Proc. Natl. Acad. Sci. USAVol. 93, pp. 3132-3137, April 1996Microbiology

An integrated map of the genome of the tubercle bacillus,Mycobacterium tuberculosis H37Rv, and comparison withMycobacterium leprae

(genome mapping/contig mapping/ordered libraries/bacterial genomics/tuberculosis)

WOLFGANG J. PHILIPP*, SYLVIE POULET*, KARIN EIGLMEIER*, LISA PASCOPELLAt, V. BALASUBRAMANIANt,BEATE HEYM*, STAFFAN BERGH*$, BARRY R. BLOOMt, WILLIAM R. JACOBS, JR.t, AND STEWART T. COLE**Unit6 de Genetique Moleculaire Bact6rienne, Institut Pasteur, 28 rue du Docteur Roux, 75724 Paris Cedex 15, France; tHoward Hughes Medical Institute,Department of Microbiology and Immunology, Albert Einstein College of Medicine, New York, NY 10461; and *Department of Biochemistry and Biotechnology,Royal Institute of Technology, 100 44 Stockholm, Sweden

Contributed by Barry R. Bloom, December 12, 1995

ABSTRACT An integrated map of the genome of thetubercle bacillus, Mycobacterium tuberculosis, was constructedby using a twin-pronged approach. Pulsed-field gel electro-phoretic analysis enabled cleavage sites for Asn I and Dra I tobe positioned on the 4.4-Mb circular chromosome, while, inparallel, clones from two cosmid libraries were ordered intocontigs by means of fingerprinting and hybridization map-ping. The resultant contig map was readily correlated with thephysical map of the genome via the landmarked restrictionsites. Over 165 genes and markers were localized on theintegrated map, thus enabling comparisons with the leprosybacillus, Mycobacterium leprae, to be undertaken. Mycobacte-rial genomes appear to have evolved as mosaic structuressince extended segments with conserved gene order andorganization are interspersed with different flanking regions.Repetitive sequences and insertion elements are highly abun-dant in M. tuberculosis, but the distribution of IS6110 isapparently nonrandom.

In spite of the availability of effective short-course chemo-therapy, and the bacillus Calmette-Guerin (BCG) vaccine,Mycobacterium tuberculosis still accounts for more deathsworldwide than any other single infectious agent (1). Recentincreases in tuberculosis in both developing and industrializedcountries, together with the emergence of drug-resistantstrains and synergy with the human immunodeficiency virus(HIV) pandemic, have combined to raise considerable publicconcern and to highlight the need for radical improvements incontrol strategies (2). The development, improvement, and useof genetic tools for mycobacteria lie at the center of currentresearch programs (3, 4). Paradoxically, in the last few years,there has been a quantum jump in our understanding of therelated leprosy bacillus, Mycobacterium leprae, as a result of theapplication of genome research and systematic DNA sequenceanalysis (5), and this has opened new avenues for research inimmunology, therapeutics, and drug development. A similarapproach for M. tuberculosis was thus urgently required.The specific objectives of this project were to construct,

characterize, and maintain ordered clone libraries correspond-ing to the chromosome of M. tuberculosis and to establish acontig map on which the positions of all known genes andmarkers were established. In parallel, a physical map of thegenome was determined by means of pulsed-field gel electro-phoresis (PFGE) of macro-restriction fragments, and this wascorrelated with the contig map to produce an integrated map(6) suitable for dissemination through the dedicated myco-bacterial database, MycDB (7).

The publication costs of this article were defrayed in part by page chargepayment. This article must therefore be hereby marked "advertisement" inaccordance with 18 U.S.C. §1734 solely to indicate this fact.

MATERIALS AND METHODS

Analysis of M. tuberculosis DNA by PFGE. To preparechromosomal DNA suitable for PFGE, M. tuberculosis strainH37Rv was inoculated into Dubos medium (Pasteur Diagnos-tics) supplemented with OADC (oleic acid, albumin, dextrose,catalase) (Difco), and incubated for 10 d at 37°C. In some casesD-cycloserine was then added (1 mg/ml) and incubationcontinued for an additional 24 h prior to cell harvesting,enclosure in low melting point agarose (GIBCO/BRL), andfurther processing to release intact genomic DNA, as describedpreviously (8).

