INTERNATIONAL JOURNAL OF SYSTEMATIC BACTERIOLOGY,
OCt. 1990, p. 323-330
0020-7713/90/040323-08$02.00/0
Copyright 0 1990, International Union of Microbiological Societies
Vol. 40,No. 4
Towards a Phylogeny and Definition of Species at the Molecular
Level within the Genus Mycobacterium
TILL ROGALL,l JORN WOLTERS,* THOMAS FLOHR,' AND ERIK C. BOTTGER1*
Institut fur Medizinische Mikrobiologie, Medizinische Hochschule Hannover, Konstanty-Gutschow-Strasse 8,
3000 Hannover 61,l and Institut fur Allgemeine Mikrobiologie, Christian-Albrechts- Universitat,
2300 Kiel,2 Federal Republic of Germany
16s rRNA sequences from Mycobacterium tuberculosis, M. avium, M. gastri, M. kansasii, M. murinum, M.
chelonue, M . smegmatis, M. terrae, M. gordonae, M. scrofuluceum, M . szulgai, M . intracellulare, M.
nonchromogenicum,M. xenopi, M. malmoense,M . simiae, M. flavescens, M . fortuitum, and M. paratuberculosis
were determined and compared. The sequence data were used to infer a phylogenetic tree, which provided the
basis for a systematic phylogenetic analysis of the genus Mycobacterium. The groups of slow- and fast-growing
mycobacteria could be differentiated as distinct entities. We found that M. simiae occupies phylogenetically an
intermediate position between these two groups. The phylogenetic relatedness within the slow-growing species
did not reflect the Runyon classification of photochromogenic, scotochromogenic, and nonchromogenic
mycobacteria. In general, the phylogenetic units identified by using rRNA sequences confirmed the validity of
phenotypically defined species; an exception was M. gastri, which was indistinguishable from M. kunsasii when
this kind of analysis was used.
tribution (7,35). We recently developed a general procedure
for the isolation and direct complete nucleotide determination of entire genes coding for 16s rRNA in which the
polymerase chain reaction is used (4, 5). In contrast to
sequence determinations of 16s rRNAs in which reverse
transcriptase is used (15), a procedure which is frequently
prone to sequencing anomalies resulting in a level of inaccuracy of the sequence information of roughly 5% (ll), our
method allows contiguous and highly accurate sequence
determinations. The inaccuracy of the 16s rRNA sequence
data obtained when sequences are determined by using
reverse transcriptase makes the definition of phylogenetic
relationships difficult for species which are related at a rRNA
sequence similarity level greater than 95%. To prove the
feasibility, accuracy, and usefulness of the sequencing strategy based on the polymerase chain reaction, we applied this
procedure to the genus Mycobacterium.
In this study, which is based on the almost complete 16s
rRNA sequences of 19 species of mycobacteria, we demonstrate high levels of 16s rRNA sequence similarity within the
genus Mycobacterium (greater than 94.3%); our data provide
the basis for a phylogenetically valid taxonomy of this genus.
Mycobacteria are aerobic, nonmobile bacteria that are
characteristically acid fast. The property of acid fastness,
which is due to waxy materials in cell walls, is particularly
important for recognizing mycobacteria. Members of the
genus Mycobacterium are widespread in nature and range
from soil-dwelling saprophytes to pathogens of humans and
animals (22, 34). A major descriptive division of mycobacteria is related to growth rate and pigmentation. On the basis
of these criteria, the genus Mycobacterium has been divided
into four groups. Group I consists of the photochromogenic
(pigmented) species of slow growers; members of group I1
are scotochromogenic slow growers; group I11 contains the
nonchromogenic slow growers; and group IV consists of
rapid growers (defined as maturing in less than 1 week) (22,
34).
Taxonomic analysis of the genus Mycobacterium is complicated by the fact that a variety of specialized and complex
tests must be used. Numerical taxonomic analysis, which
requires that some dozens of characters be tested (e.g.,
enzymatic activity, growth, morphology, and drug susceptibility), is now being applied to circumscription of clusters
and description of strains. Early on, the problems and
difficulties of traditional taxonomy with respect to mycobacteria were recognized, prompting a number of investigators
in the field to organize themselves into the International
Working Group on Mycobacterial Taxonomy and to undertake a number of cooperative taxonomic studies (18, 29,
31-33).
Attempts to subdivide mycobacterial species by using
immunological approaches, DNA composition, and similar
characteristics (1-3,9,10) proved to be taxonomically useful
but gave little phylogenetic information. The use of macromolecular comparisons to infer phylogenetic relationships is
generally accepted and well established. Of the macromolecules used for phylogenetic analysis, the rRNAs, in particular 16s rRNA, have proven to be the most useful for
establishing phylogenetic relationships because of their high
information content, conservative nature, and universal dis-
MATERIALS AND METHODS
Bacterial strains. Table 1 shows the strains whose 16s
rRNA sequences were determined for this study.
