JOURNAL OF BACTERIOLOGY,0021-9193/00/$04.0010

Nov. 2000, p. 6322–6330 Vol. 182, No. 22

Copyright © 2000, American Society for Microbiology. All Rights Reserved.

Comparative Genetic Analysis of Mycobacterium ulcerans andMycobacterium marinum Reveals Evidence of

Recent DivergenceTIMOTHY P. STINEAR,1* GRANT A. JENKIN,1 PAUL D. R. JOHNSON,1,2,3 AND JOHN K. DAVIES1

Bacterial Pathogenesis Research Group, Department of Microbiology, Monash University, Clayton,1

Microbiology Research Unit, Royal Children’s Hospital,2 and Department of InfectiousDiseases and Clinical Epidemiology, Monash Medical Centre,3 Victoria, Australia

Received 19 June 2000/Accepted 30 August 2000

Previous studies of the 16S rRNA genes from Mycobacterium ulcerans and Mycobacterium marinum havesuggested a very close genetic relationship between these species (99.6% identity). However, these organismsare phenotypically distinct and cause diseases with very different pathologies. To investigate this apparentparadox, we compared 3,306 nucleotides from the partial sequences of eight housekeeping and structural genesderived from 18 M. ulcerans strains and 22 M. marinum strains. This analysis confirmed the close geneticrelationship inferred from the 16S rRNA data, with nucleotide sequence identity ranging from 98.1 to 99.7%.The multilocus sequence analysis also confirmed previous genotype studies of M. ulcerans that have identifieddistinct genotypes within a geographical region. Single isolates of both M. ulcerans and M. marinum that wereshown by the sequence analysis to be the most closely related were then selected for further study. One- andtwo-dimensional pulsed-field gel electrophoresis was employed to compare the architecture and size of thegenome from each species. Genome sizes of approximately 4.4 and 4.6 Mb were obtained for M. ulcerans andM. marinum, respectively. Significant macrorestriction fragment polymorphism was observed between the spe-cies. However, hybridization analysis of DNA cleaved with more frequently cutting enzymes identified signif-icant preservation of the flanking sequence at seven of the eight loci sequenced. The exception was the 16S rRNAlocus. Two high-copy-number insertion sequences, IS2404 and IS2606, have recently been reported in M. ul-cerans, and significantly, these elements are not present in M. marinum. Hybridization of the AseI restrictionfragments from M. ulcerans with IS2404 and IS2606 indicated widespread genome distribution for both of theserepeated sequences. Taken together, these data strongly suggest that M. ulcerans has recently diverged fromM. marinum by the acquisition and concomitant loss of DNA in a manner analogous to the emergence ofM. tuberculosis, where species diversity is being driven mainly by the activity of mobile DNA elements.

Mycobacterium ulcerans is an emerging human pathogen thatcauses a chronic, necrotic skin lesion in humans. Its prevalencethroughout West Africa appears to have increased dramati-cally since the late 1980s (35). The organism is unlike othermycobacterial pathogens in that it appears to maintain anextracellular location during infection (23). The disease is usu-ally treated by surgical excision of infected and surroundingtissue, as the organism in situ is unresponsive to drug therapy(31). Possible explanations for the increased occurrence of thisdisease include environmental changes that have led to prolif-eration of the organism followed by increased human contact(22, 30) and adaptation of the organism to a changed environ-ment and coincidental acquisition of increased virulence. De-spite several extensive investigations over the past 30 years, themode of transmission of M. ulcerans has not been determined(2, 46). Recent detection of M. ulcerans-specific DNA se-quences in water from swamps in southeastern Australia andaquatic insects in Benin have confirmed that it is an environ-mental organism (47, 53, 60).

The etiology and epidemiology of Mycobacterium marinumare much better understood. It has long been recognized as afish pathogen and has been isolated from swimming pools, fishaquaria, and marine environments worldwide (12, 15, 25). It is

an intracellular pathogen, and in humans it usually causes alimited granulomatous skin infection at the extremities,probably via direct inoculation at the site of minor cuts andabrasions (15, 17). The infection can usually be treated withantimycobacterial drugs (19). M. marinum is relatively fastgrowing, has nonfastidious growth requirements, and producesa light-inducible pigment, presumably for protection againstincident UV irradiation (50). The picture built up from thesefindings is one of a widespread and robust environmental or-ganism which is capable of withstanding some of the extremesof aquatic environments such as sunlight exposure, varyingtemperatures, and nutrient limitation. Conversely, the profileof M. ulcerans includes a worldwide but highly focal environ-mental distribution, slow growth, UV sensitivity, optimal growthunder microaerophilic conditions, and the production of anunusual cytotoxic type I polyketide (18, 40, 45; W. M. Meyers,personal communication). These characteristics suggest an or-ganism that has adapted to a specific environmental niche.

