Vol. 171, No. 9

Conservation of Genomic Sequences among Isolates ofMVycobacterium leprae

JOSEPHINE E. CLARK-CURTISS'* AND GERALD P. WALSH2

Department of Microbiology and Immunology and Department of Biology, Washington University, St. Louis,Missouri 63130,1 and Leonard Wood Memorial, Center for Leprosy Research, Cebu City, The Philippines2

Received 13 March 1989/Accepted 14 June 1989

Restriction fragment length polymorphism analysis has been used to assess relatedness among the genomes

of four isolates of Mycobacterium leprae, the causative agent of leprosy. The M. leprae isolates were from humanpatients from India, a Mangabey monkey from West Africa, and an armadillo from Louisiana. A total of 16probes were used; these were insert fragments of M. leprae DNA from plasmid recombinant libraries, 5 ofwhich had genes with identifiable functions and 11 of which were randomly chosen recombinant molecules. Inspite of the widely diverse origins of the isolates, restriction fragment length polymorphism analysisdemonstrated that less than 0.3% of the nucleotides differ among the genomes.

Over 100 years ago, G. A. Hansen presented evidence thatimplicated an acid-fast bacillus as the causative agent of thedisease of leprosy (14). That organism, Mycobacteriumleprae, remains a scientific enigma even today, for it is theonly known human pathogenic bacterium that has neverbeen cultivated in the laboratory outside of living animals (8,12, 18, 19, 27, 29). A consequence of the inability to easilycultivate M. leprae has been the great difficulty in determin-ing the basic physiological capabilities of the bacillus, whichhas precluded the design of simple assays to distinguish M.leprae from other mycobacteria and, more importantly, tounderstand the mechanism(s) of pathogenicity of M. leprae.

At the present time, any noncultivable acid-fast bacillusisolated from a lesion characteristic of leprosy is classified asM. leprae. Although certain cellular constituents of M.leprae are similar to those in other mycobacteria, many M.Ieprae components are unique in their chemical composition(e.g., phenolic glycolipid I [15], the presence of glycine inplace of L-alanine in the peptide portion of peptidoglycan[10], and the 56% guanine-plus-cytosine content of M. lepraechromosomal DNA [7, 16]). Moreover, hybridization analy-ses between total chromosomal DNA from M. leprae andDNA from 11 cultivable mycobacteria (including both slow-and fast-growing mycobacteria) have indicated that the M.leprae genome is not closely related to that of any of thewell-characterized mycobacteria (2, 13, 24).Although M. Ieprae is apparently unrelated to other bac-

teria, there have been few attempts to determine the extentof relatedness among the acid-fast bacilli isolated fromleprosy patients in different parts of the world. Moreover, itis presumed that the different manifestations of the diseaseof leprosy are a consequence of the host immune response toinfection, but there has been no way to definitively provethat the organism that causes lepromatous leprosy in someindividuals is the same as the organism that causes tubercu-loid leprosy in other individuals.

In addition to leprosy in humans (14), there have also beenreports of naturally occurring leprosy-like infections in ar-madillos (31), chimpanzees (9), and Mangabey monkeys(22). By all available criteria, including total genomic DNAhybridizations, the organisms that cause the natural leprosy-

* Corresponding author.

like infections are the same as M. leprae of human origin (2,22).The technique of restriction fragment length polymor-

phism (RFLP) analysis was introduced in 1980 as a powerfultool for detecting small differences (i.e., single base pairchanges) among chromosomes of organisms that are veryclosely related as determined by conventional genomic anal-yses (5). This technique is being used with increasing fre-quency for population genetics studies in a variety of plant,animal, and bacterial species (23). We therefore used RFLPanalysis to determine genomic relatedness among chromo-somal DNAs of M. leprae isolates. By using 16 differentrecombinant DNA probes, some of which contained knowngenes and others of which included chromosomal DNAfragments of unknown function, a remarkable conservationof fragment sizes was observed among the genomes of M.leprae isolates from three continents and three infectedhosts; only an estimated 0.02 to 0.26% of the nucleotides inthe genomes of these isolates have diverged.

MATERIALS AND METHODS

Bacterial strains. The bacterial strains from which chro-mosomal DNA was extracted and analyzed in the RFLPanalyses are listed in Table 1. These included four isolates ofM. leprae from geographically diverse origins and fromdifferent infected hosts. In addition, DNA from several othermycobacterial species were included in the experiments:Mycobacterium lufu and Mycobacterium vaccae, two culti-vable mycobacterial species with phenotypic similarities toM. leprae (see reference 7 for a discussion); and five group 3armadillo-derived mycobacterial (ADM) strains, which havebeen described by Portaels et al. (24). The ADM isolateswere recovered in addition to M. leprae from armadillosexperimentally infected with M. leprae; these isolates werecultivable and were phenotypically distinct from all pres-ently known species of mycobacteria (24). The ADM isolatesform four homogeneous groups on the basis of DNA-DNAhybridization and phenetic analyses, but they are clearlydifferent from M. leprae by a variety of criteria, includingtotal genomic DNA hybridization analyses (24). ControlDNA preparations included DNA from Escherichia coliK-12, since the genomic libraries were prepared therein;uninfected armadillo DNA, to be certain that the clones were

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TABLE 1. Bacterial strainsStrain Source

M. Ieprae human-29 (H-1) ...................................... Armadillo no. 29, experimentally infected with M. Ieprae in biopsy material

pooled from seven lepromatous leprosy patients from India; received fromC. C. Shepard, Centers for Disease Control, Atlanta, Ga.

