A putative transposase gene in the 16S-23S rRNA intergenic spacer region of Mycoplasma imitans

Mycoplasma imitans was first isolated from the turbinates of mule (broiler) ducks in southwest France, and tentatively identified as Mycoplasm gallisepticum by immunofluorescence and growth inhibition tests (Dupiellet, 1984). Similar strains were thereafter also isolated from geese in France (Buntz et al., 1986). These duck and goose isolates were found to be closely related to each other, but distinct from M. gallisepticum, by additional serological tests (Dupiellet et al., 1988). Further biochemical and molecular analyses, including SDS-PAGE profiles of the cellular proteins, restriction endonuclease cleavage patterns of the genomic DNAs, the G+C contents, and Southern blot hybridization using rRNA and tuf gene probes, also supported the observation that duck and goose strains were similar to one another but different from the reference strains of M. gallisepticum (Dupiellet, 1988; Yogev et al., 1988a, b). In addition, it was shown by DNADNA hybridization that there was a very close genetic relationship between the duck and goose strains but that the levels of homology of these strains with M. gallisepticum were only 4046 % (Dupiellet et al., 1988, 1990). Based on the accumulated data, a new species, M. imitans, was established for the duck and goose strains (Bradbury et al., 1993) and it was recognized that M. imitans and M. gallisepticum are distinct species although their rRNA sequences are nearly identical, differing by only two nucleotides (GenBank accession numbers M22441 and L24103). Comparisons of 16S rRNA sequences have been widely used in taxonomic and phylogenetic studies of prokaryotes (Swofford & Olsen, 1990), but 16S rRNA data alone may not be sufficient for defining very recently diverged species (Fox et al., 1992). Therefore in this study the 16S23S intergenic transcribed spacer (ITS) region of M. imitans and M. gallisepticum was examined in order to determine species divergence in these sequences.

The ITS region is an important tool for the development of DNA-based classification because it shows a significant degree of variation in length and sequence from one species to another (Gürtler & Stanisich, 1996), and it can also be used to determine the relationships between genetically related bacterial species because of its high rate of divergence (Harasawa et al., 1996). The cistrons for rRNA molecules of most mycoplasmas are organized in an operon and arranged in the order 5'-16S23S5S-3', in which the individual rRNA genes are separated by the ITS regions, which occupy about 25 % of the operon (Razin, 1985). Two copies of the rRNA operon have been detected in M. imitans and M. gallisepticum (Dupiellet,1988). The rRNA operon is transcribed in a monocistronic RNA transcript and ITS regions are removed from the primary RNA transcript by a series of co-ordinated nucleolytic events catalysed by rRNA-processing enzymes during maturation of the rRNA molecules. In this study, the ITS region between the 16S and 23S rRNA genes of M. imitans and M. gallisepticum was sequenced and compared. Having determined that the ITS region of these two Mycoplasma species was markedly different in size, we compared the PCR products of the ITS region of all the other recognized avian Mycoplasma species to see if the size differences had any diagnostic value.

Strains and culture.
M. imitans strains 4229^T (same progenitor as NCTC 11733), B2/85 and B35/84 and M. gallisepticum strains PG31^T (same progenitor as NCTC 10115), S6 (Zander, 1961) and F (Adler, 1960) were cultured in mycoplasma broth or on agar at 37 °C in a CO₂-rich (5 %, v/v) atmosphere until the broth showed colour change or colonies appeared on plates after 15 days, as described previously (Bradbury, 1977). The other avian Mycoplasma type strains shown in Fig. 1 were cultured under the same conditions. The identity of all the strains was confirmed by an indirect fluorescent antibody test (Rosendal & Black, 1972).

(56K): Fig. 1. Comparison of the ITS regions from 23 type strains of avian Mycoplasma species by gel electrophoresis of the PCR products. The product includes 180 bp at the 3' end of the 16S rDNA and 115 bp at the 5' end of the 23S rDNA. Markers A and B are a 100 bp ladder (Amersham Pharmacia Biotech) and a mixture of λDNA/HindIII and φX174 RF DNA/HaeI fragments (Invitrogen), respectively. The arrow indicates 800 bp. Lanes: 1, M. anatis 1340T; 2, M. anseris 1219T; 3, M. buteonis Bb/T2gT; 4, M. cloacale 383T; 5, M. columbinasale 694T; 6, M. columbinum MMP1T; 7, M. columborale MMP4T; 8, M. corogypsi BV1T; 9, M. falconis H/T1T; 10, M. gallinaceum DDT; 11, M. gallinarum PG16T; 12, M. gallisepticum PG31T; 13, M. gallopavonis WR1T; 14, M. glycophilum 486T; 15, M. gypis B1/T1T; 16, M. imitans 4229T; 17, M. iners PG30T; 18, M. iowae 695T; 19, M. lipofaciens R171T; 20, M. meleagridis 17529T; 21, M. pullorum CKKT; 22, M. sturni UCMFT; 23, M. synoviae WVU 1853T.

