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Journal of Clinical Microbiology, January 1998, p. 139-147, Vol. 36, No. 1
0095-1137/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
Differentiation of Phylogenetically Related Slowly
Growing Mycobacteria Based on 16S-23S rRNA Gene Internal Transcribed
Spacer Sequences
Andreas
Roth,1,*
Marga
Fischer,1
Mohamed E.
Hamid,1,2
Sabine
Michalke,1
Wolfgang
Ludwig,3 and
Harald
Mauch1
Institut für Mikrobiologie und
Immunologie, Krankenhaus Zehlendorf, 14109 Berlin,1 and
Institut für Botanik
und Mikrobiologie, Lehrstuhl für Mikrobiologie, Technische
Universität München, 80290 Munich,3
Germany, and
Faculty of Veterinary Science, University of
Khartoum, Khartoum, Sudan2
Received 4 August 1997/Returned for modification 17 September
1997/Accepted 17 October 1997
 |
ABSTRACT |
Interspecific polymorphisms of the 16S rRNA gene (rDNA) are widely
used for species identification of mycobacteria. 16S rDNA sequences,
however, do not vary greatly within a species, and they are either
indistinguishable in some species, for example, in Mycobacterium
kansasii and M. gastri, or highly similar, for example, in M. malmoense and M. szulgai. We
determined 16S-23S rDNA internal transcribed spacer (ITS) sequences of
60 strains in the genus Mycobacterium representing 13 species (M. avium, M. conspicuum, M. gastri, M. genavense, M. kansasii,
M. malmoense, M. marinum, M. shimoidei, M. simiae, M. szulgai,
M. triplex, M. ulcerans, and M. xenopi). An alignment of these sequences together with additional
sequences available in the EMBL database (for M. intracellulare, M. phlei, M. smegmatis,
and M. tuberculosis) was established according to primary-
and secondary-structure similarities. Comparative sequence analysis
applying different treeing methods grouped the strains into
species-specific clusters with low sequence divergence between strains
belonging to the same species (0 to 2%). The ITS-based tree topology
only partially correlated to that based on 16S rDNA, but the main
branching orders were preserved, notably, the division of fast-growing
from slowly growing mycobacteria, separate branching for M. simiae, M. genavense, and M. triplex, and
distinct branches for M. xenopi and M. shimoidei. Comparisons of M. gastri with M. kansasii and M. malmoense with M. szulgai
revealed ITS sequence similarities of 93 and 88%, respectively. M. marinum and M. ulcerans possessed identical
ITS sequences. Our results show that ITS sequencing represents a
supplement to 16S rRNA gene sequences for the differentiation of
closely related species. Slowly growing mycobacteria show a high
sequence variation in the ITS; this variation has the potential to be
used for the development of probes as a rapid approach to mycobacterial
identification.
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INTRODUCTION |
The increase of infections caused by
Mycobacterium tuberculosis and nontuberculous mycobacteria
is receiving increasing attention worldwide. Nontuberculous
mycobacteria are encountered with increasing frequency in clinical
laboratories, and phenotypic features for their identification are well
documented (6, 12, 39, 40). Numerical taxonomic matrices and
16S rRNA-based phylogenetic analyses have contributed to the
systematics of mycobacteria (26, 29, 38). 16S rRNA gene
(rDNA) sequence analysis, either by direct sequencing (17)
or by using probes (18, 22, 25), is now widely used for
rapid and accurate identification of mycobacteria. Unfortunately, the
number of polymorphic sites in the 16S rDNA in the genus
Mycobacterium is rather low, inasmuch as some species have
the same sequence (M. kansasii and M. gastri or
M. senegalense and M. farcinogenes) and others
possess a very high degree of sequence similarity (e.g., M. malmoense and M. szulgai or M. marinum and
M. ulcerans). This may lead to problems related to
cross-reactivity when oligonucleotide probes are used (5)
and makes the design of probes directed towards a broad panel of all
clinically relevant species difficult (18).
In view of this, a study of more-variable sequences in the RNA operon
of phylogenetically closely related mycobacterial species is needed.