M. leprae human-93 (H-2) ...................................... Armadillo no. 93, experimentally infected with M. leprae in biopsy material

from a single lepromatous leprosy patient from India; received from C. C.Shepard

M. Ieprae from a naturally infected armadillo (A-1) ........Wild armadillo with leprosy-like disease at time of capture in northern Louisi-

ana; received from G. P. Walsh, Armed Forces Institute of Pathology, Wash-ington, D.C.

M. leprae from a naturally infected Mangabeymonkey (M-1) ...................................... Armadillo experimentally infected with M. Ieprae in biopsy material from a nat-

urally infected Mangabey monkey; received from G. P. WalshM. Iufu ...................................... J. K. Seydel, Institut Borstel, Federal Republic of GermanyM. vaccae ...................................... C. C. Shepard, Centers for Disease Control, Atlanta, Ga.

Mycobacterium sp. strain ADM 579 ............................Group 3 ADM strain (24); received from F. Portaels, Instituut voor Tropische

Geneeskunde, Antwerp, BelgiumMycobacterium sp. strain ADM 9091 ...........................Group 3 ADM strain; received from F. Portaels

Mycobacterium sp. strain ADM 10718 .........................Mycobacterium strain of M. avium-M. intracellulare-M. scrofulaceum complex,

but with unusual lipids; received from F. PortaelsMycobacterium sp. strain ADM 10719 .........................M. gordonae with unusual lipids; received from F. Portaels

Mycobacterium sp. strain ADM 10722 .........................Group 3 ADM strain; received from F. Portaels

not fragments of armadillo DNA; and uninfected humanDNA.

Selection of probes. A genomic library of M. leprae chro-mosomal DNA had been constructed by inserting PstI-digested M. leprae DNA isolated from cells purified from anarmadillo experimentally infected with M. leprae from ahuman leprosy patient into PstI-digested pYA626 DNA (7).The average size of the M. leprae insert fragments was 3kilobases (kb). DNA was isolated from 25 recombinantcolonies chosen at random, digested with PstI, and sepa-rated by electrophoresis on 0.7% agarose gels. Ten of themolecules had insert fragments without internal PstI sites inthe range of 2 to 3.4 kb. Cross-hybridizations among theinsert fragments revealed two clones (pYA1032 andpYA1065) that were homologous by dot blot hybridization;one of these, pYA1065, has been used as a probe with

chromosomal DNAs more frequently than the other and hasbeen described in greater detail elsewhere (6). The othereight probes did not cross-hybridize with one another andthus represent separate regions of the M. leprae chromo-some. Six additional fragments were included as probes inthe experiments reported here: a 10-kb insert from apYA626: :M. Ieprae recombinant clone (also without aninternal PstI site), a 2.6-kb insert which included the M.leprae gene specifying citrate synthase (17), a recombinantclone which included an M. leprae gene that complementedthe purE mutation in E. coli, an insert which included therrnB operon from E. coli (the generous gift of H. Bercovier),and two inserts from the Agtll::M. leprae library (32) whichspecified polypeptides that reacted with antibodies in thesera of lepromatous leprosy patients (S. Mundayoor, J. E. R.Thole, R. Esser, and J. E. Clark-Curtiss, manuscript in

TABLE 2. DNA probes

Hybridization to:Clone Size of Identifiableinsert (kb) M. leprae Group I Group II phenotype (reference)

isolates mycobacteriaa mycobacteriab

pYA1026 2.3 + + + NDCpYA1027 -10 + ND ND NDpYA1031 2.8 + - - NDpYA1032 2.6 + - - NDpYA1036 2.4 + + + Citrate synthase (17)pYA1038 3.4 + - - NDpYA1053 2.4 + - - NDpYA1064 3.3 + - - NDpYA1065 2.2 + - - NDpYA1066 3.0 + - - NDpYA1068 2.5 + - + NDpYA1070d 3.0 + +-e+- Phosphoribosylaminoimidazole

carboxylase (3)Xgt1l 3.2 2.5 + ND ND Antigenic determinantXgtll 7.8 1.0 + ND ND Antigenic determinantrrnB (E. coli operon) 2.5 + + + Ribosomal RNA operon (26)

a Group I mycobacteria include M. vaccae and ADM strains 579, 9091, 10718, and 10719.b Group II mycobacteria include M. lufu and ADM strain 10722.c ND, Not determined.d pYA1070 complements a purE mutation in an E. coli host strain; purE codes for the enzyme phosphoriboylaminoimidazole carboxylase (3).e Very weak hybridization to at least one member of the group.

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4846 CLARK-CURTISS AND WALSH

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FIG. 1. Autoradiographs of hybridizations between the M. lep-

rae pYA1036 insert probe and restriction endonuclease-digestedchromosomal DNAs. (A) Chromosomal DNAs digested with Pstl.Lanes: 1, HindIll-digested bacteriophage X DNA (molecular sizesare indicated to the left of the gel); 2, uninfected armadillo DNA; 3,E. coli K-12 DNA; 4, M. leprae human isolate H-1 DNA; 5,pHC79::M. leprae recombinant library DNA (7); 6, M. lufu DNA; 7,M. vaccae DNA; 8 through 12, DNAs from five ADM strains. (B)Lanes: 1 through 3, chromosomal DNAs digested with BstEIl; 4through 6, chromosomal DNAs digested with Sacl; 1 and 4, M.leprae human isolate H-2 DNA; 2 and 5, DNA from M. lepraeisolate A-1 from a naturally infected armadillo; 3 and 6, DNA fromM. Ieprae isolate M-1 from a naturally infected Mangabey monkey.Approximately 500 ng of each DNA (except in lanes 8 to 11 of Fig.1A, in which approximately 200 ng of DNA was added to each well)were separated by electrophoresis through a 0.7% agarose gel.