Determination of sequences. DNA was extracted by using
standard procedures (16). Amplification of gene fragments
coding for 16s rRNA and direct sequencing of the amplified
DNA fragments were performed as described previously (4,
5). Polymerase chain reaction-mediated synthesis was performed by using oligonucleotide AGA GTT TGA TCC TGG
CTC AG (positions 8 to 28) in combination with CCC TCA
ATT CCT TTG AGT TT (positions 928 to 908) and oligonucleotide CAG CAG CCG CGG TAA TAC (positions 518 to
536) in combination with AAG GAG GTG ATC CAG CCG
CA (positions 1542 to 1522), resulting in two overlapping
DNA fragments that covered the 16s rRNA gene. The
nucleotide positions indicated above are the target sites of
the synthetic oligonucleotides in procaryotic 16s rRNAs, as
represented by Escherichia coli. The oligonucleotide prim-
* Corresponding author.
323
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INT. J . SYST.BACTERIOL.
ROGALL ET AL.
TABLE 1. Bacteria used in this study
Species or strain
M . tuberculosis
M . bovis
M . bovis BCG
M . tuberculosis H37
M . marinum
M . kansasii DSM 43224
M . simiae ATCC 2527STb
M . scrofulaceum ATCC 19981T
M . szulgai ATCC 25799=
M . gordonae ATCC 14470T
M . xenopi ATCC 19250T
M . jlavescens ATCC 14474T
M . avium DSM 43216
M . intracellulare ATCC 15985
M . paratuberculosis ATCC 19698
M . gastri ATCC 15754T
M . malmoense ATCC 29571T
M . nonchromogenicum ATCC 19530T
M . terrae ATCC 15755=
M . chelonae ATCC 14472
M . smegmatis ATCC 14468
M . fortuitum ATCC 6841T
N . asteroides ATCC 3306
Sourcea
Runyon
group
IMM
Schroder
DSM
ATCC
ATCC
ATCC
ATCC
ATCC
ATCC
DSM
DSM
Jorgensen
ATCC
ATCC
ATCC
DSM
DSM
DSM
ATCC
DSM
I
I
I
I1
I1
I1
I1
I1
I11
I11
I11
I11
I11
I11
I11
IV
IV
IV
a ATCC, American Type Culture Collection, Rockville, Md.; DSM, Deutsche Stammsammlung fur Mikroorganismen, Braunschweig, Federal Republic of Germany; IMM, Institut fur Medizinische Mikrobiologie, Hannover,
Federal Republic of Germany; Schroder, K. H. Schroder, Forschungsinstitut,
Borstel, Federal Republic of Germany; Jorgensen, J. Jorgensen, National
Veterinary Laboratory, Copenhagen, Denmark.
T = type strain.
ers used to sequence the amplified DNA fragments have
been described previously (5). The sequencing strategy
which we used resulted in almost complete nucleotide determination of the 16s rRNA gene, in which approximately 40%
of the sequence was determined for both strands. The
complete sequences are available from the European Molecular Biology Laboratory data library (accession numbers X
52917 through X 52934) or from one of us (E.C.B.).
Data analysis. The sequences of 19 mycobacteria and
Norcardia asteriodes were aligned by using the multisequence alignment algorithm of Kriiger and Osterburg (12).
This algorithm was manually adjusted to account for common secondary structure. For the phylogenetic analysis
regions of alignment uncertainty were omitted, reducing the
number of positions from 1,481 to 1,431. Pairwise distances
were calculated by weighting nucleotide differences and
insertions-deletions equally (Hamming distance). The phylogenetic tree was constructed by using the neighborliness
method (6). This method clusters sequences according to
their neighborliness in all possible quadruples. In simulation
studies this method has been shown to be superior to simple
clustering methods, such as unweighted pair group matrix
analysis, and even superior to the distance Wagner, modified
Farries, Fitch-Margoliash, and maximum parsimony methods for recovering a given tree topology from distance data
when there is a varying rate of nucleotide substitution (20,
23). The necessary algorithms are implemented in the program package SAGE (technoma GmbH, Heidelberg, Federal
Republic of Germany) designed for IBM XTIATIPS’L computers and compatibles.
RESULTS AND DISCUSSION
The sequencing strategy used in this study generated
nearly complete 16s rRNA sequences (1,481 positions,
covering 95.4% of the 16s rRNA molecule). The 16s rRNAs
of the mycobacterial species which we investigated are
shown aligned in Fig. 1. Similarity values were calculated,
and values greater than 94.3% were obtained (Table 2). The
same level of similarity was recently reported for other
mycobacterial species by Stahl and Urbance (25). To detect
varying rates of evolution, a phylogenetic analysis was
carried out by using the neighborliness method (6). A phylogenetic tree displaying the natural relationships among
mycobacteria (Fig. 2) was constructed by using equally
weighted (Hamming) distances between sequence pairs for
19 mycobacteria by using N . asteroides as an outgroup
(Table 3).
