNA from N. gonorrhoeae or N. meningitidis was cleaved withMboI, MspI, or Tsp509I, precipitated with ethanol-sodium acetate, and ligatedwith 5 nmol of the appropriate oligonucleotide adapter pair (RBam12 andRBam24, RCla12 and RCla24, or REco12 and REco24 [Table 1]) for 18 h at11°C. The mixture was gel purified on 2% low-melting-point agarose (takingfragments above 200 bp) to remove unincorporated primers, phenol purified,precipitated, and redissolved in TE buffer. This procedure results in DNA frag-ments whose two 59 ends are covalently linked to the 24-base adapter. To preparethe subtracting DNA, chromosomes of two strains of N. lactamica were shearedby repeated passage through a hypodermic needle to give fragments rangingfrom about 3 to 10 kb. The DNA was repurified by phenol extraction, precipi-tated, and redissolved in TE buffer. Equal quantities of the two were mixed tomake the subtracting DNA.

The first subtractive hybridization was performed with 40 mg of N. lactamicasubtracting DNA and 200 ng (MboI or MspI digested) or 400 ng (Tsp509Idigested) of R-adapter-linked pathogen DNA fragments. The DNA was mixed,ethanol precipitated, and redissolved in 8ml of EE buffer [10 mM N-(2-hydroxy-ethyl)piperazine-N9-(3-propanesulfonic acid), 1 mM EDTA (pH 8.0)]. The liquidwas overlaid with 30 ml of mineral oil, denatured at 100°C for 2 min, and thenplaced at 55°C. After the addition of 2 ml of 5 M NaCl, the mixture was left tohybridize at 55°C for 48 h.

The reaction mixture was then diluted 10-fold with preheated EE buffer-NaCland immediately placed on ice. A portion of the subtraction mixture (10 ml) wasdiluted into 400 ml of PCR mix (10 mM Tris-HCl [pH 9.0], 50 mM KCl, 1.5 mMMgCl2, 0.1% Triton X-100, 0.125 mM each deoxynucleoside triphosphate, 100 Uof Taq polymerase per ml) to fill in the ends corresponding to the 24-baseadapter. The reaction mixtures were diluted a further 10-fold, and PCR ampli-fications were performed on 400 ml of the dilutions. After denaturation for 5 minat 94°C and addition of the appropriate 24-base oligonucleotide, the mixtureswere amplified by PCR (30 cycles of 1 min at 70°C, 3 min at 72°C, and 1 min at94°C, followed by 1 cycle of 1 min at 94°C and 10 min at 72°C [Perkin-ElmerGeneAmp 9600 thermal cycler]). The amplified meningococcal DNA was sepa-rated by agarose gel electrophoresis from the primers and high-molecular-weightsubtracting DNA.

The first adapters (R) were cleaved from the PCR products with the appro-priate restriction enzymes, and the second-round adapters were ligated (2 mg ofsubtractive fragments and 2 nmol of adapters [JBam12 and JBam24, JCla12 andJCla24, or JEco12 and JEco24; Table 1] in a volume of 50 ml). The ligatedfragments were gel purified and phenol extracted.

The second-round subtractive hybridization was performed with 25 ng of DNAfrom the pathogens (first-round products, cleaved and religated to the J adapt-ers) and 40 mg of DNA from N. lactamica. Fragments amplified from the secondround were cleaved with the appropriate enzyme, gel purified, and cloned intopBluescript (Stratagene) cleaved with the appropriate enzyme (BamHI for theMboI fragments, ClaI for the MspI fragments, and EcoRI for the Tsp509I frag-ments). The recombinant plasmids were maintained in Escherichia coli DH5a.Subsequent manipulations all used the PCR product corresponding to the in-serted DNA, amplified between primers flanking the polycloning site of pBlue-script.