preparation). Table 2 summarizes the characteristics of theprobe molecules.RFLP analysis. All chromosomal DNAs were digested to

completion with the restriction endonuclease PstI. Chromo-somal DNAs from the M. leprae isolates and the controls (E.coli, uninfected armadillo, and human DNAs) were alsodigested to completion (in separate reactions) with BamHI,BstEII, MboI, MspI, and Sacl. The digested DNA fragmentswere separated by electrophoresis at 50 V for 16 to 18 hthrough 1.2% (for MboI- and MspI-digested DNAs) or 0.7%(for all other digested DNAs) agarose gels (Sigma ChemicalCo., St. Louis, Mo.) submerged in 100 mM Tris-100 mMborate-2.5 mM EDTA buffer. Approximately 500 ng of eachdigested DNA was loaded per well, except where noted inthe figure legends. Gels containing the separated digestionfragments were denatured, neutralized, and transferred toGeneScreenPlus (Dupont, NEN Research Products, Bos-ton, Mass.) and hybridized with denatured 32P-labeled probeDNAs as described previously (6, 7, 17).

Preparation of the probe DNAs was as follows. The M.leprae DNA insert fragment was separated from the pYA626vector DNA by agarose gel electrophoresis after completedigestion with PstI as described previously (6, 7). Thepurified probe DNA was radioactively labeled with[32P]dATP by nick translation (20). Southern hybridizations(28) of the labeled probe DNA to DNA fragments on the

filters were done under nonstringent conditions (the hybrid-ization solution was 1 M NaCl-1% sodium dodecyl sulfate-10% dextran sulfate-100 p.g of denatured salmon spermDNA per ml). The labeled, denatured probe DNA was addedat a final concentration of 10 ng per ml of hybridizationsolution, and the hybridizations were done at 65°C for 20 h.The filters were washed in 2x SSC (1x SSC is 0.15 M NaClplus 0.015 M sodium citrate) at room temperature, in 2xSSC-1% sodium dodecyl sulfate at 65°C, and in 0.1 x SSC atroom temperature (as described in references 6 and 7). Thefilters were exposed to Kodak XAR-2 film (Eastman KodakCo., Rochester, N.Y.) at -70°C for 16 to 20 h.The data obtained from the Southern hybridizations were

analyzed by using the formulae of Upholt (30) as used byMcFadden et al. (21) for quantitative analysis of nearlyhomologous DNAs (>80% homology) in which DNA rear-rangements have not occurred and in which single base pairchanges are likely to be the predominant genetic alteration.In this type of analysis, F is the fraction of conservedrestriction endonuclease digestion fragments out of the totalnumber of hybridizing fragments (21, 30). From this value,the estimated fraction of substituted base pairs (P) canbe calculated from the formula P = 1 - [-F + (F2 +8F)12/211/", where n is the number of bases in the recognitionsite of the restriction endonucleases (21, 30).

In cases in which highly homologous DNAs were com-pared, often no RFLPs were found. To determine themaximum frequency of base substitution compatible withthis finding, we have used the equation used by McFadden etal. to estimate similar values for their RFLP analyses (21),Pm, = Z21(N + Z2), where PMn is the maximum fraction ofbase substitution, N is the total number of independent basesexamined (i.e., the number of fragments examined multi-plied by the number of bases in the recognition site of therestriction endonuclease which produced the fragment pat-tern), and Z is the binomial test statistic. The value ofZ wasobtained from tables of area under the normal curve; at alevel of significance of 95%, the value of Z corresponds to1.64 (21).

RESULTS

Hybridizations with probes containing known genes. FigureIA is an autoradiograph of a hybridization between thelabeled M. leprae insert fragment from pYA1036 (whichincludes the M. leprae gene for citrate synthase [17]) andPstI-digested chromosomal DNAs from each of the myco-bacterial strains listed above. This probe hybridized to allchromosomal DNAs included on the gel, except uninfectedarmadillo DNA, under the permissive hybridization condi-tions used (see the legend to Fig. 1). However, the probehybridized to different-sized fragments in each of the dif-ferent chromosomal DNAs, as would be expected if therehad been polymorphisms in the DNA sequences in areasadjacent to the citrate synthase genes of these mycobacterialspecies and strains.

Figure 1B is an autoradiograph of the hybridization be-tween the same probe and BstEII- and SacI-digested chro-mosomal DNA from three different isolates of M. leprae(isolates H-2, A-1, and M-1). In this experiment, it is clearthat the probe hybridized to exactly the same major fragmentor fragments of chromosomal DNA from each of the threeisolates regardless of the restriction endonuclease used todigest the DNAs.

