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Phylogenetic Position of Rare Human Mycoplasmas, Mycoplasma Faucium, Mm Buccale, Mmprimatum and M

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Phylogenetic Position of Rare Human Mycoplasmas, Mycoplasma Faucium, Mm Buccale, Mmprimatum and M lnternational Journal of Systematic Bacteriology (1 998), 48, 305-309 Printed in Great Britain Phylogenetic position of rare human mycoplasmas, Mycoplasma faucium, Mm buccale, Mmprimatum and M. spermatophilum, based on 16s rRNA gene sequences G eorg es Raw ad if An nic k Du j ea nco urt- H e nry, B rigi tte Le merc ie r and Daisy Roulland-Dussoix Author for correspondence: Georges Rawadi. Tel: +33 1 45 68 87 39. Fax: +33 1 40 61 33 56. e-mail : rawadi21 @pasteur.fr lnstitut Pasteur, The nucleotide sequence of the 16s rRNA genes of four rare human Departement de mycoplasma species, Mycoplasma faucium, M. buccale, M. primatum and M. Bactkriologie et de Mycologie, La boratoi re des spermatophilum, were partially sequenced and compared to pub1ished rRNA Mycoplasmes, 25 Rue genes of mycoplasmas to determine their position in the Mollicutes Docteur Roux, 75724 Paris phylogenetic tree. Nucleotide sequence motif and overall similarities allowed Cedex 15, France positioning of these mycoplasmas in the hominis phylogenetic group, as defined by Weisburg eta/. [Weisburg, W. G., Tully, J. G., Rose, D. L. & 9 other authors (1989). J Bacteriol 171, 6455-64671. Furthermore, these mycoplasmas could be clustered into two different subdivisions of the hominis group: (i) M. faucium and M. buccale were found to be included in the M. fermentans subdivision, and (ii) M. primatum and M. spermatophilum were included in the M. hominis one. Variable regions of the 16s rRNA genes were used to determine specific PCR primers to detect and identify M. faucium. Keywords: mycoplasmas, 16s rRNA Mycoplasmas are the smallest micro-organisms able to these rare species should be clarified by further replicate in acellular media. They belong to the Gram- epidemiological studies employing sensitive diagnostic positive bacteria and are currently assigned to class tools, such as PCR. Mollicutes, and are widely distributed in human, Phylogeny of mollicutes has been determined on the animals and plants. Several mycoplasma species have basis of 16s rRNA sequences which reflect the phylo- been isolated from respiratory and genital tracts of sgenetic relationship among various genera in the humans (10). class, as well as between mollicutes and other bacteria Recently, a fastidious mycoplasma was isolated from (5, 16, 17). Furthermore, 16s rRNA gene nucleotide urogenital tracts of patients with infertility problems sequences are used to develop PCR assays for the and named Mycoplasma spermatophilum (3,4). Myco- detection and the identification of fastidious myco- plasma primatum, a species commonly present in non- plasma species. In the present study, we have partially human primates, has been isolated from human determined the 16s rRNA gene nucleotide sequences urinary tract as well (4, 1 1, 12). M. spermatophilum and of M. spermatophilum, M. buccale, M.faucium and M. M. primaturn along with Mycoplasma buccale and primatum for comparison with previously published Mycoplasma faucium appear as rare inhabitants in 16s rRNA sequences (obtained from databases) to humans. It remains unclear whether their infrequent position these human mycoplasmas in the taxonomy isolation is due to their infrequent occurrence or of mollicutes. The 16s rRNA sequences reported here whether it relates to their fastidious nutritional could be used to design specific primers for PCR requirements. These mycoplasmas are regarded as diagnosis of these mycoplasma species. commensals, although M. spermatophilum has been suspected to cause infertility (4). The pathogenicity of Amplification of 16s ribosomal RNA genes M. buccale DC-1600T strain (ATCC 2304) and M.faucium The GenBank accession numbers for the sequences reported in this paper DC-333T strain (ATCC 25293) were obtained from the are U83663, U83502, AFO13996, AFO13998 and AFO13997. American Type Culture Collection (Rockville, MD, USA). 00606 0 1998 IUMS 305 Notes Table I=Accession numbers of bacterial 16s rRNA genes used for sequence alignments and construction of phy Iogenet ic trees Species Strain Accession Reference designation no. Mycoplasma faucium DC-333T U83663 Mycoplasma buccale DC- 1600T US3502 Mycoplasma spermatop h ilum NCTC 1 1720T AFO 13996 Mycoplasma primatum NavelT AFO 13998 Mycoplasma primatum 243 AFO 13997 Mycoplasma h omin is PG2 1 M24473 Mycoplasma fermentans PGl ST M2244 1 Mycop lasma pneumon iae FHT M2906 1 Mycoplasma orale CH 19299* M24659 Mycoplasma arginini G230T M24579 Mycoplasma salivarium PG20T M2466 1 Mycoplasma lipohilum MaByT M2458 1 Mycoplasma bovigenitalium PG1 IT M2429 1 Mycoplasma arthritidis PG6T M24580 Mycoplasma mycoides* UM30847 M23943 Mycoplasma pulmonis PG34T M23941 Mycoplasma agalactiae PG2T M24290 Ureaplasma urealyticum 27 M23935 Spiroplasma citri MarocT M23942 Acholeplasma laidlawii JAl M23932 Anaeroplasma abactoclasticum 6- 1 M25050 Clostridium innocuum B-3T M23732 ' Large colony. M. primatum NavelT and 243 strains were kindly provided phoresis through 1% agarose gel. The