Several studies have highlighted an apparently paradoxicalrelationship between these two species, where their strikingphenotypic differences are contradicted by a high degree ofgenetic similarity. It has been known for some time that M. ul-cerans and M. marinum have identical signature sequencesthrough the two hypervariable regions of the 16S rRNA gene(6, 52) and that the only sequence differences within this locusare two nucleotides at the 39 end of the gene (48, 64). Fur-thermore, the nucleotide at one of these positions varies fromthat in M. marinum in only some strains of M. ulcerans (48).

* Corresponding author. Mailing address: Bacterial PathogenesisResearch Group, Department of Microbiology, P.O. Box 53, MonashUniversity, Victoria 3800, Australia. Phone: 61 3 9905 4809. Fax: 61 39905 4811. E-mail: tim.stinear@med.monash.edu.au.

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Sequence analysis of a partial groEL fragment (51) and anal-yses of cell wall mycolate composition (11, 64) have also con-firmed the close genetic relationship between these species.However, DNA-DNA hybridization studies have shown a rel-ative binding ratio of approximately 37% between M. ulceransand M. marinum strains (64). This does suggest that there is afundamental genetic basis for the significant phenotypic differ-ences observed. Recently, two high-copy-number insertion se-quences, IS2404 and IS2606, were identified in M. ulcerans(59). Neither of these elements was present in M. marinum, butthey were present in M. ulcerans isolates collected from aroundthe world (58). Thus, the presence of these sequences appearsto be a defining and important characteristic of M. ulcerans.

Our hypothesis is that M. ulcerans has recently divergedfrom M. marinum by the recruitment of foreign DNA from theenvironment. Such a scenario is in accord with the mosaicgenome structure identified within other mycobacteria (43)and their ability to evolve rapidly by the transposition of in-

sertion sequences, such as IS6110 in Mycobacterium tuberculo-sis (62), IS900 in Mycobacterium avium subsp. paratuberculosis(20), and IS1512 in Mycobacterium gordonae (44).

In the current study, our overall aim was to learn more aboutthe emergence of M. ulcerans as a pathogen by comparing itat a genetic level with M. marinum. This was accomplished byemploying multilocus sequence typing, two-dimensional pulsed-field gel electrophoresis (PFGE), and restriction fragment hy-bridization analysis to compare both structural and sequencecompositions of the genomes of these species.

MATERIALS AND METHODS

Bacterial strains. The details of the 18 M. ulcerans isolates and 22 M. marinumisolates used in this study are listed in Table 1. Culture media and conditionswere as previously described (59).

Multilocus sequence analysis. PCR was used to amplify internal fragmentsfrom eight genes in M. ulcerans and M. marinum. The oligonucleotide primers foramplification of the rrs, groEL, sod, and fbpA loci were those used previously (48,55, 61, 69) (Table 2). Primers for adk, aroE, and ppk were designed by alignment

TABLE 1. Strain information

Species Strain Yr isolated Origin Sourcea 2426 typeb Sequence type

M. ulcerans 144727 1989 Victoria, Australia VIDRL Victorian VictorianATCC 19423 1948 Victoria, Australia ATCC Victorian Victorian11878/70 1971 Papua New Guinea QDRL PNG(I)c SE Asian

MD94-1331 1994 Papua New Guinea ITM PNG(II)c SE Asian13822/70 1971 North Queensland, Australia QDRL Queensland SE Asian

MD94-1328 1994 Malaysia ITM Malaysian SE Asian186510 1992 Malaysia VIDRL Malaysian SE Asian96-658 1996 Angola ITM African African94-856 1994 Benin ITM African African97-111 1997 Benin ITM African African5152 1976 Congo ITM African African97-610 1997 Ghana ITM African African97-680 1997 Togo ITM African African98-912 1997 China ITM Asian AsianATCC 33728 1980 Japan (also called M. shinshuense) ITM Asian Asian5114 1953 Mexico ITM Mexico Mexican5143 1967 Mexico ITM Mexican Mexican842 1986 Surinam ITM Surinam SurinamNCTC 2275 1926 Saltwater fish, Philadelphia (same as ATCC 927) NCTC I