Since citrate synthase is an essential enzyme of the Krebscycle, it is perhaps not surprising that there is conservation

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FIG. 2. Autoradiographs of hybridizations between the M. leprae pYA1070 probe DNA and restriction endonuclease-digested chromo-somal DNAs. (A) Chromosomal DNAs digested with PstI. Lanes: 1, HindIll-digested bacteriophage X DNA; 2, E. coli K-12 DNA; 3,uninfected armadillo DNA; 4, M. leprae H-1 DNA; 5, M. lufu DNA; 6, M. vaccae DNA; 7 through 11, DNAs from five ADM strains. (B)Lanes: 2 through 7, chromosomal DNAs digested with PstI; 8 through 11, chromosomal DNAs digested with BamHI; 1 through 3, same DNApreparations as in lanes 1 through 3 of Fig. 2A; 4, M. leprae H-1 DNA; 5 and 9, M. leprae H-2 DNA; 6 and 10, M. leprae A-1 DNA; 7 and1i, M. leprae M-1 DNA; 8, uninfected human DNA. (C) Lanes: 2 through 6, chromosomal DNAs digested with BstEII; 7 through 9,chromosomal DNAs digested with Sacl; 1 through 3, same as lanes 1 through 3 of Fig. 1A; 4 and 7, M. leprae H-2 DNA; 5 and 8, M. lepraeA-1 DNA; 6 and 9, M. leprae M-1 DNA. (D) Lanes: 2 through 7, chromosomal DNAs digested with MboI; 8 through 10, chromosomal DNAsdigested with MspI; 1, HindIII-digested DNA plus HaeIII-digested (X174 DNA; 2, E. coli K-12 DNA; 3, uninfected armadillo DNA; 4, M.leprae H-1 DNA; 5 and 8, M. leprae H-2 DNA; 6 and 9, M. leprae A-1 DNA; 7 and 10, M. leprae M-1 DNA. The gel on which the MboI-and MspI-digested DNAs were separated was 1.2 rather than 0.7% agarose. Only 175 to 190 ng of chromosomal DNAs was added to lanes5, 6, and 9 of the 1.2% agarose gel (panel D); approximately 500 ng of DNA was added in all other lanes in panels A through D.

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4848 CLARK-CURTISS AND WALSH

TABLE 3. Fraction of conserved fragments and inferred frequency of base substitution for M. leprae isolates H-2, A-1, and M-1

Restriction endonucleases recognizing Restriction endonucleases recognizing4-base-pair sites (MboI and MspI) 6-base-pair sites (BamHI, PstI, and Sacl)

ProbeNoofN.fNo.of~~~aInferred No fInferredfragments F Inprdfragments F Ifrexamined examined

pYA1027 31 0.903 0.86 13 1.000 0.00pYA1031 22 1.000 0.00 2 1.000 0.00pYA1036 18 1.000 0.00 15 1.000 0.00pYA1038 11 1.000 0.00 14 1.000 0.00pYA1064 6 1.000 0.00 7 1.000 0.00pYA1065 ND" ND ND 229 0.996 0.02pYA1070 14 1.000 0.00 17 1.000 0.00All probes combined" 103 0.971 0.26 347 0.997 0.02

aF, Fraction of conserved restriction endonuclease digestion fragments out of the total number of hybridizing fragments (21, 30).b p = 1 - [-F + (F2 + 8F)12I/211/", where P is the estimated fraction of substituted base pairs and n is the number of bases in the recognition site of the

restriction endonucleases (21, 30).' ND, Not determined.d Number of fragments examined, F, and inferred % P for BstEII (which recognizes a 7-base-pair site) were 102, 1.000, and 0.00. respectively.

of at least some sequences in the gene among mycobacterialspecies and a very high degree of homology between citratesynthase genes from the different isolates of M. leprae.Thus, we chose a second probe of known function for thenext series of experiments: pYA1070, in which the M. lepraeinsert included a gene that was able to complement a purEmutation in E. coli. The results of the hybridizations with thepYA1070 probe are shown in Fig. 2A through D. In contrastto the pYA1036 insert, the pYA1070 probe reacted stronglywith M. Ieprae DNA (Fig. 2A) and only very weakly withDNA from any of the other mycobacterial species tested(these were detectable only after prolonged exposure of thehybridization filter to X-ray film [Table 2]). pYA1070 wasfrom a pYA804::M. leprae library (J. E. Clark-Curtiss andW. R. Jacobs, unpublished data); the insert fragments werecloned into an AsuII site rather than a PstI site as they werein the pYA626 library. Thus, the probe hybridized to twoPstI fragments of chromosomal M. Ieprae DNA (Fig. 2B).Moreover, the probe hybridized to exactly the same majorfragments of chromosomal DNA from four M. leprae iso-lates (the same sources of M. leprae DNA as in Fig. 1B, plusan additional human isolate designated H-1) regardless of therestriction endonuclease used to digest the chromosomalDNAs (Fig. 2B, C, and D). In Fig. 2D, the amounts of M.Ieprae H-2 and M. leprae A-1 loaded on the gels were 175and 190 ng, respectively, whereas 500 ng of M. leprae H-1and M. Ieprae M-1 were loaded. Therefore, we have com-pared hybridizations of the probes only to the major frag-ments (for MboI-digested DNA, the fragments are approxi-mately 1.1 and 0.7 kb; for MspI-digested DNA, fragmentsare approximately 0.35 to 0.37 kb and approximately 0.27kb).A third probe of known function (the rrnB operon of E.

coli) was used in hybridizations with DNA from M. Iepraehuman, armadillo, and Mangabey monkey isolates and withM. lufu and M. vaccae DNAs. The rRNA genes have regionsof highly conserved nucleotide sequences present in procar-yotic DNA; thus, Bercovier et al. used the rrnB operon fromE. coli as a probe to identify ribosomal RNA genes fromseveral mycobacterial species in hybridization experimentssimilar to those described here (4). The rrnB probe hybrid-ized to different-sized fragments of the latter two chromo-somal DNAs but to the same-sized fragments of M. lepraeDNA from the different isolates (26). Likewise, when theinsert fragments from two Agtll::M. leprae recombinantmolecules (32) which possess genes specifying antigenic

determinants recognized by antibodies in the sera of lepro-matous leprosy patients were used as probes, each probehybridized to the same PstI- and BamHI-digested fragmentsof chromosomal DNA from three isolates of M. leprae (H-2,A-1, and M-1; Mundayoor et al., in preparation).

