Content uploaded by Lyn Gilbert
Author content
All content in this area was uploaded by Lyn Gilbert
Content may be subject to copyright.
lnternational Journal of Systematic Bacteriology
(1 999),
49,
1
879-1 889
Printed in Great Britain
1
Centre for Infectious
Diseases and Microbiology,
institute
of
Clinical
Pathology and Medical
Research, Westmead, New
South Wales, Australia
Laboratory, Centre for
Virus Research, Westmead
Hospital, Darcy Rd,
Westmead, New South
Wales,
2145
Australia
*
Retroviral Genetics
INTRODUCTION
P
h
y
logenet
ic
ana
I
ys
is
of
Ureaplasma
urealyticum
-
support for the establishment
of
a
new species,
Ureaplasma
parvum
Kong Fanrong,’ Greg James,’ Ma Zhenfang,’ Susanna Gordon,’
Bin Wang’ and Gwendolyn
L.
Gilbert’
Author for correspondence: Gwendolyn
L.
Gilbert.
Tel:
+61
2
9845 6255. Fax:
f61
2 9893 8659.
e-mail
:
lyng@icpmr.wsahs.nsw.gov.au
~~
In
this study, the phylogenetic relationships between the
two
biovars and
14
serovars
of
Ureaplasma urealyticum
were studied using the sequences
of
four
different genes
or
genetic regions, namely:
165
rRNA genes;
16s-235
rRNA
gene spacer regions; urease gene subunits
ureA,
ureB,
partial
ureC
and
adjoining regions upstream
of
ureA, ureA-ureB
spacer and
ureB-ureC
spacer;
the 5’-ends
of
the multiple-banded antigen (MBA) genes.
U.
urealyticum
genotypes, based
on
all
four
genomic sequences, could be clearly separated
into
two
clusters corresponding
with
currently recognized biovars
1
and
2.
Sequences were generally conserved
within
each biovar. However, there was
heterogeneity
within
the 5’-end regions
of
the MBA genes
of
the
four
serovars
of
biovar
1;
the sequence
of
serovar
3
was identical
with
the previously
published sequence and differed by only three bases
from
that
of
serovar
14;
but
there were significant differences between the sequences
of
serovars
3
and
14
and those
of
serovars
1
and
6.
Based
on
the phylogenetic analysis,
support
is
given
to
previous recommendations that the
two
biovars
of
U.
urealyticum
be
classified
as
distinct species, namely
U.
parvum
and
U.
urealyticum
for
biovars
1
and
2,
respectively.
In
the future, the relationship between the new species and
clinical manifestations
of
ureaplasma infections should be studied.
Keywords:
Ureaplasma urealyticum, Ureaplasma parvum,
phylogeny
Ureaplasma urealyticum
is a recognized cause of
urethritis (Kong
et al.,
1996; Taylor-Robinson
&
Furr,
1997; Taylor-Robinson
et al.,
1985) and has been
implicated in complications
of
pregnancy and prema-
turity (Abele-Horn
et al.,
1997a, b; Cassell
et al.,
1988;
Kundsin
et al.,
1996
;
Nelson
et
al.,
1998). As a common
genital tract commensal (Tully, 1993), its pathogenic
role in individual cases is difficult to confirm. Cur-
rently,
U.
urealyticum
includes two biovars and 14
serovars (Lin
&
Kass, 1980; Naessens
et al.,
1988;
Razin
&
Yogev, 1986; Robertson
&
Stemke, 1982;
Robertson
et
al.,
1993). Some serovars have been
implicated in disease syndromes more commonly than
Abbreviation
:
M
BA,
mu Iti pl e- banded antigen.
The GenBank accession numbers for the sequences in this paper are:
AF055358-AF055367, AF056982-AF056984 (MBA
genes);
AF059322-
AF059335 (1 65-235 rRNA
gene spacer regions);
AF085720-AF085733
(urease gene subunits);
AF073446-AF073459 (1 65 rRNA
genes).
others (Abele-Horn
et
al.,
1997b; Grattard
et al.,
1995b; Naessens
et al.,
1988; Zheng
et al.,
1992), but
any differences in pathogenicity among serovars is
unproven. Investigations have been limited by tech-
nical difficulties and cross-reactions associated with
conventional serotyping methods even when mono-
clonal antibodies are used (Cheng
et al.,
1994; Naes-
sens
et al.,
1988; Quinn
et al.,
1981; Robertson
&
Stemke, 1982; Watson
et al.,
1990; Wiley
&
Quinn,
1984).
Phylogenetic analysis of the relationships between the
biovars and serovars of
U.
urealyticum
would provide
the basis for a molecular typing system and allow
further investigation of the pathogenic potential
of
individual types (Razin
&
Yogev, 1986; Robertson
&
Stemke, 1982; Robertson
et
al.,
1994; Weisburg
et
al.,
1989). The target sequences chosen for such an analysis
should be relatively conserved and have biovar-specific
and serovar-specific differences. In this study, we
sequenced four gene regions
of
all
14 serovars of
U.
urealyticum
to study the phylogenetic relationships
01133
0
1999
IUMS
1879
K.
Fanrong
and others
between them. They were: 16s rRNA genes; 16s-23s
rRNA gene spacer regions
;
the urease gene subunits
ureA, ureB,
partial
ureC
and adjoining regions up-
stream of
ureA, ureA-ureB
spacer, and
ureB-ureC
spacer; and the 5’-end region
of
the MBA (multiple-
banded antigen) genes. All have been used, individu-
ally, in previous phylogenetic studies
of other bacteria
and/or mycoplasmas, including ureaplasmas (Blanc-
hard, 1990; Harasawa
&
Cassell, 1996; Harasawa
et
al.
1996; Robertson et
al.,
1994; Zheng
et al.,
1995).
However, they have not previously been studied
together to compare the sequences of all 14 serovars
of
U.
urealyticum.
We believed that combined data from
analysis of several important genetic regions from all
serovars would provide a better understanding of the
phylogeny. The sequences obtained could also be used
to develop methods for biotyping and serotyping
of
U.
urealyticum
isolates and for detection and subtyping of
U.
urealyticurn
directly from clinical specimens.
METHODS
Bacterial strains.
Reference strains of each
U.
urealyticurn
serovar were obtained directly from the American Type
Culture Collection (ATCC reference set) as follows
:
serovars
1,
ATCC 278 13
;
2, ATCC 278
14
;
3, ATCC 278
15
;
4, ATCC
27816;
5,
ATCC 27817; 6, ATCC 27818; 7, ATCC 27819; 8,
ATCC27618; 9,ATCC33175; 10,ATCC 33699; 11,ATCC
33695;
12, ATCC 33696;
13,
ATCC 33698;
14,
ATCC
33697. In addition, a set of reference strains of serovars 1-14
were kindly provided by Dr
H.
L.
Watson, Department of
Microbiology, University of Alabama
at
Birmingham, AL,
USA (UAB reference set). These had been obtained orig-
inally from
E.
A. Freundt, Institute of Medical Micro-
biology, University
of
Aarhus, Aarhus, Denmark (serovars
1-8) and
J.
A. Robertson, Department of Medical Micro-
biology and Infectious Diseases, University of Alberta,
Edmonton, AB, Canada (serovars 9-14).
Oligonucleotide primers.
The oligonucleotide primers used
in the paper are shown in Table 1. Previously published
primers were used as follows: P1, P6, U3, U8 (Robertson
et
al.,
1993), GPO-1, Mseq-3, GPO-3 and MGSO (van Kup-
peveld
et al.,
1992) were used to amplify and sequence the
16s rRNA genes; MCGpF11, R23-1R, R16-2 and MCG-
pR21 (Harasawa
et
al.,
1993) were used to amplify and
sequence the 16s-23s rRNA gene spacer regions; UUSl,
UUA1, UlA, U1B (Blanchard, 1990),
U2B,
U2C (Ruifu
et
al.,
1997)
-
as well as additional primers designed by
us,
UUSP, UCAl and UCA2
-
were used to amplify and
Table
1.
Sequences
of
oligonucleotide primers used in the paper
Target gene/region Reference Primer
name
Primer sequence
16s
rRNA genes
Robertson
et
al.
(1993)
Robertson
et al.
(1993)
Robertson
et
al.
(1993)
Robertson
et
al.
(1993)
van Kuppeveld
et
al.
(1 992)
van Kuppeveld
et al.
(1
992)
van Kuppeveld
et al.
(1 992)
van Kuppeveld
et
al.