M. marinum ATCC 11565 1958 Human, Sweden ATCC I99/84 1999 Bilby, western Australia PC I99/88 1993 Human, western Australia PC IMon10 1996 Human, Philadelphia, Pa. RML I472 1993 Water, Norway RML IJKD2394 1998 Human, Victoria, Australia VIDRL II991831797 1999 Human, New South Wales, Australia ICPMR II471 Human, Norway RML III99/87 1996 Human, western Australia PC IV993362605 1999 Human, New South Wales, Australia ICPMR IV99/86 1993 Human, Tasmania, Australia PC V99/89 1994 Human, Tasmania, Australia PC V99/90 1997 Human, Tasmania, Australia PC VJKD2395 1998 Human, Victoria, Australia VIDRL VJKD2396 1998 Human, Victoria, Australia VIDRL VJKD2397 1998 Human, Victoria, Australia VIDRL V0500525 1999 Human, Canberra, Australia ICPMR V0412214 1999 Human, New South Wales, Australia ICPMR V1542578 1999 Human, New South Wales, Australia ICPMR V992092077 1999 Human, New South Wales, Australia ICPMR V991961552 1999 Human, New South Wales, Australia ICPMR V

a VIDRL, Victorian Infectious Diseases Reference Laboratory; QDRLMD, Queensland Diagnostic and Reference Laboratory for Mycobacterial Diseases; ITM,Institute for Tropical Medicine; PC, Western Australian Centre for Pathology and Medical Research; RML, NIH/NIAID/DIR Rocky Mountain Laboratories; ICPMR,Institute of Clinical Pathology and Medical Research.

b 2426 type, genotype designation as determined by 2426-PCR (58).c PNG(I) and PNG(II), Papua New Guinea 2426 types (I) and (II), respectively.

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of sequences obtained from the Mycobacterium leprae and M. tuberculosis ge-nome databases (10; http://www.sanger.ac.uk/Projects/M_leprae/blast_server.shtml). It was reasoned that regions of sequence conservation between these twodistantly related mycobacteria would permit the design of genus-level primers.The names of each of the eight genes, the putative gene products, and thepositions sequenced are given in Table 2. GenBank accession numbers are alsogiven in Table 2 for the sequences obtained from the type strains of M. ulceransand M. marinum. The sequences obtained from the other 38 isolates have alsobeen deposited in GenBank. The accession numbers for these additional se-quences are available from the authors or by searching GenBank.

DNA extraction and PCR. Mycobacterial DNA was extracted from 5 to 25 mg(wet weight) of cell pellet by glass bead cell homogenization in the presence ofTriton X-100 and chloroform-isoamyl alcohol (24:1) as previously described (58).A 2-ml volume of the Triton X-100 aqueous phase was then used as a templatefor PCR. Reaction conditions used for the PCR amplification of all fragmentswere as follows: each reaction mixture (50 ml) contained 13 PCR buffer II (103PCR buffer II contained 500 mM KCl, 100 mM Tris-HCl [pH 8.3]), 1.5 mMMgCl2, 0.5 mM deoxynucleoside triphosphates (dNTPs; 0.5 mM each dATP,dTTP, dCTP, and dGTP), 10% dimethyl sulfoxide, 0.5 mM each primer, and 1 Uof Ampli-Taq DNA polymerase (Applied Biosystems, Foster City, Calif.). Ther-mal cycling was performed in an FTS-960 thermal sequencer (Corbett Research,Sydney, Australia) with five cycles of 95°C for 1 min, 60°C for 1 min, and 72°C for1 min, 30 cycles of 95°C for 20 s, 58°C for 30 s, and 72°C for 45 s, followed by afinal extension step at 72°C for 5 min. The reactions were held at 4°C untilanalyzed by 1.5% agarose gel electrophoresis with ethidium bromide staining.QIAquick spin columns (Qiagen Inc., Valencia, Calif.) were used to purify thePCR products prior to cycle sequencing. The products were sequenced on bothstrands with the primers used for PCR, according to the protocols supplied withthe Prism Big Dye Terminator Cycle Sequencing Ready Reaction kit (AppliedBiosystems). Extension products were analyzed with a PE Applied Biosystemsmodel 373 automated sequencer, and the sequences were compiled with Se-quencher 3.1.1 software (Gene Codes Corporation).