The genes coding for the rRNA are arranged in the order 5'-16S-23S-5S-3', and they are separated by two noncoding spacer regions. The 16S-23S rDNA internal transcribed spacer (ITS) has been
suggested to represent a potential target within the bacterial genome
to find suitable sites for probes and from which to derive additional
phylogenetic information. This genetic locus is flanked by
well-conserved regions of the rRNA operon, contains both conserved and
highly variable signatures, and is rather small (2, 13, 19).
Previous work, particularly with respect to mycobacteria, has shown
that both the high level of spacer sequence variation and the good
reproducibility of ITS sequencing suggest the applicability of this
approach (4, 9, 10, 35). Distinct ITS sequences found in the
M. avium complex have been used to define infrasubspecific taxons, and such subspecies defined by a sequence were called a
sequevar (4, 10). Thus, ITS is suitable for differentiating strains within some mycobacterial species and has the potential to be
used as a marker for clinically relevant subspecies in these cases
(1, 11, 24).
The aim of the present study was to further investigate the suitability
of the ITS for the reliable molecular identification of mycobacteria.
The ITS sequences of a total of 60 strains comprising 13 species of
slowly growing mycobacteria were determined (i) to discover whether
spacer sequences can differentiate slowly growing mycobacterial species
which are identical or closely related on the basis of their 16S rDNA
sequences, (ii) to evaluate the degree of interspecies divergence and
intraspecies conservation of ITS sequences, and (iii) to compare the
ITS-based clustering of the organisms with the tree obtained from 16S
rDNA sequence analysis.
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MATERIALS AND METHODS |
Bacterial strains.
The sources, nucleotide sequence
accession numbers, and sequevar assignments of 17 mycobacterial species
investigated in this study are listed in Table
1. With the exception of M. conspicuum and M. triplex, more than one strain was
included within each species to explore intraspecies variability. The
following numbers of clinical isolates obtained from the strain
collection held at our institute were included: M. avium,
12; M. gastri, M. simiae, and M. xenopi, 7 each; M. marinum, 5; M. kansasii,
4; M. szulgai, 3; M. genavense, 2; and M. malmoense, M. shimoidei, M. triplex, and
M. ulcerans, 1 each. The M. shimoidei strain was
isolated from the respiratory tract of a patient with chronic lung
disease, and the M. triplex isolate was recovered from
pleural effusion from a patient with empyema. All M. avium
and M. genavense strains were isolated from the blood of
patients with AIDS. Strain S134 (M. marinum) was kindly
provided by P. Buchholz, Berlin, Germany, and strain S219 (M. ulcerans), isolated from an African patient with Buruli ulcera,
was donated by G. Márquez de Bär, Cottbus, Germany. All
strains used were identified to the species level by standard
biochemical procedures (16). The species identity of all
strains was confirmed by 16S rDNA sequencing (see below). Bacteria
grown on Löwenstein-Jensen slants or in 7H12 broth were suspended
in 1 ml Tris-HCl buffer (10 mM, pH 7.5). The cells were inactivated at
80°C for 10 min, washed twice with Tris-HCl buffer, and kept at
20°C until needed.
Sequence analysis.