(1 992)
Harasawa
et
al.
(1993)
Harasawa
et
al.
(1993)
Harasawa
et
al.
(1993)
Harasawa
et
al.
(1993)
16s-23s rRNA gene
spacer regions
Urease gene subunits
-
and adjacent regions
Blanchard (1990)
Blanchard (1990)
Blanchard (1990)
Blanchard (1 990)
Ruifu
et al.
(1997)
Ruifu
et
al.
(1997)
-
-
The
5’
end of MBA
Teng
et al.
(1994, 1995)
Teng
et
al.
(1994, 1995)
Teng
et
al.
(1994, 1995)
Teng
et al.
(1994, 1995)
Teng
et al.
(1994, 1995)
Teng
et
al.
(1994, 1995)
genes
PI
u3
U8
P6
Mseq-3
MGSO
MCGpFll
GPO-
1
GPO-3
R16-2
MCGpR2
R23-1R
UUSP
UUSl
UUAl
UlA
UlB
U2B
u2c
UCAl
UCA2
UMS- 125
UMA226
UMSSI
UMA427
UMA263
UMS- 170
AGA GTT TGA TCC TGG CTC AGG A
TAG AAG TCG CTC TTT GTG G
GAA GAT GTA GAA AGT CGC GTT TGC
GGT AGG GAT ACC TTG TTA CGA CT
ACT CCT ACG GGA GGC AGC AGT A
TGT ATT ACC GCG GCT GCT G
GGG GAG CAA ATA GGA TTA GAT ACC CT
TGC ACC ATC TGT CAC TCT GTT AAC CTC
AAA CTA TGG GAG CTG GTA AT
GTG GGG ATG GAT CAC CTC CT
CCA TTC ACC ATA AAC TCT T
CTC CTA GTG CCA AGG CAT C/TC
AAT TCT (C/T)(C/T)A (A/T)TA AGA ATA
CAC AGA TGT CCT TGA TGT AC
TAC TTC ACG AGC AGA TTG CA
GAT GGT AAG TTA GTT GCT GAC
ACG ACG TCC ATA AGC AAC T
CGA AAT TGT GAT GAA CGA AGG
CTC CTA ATC TAA CGC TAT CAC C
TTC AT(C/T) CCC ATA CCT TCA CG
GTG AAC GTG AGT ATC TAA AC
GTG AAC GTG AGT ATC TAA AC
(A/G)CA CAT
CAG CTG ATG TAA GTG CAG CAT TAA ATT C
TTC TGG GCT ATG ACA TTA GGT GTT ACC
ACC TGG TTG TGT ACT TTC AAA GTT CAC
GTA TTT GCA ATC TTT ATA TGT TTT CG
TTT GTT GTT GCG TTT TCT G
1880
International Journal
of
Systematic Bacteriology
49
Phylogeny
of
Ureaplasma
urealyticum
Table
2.
Summary of
PCR
results showing
sizes
of bands (amplicons) produced by
all
14
serovars
of
U.
urealyticum
using
11
primer pairs
to
amplify four different target geneshegions
Target genelregion
Primer pair*
Amplicon size (bp)
See Fig.
:
Reference
Biovar Biovar
It
21-
16s
rRNA genes
16s-23s rRNA
gene
spacer regions
P1, P6
P6, U3
P6, U8
MCGpFl1, R23-1R
Urease gene subunits and
adjacent regions
5’
end
of
MBA
genes
R16-2, MCGpR2
U2B, U2C
UUS1, UUAl
UUSP, UCA2
UMS51, UMA427
UMS- 125, UMA226
UMS- 170, UMA263
1488
1299
47
1
344
425
-
-
1354
403/404$
447
-
1484
1301
472
345
418
313
1350
448
476
-
-
-
Robertson et
al.
(1 993)
-
Robertson et
al.
(1993)
1
Robertson et
al.
(1993)
-
Harasawa et
al.
(1993)
Harasawa et
al.
(1993)
Ruifu et
al.
(1997)
-
-
Blanchard
(1 990)
Teng et
al.
(1994, 1995)
Teng et
al.
(1994, 1995)
Teng et
al.
(1994, 1995)
-
-
-
2
-
3
*
See text for primer sequences.
5
Serovars
1,
3
and
14
produce bands
of
403
bp
and serovar
6,
a band of
404
bp.
Biovar
1
includes serovars
1,
3, 6
and
14;
biovar
2
includes serovars
2,
4,
5,
7,
8,
9, 10, 11, 12, 13.
Fig.
1.
Results of
PCR
amplification of the 165 rRNA genes of all
14 serovars of
U.
ureafyticum using primers P6 and US. (a) ATCC
strains and (b) UAB reference strains. Biovar
1
consists of
serovars 1,
3,
6 and
14,
the other ten serovars belong
to
biovar
2. Lane M, molecular mass markers 4x174 DNNHaelll; lane
numbers in each panel correspond with
U.
urealyticum serovar
numbers.
, , ,
.
,
.
. .
. .
.
.
. .
.
.
.
.
. . . .
.
.
.
. .
. .
.
.
.
.
.
. . . .
, , ,
. . . .
.
. .
. .
. .
.
.
.
. .
.
.
.
. .
. . . .
.
. . .
.
. .
, ,
.
, ,
.
.
.
. .
.
. . . .
.
.
.
. .
.
.
.
.
,
.
,
. .
,
.
. .
.
.
.
. .
.
. .
. .
.
.
.
. .
. .
. .
.
,
.
.
.
.
.
.
. . . .
.
. . . .
.
.
.
Fig.
2.
Results of
PCR
amplification of the 5’-end of MBA genes
of all 14 serovars of
U.
urealyticum using primers UMS-125 and
UMA226. Biovar 1 consists
of
serovars
1,
3,
6
and 14, the other
ten serovars belong to biovar
2.
Lanes:
M,
molecular mass
markers (6x174 DNNHaelll; 1-14, correspond with
U.
urealyticum serovars, ATCC strains; 8’, UAB reference strain of
U.
urealyticum serovar
8.
sequence
U.
urealyticum
urease gene subunits
ureA,
ureB,
partial
ureC
and adjoining upstream region of
ureA,
ureA-ureB
spacer and
ureB-ureC
spacer. Three previously
published oligonucleotide primer pairs were used to amplify
the 5’-end region of the
MBA
genes
of
U. urealyticum
serovars
1-14
namely:
UMS-125, UMA226
for all
14
serovars;
UMS51
and
UMA427
for the four serovars of
biovar
1
(Teng et
al.,
1994); UMS-170, UMA 263
for the
ten serovars of biovar
2
(Teng et
al.,
1995).
International Journal of Systematic Bacteriology
49
1881
K.
Fanrong and others
~~~ ~
DNA
preparations.
Cells from
0-5
ml of ureaplasma broth
(
10B) cultures of each
U.
urealyticum
serovar were harvested
from late exponential growth by centrifugation at 14000
g
for 20 min. DNA was isolated from cultures by treatment
with
500
p1 digestion buffer (10 mM Tris/HCl, pH
8.0,
0.45
%
Triton X-100 and 0.45
Yo
Tween 20) and proteinase
K,
20 g l-l, at
55
"C
for
1
h, 95 "C for
20
min and then
extraction with
phenol/chloroform/isoamyl
alcohol
(25
:
24:
1,
by vol.) and chloroform/isoamyl alcohol (24: 1, v/v).
DNA was precipitated with 0.1 vol. of
3
M sodium acetate
(pH
5.2)
and
2
vols
of
ethanol. The washed and dried pellets
were hydrated in
200
pl ultrapure and sterile water.
PCR.
The
25
p1
amplification reaction mixtures contained
2.5
pl
10
x
PCR buffer (1
x
is
10
mM Tris/HCl,
pH
8.8
at
25
"C,
1.5
mM MgCl,,
50
mM KCl and 0.1
YO
Triton X-loo),
0.5
U
Taq
polymerase (Finnzymes),
200
mM of each dNTP
(dATP, dCTP, dGTP, dTTP; Boehringer Mannheim), 10
pmol each primer,
5
p1 sample DNA, and added ultrapure
sterile water to
25
pl.
The denaturation, annealing and elongation temperatures
and times used were 95 "C for
30
s,
55-62
"C (according to
the
T,
values of different primers) for
30
s
and 72
"C
for
1
min, respectively, for
40
cycles using a Perkin Elmer
thermocycler (Blanchard, 1990; Harasawa
et al.,
1993
;
Robertson
et al.,
1993; Ruifu
et al.,
1997; Teng
et al.,
1994,
1995; van Kuppeveld
et al.,
1992).