Samples were digested with restriction endonucleases andanalyzed on a clamped homogeneous electric field (CHEF)apparatus (LKB 2015 Pulsaphor Plus) using 1.2% agarose gels(in 0.5 x TBE; 1 x TBE = 89mM Tris/89mM boric acid/2mMEDTA) as described (8, 9). To separate fragments <100 kb insize, the run time was 18 h with a pulse time of 3 s and a voltageof 270 V; for fragments >100 kb in size, the run time wasextended to 21 h with a 10-s pulse time; whereas for very largefragments (>1 Mb) the run time was 48 h with a pulse time of30 s. Gels were calibrated by using concatemerized A or yeastchromosomes (Saccharomyces cerevisiae YPH80; 225-1,900kb; New England Biolabs; or Schizosaccharomycespombe, 3-6Mb) then processed for Southern blotting onto Hybond C-extra membranes (Amersham Plc), as outlined (8).To isolate linking clones carrying rare restriction sites, 400

cosmids from the pYUB18 library were screened by restrictiondigestion for the presence of Asn I or Dra I sites. Two-dimensional PFGE-analysis of reciprocal Asn I and Dra Idigests was performed exactly as described in ref. 10.Cosmid Library Construction. DNA was extracted from M.

tuberculosis strain H37Rv, then subjected to partial digestionwith Sau3AI, as described previously (3). Fragments of 30-45kb were obtained after fractionation on a 0.4% agarose gel,then ligated to either dephosphorylated, BamHI-cleavedpYUB18 (3) or later to pYUB328 (11). After in vitro packagingwith Gigapack Gold II extracts (Stratagene), the pYUB18recombinant cosmids were introduced into the Escherichia coliK-12 strain X 2819 (12) and packaged in vivo to produce a hightiter cosmid lysate. Aliquots were subsequently used to infectE. coli strain NM554 (13) and maintained either as griddedarrays of colonies or stored frozen at -80°C in microtiter platescontaining 15% (vol/vol) glycerol. The pYUB328 cosmid librarywas constructed as described, except that no in vivo packagingwasperformed prior to infection and clone preparation.

Fingerprinting. Cosmid miniprep DNA (-3 ,g) was obtainedfrom 2 ml overnight cultures and subjected to fingerprint analysiswith EcoRI and Alu I as described (14). Samples were thenlyophilized, denatured by heating, and loaded onto a 6% dena-

Abbreviation: PFGE, pulsed-field gel electrophoresis.

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Proc. Natl. Acad. Sci. USA 93 (1996) 3133

turing polyacrylamide gel calibrated with end-labeled Sau3AIfragments of A DNA (15). Fingerprints were analyzed, andoverlapping cosmids were organized into contigs with the pro-gram CONTIG9 (16).

Hybridization Mapping, Data Handling and Analysis. Tofacilitate gene mapping and the construction of contigs byhybridization mapping (17), arrays of cosmids were dot blottedonto Hybond N membranes and processed as recommended byAmersham, Plc. The probes were macrorestriction fragmentsisolated from pulsed-field gels and labeled in the gel slice,whole cosmids, repetitive sequences, or single-copy probesproduced either from cloned genes or by PCR amplification ofgenomic DNA (18).

Hybridization was performed with 32P-labeled probes, pro-duced by nick translation or random priming, under stringentconditions [16 h at 37°C in 50% (vol/vol) formamide/5x SSC(1 x SSC = 0.15 M NaCl/0.015 M sodium citrate) (19)], exceptwhen using heterologous probes (30°C in the same hybridiza-tion solution but with washes in 2x SSC instead of 0.1 x SSC).Oligonucleotide probes were labeled by using terminal deoxy-nucleotidyl transferase and [a-32P]dCTP and hybridized asdescribed (20).