In this scheme (Fig. 2) Mycobacterium fortuitum, M .
chelonae, M . smegmatis, and M . flavescens formed a tight
cluster that was separated from all of the other mycobacterial species investigated. This finding correlated with previously described growth characteristics; M . fortuitum, M .
chelonae, and M . smegmatis are defined as rapidly growing
mycobacteria (34), whereas M . flavescens has a growth rate
which is intermediate between the growth rates of the
rapidly growing and slowly growing acid-fast bacteria (13).
The segregation of M . flavescens into this cluster confirmed
previous phenotypic observations, as well as the results of
previous DNA-DNA hybridization studies, which indicated
that this species is very unlike other scotochromogens (9,13,
14).
Within the genus Mycobacteriurn all of the slow-growing
species were highly related (similarity values greater 94.8%),
and these species formed a shallow, heterogenous group that
was separated from the tight cluster defined by the fastgrowing species (Fig. 2). Interestingly, M. simiae, a slowgrowing species, occupied an intermediate position in our
phylogenetic scheme (Fig. 2); this organism exhibited rRNA
sequence elements that were characteristic of rapidly growing species, as well as elements that were characteristic of
slowly growing species (see below).
The phylogenetic relatedness between M. terrae and M .
nonchromogenicum at the small-subunit rRNA level confirmed the International Working Group on Mycobacterial
FIG. 1. Alignment of 16s rRNA sequences (total length, 1,481 positions) from M . fortuitum (M.fo.), M . chelonae (M.ch.), M . smegmatis
(M.sm.), M . flavescens (M.fl.), M . simiae (M.si.), M . terrae (M.te.), M . nonchromogenicum (M.no.), M . xenopi (M.xe), M . gordonae
(M.go.), M . bovis (M.bo.), M . marinum (M.mr.) M . scrofulaceum (M.sc.), M . gastri (M.ga.), M.kansasii (M.ka.), M . szulgai (M.sz.), M .
malmoense ( M d . ) , M . intracellulare (M.in.), M . paratuberculosis (M.pa.), and M . avium (M.av.). The sequence of the noncoding
(RNA-like) strand is shown. For uniformity, uridine residues were changed to thymidine. A dot or N indicates an undetermined nucleotide,
dashes indicate deletions, and nucleotides common to all mycobacterial species are indicated by asteriks. The sequences of M. tuberculosis,
M . bovis BCG, and M . tuberculosis H37 are identical to the sequence of M . bovis and therefore are not shown. Compared with the previously
published sequence of M . bovis (27), our sequence has an additional cytosine at position 1439; the lack of this residue in the previously
published sequence (27) was probably due to a sequencing or cloning artifact. The first and last nucleotides correspond to E. coli 16s rRNA
positions 38 and 1510, respectively. Only the relevant portions of the sequences are shown, and the positions of alignment uncertainty that
were omitted for the phylogenetic analysis are indicated by brackets.
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PHYLOGENY OF THE GENUS MYCOBACTERIUM
M. fo.
M. ch.
M. M.
M. f l .
M.
ci.
M. te.
M. no.
M. xe.
M. go.
M. bo.
M. mr.
M. sc.
M. ga.
M. ke.
M. cz.
M. nl.
M.
in.
M. pa.
M. av.
M. fo.
M. ch.
M.
sm.
11. 11.
M. s i .
M. te.
M. no.
M. xe.
M. go.
M. bo.
16. mr.
M. sc.
M. ga.
M. ka.
M.
92.
M.
ml.
M.
in.
M. pa.
M. av.
M. fo.
M. ch.
M. u.
M. f l .
M. s i .
re.
k. no.
M.
M.
M.
M.
M.
M.
xe.
go.
bo.
M.
sc.
M. 0..
M. to.
M. 82.
M. nl.
M.
in.
M. pa.
M. av.
M. fo.
M. ch.
M. M.
M. f l .
M. s i .
M. te.
M. no.
M. me.
M. go.
M. bo.
M. mr.
M. sc.
M. ga.
11. Ira.
M. 82.
M. nl.
M. in.
M. pa.
M. ev.
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325
326
INT. J. SYST.BACTERIOL.
ROGALL ET AL.
M. fo.
M. ch.
M. sm.
M. f l .
M. r l.
M.
M.
M.
M.
M.
M.
M.
te.
no.
M.
M.
M.
M.
98.