Nucleotide sequence analysis. Strains were grouped according to their com-bination of alleles, and each unique allelic pattern was identified as a sequencetype (genotype). A representative strain from each genotype was then selectedfor phylogenetic analysis. The sequences from the seven protein-encoding lociwere concatenated in frame to produce a 2,853-bp semantide for each genotype,which were aligned with Clustal W (63). Phylogenetic analysis was performedwith MEGA software version 1.1.2 (33) and Splits Tree version 3.1 (26). Pdistances were used throughout, as the overall level of sequence divergence wassmall. Values for synonymous (dS) and nonsynonymous (dN) mutation frequen-cies were calculated with Nei and Gojobori’s method (38), and standard errors ofthe means of these values were estimated by the method of Nei and Jin (39). Allcalculations of dS and dN were performed using the dSdNqw program (14). TheG1C% at each codon position was determined using Web-based software (Mur-doch University Bioinformatics Research Institute, http://arginine.it.murdoch.edu.au/research).

PFGE. Mycobacterial DNA plugs were prepared as previously described (54)with the following modifications. Ampicillin and D-cycloserine were added to theculture 24 h prior to harvesting at final concentrations of 0.1 and 1.0 mg/ml,respectively (8). The step requiring vortexing of the cells in the presence of 3-mmglass beads was omitted, and the Bio-Rad Genepath wash solution was replacedwith TE buffer (10 mM Tris, 1 mM EDTA [pH 8.0]). Restriction endoucleasedigestion of the DNA in the plugs was performed as described previously (42).For DraI digestion, MgCl2 was added to a final concentration of 10 mM. First-and second-dimension PFGE were performed using the Bio-Rad CHEF DRIIsystem (Bio-Rad, Richmond, Calif.) with 1.0% agarose in 0.53 Tris-borate-EDTA (TBE) at 200 V, with 10 to 35 s switching times for 25 h. DNA wasvisualized by staining with ethidium bromide (0.5 mg/ml) overnight at 4°C. South-ern hybridization analysis was performed as described previously (59), and DNArestriction fragment sizes from both PFGE and Southern blots were estimatedwith Sigmagel software (Jandel Scientific).

RESULTS

Multilocus sequence typing. A collection of 18 M. ulceransisolates and 22 M. marinum isolates was used in this study(Table 1). These isolates originated from a variety of sourcesand represent both temporal and geographic diversity. Themajority of the isolates were of human origin. However, amongthe M. marinum strains, one was isolated from a fish, anotherfrom a bilby (Macrotis lagotis, a small Australian native mar-supial), and another from water (Table 1). For the sequencetyping, a panel of seven unlinked genes were used (see thehybridization results below). The 39 region of the 16S rRNAgene from each isolate was also sequenced, but only the datafrom the seven protein-encoding loci were included in thesubsequent phylogenetic analyses. The allelic profiles for some

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isolates differed at more than three of the seven loci, so phy-logeny was inferred by using a distance method rather than apairwise comparison of the allelic profiles (56). The sequencesfrom the seven loci were concatenated in the order crtB, adk,fbpA, aroE, groEL, ppk, and sod to produce a 951-codon se-mantide.

The 40 isolates were represented by 11 different genotypes,where a unique combination of the seven alleles defined a par-ticular genotype. A summary of all the variable sites for eachgenotype and the division between synonymous and nonsyn-onymous substitutions is shown in Fig. 1. Five M. marinumgenotypes were identified and named types I to V (Table 1,Fig. 1). There was no obvious correlation between strain originand genotype, although no genotype IV or V isolates weredetected among the strains obtained from the Northern Hemi-sphere. There were six M. ulcerans genotypes, and in accordwith previous studies, these were named according to theirgeographic origin. There was only one variable position acrossall eight loci that discriminated between the species. This sitewas within the fbpA gene at position 1128 of the concatenatedsequences (Fig. 1). As has been reported previously, no vari-ation was detected in the 39 region of the 16S rRNA gene forany of the M. marinum isolates, and there were five alleles ofthe gene among the M. ulcerans strains (48, 64).

M. ulcerans and M. marinum have been shown by 16S rRNAanalysis to be most closely related to M. tuberculosis (64). Thepercent nucleotide identity between M. ulcerans ATCC 19423,M. marinum NCTC 2275, and M. tuberculosis H37Rv was cal-culated at each locus to indicate the general relatedness be-tween each species. Identity scores ranged from 96.3 to 99.6%(average, 98.7%) between M. ulcerans and M. marinum, com-pared to 77.2 to 99.3% (average, 86.9%) between M. ulceransor M. marinum and M. tuberculosis.