Thus, for probes containing genes with known functionsand for two probes containing genes that specify antigenicdeterminants, there appears to be a strong selection forconservation of sequences within and/or adjacent to thegenes among the different isolates of M. Ieprae, perhapsbecause at least one gene present on the probes has animportant function within the organism. This observation isconsistent with those of Nei (23) and of Anilionis and Riley(1), who used five probes from different portions of the E.coli chromosome to identify RFLPs among six laboratorystrains of E. coli.

Hybridizations with randomly chosen probes. In addition tothe recombinant molecules which contained genes of knownfunction or which specified antigenic determinants, we alsochose 10 other pYA626::M. leprae clones solely on the basisof the size of the insert DNA. When the insert fragmentsfrom these molecules were used as probes in Southernhybridizations, four patterns were observed when hybridiza-tion of the probe to M. Ieprae DNA was compared withhybridization to DNA of the other mycobacterial species(Table 2). (i) One probe (from pYA1026) hybridized to allmycobacterial DNAs tested, although the probe hybridizedto different-sized PstI digestion fragments of the differentmycobacterial DNAs. This pattern was similar to that ob-served with the pYA1036 and rrnB probes discussed aboveand shown in Fig. 1A. (ii) One probe (from pYA1068)hybridized to DNA of M. lufu and of one ADM strain as wellas to chromosomal DNA of the M. leprae isolates buthybridized to different-sized fragments of M. lufu DNA andthe ADM strain DNA than it did to the three M. lepraeDNAs (data not shown). (iii) Six probes hybridized only tosingle PstI digestion fragments of M. Ieprae DNAs, similarto the pattern for the pYA1070 probe (Fig. 2). However,even prolonged exposure of the hybridization filters to X-rayfilm did not reveal any hybridization of these probes to thechromosomal DNAs of the other mycobacterial speciestested. (iv) Two probes (from pYA1032 and pYA1065)hybridized only to M. Ieprae chromosomal DNAs, but eachof these probes hybridized to at least 19 PstI-generatedfragments (6).When hybridizations of the probe DNAs with chromo-

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FIG. 3. Autoradiographs of hybridizations between the M. leprae pYA1027 insert probe and restriction endonuclease-digested chromo-somal DNAs. (A) Lanes: 2 through 8, chromosomal DNAs digested with PstI; 9 through 12, chromosomal DNAs digested with BamHI; 1,HindIll-digested A DNA; 2 and 10, M. leprae H-1 DNA; 3, M. leprae H-2 DNA; 4 and 11, M. leprae A-1 DNA; 5 and 12, M. leprae M-1 DNA;6, uninfected armadillo DNA; 7, E. coli K-12 DNA; 8, uninfected human DNA; 9, ADM DNA. (B) Lanes: 2 through 6, chromosomal DNAsdigested with BstEII; 7 through 9, chromosomal DNAs digested with Sacl; 1, HindIll-digested bacteriophage A DNA; 2, E. coli DNA; 3,uninfected armadillo DNA; 4 and 7, M. leprae H-2 DNA; 5 and 8, M. leprae A-1 DNA; 6 and 9, M. leprae M-1 DNA. (C) Lanes: 2 through7, chromosomal DNAs digested with MboI; 8 through 10, chromosomal DNAs digested with MspI; 1, HindIll-digested bacteriophage X DNAplus HaeIII-digested fX174 DNA; 2, E. coli K-12 DNA; 3, uninfected armadillo DNA; 4 and 8, M. leprae H-2 DNA; 5, 175 ng of M. lepraeA-1 DNA; 6, 400 ng of M. leprae A-1 DNA; 9, M. leprae A-1 DNA; 7 and 10, M. leprae M-1 DNA.

somal DNAs from the different isolates of M. leprae werecompared, an unexpected pattern emerged. Most of theprobes hybridized to exactly the same-sized fragment orfragments of chromosomal DNA from each of the isolates,whether the chromosomal DNAs were digested withBamHI, BstEII, MboI, MspI, PstI, or Sacl. These data arepresented in Table 3. Table 3 presents data obtained withseven individual probes. In addition, the data obtained withall probes were combined to estimate the fraction of substi-tuted base pairs (P) for DNAs digested with restrictionendonucleases having 4-base-pair recognition sites and forDNAs digested by restriction endonucleases having 6-base-pair recognition sites. It is evident that there have been veryfew changes in the nucleotide sequences of DNAs from theM. leprae isolates. Only two of the probes detected anypolymorphisms, those from pYA1027 and pYA1065. The M.leprae insert fragment from pYA1027 was approximately 10kb (and was therefore three or more times larger than any ofthe other insert fragments used as probes). When chromo-somal DNA from the different M. leprae isolates was di-gested with BamHI, BstEII, PstI, or Sacl, the pYA1027probe hybridized to the same-sized DNA fragments from allthree M. leprae isolates, as shown in Fig. 3A and B.However, when the chromosomal DNAs were digested withMspI, a restriction endonuclease with a 4-base-pair recogni-tion sequence, a single polymorphism among the chromo-somal DNAs was observed (Fig. 3C, lanes 8 to 10). The M.leprae H-2 DNA has a fragment of approximately 1.2 kbwhich is absent from the other two DNAs, and the DNAfrom M. leprae A-1 is missing a fragment of 1.1 kb which ispresent in the other two chromosomal DNAs, but there weretwo additional lower-molecular-weight fragments of M. lep-