After autoradiography, the hybridization signals werescored manually then entered into input files (clone.file andprobe.file) prior to analysis with the programs of Mott et al.(21) on a Sun Sparc II workstation. All hybridizations were runthrough PROBEORDER, which orders probes and clones. Theresultant hybridization contigs were subsequently reanalyzedusing REORDER, and in some cases additional joins and veri-fications were done by referring to the fingerprinting datasetconstructed by CONTIG9 (16). The consensus contigs were thencorrelated with the PFGE map by using the Dra I and Asn Ilinking clones as landmarks.

RESULTSPFGE Analysis of Genomic DNA from M. tuberculosis.

Generally, the genomes of microorganisms with dG+dC-richchromosomes like the tubercle bacillus are readily cleaved intoa limited number of fragments by restriction endonucleaseswith dA+dT-rich recognition sequences, as these sites areunderrepresented. On screening a battery of restriction en-zymes, it was found that none of the enzymes with 8-bprecognition sites cut the chromosome and that the majority ofthe enzymes with 6-bp recognition sites (Cla I, Hpa I, Nde I,

A BM Dral Asnl

291242.5

194

145.5-

97

48.5 -

23.1 -

CM Dral Asnl

-Y1

-M2

-F2.sMl

FIG. 1. PFGE analysis of M. tuberculosis H37Rv. (A) Ethidiumbromide stained gel of DNA from H37Rv digested with Asn I or DraI. (B) Southern blot of gel shown in A hybridized with Asn I linkingclone T227. (C) Same blot as in B hybridized with Dra I linking cloneT551. The size markers (in kb) and relevant fragments (see Table 1)are indicated. M, marker.

Table 1. Restriction fragments of the chromosome ofM. tuberculosis H37Rv

Dra I

Fragment Size, kb

Z7 580Z6 475Z5 300Z4 260Z3 230Z2 230Z1 220Y2 190Y1 190X 165W 160V 140U 125T 120S 95R 87Q1 87Q2 87P 82O 78N 72M 65L 60K 47J 47I 40H 33G 30F 26EDCBAA1

232213762.2

4394.2

Asn I

Fragment Size, kb

V 700U 340T 240S 235R 180Q 170P 15503 13702 13501 133N 130M1/2 125 (x2)L 115K 105J1/2 100 (x2)I 78H 73G8 65G5/6/7 63 (X3)G4 61G2/3 59 (x2)G1 57F1/2 50 (X2)E4 47E3 46E2 45E1 44D 37C4 33C3 31C1/2 30 (X2)B6 18B4/5 15 (x2)B3 13B2 11B1 9A4 4.5A3 4A2 3.5A1 3

4405

Ssp I, Xba I, etc.) yielded more than 50 fragments and werethus of limited use for map construction. By contrast, digestionof DNA from strain H37Rv with Dra I (TTTAAA) or Asn I(ATTAAT) gave a manageable number of fragments, 35 and47, respectively (Fig. 1). Sixteen of the Dra I sites wereassociated with IS6110, as this insertion element contains aunique Dra I site (22, 23).To determine the exact number of fragments and obtain an

estimate of the chromosome size, samples were analyzed undera variety of electorphoretic conditions and pulse times. Dra Idigestion gave rise to 35 fragments ranging in size from 2.2 to580 kb (Fig. 1 and Table 1), whereas Asn I generated 47fragments (3-700 kb; Fig. 1 and Table 1), the majority of whichwere in the lower range (3-100 kb). The size of the genome wasestimated by summing the fragment sizes, and, in both cases,this was about 4.4 Mb (Table 1).

Construction of a Physical Map. To establish the order andcontiguity of the Dra I or Asn I restriction fragments on thechromosome, a pYUB18 cosmid library of M. tuberculosisH37Rv was screened for clones carrying Dra I or Asn Irestriction sites. The 26 independent Dra I linking clones (3 ofwhich carried two sites) that resulted from this screen were

Microbiology: Philipp et al.