Split decomposition analysis was used to examine the phy-logenetic relationship between the M. marinum and M. ulcer-ans strains. The treelike structure shown in the splits graph andthe absence of networks (Fig. 2) are clear evidence of a bifur-cating phylogeny. These observations, combined with a highlevel of statistical support for each node in the splits graph andcomplete congruence with a dendrogram derived by the neigh-bor-joining method (data not shown), provide good evidencefor an evolutionary link between M. ulcerans and M. marinumvia a series of de novo point mutations within each locus.M. marinum could be categorized into two distinct and diver-gent groups (I and II versus III, IV, and V). The discreteclustering of all M. ulcerans strains suggests that M. ulcerans isa derivative of an M. marinum type III, IV, or V ancestor.There was also significantly less sequence variation within theM. ulcerans cluster compared to M. marinum (Fig. 1), support-ing the proposition that M. marinum is the ancestral species. Aclose genetic relationship was also evident between the south-east Asian, African, and Victorian (Australian) genotypes ofM. ulcerans (Fig. 2). This observation is in accord with previousfindings based on PCR amplification of inter-IS sequences(2426-PCR) (58). No sequence differences were detectedamong any of the African isolates. Overall, there was goodcorrelation between multilocus sequence analysis and 2426-PCR, but the 2426-PCR offered additional resolution amongisolates of the southeast Asian genotype (Table 1).

Synonymous and nonsynonymous substitution frequencies.A high frequency of nonsynonymous substitutions (dN) com-pared to synonymous substitutions (dS) within a particulargene or locus can indicate the presence of positive selectionpressure (16, 65). From the data presented in Fig. 1, thisdifference (dS 2 dN) was calculated across all loci for bothspecies. For the M. marinum genotypes, the value for dS 2 dN

FIG

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iththe

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was 2.8 6 0.5 (z 5 5.57, P , 0.001, dS 5 3.0 6 0.5, dN 5 0.2 60.08). That is, the frequency of synonymous mutation wassignificantly higher than the nonsynonymous mutation fre-quency, suggesting that there is no obvious selection pressure.However, among the M. ulcerans genotypes, the value for dS 2dN of 0.32 6 0.18 (z 5 1.76, P . 0.05, dS 5 0.54 6 0.17, dN 50.22 6 0.07) was much lower, and the dS and dN values werenot significantly different. Expressed another way, the ratio ofdN to dS was 6.8 times higher in M. ulcerans than in M. mari-num, suggesting the presence of positive or purifying selectionpressure acting on M. ulcerans. This observation lends sup-port to a theory that M. ulcerans has adapted to a changed orchanging environment, particularly given that the two speciesappear to have a common genetic backbone and thereforeshould exhibit similar theoretical mutation rates. The presenceof five rrs alleles among the six M. ulcerans strains comparedwith only a single rrs allele for all the M. marinum genotypes isalso consistent with an organism in a state of evolutionary fluxand adaptation.

The evolutionary age of M. ulcerans was estimated by deter-mining dS across the 951 codons of the seven loci (rrs exclud-ed). By using previous estimates of bacterial synonymous sub-stitution rates of 0.58 to 0.78 substitutions per 100 sites permillion years (32), the time needed to accumulate the amountof synonymous mutation observed within the M. ulcerans ge-notypes was calculated. This analysis indicated that M. ulceransemerged between 470,000 and 1,200,000 years ago. To checkthat there were no codon biases, which can indicate reducedrates of substitution (7), the GC content at the third codonposition (GC3%) was compared with the overall GC contentfor each genotype across both species. The values obtained(average GC% 5 65.5, standard deviation [sd] 5 0.1; averageGC3% 5 85.9, sd 5 0.1) were very similar to those reported forM. tuberculosis, suggesting that the rate at which M. ulceransand M. marinum accumulate synonymous substitutions is thesame as that observed in M. tuberculosis (4). This estimateassumes that there are no significant in vivo growth rate dif-ferences between species. However, fluctuations in growthrates have been suggested to be inconsequential over a geo-logical time scale and given actual environmental generationtimes (37).

Comparisons of genome structure. To further investigatethe hypothesis that M. ulcerans has recently diverged fromM. marinum, a southeast Asian isolate of M. ulcerans (isolate13822/70) and a type V isolate of M. marinum (isolate 99/86)were selected for genome structure comparisons.