Cloned DNA fragments were tested first by Southern blotting for their reac-tivity with N. meningitidis and/or N. gonorrhoeae and absence of reactivity witheither of the strains of N. lactamica; this also permitted the elimination ofobvious duplicate clones. Sequences were compared against other subtractiveclones and against public-domain databases by using the BLAST algorithm(National Center for Biotechnology Information, Bethesda, Md.) (2). The loca-tions of the genes on the published macrorestriction maps of N. meningitidisZ2491 and of N. gonorrhoeae FA1090 were determined as described previously(44). The sequences were also used to extract the sequence of the chromosomalDNA surrounding the subtractive clones from the databases of the Z2491 ge-nome sequencing project (46a) and FA1090 (44a). From this data, open readingframes (ORFs) were predicted by using the programs MacVector (Oxford Mo-lecular Group, Oxford, United Kingdom) and CodonUse (Conrad Halling, Mon-santo Corp.). These were also compared to sequences in public-domain data-bases by using BLAST.

RESULTS AND DISCUSSION

Production of libraries of clones specific to the pathogenicspecies. In a first experiment, three banks of N. meningitidis-specific clones were prepared by subtracting the chromosomeof N. lactamica from meningococcal chromosomal DNA,cleaved with three restriction enzymes. Meningococcal DNAfrom strain Z2491 was cleaved with MboI (GATC, compat-ible with BamHI), MspI (CCGG, compatible with ClaI) andTsp509I (AATT, compatible with EcoRI) and subjected to tworounds of subtraction by using DNA mixed from two strains ofN. lactamica. The use of two strains of N. lactamica ensuredthat clones isolated were not taken as being N. meningitidisspecific due to their absence from one particular strain ofN. lactamica. The N. meningitidis-specific fragments werecloned into pBluescript. PCR products corresponding to theinserts, were radiolabelled and used in an initial screening bySouthern blotting against chromosomal DNA from the menin-gococcus Z2491, the gonococcus FA1090, and the two strainsof N. lactamica used for subtraction, each cleaved with ClaI. Of237 clones initially isolated, 41 showed a double specificity forN. gonorrhoeae and N. meningitidis and no reactivity withN. lactamica. These were chosen for further study.

Pathogen-specific DNA sequences should be equally attain-able by the subtraction of N. lactamica DNA from gonococcalDNA. To test the completeness of the bank obtained by sub-traction of N. lactamica from N. meningitidis and to increasethe representativity of the subtractive clones, another threebanks were produced as above, but this time subtracting theDNA of the two strains of N. lactamica from N. gonorrhoeaeFA1090 DNA. Again, 20 of 83 clones showing reactivity withboth N. meningitidis and N. gonorrhoeae were kept.

Clones derived from the subtraction involving meningococ-cal MboI fragments were designated Bm001, Bm002, etc.;those involving the MspI fragments were named Cm001,etc., and those involving the Tsp509I fragments were namedEm001, etc.; the letters B, C, and E refer to the correspondingBamHI, ClaI, and EcoRI sites used for their cloning, respec-tively, and the letter m refers to the originating species N.meningitidis. Clones derived from N. gonorrhoeae were desig-nated Bg001, Cg001, Eg001, etc. The positions of the 61 cloneswhich were retained were determined in relation to the pub-lished macrorestriction maps of N. gonorrhoeae FA1090 (10)and N. meningitidis Z2491 (11) by probing Southern blots ofchromosomal DNA cleaved with infrequently cutting restric-tion enzymes and subsequent comparison of the reactive bandswith their published maps. In addition, the subtractive cloneswere sequenced, and, following BLAST searches of the par-tially sequenced chromosomes of N. gonorrhoeae FA1090 andN. meningitidis Z2491, the corresponding contigs were extract-