The cell pellet from a 100-µl aliquot
of the slightly turbid bacterial suspensions mentioned above was
sonicated with 50 µl of glass beads (100-µm diameter; Sigma,
Diesenhofen, Germany) for 10 min in a water bath sonicator (Sonorex RK
156; Bandelin, Berlin, Germany). The lysate obtained after settlement
of the glass beads containing the genomic DNA was then heated at 94°C for 10 min. Two independent DNA extractions were performed for each
strain to confirm the results. Amplification of the 16S rDNA and ITS
sequences was performed with the following primers. (i) For the 16S
rDNA sequences, primers modified according to recently published
sequences (33) were used, namely, Seq1 (identical to KY18),
which is biotin-5'-CAC ATG CAA GTC GAA CGG AAA GG-3', and Seq2 (which
corresponds to the modified primer KY75), which is 5'-GCC CGT ATC GCC
CGC ACG CT-3'. (ii) For the ITS sequences, primers Ec16S.1390p,
biotin-5'-TTG TAC ACA CCG CCC GTC A-3', and Mb23S.44n, 5'-TCT CGA TGC
CAA GGC ATC CAC C-3' (10), were used. The amplification was
done with a 50-µl reaction mix containing 10 mM Tris-HCl (pH 8.3), 50 mM KCl, 1.5 mM MgCl2, 0.1% Triton X-100, 200 µM each
deoxynucleoside triphosphate, i.e., dATP, dGTP, dCTP, and dUTP, 20 pmol
of the unbiotinylated primer, 10 pmol of the biotinylated primer, 1 U
of Thermus aquaticus DNA polymerase (all reagents from
Pharmacia Biotech, Freiburg, Germany), and 5 µl of DNA. The thermal
profile for both 16S rDNA and ITS amplification involved 38 cycles with
the following steps: initial denaturation for 5 min at 95°C followed
by 1 min of denaturation at 94°C, annealing at 62°C, and extension
at 72°C. Amplified products were analyzed by 1.8% agarose gel
electrophoresis.
The amplified PCR products were captured and purified with
streptavidin-coated Dynabeads as described in the instructions of the
manufacturer (Dynabeads M-280 streptavidin; Dynal AS, Oslo, Norway).
Sequencing reactions were done by standard dideoxy sequencing methods
with a DNA sequencing kit, and the procedure was done as described in
the instructions of the manufacturer (Pharmacia). The sequencing
procedure was performed with the A.L.F. DNA sequencer (Pharmacia). The
primers used for sequencing were as follows: (i) for the 16S rDNA
(including helix 10 and helix 18), 5'-fluorescein-labelled primers 244 and 259 (17); (ii) for the ITS, 5'-fluorescein-labelled primers complementary to the PCR primers for sequencing of both the
sense and the antisense strands.
Data analysis.
New and additional database ITS sequences
were aligned by using the respective tools of the ARB software package
(30) according to primary-structure, as well as predicted
secondary-structure, similarity. Secondary-structure prediction was in
analogy to that proposed elsewhere (14, 15). Comparative
analyses of ITS sequences were performed with distance matrix,
maximum-parsimony, and maximum-likelihood methods (32) as
implemented in the ARB package. The significance of the resulting tree
topologies was tested by performing bootstrap analyses and varying the
composition of the data sets by successively removing or including
alignment positions according to their degree of evolutionary
conservation. Conservation profiles were established by using the
respective ARB subroutines. For comparison, we included a 16S
rRNA-based tree. This tree was reconstructed by performing a distance
matrix analysis of all available, at-least-90%-complete (in comparison
with Escherichia coli 16S rRNA sequences), homologous primary structures from gram-positive bacteria with a high DNA G+C
content as well as a selection of reference strains from the other
major bacterial lines of descent. Evaluation and correction of the tree
topology were done as described for the ITS-based tree.
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RESULTS |
PCR amplification with primers Ec16S.1390p and Mb23S.44n resulted
in the detection of a single band of approximately 480 bp in all 60 strains investigated. The variation in product length was not
considerable between the different species of slow growers, but a
smaller product of approximately 430 bp was noted for M. xenopi. The sequence alignment and the ITS sizes for all species studied are shown in Fig.
1. The
sizes of the spacers of slow growers ranged from 235 nucleotides (nt)
for M. xenopi to 285 nt for M. gastri, showing
that the spacer sequences of slow growers are approximately 75 nt
shorter than those of rapid growers. This is due to features that
clearly differentiate slow growers from fast growers (14,
15), notably, a missing expansion of helix 4 and a shorter helix
5 in slowly growing mycobacteria, a finding which was confirmed here
for all strains studied (Fig. 1). Longer stretches of conserved
noncoding regions which could be characteristic of the genus were
limited to only a few sites, for example, positions 1 to 10 and parts
of the two stem-loop sequences (Fig. 1). In contrast, a high degree of
sequence variability was found dispersed over the whole spacer
sequence, with the highest degree of diversity found in the
antitermination elements (referred to as boxes [Fig. 1]) and helices
5 and 6. As a result of this variability, we found an intraspecies
sequence polymorphism in 4 of 11 species for which multiple strains
within one species were analyzed. M. gastri and M. avium each split into two distinct sequevars, designated Mga-A and
Mga-B and Mav-A and Mav-B, respectively, based on the nomenclature proposed by Frothingham and Wilson (10) and De Smet et al.