DNA extraction.
DNA of M. imitans strain 4229^T and M. gallisepticum strain PG31^T was extracted from 450 µl broth culture (10⁷ c.f.u. ml¹) as described previously (Harasawa, 1996). Type strain cultures of the other avian Mycoplasma species and the other strains of M. imitans and M. gallisepticum (1·5 ml) at the end of the exponential growth phase were transferred to a 1·5 ml sterile Eppendorf tube and centrifuged at 10 000 g for 10 min. The pellet was washed twice in sterile PBS and resuspended in 200 µl sterile water (Sigma) by vigorous vortexing. A 25 % suspension of Chelex 100 (Bio-Rad) (200 µl) was added and the mixture vortexed again. The mixture was incubated at 56 °C for 30 min, and vortexed again. After placing in boiling water for 8 min, the mixture was cooled on ice, vortexed again, and centrifuged at 10 000 g for 5 min. The supernatant fluid was transferred to a fresh tube and was stored at 20 °C before use.

PCR.
DNA amplifications were performed with a forward primer (5'-GGG ATG ACG TCA AAT CAT CAT GCC-3') and a reverse primer (5'-TAG TGC CAA GGC ATC CAC C-3') in a DNA thermal cycler (Perkin-Elmer). Reaction mixtures contained 2·5 U Taq DNA polymerase (AB Gene), 0·2 µM of each primer, 1x reaction buffer, 1·75 mM MgCl₂, 0·2 mM dNTPs, and water to a volume of 50 µl. DNA amplification was achieved with 5 cycles of denaturation at 94 °C for 15 s, renaturation at 60 °C for 30 s, and elongation at 72 °C for 2 min, followed by 30 cycles with the same parameters, except that there was an extension of 2 s per cycle in the elongation step. A 5 µl volume of each amplification reaction was subjected to electrophoresis in a 1·5 % agarose gel. Gels were stained with ethidium bromide (0·3 µg ml¹) and DNA visualized with ultraviolet light.

Sequencing.
The 16S23S rRNA intergenic spacer region was amplified as described elsewhere (Harasawa, 1999) using a pair of universal primers, 16S-1359F (5'-GGGTCTTGTACACACCG-3') and 23S-115R (5'-GGGTTBCCCCATTCGG-3') (Lane, 1991), with denaturation at 94 °C for 30 s, annealing at 55 °C for 100 s, and extension at 72 °C for 100 s. Amplified DNA products of M. imitans strain 4229^T and M. gallisepticum PG31^T were extracted from agarose gels and subjected to direct sequencing twice on each strand in an ABI Prism 310 Genetic Analyser (Perkin Elmer-Cetus) by primer walking.

The ITS regions of the 23 type strains of avian Mycoplasma species were amplified by PCR (Fig. 1). The nucleotide sequences of the type strains of M. imitans and M. gallisepticum revealed that their ITS regions are 2488 nt and 645 nt, respectively, in size (Fig. 2). The ITS sequences from the two species were aligned using CLUSTAL X (Thompson et al., 1997). The first 153 nt of the ITS region showed 96 % identity between M. imitans and M. gallisepticum (Fig. 2); this region is believed to form an RNARNA double strand with the leader sequence of the 16S rRNA (Chiaruttini & Milet, 1993; Liiv et al., 1998). Similarly, the last 138 nt of the ITS region were almost identical (98 % identity) between the two species (Fig. 2); this region is believed to form an RNARNA double strand with the 3' flanking region of the 23S rRNA. These double-stranded regions contain a recognition site for the rRNA-processing enzyme RNase III (Chiaruttini & Milet, 1993; Liiv et al., 1998). Our data suggest that the rRNA processing in M. imitans and M. gallisepticum is conducted by a similar RNase III, since the two species share common features at the 5' and 3' ends of the ITS region. A putative boxA sequence was found at 90 nt upstream from the 23S rRNA gene of M. imitans and M. gallisepticum. The boxA sequence is considered to be either an anti-terminator or an internal promoter (Berg et al., 1989). Several palindromic sequences, some of which were nested, were detected in the ITS region of both the species (Fig. 2). These palindromic sequences may be responsible for rearrangement of the ITS region (Gürtler, 1999). The most common insertional genes found in bacterial ITS regions are tRNA genes, but neither tRNA genes nor their pseudogenes were found in the ITS of these two Mycoplasma species. This finding is common to all mycoplasmal ITS regions that have been sequenced (Harasawa, 1999). Instead of tRNA genes, the M. imitans genome has an ORF of 1260 nt on the complementary strand of the ITS region. The amino acid sequence predicted from this ORF showed a high similarity score with bacterial transposases of Yersinia enterocolitica (72 bits), Bacillus halodurans (65 bits), Escherichia coli (61 bits) and Yersinia pestis (56 bits) using the BLAST algorithms (Altschul et al., 1997). Although other M. imitans strains have not yet been sequenced, this is likely to be a common property among M. imitans strains, as the PCR products of the three strains all had a similar size by gel electrophoresis (Fig. 3), which was distinct from that of all the M. gallisepticum strains tested (Fig. 3). These sizes are relatively large among the Mycoplasma species, as most ITS regions are smaller than 500 bp. They are also larger than those of the other avian Mycoplasma species examined here. These size differences may prove helpful in discriminating between M. imitans and M. gallisepticum and also for distinguishing between these two species and all the other avian species.