TABLE 1. Oligonucleotides used in this study

Name Sequence

RBam12 ..............................................GATCCTCGGTGARBam24 ..............................................AGCACTCTCCAGCCTCTCACCGAGJBam12................................................GATCCGTTCATGJBam24................................................ACCGACGTCGACTATCCATGAACGRCla10 ................................................CGGTCGGTGARCla24 ................................................AGCACTCTCCAGCCTCTCACCGACJCla10..................................................CGGGTTCATGJCla24..................................................ACCGACGTCGACTATCCATGAACCREco12................................................AATTCTCGGTGAREco24................................................AGCACTCTCCAGCCTCTCACCGAGJEco12.................................................AATTCGTTCATGJEco24.................................................ACCGACGTCGACTATCCATGAACG

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FIG. 2. Position of the pathogen-specific clones on the chromosomal map of N. meningitidis Z2491. Clones were mapped by Southern blotting and by comparison with thepublished partial genome sequence. Those derived from the N. gonorrhoeae-minus-N. lactamica subtraction are shown on the left (G-L libraries), and those from the N. men-ingitidis-minus-N. lactamica subtraction are shown on the right (M-L libraries). Clones from the two libraries derived from the same pathogen-specific region are marked withthe same shading. Some clones were present in multiple copies (generally insertion sequences), and these are mapped only where they coincide with another identified locus.

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FIG. 3. Comparison of the positions of the pathogen-specific clones on the chromosomes of N. gonorrhoeae FA1090 and N. meningitidis Z2491. The relativepositions follow the lines of dislocation between the two chromosomes as previously described (11), with certain exceptions.

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ed from the genome sequence data of N. meningitidis Z2491and analyzed to permit a tentative mapping of the subtractiveclones on a smaller scale, relative to one another and to otherdefined genes. Figures 2 and 3 show the positions of the cloneson the chromosome of N. meningitidis Z2491 and N. gonor-rhoeae FA1090. In addition, in some cases the sequences sur-rounding these contigs were annotated and are shown in Fig. 4.

It is noticeable that these clones are not scattered through-out the chromosomes of N. gonorrhoeae and N. meningitidis butare clustered only in some regions (Fig. 2). Furthermore, com-paring the maps of N. gonorrhoeae and N. meningitidis (Fig. 3),it is seen that in general the relative positions of the cloneson the two chromosomes follow the lines linking the previ-ously mapped markers (11), whose positions have presumablychanged following the chromosomal rearrangements whichhave occurred since the divergence of these two species. Ofparticular interest is the group of clones Em085, Cm024, andEm029, mapping near argJ and regF at 0.45 Mb and separat-ed one from another by about 25 kb in N. meningitidis. In thegonococcus, clone Em029 and regF map around 0.45 Mbwhereas the other clones map at about 1.65 Mb, indicatingthat a large-scale genetic rearrangement has occurred to sep-arate these genes in N. gonorrhoeae or to bring them togetherin N. meningitidis.

It should be pointed out that the clones cluster in the sameregions of the chromosome, whether they have been obtainedby subtracting the N. lactamica genome from either N. menin-gitidis or N. gonorrhoeae, thus suggesting the completeness ofthe bank and validating the hypothesis that these clones des-ignate regions which are specific for pathogenic neisseriae.

Functional classification of ORFs corresponding to the N.meningitidis- and N. gonorrhoeae-specific clones. To get someinsight into the function of these regions specific for patho-genic Neisseria species, the homologies at the protein levels ofthe ORFs corresponding to the resulting subtractive cloneswere noted after a BLAST search of the gene and proteindatabases. The results are summarized in Table 2, where thevarious homologies are divided into groups based on the func-tionality of the homologous proteins.

(i) Sequences having homologies to known virulence factors.A few clones were located in sequences containing geneswhose function has been established as playing a role in thecolonization and survival of the port of entry, such as the IgAprotease Iga (23, 29), and the pilus-associated adhesion mol-ecule PilC (40). The fact that these genes are N. meningitidisand N. gonorrhoeae restricted confirms the original hypothesisthat these regions may encode virulence factors which areimportant in the first step of pathogenesis, i.e., the colonizationof the epithelium and survival at the port of entry. Further-more, it suggests that the other, as yet uninvestigated potentialvirulence factors (Table 2) which have been identified on thebasis of homology could be involved in common steps of thedisease.