(1.
p..
X.
go.
bo.
IK.
ac.
ka.
az.
InI.
M. in.
M. w .
M.
M.
M.
M.
fo.
ch.
m.
11.
M. ri.
M. te.
M. m.
M. xe.
M. go.
M. bo.
M. w.
M. sc.
M. ga.
ti. ka.
M. 61.
M. m l .
M. in.
M. pa.
M. av.
FIG. 1-Continued.
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PHYLOGENY OF THE GENUS MYCOBACTERKJM
327
TABLE 2. Homology values derived from 16s rRNA sequences"
% Homology with:
M. paratuberculosis
M. intracellulare
M. malmoense
M. smlgai
M. kansasii
M. gastn'
M. scrofulaceum
M. marinum
M. bovis
M. gordonae
M. xenopi
M. nonchromogenicum
M. terrae
M. simiae
M. fravescens
M. smegmatis
M. chelonae
M. fortuitum
N. asteroides
a
99.9
99.4
99.1
99.1
99.0
99.0
98.5
98.6
98.6
98.0
95.6
96.3
96.6
96.3
94.5
95.0
95.0
95.0
92.2
99.5
99.2
99.2
99.0
99.0
98.5
98.7
98.7
98.0
95.7
96.4
96.7
96.4
94.6
95.0
95.0
95.0
92.3
99.1
99.2
99.0
99.0
98.7
98.5
98.5
97.9
95.8
96.2
96.6
96.4
94.6
95.0
94.9
94.9
92.2
99.9
99.2
99.2
98.7
98.6
98.7
98.2
95.8
96.3
96.6
96.8
94.9
95.2
94.9
95.2
92.2
99.3
99.3 100
98.7 98.8
98.7 98.7
98.9 98.8
98.2 98.1
95.9 95.7
96.3 96.1
96.8 96.7
96.8 96.8
94.9 94.7
95.2 95.1
94.9 95.0
95.2 95.1
92.3 92.2
98.8
98.7
98.8
98.1
95.7
96.1
96.7
96.8
94.7
95.1
95.0
95.1
92.2
98.2
98.0
97.6
95.4
96.0
96.2
97.0
94.9
95.4
95.3
95.4
92.4
99.4
97.9
96.1
96.7
97.5
96.0
94.9
95.4
95.6
95.9
92.3
97.8
96.1
96.6
97.5
96.1
95.1
95.5
95.4
95.7
92.2
95.6
96.5
97.3
96.7
95.2
95.5
95.3
95.4
92.9
95.8
96.3
94.8
94.5
94.7
94.3
94.8
91.7
98.4
95.5
96.1
96.0
96.2
94.4
92.5
96.0
96.3
96.4
96.1
96.7
92.9
96.9
96.8
96.6
96.8
94.3
99.2
97.6 97.4
98.2 97.9 99.0
94.3 93.9 95.0 94.8
Values were based on the data shown in Fig. 1; 1,431 nucleotides were used.
M. scrofulaceum could not be clearly separated from the
M. avium group by numerical classification; thus, the M.
avium-M. intracellulare-M. scrofulaceum complex was
formed (28). Phylogenetically, M. scrofulaceum is clearly
separated from M. avium and M. intracellulare.
M. malmoense has been described as a species that is
different from other species (10, 30, 33). Phylogenetically,
M. malmoense, a nonchromogen, is closely related to M.
szulgai, a scotochromogen, as demonstrated by a level of
16s rRNA similarity of 99.9%. Consequently, these two
species form a tight subgroup.
Taxonomy finding that these two species resemble each
other but are distinguishable (18). M. terrae and M. nonchromogenicum formed a tight subgroup with individual lines of
descent and a common root. M. xenopi emerged phylogenetically as a clearly distinct species, as indicated by its
isolated position among the slow-growing species.
The remaining slow-growing mycobacteria (M. gordonae,
M. bovis, M. marinum, M. scrofulaceum, M. gastri, M.
kansasii, M. szulgai, M. malmoense, M. intracellulare, M.
paratuberculosis, M. avium) were grouped together and had
a common line of descent.
I
//
M. foRuitum
M. chelonae
RAPID GROWERS
- - - - - - - -ns
-----------
-
M. simiae
a
SLOW
GROWTH
-
I
M. terrae
SLOW GROWERS
M. nonchromogenicum
M. xenopi
a
LONG
HELIX
(Pas. 390-W)
1
M. Qordonae
M. bovis
M. marinum
M. scrofuiaceum
M. Qastri / M. kansasii
-
M. lntracelluiare
M. paratuberculosis
FIG. 2. Phylogenetic tree showing the relationships of species belonging to the genus Mycobacterium. This tree was constructed by using
the neighborliness method (6). The tree was rooted by using N. asteriodes as an outgroup. Bar = 10 nucleotide differences. pos., Positions.