(4). The base difference between Mga-A and Mga-B was found
to be 5 nt, and that between Mav-A and Mav-B was 1 nt; the latter
finding was described previously (10). Members of M. simiae exhibited a bigger variation, with the formation of four
distinct sequevars. M. xenopi sequevars distinguishable from
the type strain sequevar exhibited a 4- or 5-base-long insertion at
position 28. M. triplex and M. genavense were the
two species with the highest ITS similarity values (Table
2). Hence, the lowest level of ITS
sequence divergence between any two species in this setting of strains
was at least 13 nt (4%). M. marinum and M. ulcerans had the same ITS sequence. Much lower levels of
similarity were obtained for ITS than for 16S rDNA sequences (Table 2).
ITS base changes between species closely related on the basis of their
16S rDNA sequences (similarity greater than 99%) accounted for the 7 to 8% difference between M. gastri and M. kansasii and for the 12% difference between M. malmoense and M. szulgai. ITS similarity values as low
as 63% could be found when the sequences of slow-growing
mycobacteria were compared with those of two representatives of
fast-growing mycobacteria. M. xenopi occupied an
intermediate position in its sequence similarity with rapid growers,
but its short ITS sequence and the missing expanded helix 4 indicate
that this species belongs to the group of slow growers.
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FIG. 1.
Alignment of 16S-23S rDNA ITS sequences including those
of 13 mycobacterial species investigated in this study together with
sequences of 4 other species published elsewhere (9, 10, 14,
34) (Table 1). Sequevar designations are shown in parentheses.
The sequence of M. simiae (Msi-B) was published by De Smet
et al. (4). The length of the ITS is indicated at the end of
the sequences in nucleotides. The complete ITS sequence between the end
of the 16S rRNA gene and the beginning of the 23S rRNA gene is shown.
With the exception of M. conspicuum, whose ITS ends with GG,
the 3' end of the ITS was inferred to be GTGT. Dots indicate identity,
and hyphens represent alignment gaps. Possible features of secondary
structures predicted in analogy to those previously proposed for
pre-rRNA transcripts of mycobacteria are indicated by labelled arrows,
whereas the stem-loop structures designated leader and spacer 2 interact with leader and 23S-5S rRNA spacer regions, respectively
(14, 15).
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TABLE 2.
Similarity values for 16S-23S rDNA spacer (ITS) sequences
(lower left) and 16S rRNA sequences (upper right) for representatives
of the genus Mycobacterium
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A dendrogram reflecting the ITS sequence-based clustering of all test
strains is shown in Fig. 2. Within the
consensus tree, five clusters among the slowly growing mycobacteria
could be defined; of these clusters, M. shimoidei and
M. xenopi showed sufficient sequence variation to emerge as
distinct branches. The biggest cluster was composed of more than one
closely related species, namely, M. conspicuum, M. gastri, M. kansasii, M. marinum, M. szulgai, M. tuberculosis, and M. ulcerans.
The somewhat-separated position of M. conspicuum was
supported by the majority of the different analyses; however, its
significance was less than that of the other branchings. Two additional
clusters comprised M. simiae, M. genavense, and
M. triplex and M. avium, M. intracellulare, and M. malmoense. The significance of
the branching orders was estimated by applying alternative treeing
methods to various data sets which differed in sequence as well as in
alignment position and by performing bootstrap analyses. The
multifurcations indicate that a significant relative branching order
could not be determined or that a common branching order was not
supported by the results obtained by applying different treeing
methods. Depending on the treeing method used, the corresponding
bootstrap values were 25% and lower. A 16S rRNA-based tree is given in
Fig. 3 for comparison. The overall
pictures of both trees are similar with regard to the separation of
fast and slow growers, the positions of M. shimoidei and
M. xenopi, the clustering of M. avium together
with M. intracellulare, and the grouping of M. genavense, M. triplex, and M. simiae.