Eight microlitres of PCR products were analysed by elec-
trophoresis on
2.0
YO
(w/v) agarose gels which were stained
with
0.5
pg
ethidium bromide ml-'.
A
visible band with
appropriate size on
UV
translumination was considered a
positive result.
Sequencing
and
analysis.
The PCR products were sequenced
with Applied Biosystems (ABI)
Taq
DyeDexoy terminator
cycle-sequencing kits according to standard protocols. The
multiple sequence alignments were performed with
PILEUP
and
PRETTY
programs from the Multiple Sequence Analysis
program group, provided in WebANGIS, ANGIS (Austra-
lian National Genomic Information Service) version 3.
Phylogenetic relationships were studied using
CLUSTAL
and
trees were bootstrapped with 100 replications.
Fig.
3.
Results
of
PCR amplification
of
the 5'-end
of
MBA genes
of
all 14 serovars
of
U.
urealyticum
using primers UMS-170 and
UMA263. Biovar
1
consists
of
serovars 1,
3,
6
and 14, the other
ten serovars belong to biovar 2. Lanes:
MI
molecular mass
markers 4x174
DNNHinfl;
1-14 correspond with
U.
urealyticum
serovars, ATCC strains;
8',
UAB reference strain
of
U.
urealyticum
serovar
8.
RESULTS
PCR
The results
of
PCR
for all
14
U.
urealyticum
serovars,
using
11
primer pairs to amplify the four different gene
regions are summarized in Table
2
and representative
examples are shown in Figs
1-3. Five primer pairs were
specific for either biovar
1
or biovar
2
and the others
produced different sized bands for each biovar. With
one exception, band sizes produced by individual
primer pairs were consistent for all serovars within
Table
3.
Comparative study
of
the sequences
of
165
rRNA
genes
of
14
serovars
of
U.
urealyticum
(ATCC
st
r
a
i
ns)
Numbers
in
parentheses are serovars affected
by
changes.
~_________
____~
___~ ___~
~
Site Biovar 1 Biovar 2 Biovar 1/2
~~
87
176
177
180-1
83
200
3 50
430
807
808
812
8 14
974
1090
1270
1407
C
G
T
G
-
A
(5)
T
C
A
C
G
C
T
(5,
7")
C
G
C
C
*For serovar
7
UAB
reference strain, the base position 1407
was
C
not T.
SPUU.msf(biovar
1)
SPUU.msf(biovar
2)
Consensus
SPUU.msf(biovar
1)
SPUU.msf(biovar 2)
Consensus
SPUU.msf{biovar
1)
SPUU.msf(biovar
2)
Consensus
SPUU.msf{biovar
1)
SPUU.msf{biovar 2)
Consensus
16-239 rRNA gene spacer123S
rRNA
SPUU.msf(biovar
1)
----------
--
SPUU.msf(biovar
2)
----------
--
Consensus TATAATAAGT TA
.
. . .
.
.
.
,
.
, ,
.
,
. . . . .
.
.
.
. . .
. . .
.
.
.
. .
.
.
.
.
.
.
.
.
.
. .
. . .
. .
.
.
. . .
.
.
.
.
.
. . .
.
. .
.
,
.
.
. .
.
. . .
.
. .
.
. .
. . .
.
. .
. . .
.
. .
.
.
.
.
.
. .
. . .
. . . .
. . .
. .
. . .
.
. . .
.
.
.
.
. .
.
.
.
.
.
. . . . .
.
.
.
.
.
.
.
.
.
.
.
Fig.
4.
Multiple sequence alignment
of
the sequences
of
the
165-235 rRNA gene spacer region
of
14 serovars
of
U.
urealyticum
(ATCC strains).
1882
International Journal
of
Systematic Bacteriology
49
Phylogeny
of
Ureuplusma urealyticum
UuU.msf(biovar
1)
uuu.msf{biovar
2)
consensus
Um.msf{biovar
1)
uuu.msf(biovar
2)
consensus
UUU.msf{biovar
1)
UUU.rnsfibiovar
2)
Consensus
UUU.msf(biovar
1)
UW.msf(biovar
2)
Consensus
UUU.msf(biovar
1)
um.msf(biovar
2)
consensus
uuu.msf{biovar
1)
UUU.msf(biovar
2)
Consensus
UUU.msf(biovar
1)
UUU.ms€(biovar
2)
Consensus
UUU.msf{biovar
1)
Uuu.msf(biovar
2)
Consensus
UUU.msfibiovar
11
UUu.msf(biovar
2)
Consensus
UUU.msf{biovar
1)
UWLJ.msf(biovar
2)
Consensus
uuu.msf{biovar
1)
uuu.msf{biovar
2)
Consensus
UUU.msf{biovar
11
UUU.msf(biovar
2)
Consensus
UUU.msf(biovar
1)
wu.msf(biovar
2)
Consensus
UUU.msf(biovar
1)
UUU.msf(biovar
21
Consensus
UUU.msf{biovar
1)
UUu.msf(biovar
2)
Consensus
UUU.msf(biovar
1)
UUu.msf{biovar
2)
Consensus
UUU.msf{biovar
1)
UUU.msf(biovar
2)
Consensus
UUU.msf(biovar
1)
WU.msf(biovar
21
Consensus
WU.msf{biovar
1)
uuu.msf{biovar
2)
Consensus
UUU.msf{biovar
1)
UUU.msf(biovar
2)
Consensus
uuU.msf(biovar
1)
UUU.msf(biovar
2)
Consensus
UUU.msf(biovar
1)
UUU.msf(biovar
2)
Consensus
UUU.msfibiovar
1)
UUU.msf{biovar
2)
Consensus
UUU.msf(biovar
1)
UUU.msf(biovar
2)
Consensus
UUU.msf(biovar
1)
UUU.msf(biovar
2)
Consensus
UUU.msf{biovar
11
UUU.msf(biovar
2)
Consensus
UUU.msfIbiovar
1)
UUU.msf{biovar
2)
Consensus
fig.
5.
Multiple sequence alignment of the sequences
of
urease
gene subunits of
14
serovars of
U.
urealyticum
(ATCC
strains).
*,
Base for serovar
2
is
G
instead
of
A;
#,
base for serovar
2
is
T,
for
the other serovars
of
biovar
2
it
is
C.
each biovar. The exception was the primer pair UMS-
125 and UMA226, which gave fragments of 403 bp for
serovars 1,3 and 14 and of 404 bp for serovar 6 (due to
a single base insertion in serovar 6 at position -46)
(Fig. 7).
Comparative study of the sequences of four genetic
regions
165
rRNA genes.
There were 14 (14/ 1439
=
0.97
YO)
base
differences in the sequences of 16s rRNA genes
between the two biovars. Heterogeneity was found at
two sites among four serovars of biovar
1
and at two
sites among ten serovars of biovar 2 (Table
3).
UAB reference isolates of
U.
urealyticum
serovars 1,2,
3,
5,6,7,8 and 14 were sequenced and the results were
identical to those of the corresponding ATCC serovar
strains, with the exception of serovar 7. In the UAB
reference strain
of
serovar
7,
the base at position 1407
International Journal
of
Systematic Bacteriology
49
was
C,
as it is for the other eight serovars of biovar 2,
rather than T as it was for ATCC serovars
5
and 7.
165-235
rRNA gene spacer regions.
The DNA sequence
alignment for the sequences of 16s-23s rRNA gene
spacer regions showed 14 (14/312
=
4.5
Yo)
base
differences between the two biovars (Fig. 4),
but
sequences were similar among serovars within each
biovar.
Urease subunits ureA, ureB, partial ureCgenes and adjoining
upstream
of
ureA, ureA-ureB spacer and
ureB-ureC
spacer.
The sequences of the four serovars of biovar
1
were
identical. Sequences of nine of the ten serovars
of
biovar 2 were identical, but serovar
2
differed by two
bases, at positions 126
(G
instead of A) and 248 (T
instead of C). There were 141 base differences
(141/ 1320
=
10.7
YO)
between the sequences of biovars
1
and 2. There were
25
base differences (25/149
=
16.8
YO)
in the region upstream of
ureA
;
19
(1
9/306
=
6-2
%)
in
ureA;
11
(1 1
/51
=
20.4
YO)
in the
ureA-ureB
1883
K.
