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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:
[email protected].
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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
TA
BL
E2.
Olig
onuc
leot
ides
used
for
PCR
ampl
ifica
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and
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ese
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tern
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.mar
inum
Olig
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tide
Sequ
ence
,593
39E
xpec
ted
PCR
prod
uct
size
and
puta
tive
gene
func
tion
Ref
er-
ence
Nuc
leot
ide
posi
tions
sequ
ence
da
Gen
Ban
kac
cess
ion
no.b
adk-
P1G
(GT
)AT
CC
CG
CA
GA
TC
TC
CA
CC
adk-
P11
adk-
P2,a
mpl
ifica
tion
ofa
442-
bppr
oduc
tfr
omad
k(a
deny
late
kina
se)
Thi
sst
udy
114–
486
AF
2710
93ad
k-P2
CA
C(C
T)T
CG
TC
CA
TG
GT
GC
CG
AA
F27
1342
aroE
-P1
CC
CG
GT
GA
AC
TG
CT
CC
AC
CT
aroE
-P1
1ar
oE-P
2,am
plifi
catio
nof
a46
7-bp
prod
uct
from
aroE
(shi
kim
ate
dehy
drog
enas
e)T
his
stud
y30
4–74
8A
F27
1094
aroE
-P2
TG
GC
GG
GC
CG
AC
AA
CA
CC
GA
AF
2713
43cr
tB-P
1C
GA
CG
AC
AT
TC
TG
GA
CT
CC
Tcr
tB-P
11
crtB
-P2,
ampl
ifica
tion
ofa
469-
bppr
oduc
tfr
omcr
tB(p
hyto
ene
synt
hase
)T
his
stud
y18
4–63
8A
F27
1095
crtB
-P2
GA
CA
CC
AC
AT
CA
GC
AC
AT
CC
AF
2713
44M
T1
TT
CC
TG
AC
CA
GC
GA
GC
TG
CC
GM
T1
1M
T2,
ampl
ifica
tion
ofa
508-
bppr
oduc
tfr
omfb
pA(3
2-kD
asu
rfac
ean
tigen
)55
476–
893
AF
2710
92M
T2
CC
CC
AG
TA
CT
CC
CA
GC
TG
TG
CA
F27
1345
Tb1
1A
CC
AA
CG
AT
GG
TG
TG
TC
CA
TT
b11
1T
b12,
ampl
ifica
tion
ofa
439-
bppr
oduc
tfr
omgr
oEL
(65-
kDa
heat
shoc
kpr
otei
n)61
159–
540
AF
2710
96T
b12
CT
TG
TC
GA
AC
CG
CA
TA
CC
CT
AF
2713
4610
04R
AG
GA
AT
TC
TG
GG
TT
TG
AC
AT
GC
AC
AG
GA
1004
R1
rRog
,am
plifi
catio
nof
a51
7-bp
prod
uct
from
rrs
(39
regi
onof
the
16S
rRN
Age
ne)
4810
38–1
491
AF
2730
2rR
ogA
AG
GA
GG
TG
AT
CC
AG
CC
GC
AA
F27
1347
ppk-
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GT
TG
CT
GC
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CG
TG
AG
Cpp
k-P1
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,am
plifi
catio
nof
a42
1-bp
prod
uct
from
ppk
(pol
ypho
spha
teki
nase
)T
his
stud
y99
9–13
95A
F27
1097
ppk-
P2G
AT
GT
TG
GC
CT
GC
TC
GT
CA
F27
1348
Z21
2T
CG
(GT
)CC
CA
GT
TC
AC
GA
C(G
A)T
TC
CA
Z21
21
Z26
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plifi
catio
nof
a43
4-bp
prod
uct
from
sod
(sup
erox
ide
dism
utas
e)69
144–
534
AF
2710
98Z
261
CC
AA
(AG
)CT
CG
AA
GA
GG
CG
CG
(CG
)GC
CA
AA
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1349
aN
umbe
ring
base
don
M.t
uber
culo
sis
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exce
ptfo
rcr
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hich
was
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don
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ce(a
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9207
5)an
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hich
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NA
.b
Acc
essi
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ovid
edfo
rth
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ies,
M.u
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pper
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and
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.
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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
.1.
Alignm
entofthe2,853-bp
sequencesderived
fromthe
sevenconcatenated
protein-encodinglocifor
eachofthe
11genotypes.O
nlyvariable
nucleotidesare
shown,and
thenum
bersatthe
topoffigure
indicatetheir
positionsin
thesequence.A
periodindicates
identityw
iththe
M.ulcerans
Surinamstrain,and
nonsynonymous
mutations
arehighlighted
with
grayshading.
VOL. 182, 2000 GENETIC COMPARISON OF M. ULCERANS AND M. MARINUM
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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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