Copyright 0 1996, International Union of Microbiological Societies
OF SYSTEMATIC BACTERIOLOGY, Oct. 1996, p. 1078-1082
Vol. 46, No. 4
Reassessment of the Phylogenetic Position of the Bacterium
Associated with Whipple’s Disease and Determination of
the 16s-23s Ribosomal Intergenic Spacer Sequence
MATTHIAS MAIWALD,’” HANS-JURGEN DITTON,’ AXEL VON HERBAY,2
FREDERICK A. RAINEY,3 AND ERKO STACKEBRANDT3
Hygiene-Institut der Universitat, Abteilung Hygiene und Medizinische Mikrobiologie, and
Pathologisches Institut der Universitat, 691 20 Heidelberg, and Deutsche Sammlung
von Mikrooiganismen und Zellkulturen GmbH, 38124 Braunschweig, Germany
Whipple’s disease is a rare chronic illness associated with an unculturable bacterium that is constantly
present in affected tissues. This bacterium was previously characterized at the molecular level by PCR and
sequencing of the 16s rRNA gene. On the basis of 1,321 nucleotides of the sequence of its gene coding for 16s
rRNA (16s rDNA), a phylogenetic relationship to the actinomycetes was established. In this study, we deter-
mined an almost complete 16s rDNA sequence (1,495 nucleotides), the 16s-235 ribosomal intergenic spacer
sequence, and 200 nucleotides of the 23s rRNA gene. The 16s rDNA sequence was compared with the large
number of actinomycete sequences that have been added to the database since the original study. Phylogenetic
analysis revealed a branching position as the deepest branch of the cluster comprising the actinomycetes with
group B peptidoglycan between this group and the family Cellulomonadaceae. This provides additional infor-
mation on the phylogenetic position of this bacterium and some clues as to its characteristics. The spacer
region between the 16s and 23s rRNA genes is 294 nucleotides long and does not contain tRNA genes. As
has been shown in other instances, the increased variability of the ribosomal intergenic spacer compared with
the 16s rRNA gene makes it a potential target for use in the differentiation of strains of the bacterium
associated with Whipple’s disease.
Whipple’s disease is a rare chronic illness with intestinal and
extraintestinal manifestations. A constant feature of the dis-
ease is the appearance of periodic acid-Schiff stain-positive
cellular inclusions detected by histology. These periodic acid-
Schiff stain-positive inclusions contain bacteria visible by elec-
tron microscopy that are approximately 0.2 pm wide by 1.5 to
2.5 p,m long and have a typical trilaminar appearance of the
cell wall (4). Numerous attempts to culture these bacteria on
artificial media or in cell culture have failed or have yielded
contaminants (3). The bacterium associated with Whipple’s
disease (Whipple’s disease bacterium) was characterized at the
molecular level by PCR and universal bacterial primers for the
16s rRNA gene; the resulting PCR products were sequenced
(36,48). In a first investigation, less than 50% (645 nucleotide
positions) of the bacterial 16s rRNA gene was sequenced and
a relationship to the actinomycetes was established, with the
closest relative reported to be Rhodococcus equi (48). Later,
approximately 85% of the 16s rRNA gene was sequenced (36).
Analysis of this sequence comprising 1,321 bp confirmed a
phylogenetic relationship to the actinomycetes (36). The clos-
est phylogenetic relatives within the order Actinomycetales
were members of a group designated the “actinobacteria”
comprising representatives of the genera Actinomyces, Rothia,
Arthrobacter, Micrococcus, Terrabacter, and Dewnatophilus for
which sequence data had been previously determined and
rnade available via the public databases and the Ribosomal
Database Project (25, 36). On the basis of the novelty of the
* Corresponding author. Mailing address: Hygiene-Institut der
IJniversitat Heidelberg, Im Neuenheimer Feld 324, 69120 Heidelberg,
Germany. Phone: 49-6221-567815. Fax: 49-6221-564343. Electronic
mail address: firstname.lastname@example.org.
sequence of the gene coding for 16s rRNA (16s rDNA) of the
Whipple’s disease bacterium and its lack of close relationship
to any actinomycete for which 16s rDNA sequence data were
available, the name “Tropheiyma whippelii” was proposed (36).