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INT. J. SYST.BACTERIOL.
ROGALL ET AL.
TABLE 3. Hamming distances derived from 16s rRNA sequencesa
Hamming distances with:
Species
N. asteroides
M. fortuitum
M. chelonae
M. smegmatis
M. flavescens
M. simiae
M. terrae
M. nonchromogenicum
M. xenopi
M. gordonae
M. bovis
M. marinum
M. scrofulaceum
M. gastri
M. kansasii
M. szulgai
M. malmoense
M. intracellulare
M. paratuberculosis
75
72
15
88
30
37
82
26
34
12
81
46
49
45
44
102
47
56
51
52
57
108
51
54
57
56
64
23
119
75
81
76
79
74
52
60
102
66
67
64
69
47
38
50
63
111
61
66
64
70
55
36
48
55
31
110
58
63
66
72
57
36
47
56
30
8
109
66
67
66
72
42
54
57
65
34
28
26
112
70
71
70
76
46
47
56
61
27
17
19
17
112
70
71
70
76
46
47
56
61
27
17
19
17
0
110
68
68
69
73
45
46
52
58
26
16
18
19
10
10
112
68
68
69
73
45
48
52
60
26
18
20
19
12
12
2
111
73
73
72
77
51
48
54
60
30
22
22
19
14
14
12
13
110
71
71
71
77
51
47
51
62
28
19
19
21
14
14
12
12
7
112
72
72
72
78
52
48
52
63
29
20
20
22
15
15
13
13
8
1
Values were based on the data shown in Fig. 1; 1,431 nucleotides were used.
Numerical taxonomic analysis frequently differentiatesM.
gastri and M, kansasii as distinct species. M. gastri is
classified as a nonchromogenic slow grower, and M. kansasii
is classified as a photochromogenic slow-growing species,
although nonpigmented M. kansasii strains have been described (18). At the 16s rRNA sequence level M. gastri is
identical to M. kansasii. The close phylogenetic relationship
between these two taxa is reflected by the results of an
immunodiffusion analysis of cell extracts which identified
four antigens that M. kansasii shares with no other species
but M. gastri (26), as well as by the results of studies which
indicated that the T-catalase of M. gastri is closely related to
the T-catalase of M. kansasii (30).
M. intracellulare, M. avium, and M. paratuberculosis
form a discrete cluster. In many characteristics, M. paratuberculosis resembles M. avium (19), and a close genetic
relationship between M. paratuberculosis and M. avium has
been suspected previously (8). The phenotypic diversity of
organisms in the M. avium complex was illustrated by the
results of recent DNA studies which indicated that M.
paratuberculosis may be synonymous with M. avium (17,
21). Our sequence analysis confirmed and reinforced this
notion by finding a level of 16s rRNA similarity of 99.9%.
The sequence divergence among 16s rRNAs is not random but is confined to certain areas, as comparative analysis
of 16s rRNAs revealed regions of highly conserved primary
sequences and other regions with a considerable amount of
variability (35). The variable portions of rRNA characterize
taxa at a genus, group, or species level (35), and natural
relationships are also reflected in common sequence patterns
or structures among members of a common line of descent
(7)Stackebrandt and Smida found that there is a substantial
difference among fast- and slow-growing mycobacteria (24)
between positions 370 and 450 (in the numbering system
shown in Fig. 1). These authors found that while fast
growers consistently form a 21-base-long loop, all slow
growers exhibit a longer version in which a 15-base-longloop
is interrupted by a 7-base-pair-long stem and a 4-base-long
loop. This observation was recently confirmed by Stahl and
Urbance (25). However, the data show that there is no exact
correlation between the length of the helix comprising positions 390 to 430 and the growth rate since M. simiae, which
is characterized as a slow-growing species (33), has the short
21-base-long loop version in this region. These seemingly
inconsistent attributes of M. simiae are no contradiction in
phylogenetic terms. From a phylogenetic standpoint only
groups defined by synapomorphic (shared derived) characters are monophyletic (natural) groups. As Fig. 2 shows,
species with a long helix, including M. terrae, M. nonchromogenicurn, M. xenopi, M. gordonae, M. bovis, M. marinum, M. scrofulaceum, M. gastri, M. kunsasii, M. szulgai,
M. malmoense, M. intracellulare, M. paratuberculosis, and
M. avium, form one monophyletic group. With respect to
growth as a different character, fast growth is the plesiomorphic (primitive) condition, and slow-growing mycobacteria,
including M. simiae and the members of the group described
above, represent another monophyletic group. Inspection of
the collection of mycobacterial small-subunit rRNA sequences shown in Fig. 1 for sequences that differentiate
between fast- and slow-growing species did not reveal substantial differences; instead, both groups could be distinguished by the composition of a certain few nucleotides
(Table 4).
