Journal of Clinical Microbiology, December 1999, p. 4135-4138, Vol. 37, No. 12
0095-1137/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
Differentiation of Two Biovars of Ureaplasma
urealyticum Based on the 16S-23S rRNA Intergenic Spacer
Region
Ryô
Harasawa1,* and
Yasuo
Kanamoto2
Animal Center for Biomedical Research,
Faculty of Medicine, The University of Tokyo, Bunkyo-ku, Tokyo
113-0033,1 and Department of Nutrition,
Hiroshima Chuo Women's Junior College, Asaminami-ku, Hiroshima
731-0138,2 Japan
Received 9 June 1999/Returned for modification 21 July
1999/Accepted 20 August 1999
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ABSTRACT |
The 16S-23S rRNA intergenic spacer regions of 14 strains
representing the 14 serovars of Ureaplasma urealyticum were
amplified by PCR and sequenced for genetic differentiation between the
two biovars Parvo and T960. Although the spacer region of the Parvo and
T960 biovars comprised 302 nucleotides and lacked spacer tRNA genes, 15 nucleotides were different between the two biovars. The four nucleotide
sequences of the 16S-23S rRNA intergenic spacer region of serovars 1, 3, 6, and 14 in the Parvo biovar were found to be identical. Similarly,
the 10 nucleotide sequences of the 16S-23S rRNA intergenic spacer
region of serovars 2, 4, 5, and 7 to 13 in the T960 biovar were found
to be identical. The nucleotide sequence of the T960 biovar contains
multiple restriction sites for restriction endonuclease
SspI, which allows differentiation of the T960 biovar from
the Parvo biovar.
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TEXT |
Ureaplasma urealyticum is
commonly known as a commensal in the female lower urogenital tract, yet
it has been suspected as a causative agent of choriamnionitis
(3), respiratory disease (4), meningitis
(27), and death in premature infants (2). U. urealyticum strains have been divided into 14 serovars by
a number of serological tests (21, 24). Although there is no definite evidence that a specific serovar is pathogenic, some studies
have suggested that certain serovars, especially serovars 4 and 8, are
frequently associated with diseases (16, 18). The
accumulated data suggest that the 14 serovars of U. urealyticum are divided into two biovars, the T960 and Parvo
biovars, which are distinguishable on the basis of polyacrylamide gel
electrophoresis of cellular proteins (17, 25), DNA-DNA
homology (5), restriction endonuclease cleavage patterns
(19), restriction fragment length polymorphism
(11), manganese susceptibility (23), DNA
modification system (6), enzyme profiles (8), and
genome size (14). The T960 biovar includes serovars 2, 4, 5, and 7 to 13, and the Parvo biovar comprises four serovars, 1, 3, 6, and
14. Although U. urealyticum strains are most commonly
identified by serology, serotyping of ureaplasmas has been hampered
because there are no commercially available antisera. Genetic analysis
has allowed two biovars to be distinguished on the basis of differences
in nucleotide sequences of ureaplasma genes (1, 10, 15, 20, 22,
26). For example, PCR-based methods have been used to analyze the
mba and 16S rRNA genes in U. urealyticum
(10, 20). In the present study, we examined the 16S-23S rRNA
intergenic spacer regions of the 14 serovars of U. urealyticum and showed that this region can also be used for
biovar identification.
Strains 7, 23, 27, 58, 354, Pi, Co, and T960 (serovars 1 to 8) of
U. urealyticum were obtained from D. K. Ford,
University of British Columbia, Vancouver, British Columbia, Canada.
Strains Vancouver, Western, K2, U24, U38, and U26 (serovars 9 to 14) of U. urealyticum were obtained from J. A. Robertson,
University of Alberta, Edmonton, Alberta, Canada. All the strains were
grown aerobically in the liquid medium at 37°C as described
previously (12). Ureaplasma broth cultures diluted 1:500
with sterile distilled water were subjected to PCR without DNA extraction.
The spacer regions of the 16S and 23S rRNA genes were amplified by PCR,
which was performed in a DNA thermal cycler (Perkin-Elmer Cetus,
Norwalk, Conn.) by using a pair of universal primers, F1 [5'-ACACCATGGGAG(C/T)TGGTAAT-3'] and R1
[5'-CTTC(A/T)TCGACTT(C/T)CAGACCCAAGGCAT-3'], based on the
rrnB operon of mycoplasmas as described elsewhere (11). These primers amplified 418 nucleotides, including the nucleotide sequences flanking the 16S and 23S rRNA genes of the 14 Ureaplasma strains. The amplified DNA was directly sequenced in an ABI Prism 310 genetic analyzer (Perkin-Elmer Applied Biosystems, Foster City, Calif.). Nucleotide sequences of the 16S-23S rRNA intergenic spacer regions of the 14 serovars of U. urealyticum were aligned by the method of Higgins et al.
(13) by using the DNASIS software package (Hitachi Software
Engineering, Co., Yokohama, Japan) (Fig.
1). Although the 16S-23S rRNA intergenic
spacer region of both the Parvo and T960 biovars comprised 302 nucleotides and lacked spacer tRNA genes, 15 nucleotides were different
between the two biovars. Thus, the 16S-23S rRNA intergenic spacer
region was more conserved than the mba gene, which can be
used for phylogenetic analysis of the U. urealyticum strains
(15). The nucleotide sequence of the T960 biovar contains
restriction sites for restriction endonuclease SspI, which
should allow differentiation of the T960 biovar from the Parvo biovar
by restriction enzyme analysis. No mismatched nucleotide was found in
the flanking region of the 16S and 23S rRNA genes. The four nucleotide
sequences of the 16S-23S rRNA intergenic spacer region of serovars 1, 3, 6, and 14 in the Parvo biovar were found to be identical. Similarly,
the 10 nucleotide sequences of the 16S-23S rRNA intergenic spacer
region of serovars 2, 4, 5, and 7 to 13 in the T960 biovar were found
to be identical. The box A (5'-GATCTTTG-3') and box B
(5'-GAGAGTTTATTCTCTC-3') sequences were assigned in both
biovars.
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FIG. 1.
Sequence alignment for the 14 strains from the 14 serovars of U. urealyticum. The nucleotide sequence numbers
are given from a consensus alignment. Nucleotides that are identical in
two out of three sequences are shown as white letters on a black
background. Dashes indicate spacers between adjacent nucleotides
introduced for maximum alignment. Box A and box B are underlined. Three
SspI (5'-AATATT-3') sites on the T960 biovar sequences are
indicated by asterisks.
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A typical secondary structure was predicted from the 16S-23S rRNA
spacer regions of the Parvo and T960 biovars by computer analysis
according to the Zuker-Stiegler algorithm (28) (Fig. 2). The folding energy for the secondary
structures was calculated by the method of Freier et al.
(9). Substantial negative free energies of the hypothetical
secondary structures in the 16S-23S rRNA spacer region of the Parvo and
T960 biovars were calculated to be 
97.81 and 87.62 Kcal/mol,
respectively. The box B sequence forms a stable stem-loop structure.
The box A sequence, which has been known to exist for other mycoplasmas
(12), is located on a bulge loop in the secondary structure.
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FIG. 2.
Proposed secondary structures for the 16S-23S rRNA
intergenic spacer regions of the two biovars of U. urealyticum. Minimum free energies for the Parvo (left) and T960
(right) biovars were calculated to be 97.81 and 87.62 Kcal/mol,
respectively. Box A and box B regions are enclosed by solid lines.
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