Interestingly, both the ITS and the 16S rRNA sequences of M. xenopi contain a number of unusual residues in comparison to those
of the other organisms (as evidenced by the longer branches in both
trees), which indicates a higher rate of substitution. However, there can also be seen remarkable differences in the substructures of the two
trees. No further subgrouping was supported by the 16S rRNA data for
the group comprising M. avium, M. intracellulare, M. tuberculosis, M. ulcerans, M. marinum, M. kansasii, M. gastri, M. malmoense, M. szulgai, and the slightly deeper
branching M. conspicuum. In contrast, the ITS analyses
allowed the definition of two subclusters, one containing M. avium, M. intracellulare, and M. malmoense
and one containing M. kansasii, M. ulcerans-M. marinum, M. gastri, M. szulgai, M. tuberculosis, and the somewhat more distant M. conspicuum. A closer relationship between M. tuberculosis and M. ulcerans-M. mannum such as that
shown in the 16S rRNA-based tree was not supported by the ITS data.
M. malmoense and M. szulgai are members of
different ITS clusters, while a closer relationship of these two
species had been reported on the basis of 16S rRNA data (7, 26,
28). The latter finding could be determined with only low
significance when distance methods were applied; however, this was not
supported by maximum-parsimony or maximum-likelihood analyses (Fig. 3).
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FIG. 2.
Distance matrix tree showing the divergence of ITS
sequences of the investigated mycobacteria. All alignment positions
which are occupied by residues were used for the calculation of binary
distance values. The topology of the tree was evaluated and corrected
according to the results of maximum-parsimony and maximum-likelihood
analyses. Multifurcations indicate that a relative branching order
could not be unambiguously determined or that a common branching order
was not supported by the different treeing methods. The corresponding
sequences of the fast-growing mycobacteria M. phlei and
M. smegmatis were used as outgroup references. Numbers in
brackets indicate the numbers of strains sequenced. The bar represents
10% estimated sequence divergence.
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FIG. 3.
16S rRNA-based distance matrix tree for a selection of
mycobacterial species. The tree was reconstructed with all available,
at-least-90%-complete (with respect to the homologous E. coli molecule), 16S rRNA primary structures of gram-positive
bacteria with a high DNA G+C content as well as from a selection of
reference organisms of the other major bacterial phyla. The topology of
the tree was evaluated and corrected by the methods applied to ITS
sequences (Fig. 2). The bar indicates 5% estimated sequence
divergence.
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DISCUSSION |
Sequence-specific differentiation of mycobacteria by using the
ITS.
The conventional methods for identifying mycobacteria based
on growth characteristics and biochemical tests are time-consuming and
often not unambiguous in their interpretation. Currently, the widely
accepted strategy formulated to improve methods of mycobacterial strain
identification includes analysis of the gene encoding 16S rRNA (3,
17, 18, 22, 23, 25, 26, 33). Furthermore, the potential utility
of alternative targets such as the gene encoding a 65-kDa heat shock
protein (hsp65) has been described (31). Each
technique has several advantages and disadvantages. The small number of
polymorphic positions within the 16S rDNA obviates the need of
nucleotide sequencing (17, 23). The limited availability of
commercial probes is a reflection of this problem, and only a few
studies have highlighted the potential applicability of a wider panel
of probes directed to 16S rDNA sequences (3, 18, 22). In the
clinical laboratory, easy and cost-effective identification of
mycobacteria is of high priority. Undoubtedly, a target with a higher
level of variability would considerably facilitate the wide
introduction of hybridization methods or even simpler methods such as
multiplex PCR into the routine laboratory. On the other side, an
excessively high degree of variability such as that found in the
hsp65 gene (31) may be undesirable because such a
variety or instability of species-specific signatures will make
development of reliable probes that cover all strains within a species
very cumbersome. Finally, the phylogenetic information deducible from a
gene with such a low selective pressure for its sequence conservation
(as compared to coding and noncoding rDNA sequences) may result in
inconsistent conclusions. The principal goal of our study was to
further investigate the level of ITS polymorphism and thereby assess
the utility of this target for mycobacterial species identification.