rae A-1 DNA to which pYA1027 hybridized. The secondprobe with which a single RFLP was detected was pYA1065,a recombinant molecule that contains an M. leprae-specificrepetitive sequence (6). This probe hybridizes to between 15and 24 different bands of M. leprae DNA, depending uponthe restriction endonuclease used to digest the DNA, but foreach restriction endonuclease used the pYA1065 probehybridized to exactly the same-sized fragments ofDNA fromeach of the isolates, except for one PstI fragment which wasobserved in the DNA of the M. leprae isolate from thenaturally infected armadillo, M. leprae A-1, and which wasnot present in the DNAs of the other isolates (6). When thedata with all of the probes were combined (Table 3), theestimated fraction of substituted nucleotides among theseisolates was 0.02 to 0.26%. There appeared to be an addi-tional polymorphism present in M. leprae A-1 DNA whichwas detectable with the pYA1027 probe when the chromo-somal DNAs were digested with MboI (Fig. 3C, lane 5),since the probe apparently did not hybridize to a fragment ofapproximately 0.6 kb, whereas it did hybridize to thisfragment in the Mbol digests of DNA from the other M.leprae isolates (Fig. 3C, lanes 4, 6, and 7). However, therewas significantly less M. leprae A-1 DNA loaded in lane 5

(175 ,ug) than in lane 6 (400 ng), and we believe that thesmaller amount of DNA precluded hybridization of theprobe to this minor band at a detectable level.When RFLP analysis was done comparing chromosomal

DNAs from the two human M. leprae isolates (H-1 and H-2),no polymorphisms were found. Thus, the equation P,1 =

Z21(N + Z2) (defined in detail in Materials and Methods) wasused to estimate the maximum frequency with which basesubstitutions could have occurred between the chromo-

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somes of isolates H-1 and H-2 and yet not be detected by theRFLP experiments described here. If the actual frequency ofsubstituted base pairs (P) between the two human isolateswas 1%, then the expected number of conserved fragmentsamong the 104 fragments examined (30 from digestion withrestriction endonucleases with 4-base-pair restriction sitesplus 71 from digestion with endonucleases with 6-base-pairrecognition sequences) would have been 89 (27 from diges-tion with endonucleases with 4-base-pair recognition sitesand 62 from digestion with endonucleases with 6-base-pairrecognition sites). Thus, there would have been 15 polymor-phic bands observed in the RFLP analyses. However, sinceno polymorphisms were detected between these two DNAsin the 104 fragments examined, the maximum frequency ofbase substitution (Pm) was estimated to be less than 0.47%,with 95% confidence limits, by using the equation describedabove (21).

DISCUSSION

The technique of RFLP analysis using random probes hasnot been widely used to analyze bacterial chromosomes.Sapienza and Doolittle (25) used this method to study twostrains of Halobacterium halobium, one of which (strain RI)was a spontaneously occurring variant of the other wild-typestrain (NRC-1). In experiments using randomly chosenprobes from genomic libraries of the two strains, Sapienzaand Doolittle observed more probes that hybridized todifferent-sized restriction fragments of the two chromosomalDNAs than probes that hybridized to the same-sized frag-ments of chromosomal DNA from the two strains (25).

Eisenach et al. (11) used eight randomly chosen recombi-nant molecules from a X1059::Mycobacterium tubercuilosisH37Rv library as probes to differentiate between five strainsof M. tubercutlosis, two strains of Mycobacterium bov is, twostrains of M. bovis BCG, and a strain of Mycobacteriumkansasii (11). Two of these probes hybridized to the samefragments (generated by four different restriction endonu-cleases) of all of the strains tested. Two other probeshybridized to some homologous fragments, but the overallRFLP patterns were different for all strains tested. Theauthors stated that the other four probes hybridized effi-ciently only to M. tiuberculosis H37Rv DNA, although thefigure used as an example of the data for this conclusionshowed that the probe used in the experiment did hybridizeto DNA from other M. tuberculosis strains (as well as to M.bovis DNA); however, there were very clear polymorphismsamong the different isolates of M. tuberciulosis and betweenM. tuberculosis H37Rv and M. bovis (11).

Recently, McFadden et al. have reported on their RFLPanalysis of isolates of an unclassified mycobacterium asso-ciated with Crohn's disease (21). These investigators did notdetect any RFLPs between the chromosomal DNAs of threeisolates of Crohn's disease-associated mycobacteria andMycobacterium paratuberculosis when they used probesselected from a recombinant library prepared from one of theCrohn's disease isolates. Data were presented which al-lowed McFadden and colleagues to estimate that the maxi-mum frequency of base pair substitution in the DNAs ofthese strains was 0.15% (20).When Anilionis and Riley (1) used portions of the chro-

mosome carrying known genes (thy, trp, tna, or lac) asprobes in RFLP analyses of laboratory strains of E. coli,there was essentially no difference in the sizes of HindIllfragments to which the probes hybridized among the sixstrains analyzed, but when portions of the chromosome that

are apparently inactive were used as probes, there was muchmore variability among the E. coli strains with respect to thesizes of the Hindlll fragments to which the probes hybrid-ized (1).