Fanrong and others
MBA.msf(MBAUU-11)
MBA.msf(MBAUU-10)
MBA.msf[MBAUU-l2)
MBA, ms f {MBAUU- 13
1
MBA.msf(MBAUU-4)
MBA.msf [MBAW-7)
MBA.msf(MBAUU-2)
MBA.msf(MBAUU-5)
MBA.msf(MBAW-9)
MBA.msf(MBAUU-8)
MBA.msf(MBAUU-14)
MBA. msf (MBAUU-3
1
MBA.rnsf(MBAUU-11
MBA.rnsf{MBAUU-6)
Consensus
MBA.msf{MBAUU-~l)
MBA.msf (MBAUU-7
MBA.msf(MBAUU-10)
MBA.msf(MBAUU-12)
MBA.msf(MBAUU-13)
MBA.msf(MBAUU-4)
MBA.msf(MBAUU-2)
MBA.rnsf(MBAUU-5)
MBA.msf(MBAUU-8)
MBA.mSf(MBAUU-9)
MBA.msf(MBAUU-14)
MBA.msf{MBAW-3)
MBA.msf(MBAUU-6)
MBA.msf(MBAUU-1)
consensus
MBA.msf(MBAUU-11)
MBA.msf(MBAUU-7)
MBA.msf(MBAUU-10)
MBA.msf(MBAUU-12)
MBA.msf(MBAUU-13)
MBA.msf(MBAUU-4)
MBA.msf(MBAUU-2)
MBA.msf(MBAUU-5)
MBA.msf(MBAUU-8)
MBA.msf{MBAUU-9)
MBA.msf(MBAUU-14)
MBA.msflMBAUU-3)
MBA.msf(MBAUU-1)
MBA.msf(MBAUU-6)
Consensus
MBA.msf(MBAUU-11)
MBA.msf(MBAUU-101
MBA.msf(MBAW-12)
MBA.msf(MBAUU-13)
MBA.msf(MBAUU-4)
MBA.msf(MBAW-2)
MBA.msf{MBAUU-5)
MBA.msf(MBAW-8)
MBA.msf{MBAUU-9)
MBA.msf(MBAUU-14)
MBA.msf(MBAUU-7)
MBA.msf(MBAUU-3)
MBA.msf{MBAUU-1)
Consensus
MBA.mSf(MBAUU-6)
MBA.msf{MBAW-11)
MBA.msf(MBAUU-7)
MBA.msf
{MBAUU-10)
MBA.msf(MBAUU-12)
MBA.msf(MBAUU-13)
MBA.msf(MBAUU-4)
MBA.msf{MBAW-2)
MBA.msf(MBAUU-5)
MBA.msf(MBAUU-8)
MBA.msf(MBAUU-9)
MBA.msf{MBAW-14)
MBA.msflMBAUU-3)
MBA.msf(MBAUU-1)
consensus
MBA.msf(MBAUU-6)
MBA.msf(MBAUU-11)
MBA.msf(MBAUU-7)
MBA.msf(MBAUU-10)
MBA
. ms f (MEAUU-
12
>
MBA.msf(MBAUU-4)
MBA.msf(MBAUU-2)
MBA.msf(MBAUU-5)
MBA.msf(MBAW-8)
MBA.msf{MBAW-9)
MBA.msf{MBAUU-14)
MBA.msf(MBAUU-3)
mA.msf(MBAUU-1)
MBA.msf{MBAUU-6)
consensus
MBA.msf{MBAUU-13)
spacer; 30 (30/375
=
8-00/,) in
ureB;
11 (11/45
=
24.4%) in the
ureB-ureC
spacer and 45 (45/404
=
11.1
YO)
in partial
ureC
(Fig.
5).
UAB reference isolates
of serovars
2,
6,
7,
10,
11,
12 and
13
were sequenced
and the results were identical
to
those of the cor-
responding ATCC serovar strains.
The
5’-end region
of
the
MBA
genes.
The amplified
fragments of the 5’-end of the MBA gene of serovars
belonging to biovar 1 were shorter than those
of
biovar
2,
mainly because
of
the deletion of a 45 bp segment of
the biovar
1
sequence upstream
of
the start codon
of
MBA.msf{MBAUU-11)
MBA.msf(MBAUU-10)
MBA.msf(MBAUU-12)
MBA.msf(MBAUU-13>
MBA.msf{MBAUU-4)
MBA.msf(MBAUU-71
MBA.msf(MBAUU-2)
MBA.msf(MBAUU-5)
m.msf imAuu-8)
MBA.Wf (MBAW-9)
MBA.mSf(MBAUU-14)
MBA.msf(MBAUU-1)
MBA.msf (MBAUU-3)
MBA.msf IMBAUU-6)
consensus
MBA.msf(MBAUU-11)
MBA.msf(mAW-7)
MBA.mf (MBAW-12)
MBA.lWf(MBAUU-13)
MBA.msf(MBAW-2)
MBA.msf(MBAUU-10)
MBA.msf{MBAUU-4)
MBA.msf(MBAW-5)
MBA.msf(MBAUU-8)
MBA.msf(MBAUU-9)
MBA
.
mSf
(&AUU- 14
)
MBA
.
m5
f
{MBAUV-
1
1
MBA.msf(MBAUU-3)
MBA.msf{MBAUL-6)
consensus
MBA.msf(MBAUU-11)
hEA.msf(MBAUU-7)
MBA.msf(MBAUU-1O)
MBA.msf{MBAUU-12>
MBA.msf(MBAUU-13)
MBA.msf(MBAUU-4)
MBA
.
ms f (MBAUU-
5
)
MBA.msf(MBAUU-2)
MBA.
rns
f (MBAUU-8 1
MBA.msf(MBAUU-9)
MBA. ms f T&AUU- 14
MBA.msf(MBAUU-3)
MBA.msf{MBAUU-1)
MBA.msf(MBAW-61
Consensus
MBA.msf(MBAUU-11)
MBA.msf(MBAUU-7)
MBA.msf(MBAUU-10)
MBA.msf{MBAUU-l2)
MBA.msf(MBAUU-13)
MBA.msf(MBAUU-4)
MBA.msf(MBAUU-2)
MBA.msf(MBAUU-5)
MBA.msf(MBAUU-8)
MBA.msf{MBAUU-14)
MBA.msf(MBAUU-9)
MBA.msf{MBAW-3)
MBA.msf{MBAUU-1)
MBA.msf{MBAUU-6)
Consensus
Fig.
6.
Multiple sequence alignment
of
the 5’-end
of
MBA gene
sequences
of
14
serovars
of
U.
urealyticum
(ATCC
strains).
the MBA genes (-87 to -43). There were 155
(155/481
=
32.2%) base differences between the se-
quences for the two biovars
-
73 (73/281
=
26.0
YO)
in
the region downstream from the start codon and 82
(82/200
=
41.0%) in the upstream region.
These
differences included deletions at 49 sites in biovar 1
and at five sites in biovar
2
(Fig.
6).
All 14 serovars of
UAB reference strains were sequenced, and the results
were identical to the corresponding ATCC serovar
strains.
There were base differences at 37 (37/603
=
6.1
O/O)
1884
International Journal
of
Systematic Bacteriology
49
Phylogeny
of
Ureaplasrna urealyticum
MBAl.msf(MBAUU-14)
MBAl.msf(MBAUU-3)
MBAl.msf{MBAUU-1)
MBAl .msf {MBAUU-6)
consensus
MBAl.msf(MBAUU-14)
MBAl.msf(Ml3AW-3)
MBAl.msf(MBAUU-11
MBAl.msf(MBAUU-6)
Consensus
MBAl.rnsf(MBAUU-14)
MBAl.msfIMBAUU-3)
MBAl.msf(MBAUU-1)
MBAl.msf(MBAUU-6)
Consensus
MBAl.msf(MBAUU-14)
MBAl.mf{MBAUU-3)
MBAl.msf(MBAUU-1)
MBAl.msf(MBAUU-6)
consensus
MBAl.msf(MBAUU-141
MBAl.msf(MBAUU-3)
MBAl.msf(MBAUU-1)
MBAl.msf(MBAUU-6)
consensus
MBAl.msf{MBAUU-14)
MBAl.msf(MBAUU-3)
MBAl.msf(MBAUU-11
MBAl.msf(MBAUU-6)
consensus
MBAl.mSf(MBAW-14)
MBAl.msf(MBAUU-3)
MBAl.msf(MBAUU-1)
MBAl.msf(MBAUU-6)
Consensus
MEAl.msf(MBAUU-14>
MBAl.msflMBAUU-3)
MBAl.msf(MBAUU-1)
MBAl.msf(MBAUU-6)
Consensus
MBAl.msf(MBAUU-14)
MBAl
.mf IMBAW-3)
MBAl.msf(MBAUU-1)
MBAl.msf(MBAUU-6)
Consensus
MBAl.msf(MBAUU-14)
MBAl.msffMBAUU-3)
MBAl.msf(MBAW-1)
MBAl.msf(MBAUU-6)
Consensus
MBAl.msf(MBAUU-14)
MBAl.msf(MBAUU-3)
MBAl.rnsf{MBAUU-1)
Consensus
MBAl.msf(MBAW-6)
MBAl.msf(MBAUU-14)
MBAl.msf(MBAW-3)
MBAl.msf(MBAUU-1)
MBAl.msf(MBAW-6)
Consensus
MBAl.msf(MBAUU-14)
MBAl.mSf(MBAUU-3)
MBAl.mSf(MBAUU-1)
MBAl.msf{MBAUU-6)
Consensus
453
Fig.