This study demonstrates that the ITS of the genus
Mycobacterium exhibits variations in length and,
importantly, shows a reasonable number of base substitutions and
insertion or deletion sites. We have shown that this higher degree of
variation is of value for discriminating closely related species such
as M. gastri and M. kansasii. Unfortunately, ITS sequence analysis failed to discriminate between M. marinum
and M. ulcerans. This disappointing, yet not surprising,
finding stands in agreement with 16S rDNA data, according to which
these two species are very closely related (22, 23). Single
base-pair variations related to three residues at the 3' end of the 16S rDNA distinguish M. marinum from M. ulcerans
(23). The only mycobacterial species so far found to have
the same ITS sequences are members of the M. tuberculosis
complex (9). A high degree of genomic relatedness between
M. marinum and M. ulcerans in DNA-DNA hybridizations and shared phenotypic characteristics strongly suggest
that these two mycobacteria represent a single species (6,
23). It is even more apparent now from the high ITS sequence conservation found that clarification of this issue warrants additional studies. This study confirms and extends previous observations that the
ITS sequence between the 16S rRNA and 23S rRNA contains sufficient
interspecific polymorphisms and intraspecific conservation to serve as
a valuable target for mycobacterial identification. Besides its greater
variability, two further advantages of this target can be pointed out.
Unlike a 16S rDNA-based PCR, where the genus-specific primers are
separated by a long stretch of target sequence (more than 500 bp)
(3, 17, 33), an ITS-based PCR would imply a smaller PCR
product, resulting in a more efficient and sensitive target
amplification. In addition, the ITS has the potential to be used for
clinically significant strain differentiation (1, 4, 11,
24). We can expect that more sequevars will be characterized when
a bigger number of strains are analyzed. We did not find any
intraspecific variation in the five M. kansasii strains
studied here, but other reports give evidence that more than one
sequevar exists (1, 41). Genetic identification of possibly
more pathogenic strains of nontuberculous mycobacteria (whose clinical
significance after isolation from patient specimens is often difficult
to assess) could be of extreme diagnostic value. In view of this,
seeking further studies on this issue, for example, on the
epidemiological significance of the M. xenopi sequevars found here, is an imperative.
ITS sequence comparison as an adjunct to Mycobacterium
phylogeny.
It is well documented that a wide range of phylogenetic
relationships (domain to species) of bacteria can be substantiated by a
sequence comparison of their rRNAs (20, 21, 27). This scientific rationale cannot be doubted, and comparative 16S rDNA sequencing has contributed largely to our understanding of
mycobacterial taxonomy of well-resolved species (26, 29,
34). However, this approach provides a rather low resolution on
the level of closely related, recently emerged species within the genus
Mycobacterium, as demonstrated by high 16S rDNA similarities
or even sequence identity of different species (8, 26).
Despite considerable phenotypic diversity, slowly growing mycobacteria
appear to have diverged over a short period. Hence, ITS sequences have
been proposed to be a useful supplement when 16S rDNA shows
insufficient diversity to differentiate recently diverged species
(10, 15). The ITS sequence does not code for a final
product, but it has an important processing function in forming
pre-RNAs; as a consequence, there is presumably some functional
selective pressure for its conservation (10, 15). This
assumption is consistent with the stability of species-specific ITS
signatures found with high reproducibility in different strains. For
example, identical sequences distinct for two M. avium
sequevars were found in three completely different geographical regions
in clinical samples from patients with AIDS in this study and by two
other working groups (4, 10).