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Proc. Natl. Acad. Sci. USA 93 (1996)

Dra I _-

FIG. 2. Two-dimensional PFGE analysis of a representative Dra I (first dimension) and Asn I (second dimension) double digest of genomic DNAfrom M. tuberculosis H37Rv. (Left) Ethidium bromide stained gel. (Right) Schematic interpretation of gel shown in Left. Fragments were identifiedand labeled in accordance with Table 1 and correspond as follows: 1, dE/aC4; 2, dE/aMI; 3, dI/aB5; 4, dI/aM2; 5, dK/aC2; 6, dK/aL; 7, dL/aI;8, dQ1/aT; 9/10, dQ2/aT/aG2; 11, dQl/aQ; 12, dS/aG2; 13, dS/aR; 14, dT/aOl; 15, dT/aI; 16/17/18/19, dV/aB6/aH/aE3/aG7; 20, dYl/aB2;21, dYl/aG6; 22, dY2/aD; 23, dYl/aF2; 24, dYl/aM2; 25, dY2/aP; 26, dZl/aC4; 27, dZ2/aH; 28/29/30/31, dZ3/aO3/aFl/aG3/aG5; 32,dZ2/aG8; 33, dZ2/aO2; 34/35/36/37/38, dZ4/aB3/aC3/aJl/aG6/aE2; 39, dZ5/aGl; 40, dZ5/aS; 41, ddZ4/aJ2; 42, dZ5/aS; 43, dZ7/aMl; 44,dZ6/aCl; 45, dZ6/aE4; 46, dZ6/aG4; 47, dZ7/aJl; 48, dZ6/aV; 49, dZ7/aK; 50, dZ6/aS; 51, dZ6/aN; and 52, dZ7/aU. Dra I fragments runningon the outermost diagonal lane (C, D, F, G, H, M, N, 0, P, R, U, W, X) have no internalAsn I restriction sites. Fragments of <10 kb were detectedindependently.

used as probes on Southern blots of DNA digested with Dra I,and all revealed unambiguous linkage of adjacent Dra Ifragments (Fig. lC), thereby accounting for 29 of the 35fragments. Similarly, linkage of many Asn I fragments wasobtained by using some of the 34 Asn I linking clones as probes(Fig. iB). To complete the map, various Asn I or Dra Irestriction fragments were isolated and used as probes inreciprocal cross-hybridization experiments. Final confirmationof topology and fragment order was obtained by performingtwo-dimensional PFGE analysis (Fig. 2) of H37Rv DNAreciprocally digested with Asn I and Dra I (10).

This enabled all of the sites to be accounted for and revealedthe presence of a single circular chromosome as found in mostbacteria (24-26). To exclude the possibility that linear andcircular forms might coexist, as in the related genus Strepto-myces (27), undigested M. tuberculosis DNA was subjected toPFGE under conditions which resolved intact Sch. pombechromosomes (data not shown). No bands in the 4-Mb rangewere observed, thus indicating that the chromosomal topologywas predominantly circular.

Construction of Cosmid Libraries and a Contig Map.Initially, -970 of the pYUB18 clones were subjected tocomputer-assisted fingerprint analysis (16, 28), but this ap-proach was later replaced by hybridization mapping (17) inwhich gridded arrays of cosmid DNAs were hybridized withsuitable probes. The arrays consisted of 970 pYUB18 clonesand 500 pYUB328 clones, and the probes were 150 geneticmarkers, 140 pYUB18 clones, and 300 pYUB328 clones. Insome experiments, Asn I, Dra I, and Xba I restriction frag-ments, isolated by PFGE, were used as probes. Hybridizationsignals were recorded and analyzed with a suite of programs(21) which constructs contigs on the basis of linkage of twoprobes by means of a common clone.

This gave 16 "hybridization contigs," which showed gener-ally good agreement with those obtained by fingerprinting,although a few of the contigs constructed by fingerprintingwere found to be spurious due to chimeric clones or a paucityof bands in the fingerprint. As systematic gap closure has notyet been undertaken, it is possible that short overlaps betweensome contigs were not detected. To obtain map integration,

the consensus contigs were then compared with the completePFGE map by using the Asn I and Dra I linking clones aslandmarks, and the current integrated map is depicted in Fig. 3.A Gene Map. The genetic markers which have been precisely