7.
Multiple sequence alignment
of
the 5’-end
of
MBA
gene
sequences
of
U.
urealyticum
serovars
1,
3,
6
and
14
(ATCC
strains).
sites among four serovars of biovar 1 (Fig. 7) (Kong
et
al.,
1999). Sequences were more conserved between the
ten serovars
of
biovar 2, with base changes at only six
(6/476
=
1.3
YO)
sites
:
base
-
112 of serovars 4, 10, 12,
13 changed from A to
G
;
base 194 of serovars 4,10,12,
13 changed from C to A; base 219 of serovars
7,
11
changed from C to T; base 223 of serovars 7, 11
changed from
G
to A; base 251 of serovars
7,
11
changed from
C
to T; base -29
of
serovars 4, 10, 12,
13 and
7,
11
changed from A to
G
(Fig. 8). Sequencing
of UAB reference strains showed identical differences
between serovars to those found with ATCC serovars.
Phylogenetic tree
of
U.
urealyticum
Because the sequences of the 16s rRNA genes, the
16s-23s rRNA gene spacer regions and the urease
gene subunits were conserved within each biovar, they
did not provide enough information to distinguish
serovars. However, all three showed enough differ-
ences between biovars to separate them clearly into
two clusters. We analysed the phylogenetic relation-
ships further using sequence data for the MBA genes,
based on the results of multiple sequence alignment
(Fig.
9).
The phylogenetic tree, based on the sequences of the
5’-end of MBA genes, confirmed that the four serovars
of
U.
urealyticum
biovar
1
and 10 serovars of biovar 2
were clearly separated into two clusters. Within biovar
1, serovars 3 and 14 formed one cluster, and serovars 1
and 6 another cluster. The ten serovars of biovar 2
could be separated into three clusters: (i) serovars 2,5,
8 and 9
;
(ii) serovars 4,10,12 and 13
;
(iii) serovars 7 and
11.
DISCUSSION
Various methods have been described previously to
distinguish the two biovars of
U.
urealyticum
including
susceptibility to manganese (Robertson
&
Chen,
1984)
;
enzyme profiles (Davis
&
Villanueva, 1990)
;
protein or antigen epitope analysis (Horowitz
et
al.,
1986; MacKenzie
et
ai.,
1996; Teng
et
al.,
1994);
DNA-DNA hybridization (Christiansen
et
ai.,
198 1);
RFLP (Harasawa
et
al.,
1991); one- and two-dimen-
sional gel electrophoresis (Swenson
et
al.,
1983);
genomic sizes (Robertson
et
al.,
1990); arbitrarily
primed PCR fingerprinting (Grattard
et
nl.,
1995a, b;
Kong
et
al.,
1996); and PCR amplification of specific
genes (Blanchard, 1990; Harasawa
et
al.,
1993;
Robertson
et
al.,
1993
;
Teng
et
al.,
1994). However, to
study the possible association of individual serovars
with clinical disease, further identification of individual
serovars is needed. The aim of this study was to
analyse the phylogenetic relationships between the 14
serovars of
U.
urealyticum
and define their genotypes,
as the basis of a new molecular typing system, using
sequence data from four genetic regions.
The 16s rRNA genes and the 16s-23s rRNA gene
spacer regions have been used extensively in taxonomic
studies of many different types of bacteria, including
the
Mollicutes
and, specifically, ureaplasmas (Barry
et
al.,
1991
;
Everett
&
Andersen, 1997
;
Harasawa
et
al.,
1991, 1996; Harasawa
&
Cassell, 1996; Perez Luz
et
al.,
1998; Robertson
et
al.,
1994; van Kuppeveld
et al.,
1992; Weisberg
et al.,
1989). Both are relatively well-
conserved but have sufficient heterogeneity to allow
some differentiation within species. In the 16s rRNA
genes, base differences occurred between biovars
1
and
2 at fewer than
1
Yo
of sites, several of which- at
positions 176,177 and 180-1 83
-
have been previously
described and used to design PCR primers to dis-
tinguish the two biovars (Robertson
&
Stemke, 1982).
The 16s rRNA genes were highly conserved within
International Journal
of
Systematic Bacteriology
49
1885
K.
Fanrong and others
MBAUU-6
99
MBAUU-1
-75
MBAUU-3
MBAUU-14
-
MBAUU-11
MBAZ .msf (MBAUU-11)
MBA2.msf{MBAUU-7)
MBA2.msfIMBAUU-10)
MBAZ.msf{MBAUU-12f
MBAZ.msf(MBAUU-13)
MBAZ.msf(MBAUU-4)
MBA2.msf(MBAUU-2)
MBA2.msf(MBAUU-5)
MBA2.msf(MBAUU-B)
MBAZ.msf(MBAUU-9)
consensus
I
.
MBA2.msf(MBAUU-11)
MBA2.msf(MBAUU-10)
MBAZ.msf(MBAUU-12)
MBAZ.msf{MBAUU-13)
MBA2.msfiMBAUU-4)
MBA2.msf(MBAW-5)
MBA2.msf(MBAW-8)
MBA2.msf(MBAUU-9)
consensus
MBAZ .msf (MBAUU-7)
MBA2.msf(MBAUU-2)
MBAUU-2
90
MBAUU-5
94
-
MBAUU-9
.
72
MBAUU-8
MBA2 .msf{MBAUU-ll)
MBAZ .msf (MBAUU-7
1
MBA2.msf(MBAW-10)
MBA2.msf(MBAUU-12)
MBAZ.msf(MBAUU-13)
MBAZ.msf(MBAUU-4)
MBAZ.msf(MBAUU-2)
MBAZ.msf(MBAUU-5)
MBAZ.msf(MBAUU-8)
MBAZ.rnsf(MBAUU-9)
Consensus
MBAZ.msf(MBAUU-111
MBA2.msf{MBAUU-71
MBA2.msf(MBAW-lO)
MBA2.msf(MBAUU-12)
MBAZ.msf(MBAUU-13)
MBAZ.msf(MBAUU-4)
MBAZ.msf(MBAUU-2)
MBA2.msf(MBAUU-5)
MBAZ.msf(MBAUU-8)
MBAZ.msf{MBAUU-9)
consensus
MBAZ.msf(MBAUU-11)
MBA2 .msf (MBAUU-7)
MBA2.msf(MBAUU-lO)
MEAZ.msf{MBAUU-12)
MBAZ.msf(MBAUU-13)
MBA2.msf(MBAUU-4)
MBAZ.msf(MBAW-2)
MBAZ .msf (MBAW-5)
MBA2.msf(MBAW-8)
MBA2.msf(MBAUU-9)
consensus
MBA2.msf(MBAW-ll)
MBAZ.wf(MBAUU-7)
MBA2.msf(MBAW-l0)
MBAZ .msf (MBAW-12)
MBAl.msf{MBAUU-13)
MBA2.msffMBAUU-4)
MBA2.msf(MBAUU-2)
MBAZ.msf(MBAUU-5)
MBAZ
.msf {MBAUU-8)
MBAZ.msf(MsAWU-9)
Consensus
MBAl.msf{MBAW-11)
MBAZ.msf{MBAUU-7)
MBA2.msf(MBAW-lO)
MBAZ.msf(MBAW-12)
MBA2.msf{MBAUU-13)
MBAZ.msf{MBAUU-4)
MBAZ.rnsf(MBAUU-2)
MBA2.msf(MBAW-5j
MBA2.msf(MBAW-8)
MBAZ.msf(MBAW-9)
consensus
MBAZ.msf(MBAW-11)
MBAZ.msf(MBAUU-7)
MBAZ .msf (MBAUU-10)
MBAZ.msf(MBAUU-12)
MBAZ.msf(MBAUU-13)
MBA2.msf(MBAUU-4)
MBAZ.msf(MBAUU-2)
MBAl.msf(MBAW-5)
MBA2.rnsf(MBAW-B)
MBA2.msf(MBAUU-91
Consensus
MBA2.msf(MBAUU-11)
MBAZ.msf(MBAW-7)
MBAl.msf(MBAUU-10)
MBA2.msf(MBAUU-12)
MBAZ.msf(MBAUU-13)
MBA2.msf(MBAUU-4)
MBA2.msftMBAW-5)
MBAZ.msf(MBAUU-9)
consensus
MBAZ .msf (MBAW-2)
MBAZ
.