(ii) Sequences related to DNA modifications and rearrange-ments, insertion sequences, and viral recombinases. The se-quences related to DNA modifications and rearrangements,insertion sequences, and viral recombinases include methyl-transferases DcmH and HgiDIIM, transposases from IS1106of N. meningitidis, IS18 of Acinetobacter, and IS150-like ofN. gonorrhoeae, Synechocystis, and Aeromonas salmonicida,the Correia sequences from N. meningitidis and N. gonor-rhoeae, and proteins from phages Cf1c of Xanthomonascampestris and CTX of Vibrio cholerae. Hence a relatively largenumber of sequences identified were related to DNA modifi-cations, insertion sequences or transposons, and phages. In theabsence of further evidence, they may be taken to be clonal in

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TABLE 2. Homologies of the pathogen-specific clones

Clone Length(kb) Positiona Homology of ORFb BLASTX

significanceInter- or

intragenicAccession

no.Refer-ence

Virulence-associatedgenes

Bm033 304 0.28 and 0.6 PilC1 (pilus-associated protein), N. meningitidis(Y13020)

0 Coding AF169442 38

Bm007 204 PilC1 Coding AF169438Cm018 276 PilC1 Coding AF169446Cm101 585 PilC1 Coding AF169457

Cm024 600 0.45 Ssa1 (serotype-specific antigen), Pasteurella hae-molytica (U07788)

1e-7 Coding AF169448 18

Em085 286 Ssa1 Coding AF169473

Em005 171 0.67 YgiY (putative sensor/kinase protien), E. coli(P40719)

2e-26 Coding AF169461 6

IrlS (two-component regulatory system protein)Burkholderia pseudomallei (AF005358)

4e-19 24

Cg014 156 0.85 Iga (IgA protease), Neisseria gonorrhoeae(X04835)

0 Coding AF169423 36

Cm122 311 Iga Coding AF169458Em033 276 Iga Coding AF169466

Bg021 444 1.55 AidA-1 (putative adhesin), E. coli (D90793) 2e-23 Coding/inergenic AF169420 1VirG (virulence-associated protein), Shigella

flexnerii (M22802)4e-23 32

Eg008 302 1.9 HlyIII (hemolysin III.), Bacillus cereus (P54176) 3e-25 Coding/intergenic AF169427 4

DNA modification,phage-related, andinsertion sequences

Eg016 326 0.18 DcmH (cytosine-specific methyltransferase),N. gonorrhoeae (AF001598)

1e-148 Coding AF169431 20

Cm130 851 0.42 HgiDIIM (cytosine-specific methyltransferase),Herpetosyphon giganteus (JT0594)

7e-49 Coding AF169459 13

Em024 271 HgiDIIM Coding AF169464Eg010 271 HgiDIIM Coding AF169428Cm040 269 HgiDIIM Coding/intergenic AF169451

Em029 268 0.45 Transposase in insertion sequence, Aeromonassalmonicida (L27157)

1e-10 Coding/intergenic AF169465 21

Cm016 280 1.17 Hypothetical protein in insertion sequenceIS150-like, N. gonorrhoeae (L36381)

2e-88 Coding AF169445 37

Cm042 203 1.75 Hypothetical protein phage Cf1c), Xanthomonascampestris (M57538)

1e-19 Coding AF169452 31

Eg003 364 1.95 RstA2 (phage CTX), Vibrio cholerae (U83796) 7e-8 Coding AF169426 45

Cg011 315 Multiple ORF1 (transposase, insertion sequence IS1106(partial; DNA homology), N. meningitidis

- Coding/intergenic AF169422 28

Cm034 381 Multiple Insertion sequence IS1016, Haemophilusinfluenzae (X58176)

4e-10 ? AF169450 30

Cg004 358 Multiple Hypothetical transposase, Synechocystis sp.(D90913, g1653459)