PFGE was used to compare macrorestriction fragment pat-terns and to obtain estimates of the genome sizes. The restric-tion enzymes PacI, PmeI and SwaI, which have eight-base AT-rich recognition sites, were tried first in an attempt to obtain asimple pattern of fragments that would permit straightforwardgenome size estimations. Unfortunately, these enzymes failedto cut the genome of either M. marinum or M. ulcerans. AseIand DraI gave the most useful array of fragments (Fig. 3). Noplasmid bands were detected in either isolate (Fig. 4A). How-ever, with these enzymes there were probable doublets and areasof significant compression that prevented accurate sizing. Theseregions could not be resolved satisfactorily with altered elec-trophoretic separation parameters. Two-dimensional PFGEwas used to improve resolution. Reciprocal AseI and DraI di-gests were performed for each organism, and these are shownin Fig. 5. Indicative genome sizes were obtained by summingthe averages of the AseI and DraI restriction fragments lengthestimates from both one- and two-dimensional pulsed-fieldarrays (Table 3). This indicated a genome size for M. ulceransof approximately 4.4 Mb and a slightly larger genome forM. marinum of approximately 4.6 Mb. This latter figure iscomparable to other genome size estimates for M. marinum(1).

From the one-dimensional pulsed-field patterns, there ap-peared to be little similarity in AseI and DraI restriction pat-terns between strains. One explanation for observing nucleo-tide sequence similarity with genomic structural diversity is the

FIG. 2. Splits graph of the phylogenetic relationship among the six M. ulcerans and five M. marinum genotypes. The vertices are labeled with each genotype. (MM,M. marinum; MU, M. ulcerans). The graph was generated from the concatenated sequences of the seven protein-encoding loci. All edges in the graph had greater than80% bootstrap support (1,000 iterations) with the exception of the edges marked with an asterisk. These edges had greater than 60% bootstrap support.

FIG. 3. PFGE analysis of genomic DNA from M. marinum 99/86 (lanes 1 and2) and M. ulcerans 13822/70 (lanes 3 and 4) digested with AseI (lanes 1 and 3) andDraI (lanes 2 and 4). Lanes M, 50-kb lambda DNA size ladder.

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presence of mobile DNA in one or both species. Insertionsequences are well known to promote genome rearrangements(34), and IS2404 and IS2606 are two elements present in M. ul-cerans but absent from M. marinum that could act as substratesfor such rearrangements. Hybridization of IS2404 and IS2606probes against M. ulcerans digested with AseI indicated thewidespread distribution of both elements around the genome(Fig. 4B and C). As expected, M. marinum did not hybridize toeither probe. All M. marinum isolates were also screened byPCR and found not to contain either IS2404 or IS2606 (datanot shown).

If, as suggested by the restricted sequence polymorphism,large-scale genome rearrangements have occurred recently,then some preservation of genomic subarchitecture could beexpected between each species. The restriction enzymes NcoI,PvuII, and PstI were predicted to cut no more than once withinthe entire coding region of each gene used for multilocussequence analysis. When full-length M. ulcerans or M. mari-num gene sequences were not available, this prediction wasbased on the M. tuberculosis genome sequences (10). Theseenzymes were then used to digest genomic DNA from M. ma-rinum and M. ulcerans. The DNA was hybridized against probes

from each of the eight loci described above, and the sizes of thehybridizing fragments were estimated and compared. All lociappeared to hybridize to different-sized fragments for all threeenzymes, indicating that none of the targets selected for mul-tilocus testing were linked. A significant degree of conservationof the DNA flanking most of the loci between the two specieswas revealed (Fig. 6). One exception was the 16S rRNA locus,for which multiple polymorphisms were detected with all threeenzymes. The presence of two hybridizing fragments with eachenzyme against M. marinum DNA suggests that M. marinummay possess at least two copies of the rRNA operon. Multiplebands also hybridized to the probes derived from the fbpA andaroE genes. However, from an analysis of the M. tuberculosisgenome, the presence of these bands is probably due to cross-hybridization with other genes of similar sequence, such asfbpC and other dehydrogenase genes.

DISCUSSION

In this study we have used multilocus sequence analysis toclearly establish for the first time the population structure ofand evolutionary relationship between M. ulcerans and M. ma-rinum. The data we have gathered suggest the recent diver-gence of M. ulcerans from an M. marinum progenitor. Overall,M. marinum and M. ulcerans have very high nucleotide homol-

FIG. 4. PFGE (A) and Southern hybridization (B and C) analyses of M. ma-rinum 99/86 (lanes 1 and 3) and M. ulcerans 13822/70 (lanes 2 and 4), probed withIS2606 (B) and IS2404 (C). Lanes 1 and 2, AseI digest; lanes 3 and 4, undigestedDNA; lane M, 50-kb lambda DNA size ladder.