The determination of the almost complete 16s rDNA se-
quence allowed the design of two taxon-specific PCR primers,
the use of which led to the detection of almost identical se-
quences in tissue from five patients with Whipple’s disease
(36). Subsequently, a diagnostic PCR system which detected
the specific DNA fragment in tissue from 30 additional pa-
tients with the disease was designed (45).
The genes coding for rRNA molecules are organized in
operons and arranged in the order 5’-16S-23S-5S-3’, in which
the individual rRNA genes are separated by spacer regions
(39). The size and sequence composition of the spacer region
between the 16s rRNA and 23s rRNA genes have been inves-
tigated in a number of bacterial taxa, and its potential use in
diagnostics and identification has been highlighted (1 1) be-
cause of the greater sequence variability of the spacer, com-
pared with that of the 16s rRNA gene, among bacterial strains
In this present study, we determined an almost complete 16s
rDNA sequence comprising 1,495 nucleotides for a Whipple’s
disease bacterium. This new sequence was compared with the
considerable number of actinomycete 16s rRNA sequences
that have been added to the database (1, 16-19,21, 29-37,40,
43, 46, 47) since the characterization of the Whipple’s disease
bacterium by Relman et al. (36). The phylogenetic analysis
provides additional information on the phylogenetic position
of this bacterium and allows some speculation as to the type of
organism. The spacer region between the 16s rRNA and 23s
rRNA genes was amplified with a primer specific for the Whip-
VOL. 46, 1996 PHYLOGENETIC POSITION OF WHIPPLE’S DISEASE BACTERIUM 1079
ple’s disease bacterium and a universal bacterial primer bind-
ing in the 23s rDNA, and the sequence was determined.
MATERIALS AND METHODS
Preparation of DNA from biopsy material. DNA from the Whipple’s disease
bacterium was extracted from the duodenal biopsy of a patient whose case was
previously reported (27). Prior to PCR, the biopsy was deparaffinized by shaking
it twice in 1 ml of n-hexane and twice in 500 pl of ethanol, each step lasting for
30 min. The sample was centrifuged for 5 min at 18,000 X g between each of
these steps. The biopsy was then dried under vacuum, subsequently digested for
2 h at 56°C in 40 pl of lysis buffer (50 mM KCI, 10 mM Tris, 1.5 mM MgCI2, 1%
Triton X-100, 200 pg of proteinase K per ml), and boiled for 10 min after the
addition of 20 pl of a 20% Chelcx suspension (biotechnology-grade chelating
resin Chelex 100; Bio-Rad Laboratories, Richmond, Calif.). Ten microliters of
the supernatant was added to the PCR.
PCR amplification. The composition of the PCR mix was the same as de-
scribed previously (26), and the cycling profile consisted of initial denaturation at
95°C for 3 min followed by 40 cyclcs of denaturation at 95°C for 45 s, annealing
at 58°C for 1 min, extension at 72°C for 1 min, and final extension at 72°C for 2
min. To amplify a 1,249-bp fragment from the 16s rRNA gene, the universal
bacterial primer p8FPL (5‘-AGTITGATCCTGGCTCAG) and the Whipple’s
disease bacterium-specific primer pW2RB (5’-ATTCGCTCCACCTTGCGA) of
Relman et al. (36) were used in a modified version, both without restriction
enzyme recognition sites. To obtain the 3‘ end of the 16s rRNA gene, the 5’ end
of the 23s rRNA gene and the intergenic spacer, the Whipple’s disease bacte-
rium-specific primer pW3FE (5’-AGAGATACGCCCCCCGCAA, without re-
striction sites) of Relman et al. (36), and the universal primer 2 for the 23s rRNA
gene (5’-GGTACCTTAGATGTTTCAGmC) of Kostman et al. (20) were used.