ms
f [MBAUU-
8
)
MBA2.msf(MBAW-ll)
MBA2.msf(MBAUU-7)
MBAZ.msf{MBAW-lO)
MBAZ.msf(MBAW-12)
MBA2.msf(MBAW-13)
MBA2.msf(MBAUU-4)
MBA2.msf(MBAUU-5)
MBn2.msf{MJ3AUU-8)
MBAZ.msf(Mf3AW-9)
Consensus
MBAZ.msf{MBAUU-2)
Fig,
9.
Phylogenetic tree
for
the
14
serovars
of
U. urealyticum
(based on the 5’-end
of
MBA gene nucleotide sequences of
U.
urealyticum ATCC strains).
CLUSTAL
was
used for alignment, and
PHYLIP
was used
for
constructing the phylogenetic tree. The tree
was formed using Methanococcus jannaschii
as
outgroup and
was bootstrapped with
100
replications.
each biovar with few differences between serovars. The
16s-23s rRNA gene spacer region is shorter but more
heterogeneous than the 16s rRNA gene. We found
differences between the sequences of biovars 1 and
2
at
4.5%
of sites, but none within the biovars.
Urease
is
a virulence factor
in
U.
urealyticum
and a
number of other bacteria (Collins
&
D’Orazio, 1993;
Ligon
&
Kenny, 1991
;
Smith
et al.,
1993; Willoughby
et
al.,
1991). The urease subunit genes have been used
to study the phylogenetic relationships of other urease-
producing bacteria (Akashi
et
al.,
1996) as well as
ureaplasmas (Blanchard, 1990; Ruifu
et al.,
1997), in
which it has been used to separate the two biovars.
Using urease subunit genes
ureA,
ureB,
partial
ureC
and adjoining upstream regions of
ureA, ureA-ureB
spacer and
ureB-ureC
spacer, we demonstrated base
pair differences between the two biovars at 10.7% of
sites. Again, sequences of these genes were well-
conserved within biovars, although serovar 2 was
distinguished from other serovars in biovar 2 by two
separate single-base mutations, which were confirmed
in a clinical isolate of the same serovar.
Urease subunit genes have been used previously to
biotype isolates (Blanchard, 1990) and to demonstrate
heterogeneity among different serovars (Ruifu
et
al.,
1997). In the former study (Blanchard, 1990), primers
used for biotyping
of
U.
urealyticum
did not amplify
urease genes of serovars 10 and
12
of
biovar 2.
Fig.
8.
Multiple sequence alignment
of
the 5’-end
of
MBA gene
sequences of
U.
urealyticum serovars
2,
4,
5,
7,
8,
9,
10, 11, 12,
13
(ATCC strains).
1886
International
Journal
of
Systematic Bacteriology
49
Phylogeny of
Ureaplasma urealyticum
However, we were able to amplify all serovars of
biovar 2 including serovars 10 and 12 (ATCC and
UAB reference strains) but none of the serovars of
biovar 1, using the same primers. Therefore these
primers can be used to biotype
U.
urealyticum.
Our
results also showed that the homologies between the
different serovars within each biovar were quite high
and minor differences between sequences were in-
adequate to distinguish all serovars of
U.
urealyticum
using this target (Ruifu
et al.,
1997).
The MBAs of
U.
urealyticum
are the major immuno-
gens recognized during infection and are thought to be
important virulence factors involved in interactions
with host cells (Watson
et al.,
1990; Zheng
et al.,
1992,
1995). Their genes, which contain both biovar- and
serovar-specific regions, were selected as appropriate
targets for this phylogenetic study
of
U.
urealyticum.
We amplified and sequenced the 5’-end of MBA genes
of all 14 serovars. The genes of biovar 2 were more
highly conserved and longer than those of biovar
1
in
the regions compared. The two biovars could be easily
distinguished from each other. There were base
changes in only six sites at the 5’-end of MBA gene of
biovar 2. all of which were confirmed in UAB reference
strains of the same serovars. They appear to be stable
differences that could be used to subtype
U.
urealy-
ticum
biovar 2 by direct sequencing.
Base differences between sequences of the four serovars
of biovar
1
were more numerous. However, serovars 3
and 14 were similar to each other, with only three base
pair differences
;
serovars
1
and 6 differed by 16 bp. In
common with others, we have shown that biovar 1
(serovars 1, 3, 6 and 14) is the predominant biovar
isolated from the urogenital tract (Abele-Horn
et al.,
1997a, b; Kong
et al.,
1996). Therefore, we have
developed a serovar-specific PCR/restriction endo-
nuclease analysis procedure to differentiate these
serovars (Kong
et al.,
1999).
Our results showed that the heterogeneity of inter-gene
spacer regions was higher than that of the genes
themselves (Nakashima
et al.,
1998). For example, the
heterogeneity of the 16s-23s rRNA gene spacer region
was 4.5Y0, compared with only 0.97% for the 16s
rRNA gene; the heterogeneity in the sequence up-
stream
of
ureA,
the
ureA-ureB
spacer and the
ureB-
ureC
spacer genes varied from 17 to 24
YO
compared
with
61
1
YO
for
ureA, ureB
and partial
ureC
genes; the
heterogeneity in the sequence upstream of the Send
region of MBA genes was 41
YO
compared with 26
YO
in
the 5’-end of MBA genes themselves. Therefore,
primers based on the inter-gene spacer regions could
be more discriminatory for biotyping than those based
on the genes themselves. Biovar-specific primers based
on the upstream region of the
ureA
subunit of the
urease gene have been used previously (Blanchard,
1990).
Our study is the first phylogenetic analysis of
U.
urealyticum
based on four important genes or DNA
sequences from all 14 serovars. We confirmed and
extended the findings of previous studies, that there
were significant differences between the two biovars of
U.
urealyticum,
which justify their being designated as
different species (Robertson
et al.,
1993; Teng
et al.,
1995). This was first suggested in 1981, based on
DNA-DNA hybridization, which showed only
40-
60
YO
homology between the biovars, enough to justify
their separation into two species (Christiansen
et al.,
198 1). Subsequently other workers have supported
this separation on the basis of differences in the
sequences of 16s rRNA (Robertson
et al.,
1993) and
MBA genes (Teng
et al.,
1994) between the two
biovars. Although, in our study, the degree of het-
erogeneity in the 16s rRNA genes was relatively low
(0.97%), it was higher in the other three gene regions
sequenced, especially the MBA gene (32%). It has
been shown previously that sequence identity of the
16s rRNA gene does not necessarily imply species
identity. For example, DNA-DNA hybridization
showed 99.5%
sequence identity between the se-
quences of 16s rRNA genes of three different bacterial
strains that were distinguishable as different
Bacillus
species on the basis of phenotypic and other genetic
differences (Fox
et al.,
1992).
Comparison
of
the sequences of the whole genomes of
each biovar and serovar would be the most accurate
way to study the phylogeny
of
U.
urealyticum
(Naka-
shima
et aE.,
1998). However, comparison of sequence
data for several important genes, from each of the
serovars, is more feasible and should provide almost as
much information. Genes other than those that we
sequenced, that are present in mycoplasmas (including
ureaplasmas), and for which sequences have been
reported by others, may be useful also for phylogenic
analysis of
U.
urealyticurn.
They include the
tuf
gene
which has been sequenced from
M.
genitalium
(Loechel
et al.,
1989),
M.
pneumoniae
(Yogev
et
al.,
1990), M.
hominis
(S.
A. Ladefoged
&
G.
