Polymorphism in genes for the enzyme arginine deiminase among Mycoplasma species

K Sugimura, T Ohno, I Azuma, K Yamamoto
1993 Infection and Immunity  
The extent of restriction fragment length polymorphism in genes for the arginine deiminase enzyme among 28 species of mycoplasmas was assessed by Southern blot analysis of DNA digested with EcoRI or TaqI nuclease probed with a 725-bp internal fragment of the arginine deiminase gene from Mycoplasma arginini. The results ndicated unexpected heterogeneity among species of a single genus. Two enzymatic pathways for the generation of ATP in nonfermentative mycoplasmas, the acetyl coenzyme A pathway
more » ... nd the arginine deiminase (AD) pathway, were previously proposed (6, 8) . AD plays an important role in catalyzing the direct conversion of L-arginine and H20 to L-citrulline and NH3, which initiates the AD pathway. On the other hand, mycoplasma infections often radically change host cell metabolism in vitro (2). It was proposed in classical studies published nearly three decades ago that an immunosuppressive substance derived from mycoplasmas might be an AD (1-3). Recently, we demonstrated by molecular cloning that a strong immunosuppressive factor derived from Mycoplasma arginini is an AD (10, 11). Moreover, we clarified the molecular mechanism of AD-induced immunosuppression (12) (13) (14) . In this study, we attempted to determine the restriction fragment length polymorphism of AD genes of 28 species of mycoplasmas. Mycoplasma cultures were obtained by the method of Barile and McGarrity with modifications as described previously (11). Mycoplasmal genomic DNA was prepared by a standard method (10). High-molecular-weight DNA was digested to completion with EcoRI or TaqI (Takara, Kyoto, Japan). One microgram of each sample was electrophoresed in a 0.7 or 2% agarose gel and blotted onto a nylon membrane (Hybond-N; Amersham) in accordance with the manufacturer's instructions. Hybridization was carried out with a radiolabeled 725-bp EcoRI internal fragment of an AD gene that consisted of 1,230 bp encoding 410 amino acids as described previously (Fig. lg) (10). Nick translation was carried out with a nick translation kit (Amersham). After hybridization, the filters were treated as follows: (i) for relaxed conditions, the filters were rinsed with 2x SSC (lx SSC is 0.15 M NaCI-0.015 M sodium citrate)-0.1% sodium dodecyl sulfate (SDS) at room temperature and then autoradiographed (Fig. lb , c, and f); (ii) for stringent conditions, the filters were rinsed with 2x SSC-0.1% SDS and then washed twice with 0.2x SSC-0.1% SDS for 15 min at 50°C and then autoradiographed ( Fig. la, d , and e). Figure 1 shows the results with EcoRI-digested DNA, and Table 1 summarizes these results in terms of the biochemical characteristics of mycoplasmas. HindIII-digested X DNA or HinfI-digested pBR322 was used for size markers. We focused on species of L-arginine-utilizing mycoplasmas in this study; two non-arginine-utilizing species, Mycoplasma pneumoniae and M. hyorhinis, were examined for purposes of comparison. No hybridized bands are detected in EcoRIdigested DNA (Fig. 1) or TaqI-digested DNA (data not shown) of these species, suggesting that their genomes do not contain an AD gene, as expected from their biochemical features (Table 1) (4, 5, 7, 9, 15 ). On the other hand, among 26 L-arginine-utilizing mycoplasma speci-es, 5 (Mycoplasma primatum, M. maculosum, M. meleag?idis, M. lipophilum, and M. muris) did not show hybridized bands in EcoRIdigested DNA (Fig. 1) or TaqI-digested DNA (data not shown) even when filters were washed under the relaxed conditions. Three species (Mycoplasma iners, M. gallinarum, and M. pirum) show hybridized bands only after the rinse under the relaxed conditions. These results suggest that mycoplasmal AD genes bear great diversity in their nucleotide sequence or that there is an enzyme other than AD that catalyzes L-arginine degradation in these three mycoplasma species. The remaining 18 species exhibited hybridized bands, including very faint bands, after the rinse under the stringent conditions. Biochemical and enzymological studies may be crucial to explain these data. Unexpected heterogeneity among the 21 AD-positive mycoplasma species with regard to the restriction fragment length polymorphism of AD genes was discovered. The AD gene is considered to be an essential enzyme that plays a key role in the ATP-generating pathway. This is somewhat contradictory to the popular notion that the more critical the biological functions of the molecules are, the more conservative their structures are. Our findings raise the question of how the AD gene was diversified during the evolutionary process of these species. The determination of the nucleotide sequences of these AD genes may answer this question. This unique feature of mycoplasmal AD genes may enable us to compare the protein structures of ADs and their enzymatic activities. 329 on May 5, 2020 by guest http://iai.asm.org/ Downloaded from
doi:10.1128/iai.61.1.329-331.1993 fatcat:4cqtmjw2lbaibavxoymkdnngdi