Williams and Gillis have also conducted RFLP analyseswith several M. leprae isolates, including some of the sameisolates we have used (from the naturally infected Mangabeymonkey and from the naturally infected armadillo) and otherdifferent isolates. As probes, they used three M. leprae DNAinsert fragments from the Agtll::M. leprae library, each ofwhich contained a gene that specified an antigenic determi-nant recognized by mouse monoclonal antibodies (32). Theywere unable to demonstrate any polymorphisms by usingthese probes hybridized to M. leprae chromosomal DNAdigested with three different restriction endonucleases(D. L. Williams and T. P. Gillis, Bull. Inst. Pasteur, inpress).The data which we have presented clearly demonstrate the

near identity of M. leprae strains from human leprosypatients, from a naturally infected armadillo, and from anaturally infected Mangabey monkey. These results areremarkable because not only were the M. leprae isolatesfrom three different groups of mammals, but the mammalsoriginated from three different continents (India, NorthAmerica, and Africa, respectively)! The fact that the ge-nomes of these isolates are nearly identical by RFLP analy-sis plus the corroborating results of Williams and Gillis implythat the M. leprae chromosome has undergone very littlealteration, in an evolutionary sense, over the course of time.Leprosy has been known to occur in humans since thebeginning of recorded history but was only detected innaturally infected armadillos in the southern United States in1975 (31) and in the Mangabey monkey in 1979 (22). Alter-natively, M. leprae may have originated recently in evolu-tionary time, the consequence of a founder effect in that theoriginal M. leprae may have been separated from an existingpopulation of bacteria that lived in humans by virtue of thefact that M. leprae was able to live within human macro-phages and Schwann cells. Occupancy within these special-ized human cells may have precluded exchange of geneticinformation with related bacteria, but M. leprae apparentlyhas also developed some means to protect its DNA frommutagenic agents (especially the oxygen radicals which areinvolved in the killing mechanisms of the macrophages) or torepair its DNA efficiently.

Is this exceptional conservation of sequences a conse-quence of the fastidious nature and extremely long (forbacteria) generation time (estimated to be approximately 2weeks in vivo [18]) of M. leprae or are the M. lepraecharacteristics of extremely slow growth and fastidiousrequirements a consequence of the apparent immutability ofits genome? Is M. leprae DNA impervious to commonlyencountered mutagens or does the organism possess somekind of super repair mechanisms? Does M. leprae lack anymeans for gene transfer among members of the species? Thecurrent state of knowledge about the genetics and physio-logical capabilities of M. leprae is too meager to allow us todo more than speculate about answers to these questions.Similarly, the scarcity of RFLP analyses among isolates of a

diversity of bacterial species, and especially of other intra-cellular pathogenic bacteria, also precludes drawing theconclusion that the phenomenon of large amounts of con-served sequences among isolates of a bacterial species mayreflect a more widespread occurrence among pathogenicbacterial species. Understanding these phenomena presentsan exciting challenge.

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GENOME STABILITY IN M. LEPRAE 4851

ACKNOWLEDGMENTS

We thank Francoise Portaels of The Institute of Tropical Medi-cine, Antwerp, Belgium, for cultures of the ADM; T. P. Gillis of theG. W. Long Hansen's Disease Research Center, Carville, La.; andthe late C. C. Shepard of the Centers for Disease Control, Atlanta,Ga., for armadillo liver tissue infected with M. leprae. We alsothank Roy Curtiss III and Alan Templeton for their critical readingof and helpful discussions about the manuscript.

This research was supported by Public Health Service grantAI-23470 from the National Institutes of Allergy and InfectiousDiseases to J.E.C.-C.

LITERATURE CITED1. Anilionis, A., and M. Riley. 1980. Conservation and variation of

nucleotide sequences within related bacterial genomes: Esche-richia coli strains. J. Bacteriol. 143:355-365.

2. Athwal, R. S., S. S. Deo, and T. Imaeda. 1984. Deoxyribonucleicacid relatedness among Mycobacterium leprae, Mycobacteriumtuberculosis, and selected bacteria by dot blot and spectropho-tometric deoxyribonucleic acid hybridization assays. Int. J.Syst. Bacteriol. 34:371-375.

3. Bachmann, B. J. 1987. Linkage map of Escherichia coli K-12,edition 7, p. 807-876. In F. C. Neidhardt, J. L. Ingraham, K. B.Low, B. Magasanik, M. Schaechter, and H. E. Umbarger (ed.),Escherichia coli and Salmonella typhimurium: cellular andmolecular biology, vol. 2. American Society for Microbiology,Washington, D.C.

4. Bercovier, H., 0. Kafri, and S. Sela. 1986. Mycobacteria pos-sess a surprisingly small number of ribosomal RNA genes inrelation to the size of their genome. Biochem. Biophys. Res.Commun. 136:1136-1141.

5. Botstein, D., R. L. White, M. Skolnick, and R. W. Davis. 1980.Construction of a genetic linkage map in man using restrictionfragment length polymorphisms. Am. J. Hum. Genet. 32:314-331.

6. Clark-Curtiss, J. E., and M. A. Docherty. 1989. A species-specific repetitive sequence in Mycobacterium leprae DNA. J.Infect. Dis. 159:7-15.

7. Clark-Curtiss, J. E., W. R. Jacobs, M. A. Docherty, L. R.Ritchie, and R. Curtiss III. 1985. Molecular analysis ofDNA andconstruction of genomic libraries of Mycobacterium leprae. J.Bacteriol. 161:1093-1102.

8. Colston, M. J., and G. R. F. Hilson. 1976. Growth of Mycobac-terium leprae and M. marinum in congenitally athymic (nude)mice. Nature (London) 262:399-401.