Christiansen, GenBank
accession no. X57136), the 23s RNA gene and the 3’-
end of the MBA gene of
U.
urealyticum
(Zheng
et al.,
1996). Like the urease and MBA genes, they may be
more useful than the 16s rRNA gene, for phylogenetic
analysis of
U.
urealyticum
(Powers
&
Noller, 1993;
Kamla
et
al.,
1996). However, we believe that the
comparative sequence data provided in our study is
adequate to justify our support for the division of
U.
urealyticum
into two species.
The combination of DNA sequence data from several
important genes with traditional methods of classifi-
cation, may better reflect the phylogenetic rela-
tionships between different biovars and serovars better
than either method alone. Our work provides further
support for establishment of two different human
Ureaplasma
species as proposed by Robertson
&
Chen
(1994) and Teng
et al.
(1994), namely
U.
parvum
(currently
U.
urealyticum
biovar 1) and
U.
urealyticum
(currently biovar
2).
In the future, clinical studies of
human ureaplasma infection should distinguish these
two new species to determine whether either is more
likely to be associated with disease. Further work
is
~~~
International Journal
of
Systematic Bacteriology
49
1887
K.
Fanrong
and
others
required to determine whether the current subdivision
of
these species into serovars requires modification,
based on genetic data.
ACKNOWLEDGEMENTS
We thank Mark Wheeler
for
assistance with sequencing and
Kirsty Hannaford for her help with culture of
UAB
reference
isolates.
REFERENCES
Abele-Horn,
M.,
Peters,
J.,
Genzel-Boroviczeny,
O.,
Wolff,
C.,
Zimmermann, A.
&
Gottschling, W. (1997a).
Vaginal
Ureapkasma
tirealj,ticum
colonization
:
influence on pregnancy outcome and
neonatal morbidity.
Infection
25,
286-291.
Abele-Horn,
M.,
Wolff, C., Dressel, P., Pfaff,
F.
&
Zimmermann, A.
(1997b).
Association
of
Ureaplasma urealyticum
biovars with
clinical outcome for neonates, obstetric patients, and gyne-
cological patients with pelvic inflammatory disease.
J
Clin
Microhiol35,
1 199-1 202.
Akashi, H., Hayashi, T., Koizuka, H., Shimoyama, T.
&
Tamura,
T.
(1996).
Strain differentiation and phylogenic relationships, in
terms
of
base sequence
of
the
ureB
gene, of
Helicobacter pylori.
J
Gastroenterol31
(suppl. 9), 1623.
Barry, T., Colleran, G., Glennon,
M.,
Dunican,
L.
K.
&
Cannon,
F.
(1991).
The 16S/23S ribosomal spacer region as a target for
DNA
probes to identify eubacteria.
PCR Methods Appl 1,
Blanchard,
A.
(1990).
Ureaplusmu ureulyticum
urease genes; use
of
a
UGA
tryptophan codon.
Mol Microbiol4,
669-676.
Cassell, G. H., Crouse, D.
T.,
Waites,
K.
B.,
Rudd,
P. T.
&
Davis,
J.
K.
(1
988).
Does
Ureaplasma urealyticum
cause respiratory
disease in newborns?
Pediatr Infect Dis 7,
535-541.
Cheng,
X.,
Naessens, A.
&
Lauwers,
5.
(1994).
Identification of
serotype 1-, 3-, and &specific antigens of
Ureuplasma ureuly-
ticum
by using monoclonal antibodies.
J
Clin Microbiol 32,
1060-1062.
Christiansen, C., Black,
F.
T.
&
Freundt,
E.
A. (1981).
Hybridiz-
ation experiments with deoxyribonucleic acid from
Ureplasmu
urealyticum
serovars
I
to
VIII.
Int
J
Syst Bacteriol31,
259-262.
Collins.
C.
M.
&
D'Orazio,
S.
E.
(1993).
Bacterial ureases: struc-
ture, regulation
of
expression and role in pathogenesis.
Mol
Microbiol9,
907-913.
Davis,
1.
W.,
Jr
&
Villaneuva,
1.
(1990).
Enzyme differences in
serovar clusters of
Ureaplasma urealyticum,
U.
diversum,
and
overine ureaplasmas.
Zentbl Bakteriol Suppl20,
66 1-665.
Everett, K.
D.
E.
&
Andersen, A. A. (1997).
The ribosomal
intergenic spacer and domain
I
of the 23s rRNA gene
are
phylogenetic markers for
Chlamydia
spp.
Int
J
Syst Bacteriol
Fox,
G.
E.,
Wisotzkey,
1.
D.
&
Jurtshuk, P.,
Jr
(1992).
How close is
close: 16s rRNA sequence identity may not be sufficient to
guarantee species identity.
Int
J
Syst Bacteriol42,
166170.
Grattard,
F.,
Pozzetto,
B.,
De Barbeyrac, B., Renaudin,
H.,
Clerc,
M.,
Gaudin,
0.
G.
&
Bebear,
C.
(1995a).
Arbitrarily-primed PCR
confirms the differentiation
of
strains
of
Ureaplasma
urealy-
ticum
into two biovars. Mol
Cell Probes 9,
383-389.
Grattard,
F.,
Soleihac, B., De Barbeyrac, B., Bebear,
C.,
Seffert, P.
&
Pozzetto, B.
(1
995b).
Epidemiological and molecular investi-
gations
of
genital mycoplasmas from women and neonates at
delivery.
Pediatr Infect
Dis
J
14,
853-858.
5
1-56.
47,
461-473.
Harasawa,
R.
&
Cassell, G. H. (1996).
Phylogenetic analysis of
genes coding for 16s rRNA in mammalian ureaplasmas.
Int
J
Syst Bacteriol46,
827-829.
Harasawa,
R.,
Dybvig,
K.,
Watson, H. L.
&
Cassell, G. H. (1991).
Two genomic clusters among 14 serovars of
Ureaplusma
urealyticum. Syst Appl Microbiol 14,
393-396.
Harasawa,
R.,
Mizusawa, H., Nozawa, K., Nakagawa, T., Asada,
K.
&
Kato,
I.
(1993).
Detection and tentative identification of
dominant
Mj'coplasma
species in cell cultures by restriction
analysis of the 16S-23s rRNA intergenic spacer regions.
Res
Microbioll44,
489493.
Harasawa,
R.,
Lefkowitz,
E.
J.,
Glass,
J.
I.
&
Cassell,
G.
H.
(1996).
Phylogenetic analysis of 1623s rRNA intergenic spacer regions
of genus
Ureaplasma.
J
Vet
Med Sci
58,
191-195.
Horowitz,
S.
A., Duffy,
I.,
Garrett,
B.,
Stephens,
J.,
Davis,
J.
K.
&
Cassell, G. H. (1986).
Can
group- and serovar-specific proteins be
detected in
Ureaplasma ureulyticum? Pediatr
Infecr
Dis
5
(suppl.
Kamla, V., Henrich, B.
&
Hadding,
U.
(1996).
Phylogeny based on
elongation factor Tu reflects the phenotypic features of myco-
plasmas better than that based on 16s rRNA.
Gene
171,
83-87.
Kong,
F.,
Zhu,
X.,
&
Zhou,
J.
(1996).
Grouping and typing of
Ureaplusma urealyticum. Chung-Hua i Hsueh
Tsir
Chih (Chin
Med
J)
76,
138-140 (in Chinese).
Kong,
F.,
Zhu,
X.,
Wang, W., Zhou,
X.,
Gordon,
S.
&
Gilbert, G.
L.
(1
999).
Comparative analysis and serovar-specific identification
of
the multiple banded antigen genes of
Ureaplusma urealyticum
biovar 1.
J
Clin Microbiol37,
538-543.
Kundsin,
R.
B., Leviton, A., Allred,
E.
N.
&
Poulin,
S.
A. (1996).
Ureaplasrna
urealyticum
infection of the placenta in pregnancies
that ended prematurely.
Obstet Gynecol87,
122-127.
van Kuppeveld,
F.
J.,
van der Logt,
J.
T.,
Angulo, A.
F.,
van Zoest,
M.
J.,
Quint, W. G., Niesters, H. G., Galama,
J.
M.
&
Melchers,
W.
J.
(1992).
Genus- and species-specific identification
of
myco-
plasmas by 16s rRNA amplification.
Appl Environ Microbiol
58,260626
1
5.
Ligon,
J.
V.
&
Kenny, G.
E.
(1991).
Virulence
of
ureaplasmal
urease for mice.