A broad group of slow-growing mycobacteria (including
M. gordonae, M. bovis, M. marinum, M. scrofulaceum, M.
gastri, M, kansasii, M. szulgai, M. rnalrnoense, M. intracellulare, M. paratuberculosis, and M. avium) can be de-
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PHYLOGENY OF THE GENUS MYCOBACTERIUM
TABLE 4. Signature nucleotides
Fast
growersb
Slow
growersb
141
272
T or C
A
G
T
373
412
A
G
G
T
413
G
C
449
C
G
450
C
G
462
G
C
505
C
G
1237
1238
1251
G
G
C
T
A, T
T, G, or deletion
Position“
Exceptions
M . phlei, M .
sphagni (T)
M . phlei (G)
M . smegmatis (C),
M . simiae (G)
M . smegmatis (A),
M . simiae (G)
M . smegmatis (T),
M . simiae (C)
M . smegmatis (G),
M . simiae (C)
M . phlei, M .
sphagni (C)
M . phlei, M.
sphagni (G)
M . komossense
(deletion)
Numbering as in Fig. 1.
The organisms which we studied are shown in Table 1. In addition, the
relevant portions of the 16s rRNA sequences from M. phlei, M . komossense,
“ M . cookii,” and M . sphagni (unpublished data) were included for this
analysis.
a
fined in terms of shared characters (synapomorphy),
CCATC at positions 409 to 413, GGTGG at positions 449 to
453, CACG at positions 574 to 577, and GA at positions 588
and 589. Signature nucleotides characteristic for certain
slow-growing species ( M . bovis, M . rnarinurn, M . scrofulaceum, M . gastri, M . kansasii, M . szulgai, M . malrnoense,
M . intracellulare, M . paratuberculosis, M . aviurn) can be
defined at positions 973 to 977 (CGTCT) and positions 984 to
988 (AGGCG).
Techniques for measuring evolutionary divergence in the
structure of semantides (i.e., large information-bearing molecules, such as rRNA, nucleic acids, and proteins) and
numerical taxonomy are complementary methods. Numerical taxonomy analyses have proven to be useful for identifying previously unrecognized strain clusters, as well as
providing information on frequency distribution of features
that can be used for strain identification. Numerical taxonomy may set the basis for a determination system, the basic
purpose of which is to identify species, but classification
based on phenotypes does not correlate well with natural
(i.e., evolutionary) relationships, as defined by macromolecular sequence comparisons (35). M. gastri and M . kansasii,
as well as M . rnalmoense and M . szulgai, are examples of
organisms that have been assigned to different groups according to the Runyon classification system (22), yet phylogenetically these pairs of species are closely related, as
shown by their 16s rRNA sequences.
Despite the high degree of similarity among mycobacterial
16s rRNAs, the collection of mycobacterial 16s rRNA
sequences provided in this paper was used to establish a
phylogenetically valid taxonomy of the genus Mycobacteriurn and should allow rapid and definitive classification of
strains of mycobacteria that do not belong to well-established or thoroughly characterized species, as well as classification of new species which are assigned to this genus.
ACKNOWLEDGMENTS
We thank K. Schroder and J. Jorgensen for providing strains, H.
Blocker for providing oligonucleotides, S. Maibom for typing the
329
manuscript, E. Stackebrandt for critical review of the manuscript,
and D. Bitter-Suermann for continuous encouragement.
J.W. was supported by a research grant from the Deutsche
Forschungsgemeinsc haft.
LITERATURE CITED
1. Baess, I. 1979. Deoxyribonucleic acid relatedness among species of slowly growing mycobacteria. Acta Pathol. Microbiol.
Immunol. Scand. Sect. B 87:221-226.
2. Baess, I. 1982. Deoxyribonucleic acid relatedness among species of rapidly growing mycobacteria. Acta Pathol. Microbiol.
Immunol. Scand. Sect. B 90:371-375.
3. Baess, I. 1983. Deoxyribonucleic acid relationships between
different serovars of Mycobacterium avium, Mycobacterium
intracellulare and Mycobacterium scrofulaceum. Acta Pathol.
Microbiol. Immunol. Scmd. Sect. B 91:201-203.
4. Bottger, E. C. 1989. Rapid determination of bacterial ribosomal
RNA sequences by direct sequencing of enzymatically amplified
DNA. FEMS Microbiol. Lett. 65171-176.
5 . Edwards, U., T. Rogall, H. Blocker, M. Emde, and E. C.
Bottger. 1989. Isolation and direct sequencing of entire genes.
Characterization of a gene coding for 16s ribosomal RNA.
Nucleic Acids Res. 17:7843-7853.