PCR products were checked on 5% polyacrylamide gels with previously de-
scribed electrophoresis conditions (26), subsequently transferred to nylon mem-
branes, and hybridized at 60°C with the 3’P-labeled oligonucleotide “whip3”
(5’-TGGTACAGAGGGTTGCAATA), which is located on the 16s rRNA of
the Whipple’s disease bacterium between the primers pW3FE and pW2RB of
Relman et a]. (36). To obtain pure DNA for sequencing, elcctrophorcsis was
performed on 1% agarose gels. DNA fragments were cut out from the agarose
gels and purified with the Jetsorb gel extraction kit (Genomed, Research Trian-
gle Park, N.C.). Sequencing was performed with the AmpliCycle Sequencing Kit
(Perkin-Elmer, Nonvalk, Conn.) with incorporation of [cx-~~PI~ATP.
tion products were electrophoresed on 6% standard sequencing gels at constant
power of 50 mA and then exposed to X-ray films. To confirm the results of
manual sequencing, the reactions were repeated with the Taq DyeDeoxy Ter-
minator sequencing kit (Applied Biosystems, Foster City, Calif.) according to the
manufacturer’s protocol. The sequence reactions were then electrophoresed with
the Applied Biosystems 373A DNA sequencer. Sequences were manually aligned
with published sequences from members of the actinomycete line of descent.
Phylogenetic analyses. The data set used for the phylogenetic analyses com-
prised 1,304 unambiguous nucleotides between positions 41 and 1449 (Esche-
richia coli numbering of positions). Phylogenetic analyses were carried out with
the range of programs provided by the ARB (“a software environment for
sequence data”) (42), the PHYLIP package (7), and the Ribosomal Database
Project (25). Phylogenetic trees were generated by the maximum-likelihood,
neighbor-joining, least squares, and maximum-parsimony algorithms. The tree
topologies were evaluated by bootstrap analyses with 1,000 resamplings of the
sequence data with SEQBOOT (6).
Nucleotide sequence accession numbers. The accession numbers of the se-
quences of the reference strains (strain designations given when available) used
in the phylogenetic analyses are as follows: Actinoplanes philippinensis DSM
43019T (X93187), Agrococcus jenensis DSM 9580T (X92492), Agromyces rarnoszis
DSM 43045T (X77447), Arthrobacter globifomis DSM 20124T (M2341 l), Atopo-
biurn rninutwn ATCC 33267T (M59059), Aureobacterium liquefaciens DSM
2063ST (X77444), “Brevibacteri‘um helvolurn” DSM 20419 (X77440), Brevibacte-
rium linens DSM 20425T (X77452), Cellulomonas biazotea DSM 20112T
(X83802), Cellulomonas cellasea DSM 201 18* (X83804), Cellulomonas cellulans
DSM 43879T (X83809), Cellulomonas fetmentans DSM 3133T (XS3805), Cellu-
lomonas flavigena DSM 20109T (X83799), Cellulomonas gelida DSM 201 1 lT
(X83800), Cellulomonas hominis CE40 (X82598), Clavibacter rnichiganense
subsp. michiganense DSM 46364T (X77435), Clavibacter .ryli subsp. cynodontis
(M60935), “Coiynebacterium aquaticum” DSM 20146 (X77450), Curtobacterium
citreum DSM 2052gT (X77436), Dermacoccus nishinorniyaensis DSM 2044ST
(X87757), Jonesia denitrificans DSM 20603T (X8381 l), Kocuria rosea DSM
20447T (X87756), Lentzea albidocapillata DSM 44073T (X84321), Microbacte-
rium lacticum DSM 20427T (X77441), Micrococcus luteiis (M38242), Nesterenko-
nia halobia DSM 20541T (X80747), Nocardia asteroides DSM 43757T (X80606),
Promicromonospora enterophila DSM 43852T (X83807), Rathayibacter ratha-yi
DSM 7485T (X77439), Ruthia dentocariosa ATCC 17931T (M59055), Sporichthyu
polymoipha DSM 46113T (X72377), Streptomyces griseus (M76388), and Strepto-
sporangium roseum DSM 43021T (X89947). The sequence determined for the
Whipple’s disease bacterium in this study and the structural features associated
with it have been deposited in the EMBL database under accession no. X99636.