PCR product was by Dr A. F. Angulo (RIVM, The Netherlands). M. then cloned into the pCR-I1 cloning vector (Invitrogen). spermatophilum NCTC 11720T strain was obtained from the National Collection of Type Cultures (London). Myco- Sequencing and phylogenetic analysis plasmas were cultured in SP-4 medium (1 3) supplemented with glucose, fresh yeast extract and 20% horse serum. Cloned PCR products were completely sequenced on both Mycoplasmas were grown by incubation at 37 "C, 5 YOCO,, strands by using an AmpliTaq FS dye terminator kit (Perkin and cultures were stopped at the mid-exponential growth Elmer) and nucleotide sequences were determined an ABI phase. Prism model 373A (Applied Biosystems). The newly deter- mined 16s rRNA gene sequences were compared to One millilitre of each of the mycoplasma cultures was previously published mollicutes 16s rRNA (obtained from pelleted by centrifugation at 14000 g, at 4 "C for 20 min. GenBank; Table 1) genes using the PILEUP program in the Harvested cells were resuspended in 100 pl lysis buffer GCG package. Pairwise nucleotide sequence similarity (10 mM Tris/HCl, pH 8.3, 50 mM KCl, 2.5 mM MgCl,, scores were computed with default gap and gap length 0.5 % Tween-20 and 0.5 YOTriton X-100) containing prot- weight of PILEUP. An unusual sequence motif, 5'-AAAC- einase K (60 pgml-l) then incubated at 60 "C for 1 h. TTAAA-3' (nt 907-915), is shared by all the members of the Afterwards, proteinase K was denatured by heating at 94 "C M. hominis phylogenetic group described by Weisburg et al. for 10 min. Two microlitres of the mycoplasma proteinase K (16). The T residue at position 912 is found in archaea and digest was used for PCR amplification of 16s rRNA genes. eukaryotes while eubacteria have a C residue at this position The following primers were used to amplify 16s rRNA genes (17). 16s rRNA gene sequences from M. faucium, M. from mycoplasma species : (i) RNA5 : 5'-AGAGTTTG- buccale, M.primatum and M. spermatophilum contained this ATCCTGGGCTCAGGA-3' (sense strand nucleotides motif, and accordingly they are positioned in the hominis 1 1-30) (9) and (ii) MGSO : 5'-TGCACCATCTGTCAC- group as shown in Fig. 1. The mollicute phylogenetic TCTGTTAACCTC-3' (antisense strand nucleotides tree compromises four phylogenetic groups, hominis, 1029-1055) (15). PCR was performed in 50 pl final volume pneumoniae, spiroplasma and anaeroplasma (14). In Fig. 1, containing 150 mM MgCl,, 200 pM each dNTP, 40 pM the representative species of each of these groups, and primers RNA5 and MGSO and 2.5 U Taq DNA polymerase Clostridium innocuum, a walled close relative Gram-positive (Perkin Elmer). Primer annealing temperature was 58 "C, bacterium, were included as well. and 30 PCR cycles were carried out. The 1 kb PCR product The hominis phylogenetic group has been further subdivided was visualized by ethidium bromide staining after electro- into three subdivisions (6) : Mycoplasma fermentans, Myco- ~ 306 International Journal of Systematic Bacteriology 48 Notes M. huccale (DC-1600T) M.Jouriutn (DC-333T) M. spermatophilum (NCTC-I 1720T) M. primaturn (NavelT) M. mycoides (UM30847) S. citri (MarocT) U. urealyticum (27) M. pneumoniae (FHT) An. abactoclarticutn (6-IT) A. labflawii (JA I) C. innocuum (B-3T) E. coli M. orale (CH19299T) (c> M. spermatophilum (NCTC-I 1720T) r M. faucium (DC-333T) M. bovtgenifaIiurn (PGIIT) - ..................................................................... I ............................... M. buccale (DC-16001) - M.fermentans (PG18T) Fig. 1. Phylogenetic trees. (a) Position of M. faucium, M. buccale, M. spermatophilum lr - M. agalactiae (PC2T) and M. primatum within the Mollicutes. The c. i sequence of Escherichia coli (1) is used as an outgroup to establish the root of the tree. 1 M. primaturn (NavelT) (b) Detailed position of M, faucium and M. buccale in the M. hominis subdivision, and -M. Iipophihm (MaByT) of (c) M. spermatophilum and M. primatum in the M. fermentans subdivision within the hominis phylogenetic group. M. mycoides served as an outgroup to establish the root M. mycoides (UM30847) M. mycoides (UM30847) of the tree. The GenBank accession numbers of the 165 rRNA sequences used are indicated in Table 1. The scale bar indicates base changes per 100 nucleotides. plasma hominis and Mycoplasma hyorhinis. The overall fermentans subdivision to M. spermatophilum 16s rRNA nucleotide similarity scores placed both M.faucium and M. gene ranged from 0.944 to 0463, the value being for M. buccale in the M. hominis phylogenetic subdivision (Fig. 1b), pulmonis. while M. primatum and M. spermatophilum were placed in the M.fermentans subdivision (Fig. lc). In the M. hominis subdivision, M.faucium showed the highest similarity to the Analysis of nucleotide sequences 16s rRNA sequence of the human mycoplasma M. orale, Variable regions have been identified within the 16s rRNA with a score of 0.977. On the basis of its 16s rRNA sequence, sequences on the basis of secondary structure modelling of M. buccale has been clustered with Mycoplasma arthritidis the rRNA gene of Escherichia coli (7). The nucleotide (score 0.964). Initially, by immunological studies, M. buccale sequences of 16s rRNA genes from M.faucium, M.
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