FIG. 5. Two-dimensional PFGE analysis of genomic DNA from M. ulcerans13822/70 (A and B) and from M. marinum 99/86 (C and D), reciprocally digestedwith the restriction enzymes AseI and DraI as indicated on each panel. Lanes 1,2, 4, and 6, first-dimension separations of genomic DNA digested with therestriction enzyme AseI; lanes 3 and 5, first-dimension separations of genomicDNA digested with the restriction enzyme DraI; lane M, 50-kb lambda DNA sizeladder.

TABLE 3. Estimated sizes of restriction fragments from AseI andDraI digests of M. marinum and M. ulcerans

AseI DraI

M. marinum M. ulcerans M. marinum M. ulcerans

Frag-ment

Size(kb)

Frag-ment

Size(kb)

Frag-ment

Size(kb)

Frag-ment

Size(kb)

A 441 A1 510 A 924 A1 1,044B1 248 A2 510 B 531 B 490B2 248 B 370 C1 420 C1 412C 245 C 330 C2 420 C2 412D1 240 D 278 D 370 D 360D2 240 E 245 E 340 E1 250E 220 F 216 F 235 E2 240F 208 G 200 G 206 F 205G 200 H1 170 H 201 G 140H 180 H2 167 I 187 H 129I 175 I 160 J 163 I 122J 170 J1 150 K 117 J 104K 160 J2 147 L 106 K 95L 155 K 134 M 99 L 87M 148 L 120 N 84 O 78N 137 M1 109 O 70 M 72O 132 M2 107 P 47 N 49P 112 N 104 Q 40 O 43Q1 103 O1 75 R1 31 P1 41Q2 102 O2 74 R2 28 P2 37R1 92 P1 63 S 20 Q 6R2 92 P2 62 T 7 Total 4,416S1 78 Q 43 Total 4,646S2 76 R1 40S3 75 R2 29T 71 R3 22U 55 S 5V 45 T 2W1 40 Total 4,442W2 36X 28Y 10Z 9

Total 4,571

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ogy. Their close genetic relationship is highlighted by the pres-ence of only one species-discriminating variable site among the3,306 bp from the eight loci (Fig. 1). The level of intraspeciesnucleotide sequence divergence was higher between M. mari-num strains than M. ulcerans strains, and this observation cor-relates well with previous DNA-DNA hybridization studies(64). An increased level of nucleotide sequence divergence andthe absence of IS2404 and IS2606 from all M. marinum strainsare the expected states for the ancestral species of M. ulcerans.

Insertion sequences and other repetitive DNA elements playan important role in mycobacterial genetics (13, 49). In M. tu-berculosis, IS6110 is responsible for the rapid evolution ofdistinct clones (57). Similarly, IS900 and IS901/902 are defin-ing characteristics for M. avium subsp. paratuberculosis andM. avium subsp. silvaticum, organisms with a high degree ofgenetic identity to the M. avium complex (20). M. ulcerans hasacquired at least two IS elements, IS2404 and IS2606, and theirpattern of widespread genome distribution and high copy num-ber indicate the potential for these elements to act as sub-strates for ongoing genome rearrangements. The detection ofvariations in inter-IS distances between strains of M. ulcerans isevidence of such rearrangements (58).

Interestingly, both IS2404 and IS2606 are related to ele-ments in the genus Streptomyces. The transposase from IS2404has 31% amino acid identity (45% amino acid similarity) withthat from IS1629, an IS associated with mobilization of thenec1 virulence determinant in plant-pathogenic strains of var-ious Streptomyces spp. (24). Recently, a homolog of IS2606 hasbeen identified in Streptomyces albus. The putative transposasefrom this IS has 47% amino acid identity (57% amino acidsimilarity) with that from IS2606 (C. M. Smith, personal com-

munication). The transposition of an IS from Streptomyces coe-licolor into a mycobacterial genome has been demonstrated (5).

While the IS elements may play an important role in pro-moting rearrangements and modifying gene expression, thepresence of the unusual type 1 polyketide mycolactone (18) inM. ulcerans means that it is unlikely that IS2404 and IS2606are the only sequences that M. ulcerans has acquired. A largeamount of specific genetic material is predicted to be requiredfor the synthesis of this molecule. From the M. tuberculosisgenome sequence data, mycobacteria are known to containseveral polyketide synthase operons, but none of these operonsresemble the predicted modular composition of the genes re-quired to synthesize mycolactone (10). It is possible that M. ul-cerans may have appropriated an additional polyketide syn-thase locus, and interestingly, the streptomycetes are a richsource of these enzymes (68). We are currently performinggenomic subtractions between M. ulcerans and M. marinum toidentify additional M. ulcerans-specific sequences.