2e-18 Coding/intergenic AF169421 25

Em034 (365)c Multiple Correia repetitive element (partial; DNAhomology), N. gonorrhoeae (M19676)

8e-80 Coding/intergenic AF169467 8

Metabolic and trans-porter genes

Bg012 363 0.7 CvaB (ATP-binding colicin secretion protein),E. coli (U47048)

8e-16 Coding AF169419 17

Bm026 181 0.7 GluP (glucose/galactose transporter), Brucellaabortus (Q44623)

1e-104 Coding AF169441 14

Eg028 251 1.22 Ggt (g-glutamyltranspeptidase), Bacillus subtilis(P54422)

2e-9 Coding AF169435 48

Bm003 (360)c 1.6 PorA (class 1 porin), N. meningitidis 0 Coding AF169437 5Bg004 363 PorA Coding AF169417

Cm029 248 2.0 SpeA1 (arginine decarboxylase), E. coli (M31770) 1e-138 Coding AF169449 34Bm011 347 SpeA1 Coding AF169439

Continued on following page

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their distribution between the species, reflecting the closerrelationship between the gonococcus and the meningococcusrather than genetic differences maintained by natural selection.

(iii) Sequences with homologies to proteins involved in met-abolic pathways or transporters. The fact that metabolic genesmay be specific for pathogenic Neisseria species could be re-lated to the specific environment they both have to encounter.The outer membrane porin PorA (5) belongs to this category.PorA is found only in N. meningitidis, and the gene was initiallythought to be N. meningitidis specific; however, in N. gonorrho-eae the porA gene is not expressed, being a pseudogene (15).

(iv) Sequences with weak homologies and homologies to hy-pothetical proteins typically derived from genome-sequencing

projects. The significance of sequences with weak homologiesand homologies to hypothetical proteins remains to be inves-tigated.

Genetic arrangement of the pathogen-specific regions. Theorigin of the pathogenic Neisseria sequences is another impor-tant question. In several bacterial species, which contain moreor less virulent variants (for example, E. coli, Helicobacter py-lori, Salmonella typhimurium, and Yersinia enterocolitica), genesspecifying the attributes of increased pathogenic potential areclustered in so-called pathogenicity islands (PAIs) (22). PAIsare usually large (50 to 200 kb), often having a G1C contentdifferent from that of the host chromosome. None of the re-gions had the characteristics typical of PAIs, of bacteriophages,

TABLE 2—Continued

Clone Length(kb) Positiona Homology of ORF BLASTX

significanceInter- or

intragenicAccession

no.Refer-ence

Eg022 281 SpeA1 Coding/intergenic AF169433Em061 261 SpeB (agmatinase), E. coli (M32363) 1e-134 Coding AF169470 43

Cm062 212 2.0 YflS (hypothetical protein), B. subtilis (D86417,g2443241)

1e-130 Coding AF169456 49

SODiT1 (dicarboxylate translocator), Spinaciaoleracea (U13238)

3e-97 46

Eg040 276 2.04 CppM (putative CPEP phosphomutase), E. coli(P77541)

8e-91 Coding AF169436 12

Em078 308 CisZ (citrate synthase), E. coli (P31660) 8e-28 Coding AF169472 35Cm047 224 CisZ Coding AF169455

Genes of unknownfunction

Cg023 470 2.0 Hypothetical protein ‘ORF1’, N. meningitidis(AJ010115)

6e-23 Coding AF169425 47

Em103 (375)c 2.04 HI1005 (hypothetical protein), H. influenzae(U32781)

3e-56 Coding AF169476 16

Cm045 206 HI1005 Coding AF169454Cg015 267 HI1005 Coding AF169424Em023 273 HI1005 Coding AF169463

Em094 196 2.04 Ycb9 (hypothetical protein), Pseudomonasdenitrificans

6e-9 Coding AF169475 9

Bm040 243 2.04 YraM (hypothetical protein), Bacillus subtilis(U93875, g1934631)