6. Fitch, W. M. 1981. A non-sequential method for constructing
trees and hierarchical classifications. J. Mol. Evol. 18:30-37.
7. Fox, G. E., E. Stackebrandt, R. B. Hespell, J. Gibson, J.
Maniloff, T. A. Dyer, R. S. Wolfe, W. E. Balch, R. S. Tanner,
L. J. Magrum, L. B. Zablen, R. Blakemore, R. Gupta, L. Bonen,
B. J. Lewis, D. A. Stahl, K. R. Luehrsen, K. N. Chen, and C. R.
Woese. 1980. The phylogeny of prokaryotes. Science 209:
457-463.
8. Grange, J. M. 1984. Mycobacterium avium. Eur. J. Respir. Dis.
65399-401.
9. Gross, W. M., and L. G. Wayne. 1970. Nucleic acid homology in
the genus Mycobacterium. J. Bacteriol. 104:630-634.
10. Imaeda, T., G. Broslawski, and S. Imaeda. 1988. Genomic
relatedness among mycobacterial species by nonisotopic blot
hybridization. Int. J. Syst. Bacteriol. 38:151-156.
11. Johnson, A. M., and P. R. Bavenstock. 1989. Rapid ribosomal
RNA sequencing and the phylogenetic analysis of protists.
Parasitol. Today 5102-105.
12 Kriiger, M., and G. Osterburg. 1983. On the alignment of two or
more molecular sequences. Comput. Programs Biomed. 16:
61-70.
13. Kubica, G. P., I. Baess, R. E. Gordon, P. A. Jenkins, J. B. G.
Kwapinski, C. McDurmont, S. R. Pattyn, H. Saito, V. Silcox,
J. L. Stanford, K. Takeya, and M. Tsukamura. 1972. A cooperative numerical analysis of rapidly growing mycobacteria. J.
Gen. Microbiol. 7355-70.
14. Kubica, G. P., V. A. Silcox, and E. Hall. 1973. Numerical
taxonomy of selected slowly growing mycobacteria. J. Gen.
Microbiol. 74:159-167.
15. Lane, D. J., B. Pace, G. J. Ohlsen, D. A. Stahl, M. L. Sogin, and
N. R. Pace. 1985. Rapid determination of 16s ribosomal RNA
sequences for phylogenetic analysis. Proc. Natl. Acad. Sci.
USA 82:6955-6959.
16. Maniatis, T., E. F. Fritsch, and J. Sambrook. 1982. Molecular
cloning. A laboratory manual. Cold Spring Harbor Laboratory,
Cold Spring Harbor, N.Y.
17. McFadden, J. J., P. D. Butcher, J. Thompson, R. Chiodini, and
J. Hermon-Taylor. 1987. The use of DNA probes identifying
restriction-fragment-length polymorphisms to examine the Mycobacterium avium complex. Mol. Microbiol. 1:283-291.
18. Meissner, G., K. H. Schroder, G. E. Amadio, W. Anz, S.
Chaparas, H. W. B. Engel, P. A. Jenkins, W. Kappler, H. H.
Kleeberg, E. Kubala, M. Kubin, D. Lauterbach, A. Lind, M.
Magnusson, Z. Mikova, S. R. Pattyn, W. B. Schaeffer, J. L.
Stanford, M. Tsukamura, L. G. Wayne, I. Willers, and E.
Wolinsky. 1974. A cooperative numerical analysis of nonscotoand nonphotochromogenic slowly growing mycobacteria. J.
Gen. Microbiol. 83:207-235.
19. Merkal, R. S. 1984. Paratuberculosis, p. 1237-1249. In G. P.
Kubica and L. G. Wayne (ed.), The mycobacteria. A source<
Downloaded from www.microbiologyresearch.org by
IP: 88.99.165.207
On: Sat, 17 Jun 2017 12:45:27
330
INT. J. SYST.BACTERIOL.
ROGALL ET AL.
book, part B. Marcel Dekker, Inc., New York.
20. Saitou, N., and T. Imanishi. 1989. Relative efficiencies of the
Fitch-Margoliash, maximum-parsimony, maximum-likelihood,
minimum-evolution, and neighbor-joining methods of phylogenetic tree construction in obtaining the correct tree. Mol. Biol.
Evol. 6514-525.
21. Saxegaard, F., and I. Baess. 1988. Relationship between Mycobacterium avium, Mycobacterium paratuberculosis and “wood
pigeon mycobacteria.” Acta Pathol. Microbiol. Immunol.
Scand. Sect. B 96:37-42.
22. Sommers, H. M., and R. C. Good. 1985. Mycobacterium, p.
216-248. In E. H. Lennette, A. Balows, W. J. Hausler, Jr., and
H. J. Shadomy (ed.), Manual of clinical microbiology, 4th ed.
American Society for Microbiology, Washington, D.C.