RESULTS AND DISCUSSION
By using the combination of primers described above, two
PCR products were obtained; both were slightly larger than 1
kbp on polyacrylamide gels and both hybridized with the oli-
gonucleotide whip3. The sequences of both PCR products
contained an identical overlapping fragment of 230 bases
which is located between the Whipple’s disease bacterium-
specific primers pW3FE and pW2RB of Relman et al. (36).
The whole sequence is 1,989 nucleotides long and ranges from
position 28 in the 16s rRNA to position 188 in the 23s rRNA
of the corresponding genes of E. coli (accession no. 501695).
The sequence contains 1,495 nucleotides of the 16s rRNA
gene and 200 nucleotides of the 23s rRNA gene of the Whip-
ple’s disease bacterium. The first 1,321 nucleotides are identi-
cal to the sequence of the Whipple’s disease bacterium that
was reported earlier by Relman et al. (36).
All methods of phylogenetic analysis used in this study gave
identical branching patterns with respect to the phylogenetic
position of the Whipple’s disease bacterium. Differences in the
positions and relationships of the deep branching organisms
were observed between the different analyses. In all phyloge-
netic analyses, the branching position of the Whipple’s disease
bacterium was as the deepest branch of the cluster composed
of the actinomycetes with group B peptidoglycan, between this
group and the members of the family Cellulomonadaceae. A
phylogenetic tree was calculated with the 16s rRNA sequences
of representatives of the major phylogenetic groups within the
order Actinomycetules (Fig. 1). Pairwise evolutionary distances
were computed with the correction of Jukes and Cantor (13).
The phylogenetic dendrogram shown in Fig. 1 was recon-
structed from the distance matrices by the neighbor-joining
method (38). Although the position of the Whipple’s disease
bacterium between the actinomycetes with group B peptidogly-
can and the cellulomonads was recovered in all phylogenetic
analyses, the bootstrap analyses do not indicate a very high
level of confidence at 83% associated with this position. The
bootstrap values indicated in Fig. 1 show that the cellulomonad
group was also recovered in 83% of the resamplings while the
cluster of actinomycetes with group B peptidoglycan was re-
covered in only 81% of the analyses. When the 16s rDNA
sequence similarities of the Whipple’s disease bacterium, the
representatives of the actinomycetes with group B peptidogly-
can, and the cellulomonads shown in Fig. 1 were calculated,
the 16s rDNA similarities of the Whipple’s disease bacterium
and the actinomycetes with group B peptidoglycan are in the
range of 90.0 to 91.6% compared with a range of 89.9 to 91.6%
for the cellulomonads. The highest similarities are found to the
two species “Coiynebacterium aquaticum” and Cellulomonas
cellasea, both at 91.6%. The peptidoglycan types of the organ-
isms within the clusters comprising the phylogenetic neighbors
of the Whipple’s disease bacterium are very different in struc-
ture. On one hand, there is the rare group B peptidoglycan
which is possessed by all members of one of the neighboring
clusters, whiIe the other cluster, the cellulomonads, has group
A peptidoglycan. Most of the group B peptidoglycan-contain-
ing actinomycetes are environmentally occurring or are plant
pathogens (2) and have only rarely been encountered in clin-
ical specimens (8). Members of the family Cellulomonadaceae
have generally been isolated from soil, but strains of Oerskovia
turbatu (shown to be highly related to Promicromonospora en-
terophilu ) have been isolated from clinical sources (41).
Recently, Funke et al. (9) have described Cellulomonus homi-
nis isolated from clinical samples.
The phylogenetic position determined in this study for the
Whipple’s disease bacterium based on comparison of almost
MAIWALD ET AL.
INT. J. SYST. BACTERIOL.