1e-50 Coding AF169443 42

Em059 (515)c YraM Coding AF169469

Genes with nohomologies

Em110 217 0.6 None Coding AF169477Bm013 349 None Coding AF169440Cm043 229 None Coding AF169453Bg007 400 None Coding AF169418Em045 260 None ? AF169468

Em070 323 0.6 ? AF169471

Em087 238 0.7 None Coding AF169474

Eg014 293 1.75 None Intergenic AF169430

Cm008 236 2.05 None Coding AF169444Eg012 309 None Coding AF169429Em002 308 None Coding AF169460

Cm020 354 2.25 None Coding AF169447Eg024 345 None Coding AF169434

Em022 (305)c Multiple None ? AF169462

a Positions were determined by comparison of the pattern of reactivity on Southern blots with those of the macrorestriction map of N. meningitidis Z2491 (11) andby analysis of contigs from the genome-sequencing projects which contained the clones. The position is given in megabases, according to reference 11.

b Homologous proteins or ORFs are those giving the best BLASTX scores, using the corresponding ORF or intergenic region as query sequence.c Sizes of clones in parentheses are approximate, where the sequence information was not considered sufficiently reliable to give an exact number of bases.

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or of compound transposons, structures which are associatedwith the introduction into bacterial chromosomes of foreignDNA coding for virulence factors. Several of the regions were,however, of particularly low G1C content (Fig. 4) and wereassociated with transposase and integrase genes, suggestingthat at some time in the genetic history of the species, theregions were the result of recombinational events with DNAfrom other species. For example, the region containing Cm016,Em024, and Cg004 at 1.17 Mb (Fig. 4A) contains a region witha particularly low G1C content (46%, compared with 52% forthe chromosome in general) with no homologies to genes inthe databases, surrounded by ORFs with homologies to se-quences encoding transposases and a phage integrase, and maywell represent DNA, as yet unknown, acquired from anotherorganism. A similar situation is seen with the region corre-sponding to clones Cm020 and Eg024 (Fig. 4B).

The region between the clones Em085 and Cm024 and theregF gene is the site of one of the large chromosomal translo-cations discovered by Dempsey et al. (11). The surroundingregion (Fig. 4C) contains several copies of the Correia se-quence, singly or in pairs, and these sequences are likely to beimportant in intrachromosomal rearrangements, as has beensuggested previously (28).

Another striking feature of these regions is the associationof many of the clones with the previously described N. menin-gitidis-specific regions (44). This suggests that previously dis-covered N. meningitidis-specific islands, at least in regions 2and 7 (Fig. 4D and E), have inserted into preexisting patho-gen-specific sequences. Together, these data suggest thatthese N. meningitidis and N. gonorrhoeae regions correspondto islands of pathogen-specific DNA, as was seen to be thecase in the N. meningitidis-N. gonorrhoeae subtraction.

Conclusion. Our data demonstrate that even though N. men-ingitidis and N. gonorrhoeae display very different pathogene-ses, they have regions of their chromosomes in common whichare not found in the nonpathogenic N. lactamica and which areprobably involved in common aspects of their life cycle, i.e.,colonization and survival at the port of entry. The subtractivetechnique has enabled us to identify novel candidate genes andregions involved in these common steps. A further understand-ing of these steps will require systematic mutagenesis of thegenes located in these regions. The postgenomic era has begunfor many bacterial pathogens; our data have confirmed that thetechnique of genomic subtraction has the potential to pinpointregions of chromosome that are most likely to be involved inthe differential virulence of bacterial pathogens. This tech-nique has therefore the potential to identify from among thethousands of ORFs brought to light by genome sequencing anumber of potential targets for new therapies and vaccineproduction.

ACKNOWLEDGMENTS

This work was supported by the INSERM, the Universite Paris VRene Descartes and the Fondation pour la Recherche Medicale.

Thanks are due to N. meningitidis and N. gonorrhoeae sequencingteams at the Sanger Centre and the University of Oklahoma, whomade their sequences publicly available throughout the progress of thegenome projects.

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