23. Sourdis, J., and M. Nei. 1988. Relative efficiencies of the
maximum parsimony and distance-matrix methods in obtaining
the correct phylogenetic tree. Mol. Biol. Evol. 5298-311.
24. Stackebrandt, E., and J. Smida. 1988. The phylogeny of the
genus Mycobacterium as determined by 16s rRNA sequences,
and development of DNA probes, p. 244-250. In Biology of
actinomycetes. Japan Scientific Societies Press, Tokyo.
25. Stahl, D. A., and J. W. Urbance. 1990. The division between
fast- and slow-growing species corresponds to natural relationships among the mycobacteria. J. Bacteriol. 172:116-124.
26. Stanford, J. L., and J. M. Grange. 1974. The meaning and
structure of species as applied to mycobacteria. Tubercle 55:
143-152.
27. Suzuki, Y., A. Nagata, Y. Ono, and T. Yamada. 1988. Complete
nucleotide sequence of the 16s rRNA gene of Mycobacterium
bovis BCG. J. Bacteriol. 170:2886-2889.
28. Tsukamura, M. 1976. Numerical classification of slowly growing mycobacteria. Int. J. Syst. Bacteriol. 26:409420.
29. Wayne, L. G., L. Andrade, S. Froman, W. Kappler, E. Kubala,
G. Meissner, and M. Tsukamura. 1978. A cooperative numerical
analysis of Mycobacterium gastri, Mycobacterium kansasii and
Mycobacterium marinum. J. Gen. Microbiol. 109:319-327.
30. Wayne, L. G., and G. A. Diaz. 1985. Identification of mycobac-
31.
32.
33.
34.
35.
teria by specific precipitation of catalase with absorbed sera. J.
Clin. Microbiol. 21:721-725.
Wayne, L. G., R. C. Good, M. I. Krichevsky, R. E. Beam, Z.
Blacklock, S. D. Chaparas, D. Dawson, S. Froman, W. Gross, J.
Hawkins, P. A. Jenkins, I. Juhlin, W.Kapper, H.H. Kleeberg,
I. Krasnow, M. J. Lefford, E. Mankiewicz, C. McDurmont, G.
Meissner, P. Morgan, E. E. Nel, S. R. Pattyn, F. Portaels, P. A.
Richards, S. Rusch, K. H. Schroder, V. A. Silcox, I. Szabo, M.
Tsukamura, and B. Vergmann. 1981. First report of the cooperative, open-ended study of slowly growing mycobacteria by
the International Working Group on Mycobacterial Taxonomy.
Int. J. Syst. Bacteriol. 31:l-20.
Wayne, L. G., R. C. Good, M. I. Krichevsky, R. E. Beam, Z.
Blacklock, H. L. David, D. Dawson, W. Gross, J. Hawkins, P. A.
Jenkins, I. Juhlin, W. Kappler, H. H. Kleeberg, I. Krasnow,
M. J. Lefford, E. Mankiewicz, C. McDurmont, E. E. Nel, F.
Portaels, P. A. Richards, S. Riisch, K. H.Schroder, V. A. Silcox,
I. Szabo, M. Tsukamura, L. Van den Breen, and B. Vergmann.
1983. Second report of the cooperative, open-ended study of
slowly growing mycobacteria by the International Working
Group on Mycobacterial Taxonomy. Int. J. Syst. Bacteriol.
33:265-274.
Wayne, L. G., R. C. Good, M. J. Krichevsky, Z. Blacklock,
H. L. David, D. Dawson, W. Gross, J. Hawkins, P.A. Jenkins, I.
Juhlin, W. Kiippler, H. H. Kleeberg, V. Levy-Frebault, C.
McDurmont, E. E. Nel, F. Portaels, S. Rusch-Gerdes, K. H.
Schroder, V. A. Silcox, I. Szabo, M. Tsukamura, L. Van den
Breen, B. Vergmann, and M. A. Yakrus. 1989. Third report of
the cooperative open-ended study of slowly growing mycobacteria by the International Working Group on Mycobacterial
Taxonomy. Int. J. Syst. Bacteriol. 39:267-278.
Wayne, L. G., and G. P. Kubica. 1986. The mycobacteria, p.
1435-1457. In P. H. A. Sneath, N. S. Mair, M. E. Sharpe, and
J. G. Holt (ed.), Bergey’s manual of systematic bacteriology,
vol. 2. The Williams & Wilkins Co., Baltimore.
Woese, C. R. 1987. Bacterial evolution. Microbial. Rev. 51:
221-271.
Downloaded from www.microbiologyresearch.org by
IP: 88.99.165.207
On: Sat, 17 Jun 2017 12:45:27