Clavibacter xyli subsp. cynodontis
‘Coryne bacterium aquatic urn
Clavibacter michiganense subsp. michiganense
Whipple’s disease bacterium
Arthro bacter glo biformis
Dermacoccus n is hinom iyaens is
Brevi bacterium linens
Atopo bium minutum
FIG. 1. Phylogenetic dendrogram displaying the relationship of the Whipple’s disease bacterium to other representatives of the actinomycetes. The scale bar
represents five inferred nucleotide substitutions per 100 nucleotides. Numbers at branching points indicate bootstrap values.
complete 16s rDNA sequences of more than 25 reference
organisms from a group that Relman et al. (36) designated
“actinobacteria” is Merent from the position previously shown
(36). The Whipple’s disease bacterium is not the deepest
branching organism of the actinobacteria group but branches
within this group. The phylogenetic analysis presented here
allows us to eliminate the possibility that the Whipple’s disease
bacterium is highly related to any of the actinomycete taxa for
which sequences have become available since the analysis of
€?,elman (36) with the then-available limited database. The
determination of the phylogenetic position of the Whipple’s
disease bacterium 16s rDNA sequence between the actinomy-
cetes with group B peptidoglycan and the cellulomonads may
provide some clues as to its characteristics. Future studies
should now be aimed at the determination of the peptidogly-
can type of the Whipple’s disease bacterium in order to deter-
mine its affiliation to either neighboring taxon at the chemotaxo-
nomic level. Such data would complement the phylogenetic
data available and indicate the significance of the intermediate
branching point of the Whipple’s disease bacterium demon-
strated in this study.
The spacer region between the 16s and 23s rRNA genes
determined in this study is 294 nucleotides long and does not
contain tRNA genes. Searches for sequence similarity to the
spacers of other actinomycetes (Frunkiu spp., accession num-
bers M55343 and M88466; Streptumyces sp., M27245; Chi-
bucter spp., LA3095 and U09379) revealed an overall low ho-
mology (approximately 40%) to the respective genes of these
species, indicating the lack of comparability of spacer regions
between phylogenetically distinct taxa. A region of higher sim-
VOL. 46, 1996 PHYLOGENETIC POSITION OF WHIPPLE’S DISEASE BACTERIUM
ilarity can be found. The highest similarity of 87% is found in
a 31-nucleotide-long stretch within the 3’ end of the spacer re-
gion of the Whipple’s disease bacterium and Cluvibucter mich-
igunense. This stretch contains the conserved motifs of a puta-
tive box A (CGATAmGA) and box C (GTGGACGCG
AG), as well as an interbox stretch (GAACTGCACA). The
length of the intergenic spacer between the 16s rRNA and 23s
rRNA genes of the Whipple’s disease bacterium (294 bp) is
within the size range reported for the majority of other gram-
positive bacteria with high G+C content. For example, the
spacers of mycobacteria have been found to vary in length
between 276 and 362 bp (12,14), those of Streptomyces species
vary between 277 and 304 bp (15), and a size of 411 bp has
been determined for the Frunkiu sp. (28). A recent report of
the spacer size for 29 strains of 18 Bifidobucterium species
showed them to range in size from 274 to 552 bp (21). The
spacer regions of Cluvibucter rnichigunense subspecies, organ-
isms with group B peptidoglycan, have been determined to be
approximately 500 bp (22). The 16s-23s intergenic spacer of
the Whipple’s disease bacterium did not contain tRNA genes
which are commonly found in the spacers of gram-negative
bacteria (5) and of low-G+C gram-positive bacteria (24, 44).
This is also in concordance with the findings of Liesack et al.
(23), who assumed that the absence of tRNA genes and an
approximate length of 300 nucleotides are general features of
16s-23s intergenic spacers of actinomycetes.
The 16s-23s intergenic spacer is a potential target for spe-
cies identification and strain differentiation, because it has
greater variability than the 16s rRNA gene. The spacer has
been successfully used for strain differentiation in some bacte-
rial species, such as Burkholderiu (Pseudomonus) cepuciu (20)
or Staphylococcus uureus (10). The sequence of the spacer of
the Whipple’s disease bacterium should be determined for
additional strains to check whether there are differences at the
spacer level between strains from individual patients. The se-
quence data and the results of its analysis presented here
provide additional information on the phylogenetic position of
the Whipple’s disease bacterium which can be used to further
investigate the characteristics of this organism. The possibility
of determining the 16s to 23s rRNA spacer region sequence
has been demonstrated, and this should now be applied to a
number of patients in order to investigate its use in strain
Part of this work was supported by a grant from the Deutsche
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