located on the integrated map fall into three groups: knowngenes or probes obtained by using PCR to amplify mycobac-terial genes of conserved sequence as targets (18), sequencesencoding protein antigens isolated from expression libraries byusing antibodies, and repetitive DNA such as insertion ele-ments (29, 30). There are >95 known genes (Table 2) and 20loci encoding protein antigens recognized by monoclonalantibodies or sera from patients (data not shown). The inser-tion sequences which have been mapped are IS6110 (22, 23),which is present in 16 copies, IS1081 (31), present in six loci,and two copies of a recently identified IS-like element (32). Atleast 26 loci contain copies of the polymorphic G+C-richrepetitive sequence (PGRS; refs. 20 and 30), whereas themajor polymorphic tandem repeat, MPTR, was so abundantthat accurate localization could not be achieved. Furtherdetails of the nature of the genes that have been mapped maybe found in Table 2 and in MycDB (7).Comparison of Mycobacterial Genome Maps. The genome

of M. leprae is currently represented by four contigs of orga-nized cosmids (14), and -1.6 Mb of its genome sequence, orroughly half, is available. As the positions of several hundredM. leprae genes were known from either sequencing or map-ping experiments (5-7) and as many of these had also beenmapped in M. tuberculosis, it was of interest to perform mapcomparisons to assess the extent of relatedness.Of the 95 known genes on the M. tuberculosis genome map,

61 have also been either physically mapped in M. leprae orpositioned as a result of sequencing of the correspondingcosmid. However, meaningful comparisons were only possiblefor gene clusters containing two or more genes. It was thusestablished that the largest conserved stretch was a chromo-somal region of -240 kb delimited by dnaB andfbpA that spansoriC, the chromosomal origin of replication. Likewise, a sec-ond cluster of over eight genes between hemE and lexAencoding two sigma factors and two members of the SOSregulon (RecA and LexA), is found in both cases (Fig. 3). At

3134 Mcoilg:Piipe l

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Microbiology: Philipp et al. Proc. Natl. Acad. Sci. USA 93 (1996) 3135

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Canonical shuttlecosmid

T830T854T453T183T292T407T333T146T130T149TS35T403T606T449T407T485T-/-T483T776T832T369T267T144T663T174T366T336T464T597T588T407T521T721T721T440T728T68ST144T20TSOOT366T116T224T616T316T192T902T148T174T918T776T172T808T692T369T258T54T93T172T167T460T832T441T465T3S5T49./.T2S2T449T13T311T167T73T663T198T184T685T264T333T441T240T31T776T663T112T407T776T1S6TS00T170T527T258T825T173T750

Proc. Natl. Acad. Sci. USA 93 (1996)

own genetic markers mapped in M. tuberculosis

Source/GenBank Mapped inDescription of probe Origin accession no. M. leprae

biotin binding protein Mtalcohol deyd enase Mbalohol dehydrogenase Mtalkylhydroperodde reductase MtL-alanine dehydrogenase Mtsecreted alanine-protine-rich antigen Mtomithin carbamyl tnsferase MtS-enolpyruvylshkikmte-3-P synthase Mt3-dehydroquinate synthase, 3-dehydroquinase Mtasptokne MtATlOSgene MtAT9S gene MttRNApro; tRNAgy;, attachment site for L5 Msputative tRNApro; attachment site for pSAM2 Mlbacterioferrtin Mtproteolysis regulator; chaperone NMcycloprane mycolic acid synthase Mtcytochrona 0 ubiquinol oMdase Mtinitiation ofDNA biosynthesis Mlreplication Mlchaperone MtHSP-70, chaperone Mtsimilar to diphtheria todn repressor Mtelongation factor G MlESAT6 T-cell antigen Mtfibronectin binding protein - 85A Mt