9. Donham, K. J., and J. R. Leininger. 1977. Spontaneous leprosy-like disease in a chimpanzee. J. Infect. Dis. 136:132-136.

10. Draper, P. 1976. Cell walls of Mycobacterium leprae. Int. J.Lepr. 44:95-98.

11. Eisenach, K. D., J. T. Crawford, and J. H. Bates. 1986. Geneticrelatedness among strains of Mycobacterium tuberculosis com-plex. Am. Rev. Resp. Dis. 133:1065-1068.

12. Fieldsteel, A. H., and L. Levy. 1976. Neonatally thymectomizedLewis rats infected with Mycobacterium leprae: response toprimary infection, secondary challenge and large inocula. In-fect. Immun. 14:736-741.

13. Grosskinsky, C., W. R. Jacobs, Jr., J. E. Clark-Curtiss, andB. R. Bloom. 1989. Genetic relationships between Mycobacte-rium leprae, Mycobacterium tuberculosis, and candidate lep-rosy vaccine strains by DNA hybridization: identification of anM. leprae-specific repetitive sequence. Infect. Immun. 57:1535-1541.

14. Hansen, G. A. 1874. Causes of leprosy. Norsk Mag. Laegervi-denskaben 4:76-79.

15. Hunter, S. W., and P. J. Brennan. 1981. A novel phenolicglycolipid from Mycobacterium leprae possibly involved inimmunogenicity and pathogenicity. J. Bacteriol. 147:728-735.

16. Imaeda, T., W. F. Kirchheimer, and L. Barksdale. 1982. DNAisolated from Mycobacterium leprae: genome size, base ratio,and homology with other related bacteria as determined byoptical DNA-DNA reassociation. J. Bacteriol. 150:414-417.

17. Jacobs, W. R., M. A. Docherty, R. Curtiss III, and J. E.Clark-Curtiss. 1986. Expression of Mycobacterium leprae genesfrom a Streptococcus mutans promoter in Escherichia coliK-12. Proc. Natl. Acad. Sci. USA 83:1926-1930.

18. Jopling, W. H. 1984. Handbook of leprosy. William HeinemanMedical Books, Ltd., London.

19. Kirchheimer, W. F., E. E. Storrs, and C. H. Binford. 1972.Attempts to establish the armadillo (Dasypus novemcinctusLinn.) as a model for the study of leprosy. lI. Histopathologicand bacteriologic postmortem findings in lepromatoid leprosy inthe armadillo. Int. J. Lepr. 40:229-242.

20. Maniatis, T., E. F. Fritsch, and J. Sambrook. 1982. Molecularcloning: a laboratory manual. Cold Spring Harbor Laboratory,Cold Spring Harbor, N.Y.

21. McFadden, J. J., P. D. Butcher, R. Chiodini, and J. Hermon-Taylor. 1987. Crohn's disease-isolated mycobacteria are identi-cal to Mycobacterium paratuberculosis as determined by DNAprobes that distinguish between mycobacterial species. J. Clin.Microbiol. 25:796-801.

22. Meyers, W. M., G. P. Walsh, H. L. Brown, C. H. Binford, G. D.Imes, Jr., T. L. Hadfield, C. J. Schlagel, Y. Fukunishi, P. J.Gerone, R. H. Wolf, B. J. Gormus, L. N. Martin, M. Harboe,and T. Imaeda. 1985. Leprosy in a Mangabey monkey-natu-rally-acquired infection. Int. J. Lepr. 53:1-14.

23. Nei, M. 1987. Molecular evolutionary genetics. Columbia Uni-versity Press, New York.

24. Portaels, F., C. Asselineau, I. Baess, M. Daffee, G. Dobson, P.Draper, D. Gregory, R. M. Hall, T. Imaeda, P. A. Jenkins,M. A. Laneelle, L. Larsson, M. Magnusson, D. E. Minnikin,S. R. Pattyn, G. Wieten, and P. R. Wheeler. 1986. A cooperativetaxonomic study of mycobacteria isolated from armadillos in-fected with Mycobacterium leprae. J. Gen. Microbiol. 132:2693-2707.

25. Sapienza, C., and W. F. Doolittle. 1982. Unusual physicalorganization of the Halobacterium genome. Nature (London)295:384-389.

26. Sela, S., J. E. Clark-Curtiss, and H. Bercovier. 1989. Character-ization and taxonomic implications of the rRNA genes ofMycobacterium leprae. J. Bacteriol. 171:70-73.

27. Shepard, C. C. 1960. The experimental disease that follows theinjection of human leprosy bacilli into the footpads of mice. J.Exp. Med. 112:445-454.

28. Southern, E. M. 1975. Detection of specific sequences amongDNA fragments separated by gel electrophoresis. J. Mol. Biol.98:503-517.

29. Storrs, E. E. 1971. The nine-banded armadillo: a model forleprosy and other biomedical diseases. Int. J. Lepr. 39:703-714.

30. Upholt, W. B. 1977. Estimation of DNA sequence divergencefrom comparison of restriction endonuclease digests. NucleicAcids Res. 4:1257-1265.

31. Walsh, G. P., E. E. Storrs, H. P. Burchfield, W. M. Meyers, andC. H. Binford. 1975. Leprosy-like disease occurring naturally inarmadillos. J. Reticuloendothel. Soc. 18:347-351.

32. Young, R. A., V. Mehra, D. Sweetser, T. Buchanan, J. E.Clark-Curtiss, R. W. Davis, and B. R. Bloom. 1985. Genes forthe major protein antigens of the leprosy parasite Mycobacte-rium leprae. Nature (London) 316:450-452.

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