Infect
Zmmun
59,
1170-1 171.
Lin,
1.
5.
&
Kass,
E.
H. (1980).
Fourteen serotypes
of
Ureaplasma
urealyticum
(T-mycoplasmas) demonstrated by the comple-
ment-dependent mycoplasmacidal test.
Infection
8,
152-1
55.
Loechel,
S.,
Inamine, J.
M.
&
Hu, P.
C.
(1989).
Nucleotide sequence
of
the
tuf
gene from
Mycoplasma genitalium. Nucleic Acids
Res
17,
10127.
MacKenzie, C.
R.,
Henrich, B.
&
Hadding,
U.
(1996).
Biovar-
specific epitopes of the urease enzyme
of
Ureuplasma urealy-
ticum.
J
Med Microbiol45,
366-371.
Naessens, A., Foulon, W., Breynaert,
1.
&
Lauwers,
S.
(1988).
Serotypes
of
Ureaplasma urealyticum
isolated from normal
pregnant women and patients with pregnancy complications.
J
Clin Microbiol26,
319-322.
Nakashima, H., Ota,
M.,
Nishikawa, K.
&
Ooi, T. (1998).
Genes
from nine genomes are separated into their organisms in the
dinucleotide composition space.
DNA
Res
5,
25
1-259.
Nelson,
S.,
Matlow, A., Johnson, G., Th'ng,
C.,
Dunn,
M.
&
Quinn,
P.
(1
998).
Detection of
Ureaplasma urealyticum
in endotracheal
tube aspirates from neonates by PCR.
J
Clin Microbiol 36,
1236-1239.
Perez Luz,
S.,
Rodriguez-Valera,
F.,
Lan,
R.
&
Reeves,
P.
R.
(1998).
Variation
of
the ribosomal operon 16S-23s gene spacer region
in representatives of
Salmonella
enterica
subspecies.
J
Bncteriol
180,
21462151.
6), S325-S33 1.
1888
International
Journal
of
Systematic Bacteriology
49
Phylogeny
of
Ureaplasma urealyticum
Powers, T.
&
Noller, H.
F.
(1993).
Allele-specific structure probing
of plasmid-derived
16s
ribosomal RNA from
Escherichia coli.
Gene 123,75-80.
Quinn, P.A., Arshoff, L. U.
&
Li,
H.
C.
(1981).
Serotyping of
Ureaplasma urealyticum
by immunoperoxidase assay.
J
Clin
Microbiol 13,
670-676.
Razin,
5.
&
Yogev, D.
(1986).
Genetic relatedness among
Ureaplasma urealyticum
serotypes (serovars).
Pediatr Infect Dis
5
(suppl.
6), S3OCLS304.
Robertson,
J.
A.
&
Chen, M. H.
(1984).
Effects of manganese on
the growth and morphology of
Ureaplasma urealyticum.
J
Clin
Microbiol 19,
857-864.
Robertson,
1.
A.
&
Stemke, G. W.
(1979).
Modified metabolic
inhibition test for serotyping strains of
Ureaplasma urealyticum
(T-strain Mycoplasma).
J
Clin Microbiol9,
673-676.
Robertson,
1.
A.
&
Stemke, G. W.
(1982).
Expanded serotyping
scheme for
Ureaplasma urealyticum
strains isolated from
humans.
J
Clin Microbiol 15,
873-878.
Robertson,
1.
A., Pyle,
L.
E.,
Stemke, G. W.
&
Finch,
L.
R.
(1990).
Human ureaplasmas show diverse genome sizes by pulsed-field
electrophoresis.
Nucleic Acids
Res
18,
145 1-1455.
Robertson,
1.
A,,
Vekris, A., Bebear,
C.
&
Stemke, G. W.
(1993).
Polymerase chain reaction using
16s
rRNA gene sequences
distinguishes the two biovars of
Ureaplasma urealyticum.
J
Clin
Microbiol31,
824830.
Robertson,
J.
A., Howard, L.
A.,
Zinner,
C.
L.
&
Stemke, G. W.
(1994).
Comparison of
16s
rRNA genes within the
T960
and
parvo biovars of ureaplasmas isolated from humans.
Int
J
Syst
Bacteriol44,
836-838.
Ruifu, Y., Minli,
Z.,
Guo, 2.
&
Wang,
X.
(1997).
Biovar diversity is
reflected by variations of genes encoding urease of
Ureaplasma
urealyticum. Microbiol Immunol41,
625-627.
Smith,
D.
G., Russell, W.
C.,
Ingledew, W.
1.
&Thirkell,
D.
(1993).
Hydrolysis
of
urea by
Ureaplasma urealyticum
generates a
transmembrane potential with resultant ATP synthesis.
J
Bacteriol 175,
3253-3258.
Swenson,
C.
E.,
VanHamont,
1.
&
Dunbar,
B.
5.
(1983).
Specific
protein differences among strains of
Ureaplasma urealyticum
as
determined by two-dimensional gel electrophoresis and a
sensitive silver stain.
Int
J
Syst Bacteriol33,
41 7421.
Taylor-Robinson,
D.
&
Furr,
P.
M.
(1997).
Genital mycoplasma
infections.
Wien Klin Wochenschr 109,
578-583.
Taylor-Robinson,
D.,
Furr,
P.
M.
&
Webster, A. D.
(1985).
Urea-
plasma urealyticum
causing persistent urethritis in a patient
with hypogammaglobulinaemia.
Genitourin
Med
61,
404-408.
Teng,
L.
J.,
Zheng,
X.,
Glass,
J.
I.,
Watson,
H.
L.,
Tsai,
1.
&
Cassell,
G. H.
(1994).
Ureaplasma urealyticum
biovar specificity and
diversity are encoded in multiple-banded antigen gene.
J
Clin
Microbiol32,
1464-1 469.
Teng, L.
J.,
Ho,
5.
W.,
Ho, H.
N.,
Liaw,
5.
J.,
Lai, H.
C.
&
Luh,
K.
T.
(1 995).
Rapid detection and biovar differentiation of
Urea-
plasmu urealyticum
in clinical specimens by PCR.
J
Formos Med
Assoc 94,
39WOO.
Tully,
1.
G.
(1993).
Current status of the mollicute flora of
humans.
Clin Infect Dis 17
(suppl.
l),
S2-S9.
Watson, H. L., Blalock, D.
K.
&
Cassell,
G.
H.
(1990).
Variable
antigens
of
Ureaplasma urealyticum
containing both serovar-
specific and serovar-cross-reactive epitopes.
Infect Immun
58,
Weisburg, W. G., Tully,
J.
G., Rose, D.
L.
&
9
other authors
(1989).
A phylogenetic analysis of the mycoplasmas: basis for their
classification.
J
Bacteriol 17 1,
645 5-6467.
Wiley,
C.
A.
&
Quinn,
P.
A.
(1984).
Enzyme-linked immuno-
sorbent assay for detection of specific antibodies to
Ureaplasma
urealyticum
serotypes.
J
Clin Microbioll9,
42 1426.
Willoughby,
1.
J.,
Russell, W.
C.,
Thirkell, D.
&
Burdon, M. G.
(1991).
Isolation and detection of urease genes in
Ureaplasma
urealyticum. Infect Immun 59,
2463-2469.
Yogev, D., Sela,
S.,
Bercovier, H.
&
Razin,
5.
(1990).
Nucleotide
sequence and codon isage of the elongation factor Tu (EF-Tu)
gene from
Mycoplasma pneumoniae. Mol Microbiol 4,
1303-
1310.
Zheng,
X.,
Watson, H. L., Waites,
K.
B.
&
Cassell, G. H.
(1992).
Serotype diversity and antigen variation among invasive isolates
of
Ureaplasma urealyticum
from neonates.
Infect Iminun 60,
Zheng,
X.,
Teng, L.
J.,
Watson, H.
L.,
Glass,
J.
I.,
Blanchard, A.
&
Cassell, G. H.
(1995).
Small repeating units within the
Urea-
plasma urealyticum
MB antigen gene encode serovar specificity
and are associated with antigen size variation.
Infect Immun 63,
Zheng,
X.,
Lau,
K.,
Frazier,
M.,
Cassell, G. H.
&
Watson, H. L.
(1996).
Epitope mapping of the variable repetitive region with
the MB antigen of
Ureaplasma urealyticum. Clin Diagn Lab
Immunol3,
774-778.
3679-3688.
3472-3474.
891-898.
International Journal
of
Systematic Bacteriology
49
1889