-85B Mt* * - 8SC Mt

putative dihydrofolate reductase Mtglucose-l-phosphate adenylytransferase Mt6-phosphoghiconate dehydrogenase MtHSP-60 chaperone MtHSP-60 hom lgue MtHSP-12; chaperone Mtglutanmate--semialdehyde 2,1-amlnot-ase MtDNAgyrase, Aand B subunits Mturoporphyrinogen decarboxylase MtRNA polymerase sigma factors Mtenoyl reductase Mtputative invasin homoiogue MtputatIve iron regulated antigen (28kD) Mtcatalase-peroxdase Mtisopropylnmlate synthas e,d , somerase MtSOS regulator Mtmyccerosic acid synthetase Mtputative mycocerosic acid synthetase isoenayme Mt? Mtresponse regulator MtquinoUnate synthetase Mlribonucleotide reductase Mtorigin of replication Mtanonymous antigen AA59 probably PhoS Mtanonymous andgen AA68 Mtanonymous antigen AA62 Mtanonymous antigen AA61 Mtencodes 33 kD antigen AA63 Mtputative phospholipase C Mtputative phosphate sensor Mtphosphate binding protein MtDNA polymerase I Mtpenicilin binding protein Mt? Mlputative 38 kD virulence regulator Mtpurine synthesis Mtpurine synthesis Mthomnolgous rcombinato Mtrecombinase Mtputative recombinase Mtresponse regulator Mt50S ribosomal protein L13 Mtbeta, beta' subunits ofRNA polymerase Ml30S ribosal protein Sl Mlribosomal protein S10 Mlribosomal protein S12 MlrRNAoperon (16S/23S/SS) MlDNA helkase Mtsuccinate dehydrogenase Mtsuperoodde dismutase Mlpyrophosphohydrolase MtlOSa RNA; small stable RNA Mttuberculin related peptide Mtputatve thymidylate synthetase Mtthioredaodn Mtelongation factor Tu Mlorotidine S'-P decarboaylase Mburease subunits ABC Mt? Mlantigen MPV64Mt19 kD lipoprotein antigen MtMPB70, secreted antigen Mt47 kD antigen Mtantigen 84 Mt14 kD antigenMt35 kD antigen Mtunique sequence for detection of M. tuberclosis Mtcomnlex

*Mt, M. tuberculosis; Mb, Mycobacterium bovis; Ms, Mycobacterium smegmatis; Ml, M.Bact6rienne.

J. Dale/Z19549J. Content/X63450GMsGMB/U18264A. Andersen/X63069A. LaqueyrerieGMBD.B. Young/M62708DS. Young/X59509GMBGMB/D17369GMB/D14355M65195GMB/X60720GCBGMB/M67510GMB/U27357

IL E Taklff/L39923G. Riccardi/L39923DS. Young/X58406D. Young/X584061. SmithGMB/Z14314B. Gilcquel/X79562J. Thole/M27016J. Thole/X62398J. Thole/X57229J. Dale/X59271

B GicquelD.B. Young/M15467GMB/X603S0T. Shinnick/X60350GCBH.IL . Takiff/L27512GOBGMBW.R. Jacobs/U02492S. PorterB. GicquelB. Heym/X68081W.R. JacobsQvB

GMBW.R. JacobsV. Deretc/U01971+U14909GMB/UO0010GMB/L34407H. SchrempfA. Andersen/M30046A. AndersenA. AndersenA. AndersenA. AndersenP. del PortUl/L11868?D.B. YoungV. Mizrahi/Ll 1920GBE de RossiGMB/X68281B. GicquelB. Gicquel!4J. Colstn/XS8485GbeS. NairM. PaUlen/X66591GMBGMB/Z46257GMB/Z14314GMB/Z14314X56657GMBCGbX16453GMBGMB/X60301D00815J. Dale/X59273B. WielesGMB/Z14314R.A. Young/U01072B. Glcquel/Al 141E de RossiK. Matsuo/X75361D.B Young/J03838K. Matsuo/D37968M.J. ColstonP. Hermans/X77129D.BYoung/M7671201MBS9187GMB/M75726

losis map do not have counterparts at the same locus in M. lepraeand, in some cases, additional genes are present within the cluster.