Environmental PCR-based surveys have shown that M. ul-cerans is present in water and detrital material from swamps inM. ulcerans-endemic areas in southeastern Australia (53, 60).In West Africa, aquatic insects appear to be a source of theorganism rather than water or plant material (47). These datasuggest that M. ulcerans may occupy different environmentalniches in different geographical regions. The multilocus se-quencing data (Fig. 1 and 2) and previous molecular typingstudies (29, 48, 58) have demonstrated unique genotypes with-in a geographic region. Variations in genotype according to lo-cale also correlate with phenotypic differences between strains.For example, there are consistent growth rate differences be-tween the African and Australian isolates (41). Combining the

FIG. 6. Southern hybridization analysis of genomic DNA from M. marinum 99/86 (lanes 1, 2, and 3) and from M. ulcerans 13822/70 (lanes 4, 5, and 6). The DNAwas digested with the restriction enzymes NcoI (lanes 1 and 4), PvuII (lanes 2 and 5), and PstI (lanes 3 and 6) and then probed with sequences derived from each locusas indicated. Lane M, lambda HindIII-digested DNA size markers.

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findings from the environmental surveys, the genotype data,and the phenotype data, it appears likely that M. ulcerans isadapting to the unique conditions of a particular region. Thepresence of multiple 16S rRNA alleles also suggests that strainsmay be in the process of local adaptation. Point mutationswithin the rRNA operon of mycobacteria that have only asingle copy of this operon can confer significant biologicaleffects, such as antibiotic resistance (66).

The PFGE data demonstrated that the M. ulcerans genomewas approximately 200 kb smaller than that of M. marinum.Considering that the M. ulcerans genome contains approxi-mately 180 kb of DNA not present in M. marinum (based on 40copies of IS2606 and 50 to 100 copies of IS2404) (59), there islikely to be at least 380 kb of difference in genetic materialbetween these species. Therefore, in addition to M. ulcerans’shaving acquired DNA, it may have also undergone a deletionevent(s). Other evidence that might suggest deletion of geneticmaterial includes the presence of only a single copy of the 16SrRNA gene in M. ulcerans compared to two copies in M. ma-rinum. This observation may also explain the substantialgrowth rate differences observed between these species. Italso suggests that slow growth may be of selective advantageto M. ulcerans. These advantages may include facilitation ofgrowth as an endosymbiont (9, 28) and survival under nutrient-poor conditions (27). The presence of two copies of the rRNAoperon in M. marinum also has taxonomic implications for itscurrent classification as a slow-growing species (67).

M. ulcerans may perhaps best be thought of as an ecotype ofM. marinum, that is, an M. marinum progenitor genotype thathas adapted to a particular ecological niche (36). The presenceof unique M. ulcerans genotypes or subecotypes based on geo-graphic origin represents the continuing evolution and adap-tation of the organism to varying environments. This wouldexplain the general process by which isolates from temperateregions of southeastern Australia have evolved differently fromstrains inhabiting tropical regions.

It has been proposed that M. ulcerans is a legacy of themicrobial ecology from the Jurassic Period and that its globaldistribution can be attributed to the breakup of the supercon-tinents 150 million years ago (21). However, the global historyof M. ulcerans suggested by this study is one of the organism’soriginating less than 1.2 million years ago and then spreadingthroughout the world. The absence of any sequence differencesor inter-IS variation (58) among African strains of M. ulceransis evidence of even more recent distribution of the organismacross this continent. The level of nucleotide sequence varia-tion observed among isolates from Africa is the same as thatreported for M. tuberculosis globally (57), and thus it appearsthat the African strain may have arisen in the past 18,000 years.Multilocus analysis of more strains from Africa would confirmthis proposition.

Future work should now be directed towards whole-genomestudies of M. ulcerans and M. marinum using microarray-basedcomparative techniques similar to those recently applied tostrains of Mycobacterium bovis BCG (3). Whole-genome com-parisons should reveal the fundamentals of pathogenesis ineach of these species, particularly given their close geneticrelationship and contrasting phenotypes.

ACKNOWLEDGMENTS

We are grateful to Françoise Portaels, Pam Small, William Chew,David Dawson, Aina Sievers, and Frank Haverkort for the provision ofmycobacterial isolates. We also thank Carol Smith and Wayne Meyersfor the provision of unpublished data.

This work was supported by a grant from the Australian ResearchCouncil.

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