As shown with other taxa, the evolutionary rate of the ITS is higher
than that of 16S rRNA, and rearrangements in the central
region are
relatively recent. Consequently, phylogenetic information
on the
ancestry of only moderately related species may not be
maintained
(
19). Therefore, we must expect that the two molecules
provide different levels of phylogenetic resolution. High levels
of
relatedness and identical ITS- and 16S rRNA-based tree topologies
were
found for
M. genavense and
M. triplex, for
M. simiae and
both
M. genavense and
M. triplex, and for
M. avium and
M. intracellulare.
Comparison of the sequences of
M. gastri with
M. kansasii can
be regarded as an example
of the higher resolution of ITS data
at the species level. Our ITS data
suggest a divergence between
M. gastri and
M. kansasii. Phenetic data supported the resolution
of these two
bacteria as distinct species (
36). At the 16S rDNA
sequence
level, these two species are identical (
26). The finding
that the T-catalases of these two species are related is not useful
in
this context since divergence of T-catalase is not an accurate
reflection of natural (evolutionary) relationships (
37).
The ITS-based subclustering of
M. avium,
M. intracellulare, and
M. malmoense compared with that of
M. kansasii,
M. ulcerans,
M. marinum,
M. gastri,
M. szulgai,
M. tuberculosis, and
M. conspicuum,
which is not
significantly evident from rRNA data, is another
example of different
resolution. A closer relationship of
M. tuberculosis and
M. marinum-M. ulcerans is not supported by ITS sequence data
but is rather stable in 16S rRNA-based trees. However, given the
high
sequence similarity (99.2%) of these organisms and the short
distance
to the next neighbors (1.2 to 2.2%), it cannot be excluded
(and can
hardly be tested) that the identities at the highly variable
positions
responsible for the clustering of
M. tuberculosis and
M. marinum-M. ulcerans may result from multiple base changes
during
the course of evolution and thus may represent false identities
(
20).
A closer relationship was described for
M. malmoense and
M. szulgai based on a 16S rRNA sequence comparison
(
26). As mentioned
above, this grouping can be determined
with low significance by
distance methods analyzing the currently
available data set but
is not supported by maximum-parsimony nor
maximum-likelihood analysis.
The assignment of
M. malmoense
and
M. szulgai to different subclusters
according to their
ITS sequence similarities is supported by other
data. Numerical
taxonomy characterizes
M. szulgai as a species
that emerges
as a discrete cluster with the second highest matching
score to
M. kansasii (
38). In contrast, a clear phenotypic
resolution
(with some overlaps with the
M. avium complex)
placing
M. malmoense far from
M. szulgai was
found. Therefore, by taking into account
that numerical taxonomy is
relevant and complementary to semantide
studies (
34,
38),
the position of
M. malmoense in the ITS
phylogeny appears
reasonable and contributes to satisfying the
claim that a
phylogenetically based scheme should be accompanied
by phenotypic
consistency (
34). The
M. malmoense-M. szulgai case nicely illustrates the limitations of the rRNA approach and
the
demand for a polyphasic approach for taxonomic analyses at
and above
the species level (
34).
In conclusion, comparative ITS sequencing represents a useful tool for
species (strain) differentiation and identification
if the primary
structures contain polymorphic and diagnostic residues
or stretches,
respectively. The occurrence of conserved primary-
and
secondary-structure elements among mycobacterial ITS sequences
indicates a potential for phylogenetic investigations. However,
as
demonstrated by the high divergence of the
M. xenopi
sequence
from other ITS sequences of slowly growing mycobacteria, the
rate
of base changes in ITS sequences may vary to a large scale within
different (phylogenetic) groups. Consequently, whether ITS sequences
contain useful phylogenetic information for the particular group
of
interest must be carefully determined. In the case of rapidly
growing
mycobacteria which carry multiple rRNA operons, the analysis
may be
complicated by interoperon heterogeneities.
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ACKNOWLEDGMENT |
M. E. Hamid was supported by a fellowship from the Alexander
von Humboldt Foundation.
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FOOTNOTES |
*
Corresponding author. Mailing address: Institut
für Mikrobiologie und Immunologie, Lungenklinik
Heckeshorn-Zehlendorf, Zum Heckeshorn 33, D 14109 Berlin, Germany.
Phone: 49-30-8002 2254. Fax: 49-30-8002 2299.
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Abstract
Introduction