3136 Microbiology: Philipp et al.

Table 2. Identity and source of kn,

Locus

accAadbadhA

aidamweFarowuoDasksAMaBatSB-L5amo-pSAM2bfrcpCcamlcyEdnaA

dnomdnaKdraRetSfhp4

fbpC

-Cod-EL-groEL-2groS

hemEhrdABibhAinvIra

keuABCDlexAmasADmsBwetmnrABnadBnrdAoCrpabpadpaeAjaMepofA

phaPphoSpolAponApavt

purLrecArecNrecSrgXrplMrpArpsjrpsLrrnAruvBsdhAsod

ssrAtbpthyXOXABntufwaAureABCvirE

W13

wqg25wgW31W36aq9"Is

+

+

+

+

+

++++

+++++++++++

+

++

+

+

+

+

+

+

+

++

+

+

+

+++

+

++

+

++++

++

4-

++

+

+

+

+

least seven other regions of locally conserved linkage appear toexist (Fig. 3) although the neighboring genes on the M. tubercu-

leprae; GMB, Genetique Moleculaire6%MW^

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Microbiology: Philipp et al. Proc. Natl. Acad. Sci. USA 93 (1996) 3137

possible that only limited constraints are imposed on chromo-some organization and gene order in these exceptionallyslow-growing microbes.

We wish to thank the mycobacterial research community and peoplelisted in Table 2 for kind gifts of probes, and Doug Smith for accessto M. leprae genomic sequences prior to publication. We give specialthanks to Thierry Garnier for cheerfully helping with the computeranalysis, Burkhard Tummler and Ute Romling for valuable advice, andGerard Gugliemini and Brigitte Gicquel for useful discussions. Thisinvestigation received financial support from the World Health Or-ganization, the Association Franqaise Raoul Follereau, the InstitutPasteur, and from National Institutes of Health Grants AI26170 andAI23545.

1.2.3.

I "1S6110IS611I0 is6 IS6110

FIG. 4. Distribution of IS6110 elements on the genome of H37Rvwith respect to oriC and the putative terminus terC. The positions ofthe rrn operon, recA, and the DR locus are indicated.

DISCUSSIONThe goal of this study was to elucidate the genomic organiza-tion of M. tuberculosis and to establish a set of ordered DNAfragments, a valuable genetic resource. The clones based onthe shuttle vector pYUB18 should facilitate the dissection ofthe pathogenicity of the tubercle bacillus, as they can beintroduced into easily manipulated surrogate hosts, such as

Mycobacterium smegmatis, where faithful gene expression can

be obtained (3). Several recent examples (11, 33, 34) leadingto the identification of genes involved in drug resistance or

encoding new therapeutic targets testify to the power of thisapproach. Clones from the pYUB328 library will undoubtedlyfind application in allelic exchange (35, 36) and systematicDNA sequence analysis (6). Although a whole genome shot-gun sequencing approach appears to be the most efficient andcost-effective means of achieving this objective (37), the clonesdescribed here will greatly simplify analysis as they provideinstant access to difficult regions, such as those harboringIS6110 or other extended repetitive elements.

Analysis of the distribution of copies of IS6110 around thegenome was instructive as both clustering and bias in theirlocation was observed (Figs. 3 and 4). In BCG, the sole IS6110element is located in the DR region, consisting of a series ofshort interspersed repeats (38). From the map of the M.tuberculosis genome it is clear that DR is situated roughlymidway between oriC and the putative replication terminusterC. As the DR site is invariably occupied by IS6110 in M.tuberculosis, it is probable that the original tubercle bacillusalso had a single copy of IS6110 in DR and that this subse-quently migrated outwards by transposition in a stepwisemanner, as suggested by the clustering seen in H37Rv (Fig. 4).Comparison of the genome maps of M. leprae and M.

tuberculosis revealed the absence of global similarity, althoughlocalized regions of conserved organization appear to exist.This suggests that, although mycobacterial genomes may havehad a common origin, they have subsequently undergoneextensive diversification. The current mosaic structure reflectsgroups of related genes which were primordially linked now

being surrounded by novel chromosomal segments. It is con-ceivable that, in contrast to M. leprae, the large number ofinsertion sequences and the extensive amount of repetitiveDNA present in M. tuberculosis may have contributed togenomic rearrangements (29, 30). Further analysis of thegenomes of other pathogenic mycobacteria is required to see

whether the mosaic arrangement is a general trend, for it is

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34.

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37.

38.

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