(109K): Fig. 2. Sequence alignment of the ITS regions of M. gallisepticum strain PG31T (upper line) and M. imitans strain 4229T (lower line). The nucleotide sequence numbers are from the consensus alignment. Identical nucleotides between the two species are shown as white letters on a black background. Dashes indicate spacers between adjacent nucleotides introduced for maximum alignment. Palindromic sequences are indicated by arrows pointing in opposite directions. The open reading frame on the complementary strand is shown in a box. The P75 gene homologue is underlined.

(88K): Fig. 3. Comparison of the ITS regions of M. gallisepticum and M. imitans by gel electrophoresis of PCR products. M. gallisepticum (lanes 24) and M. imitans (lanes 57) produced bands of 940 bp and 2783 bp, respectively. Lanes: 1, 100 bp ladder (Invitrogen); 2, M. gallisepticum PG31T; 3, M. gallisepticum S6; 4, M. gallisepticum MgF; 5, M. imitans 4229T; 6, M. imitans B2/85; 7, M. imitans B35/84; 8, 1 kbp ladder (Invitrogen).

Nucleotide sequences of a number of putative transposase genes in mycoplasma genomes have been deposited in GenBank, including insertion sequences in M. pulmonis strain KD735-26 [1203 bp of IS1138, GenBank accession Z16416] (Bhugra & Dybvig, 1993), M. agalactiae strain 3990 [1029 bp, GenBank accession AJ311887] (Pilo et al., 2003), M. fermentans strain PG18 [1101 bp of IS1630, GenBank accession AF100324] (Calcutt et al., 1999), M. mycoides subsp. mycoides SC strain Afade [1602 bp of IS1634, GenBank accession AF062493] (Vilei et al., 1999), M. hyopneumoniae [1659 bp, GenBank accession AF272977] (M. J. Calcutt & E. M. Wise, unpublished), M. hyorhinis strain GDL-1 [1425 bp of IS1221, GenBank accession U01217] (Zheng & McIntosh, 1995) and M. penetrans strain HF-2 [402 aa, GenBank accession AP004174-71] (Sasaki et al., 2002). A partial sequence of a putative insertion sequence-like transposase gene has been reported in M. orale strain ATCC 23714 [GenBank accession AY084048] (S. E. Ditty, B. Li, S. Zhang, N. Zou & S. C. Lo, unpublished). Of all the putative transposase genes reported in mycoplasmas thus far, the M. imitans transposase is the first to be discovered in an rRNA operon. Although no typical or consensus motif has been found in the transposase protein sequences thus far, the amino acid sequence of the M. imitans transposase was found to be most similar to that of M. penetrans. Recently sequences more similar to the M. imitans transposase have been deposited in the GenBank database under accession numbers AE016967AE016970 (Geary et al., 2003). Although we have not examined the copy number of the transposase genes in M. imitans, at least six copies (ORFs MGA1325 and MGA0910 in accession AE016967, MGA1081 and MGA0073 in accession AE016968, and MGA0145 and MGA0147 in accession AE016969) of a transposase which shows similarity to the M. imitans putative transposase were present in the M. gallisepticum genome. The presence of the transposase gene element in the ITS may be a useful tool for differentiating M. imitans from M. gallisepticum. Both the ITS regions of the two rRNA operons in M. imitans are likely to include the same transposase gene because a single band was produced by PCR. Of 1260 nt of the anti-sense ORF found in M. imitans, 315 nt (25 %) showed a high identity (83 %) to the P75 gene and its 3' flanking region in M. gallisepticum [GenBank accession AY037872] (Spencer et al., 2002) (Fig. 2). The P75 gene in M. gallisepticum encodes a 75 kDa protein that is recognized during natural infections (Spencer et al., 2002). A possible explanation for the presence of this inverted gene could be that non-homologous recombination has occurred, followed by reverse transcription of the P75-like mRNA. Reverse transcription has been reported in some bacterial species (Lim & Maas, 1989; Varmus, 1989). Although reverse transcriptase has not been reported in mycoplasmas, prokaryotic DNA polymerases are known to have some reverse transcriptase activity in some species (Lou et al., 1991; Myers & Gelfand, 1991). An alternative explanation is that the transposase gene may have inserted downstream of the P75 gene and been imprecisely excised, leaving behind part of its DNA sequence.

Conclusions
This study reports the discovery of a novel insertion sequence present in the rRNA operon of M. imitans. This element, a putative transposase, is the first to be described in this region of the genome of a Mycoplasma species. The presence of an unusually long ITS sequence in this species could be useful in differentiating it from M. gallisepticum and other avian mycoplasmas.

We would like to acknowledge the help and advice of Dr C. J. Naylor.

Footnotes

†Present address: Sección de Epidemiología y Medicina Preventiva, Facultad de Veterinaria, Universidad de Las Palmas de Gran Canaria, Trasmontaña s/n 35416 Arucas, Spain.