Oct 2, 1992 - Acholeplasma laidlawii. FEMS Lett. 3:143-145. 9. Lo, S.-C., J.-W. Shih, P. B. Newton, D. M. Wong, M. M. Hayes,. J. R. Benish, D. J. Wear, and ...
Vol. 61, No. 1
INFECrION AND IMMUNITY, Jan. 1993, p. 329-331
0019-9567/93/010329-03$02.00/0 Copyright X) 1993, American Society for Microbiology
NOTES Polymorphism in Genes for the Enzyme Arginine Deiminase among Mycoplasma Species KAZUHISA SUGIMURA,lt* TAKESHI OHNO,1 ICHIRO AZUMA,l AND KOSHI YAMAMOTO2 Institute of Immunological Science, Hokkaido University, Sapporo 060,1 and National Institute of Animal Health, Tsukuba, Ibaraki 305,2 Japan Received 16 July 1992/Accepted 2 October 1992
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.
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.
Two enzymatic pathways for the generation of ATP in nonfermentative mycoplasmas, the acetyl coenzyme A pathway and 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 (12-14). 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 SSC0.1% SDS and then washed twice with 0.2x SSC-0.1% SDS
* Corresponding author. t Present address: Department of Applied Chemical Engineering, Faculty of Engineering, Kagoshima University, 1-21-40, Korimoto,
Kagoshima 890, Japan. 329
330
NOTES
INFECT. IMMUN.
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0.5FIG. 1. Southern blot analysis of AD genes. The numbers indicate the species of mycoplasma presented in Table 1. Mycoplasmal DNA digested to completion with EcoRI. The 725-bp EcoRI fragment (nucleotides 30 through 754) of the M. arginini AD gene was used as a probe (10). (g) Restriction map of the AD-coding region. The dotted and solid boxes indicate the AD-coding region and the 725-bp EcoRI fragment, respectively. E, EcoRI; T, TaqI; X, XbaI; P, PstI. Filters were rinsed under the relaxed conditions (b, c, F) or under stringent conditions (a, d, e) (see the text). HindIII-digested DNA was used as size markers.
was
NOTES
VOL. 61, 1993
331
TABLE 1. Restriction fragment length polymorphism of mycoplasmal AD genes Mycoplasma no.
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28
Biochemical
Straina
M. orale CH19299 M. arginini G230 M. spumans PG13 M. arthritidis PG6 M. gateae Mart M. alkalescens PG51 M. buccale CH20247 M. subdolum TB M. canadense ATCC 29418 M. salivarium PG20 M. caviae G122 M. hyosynoviae S16 M. hyorhinis BTS7 M. hominis PG21 M. opalescens MM5408 M. equirhinis M432/72 M. iners PG30 M. gallinarum PG16 M. primatum HRC292 M. incognitus M. fermentans K7 M. fermentans PG18 M. pirum M. maculosum PG15 M. meleagidis 17520 M. lipophilum MaBy M. pneumoniae FH M. muris RIII4
Length (bp) of EcoRI-digested DNA under
featuresb
the following
Glu
Arg
Phos
Stringent
-
+
-
+ + + + + + + + +
+ + + + v w -
380 725 (725) 380 725, 250 380 380 (860) 2,100, (250) 1,800, 1,500 (1,200) 2,200 ND 3,500, 1,300 (6,800) 8,800
+ + + +
+ + -
v
+ + + + + + + + + + + + + + +
+ + +
+
+ +
-
ND (3,900) (3,900) (3,900)
ND ND ND ND ND
conditions:
Relaxed
380 725 725 380 725, 250 380 380 860 2,100, 250d 1,800, 1,500
1,200 2,200 ND 3,500, 1,300e
6,800 8,800 (380, 2,300, 1,300) (1,600) ND
3,900 3,900 3,900 (3,900f ND ND ND ND ND
a M. incognitus and M. fermentans K-7 and PG-18 were provided by S.-C. Lo (Armed Forces Institute of Pathology, Washington, D.C.) (9, 14), and M. pinu was provided by L. Montagnier (Institut Pasteur, Paris). b Glu, ability to degrade glucose; Arg, ability to degrade L-arginine; Phos, phosphatase activity (data from references 4, 5, 7, 9, and 15). v, inconsistent results; w, weak positive. A blank space indicates that the biochemical feature was not determined. c The lengths of hybridized DNA fragments were estimated by agarose electrophoresis (Fig. 1). See the text for descriptions of stringent and relaxed conditions. Values given within parentheses indicate faint bands. ND, no homologous bands detected. dIncompletely digested fragments between 3 and 4 kbp (Fig. 1). A band of 4 kbp is an incompletely digested fragment (Fig. 1). f A very faint band of 3,900 bp is visible. Four bands over 5 kbp are incompletely digested fragments (Fig. 1).
We thank Mihoko Sato for expert editorial assistance. This work was partly supported by grants-in-aid for AIDS research from the Japanese Ministry of Education, Science, and Culture and by a grant provided by the Ichiro Kanehara Foundation. REFERENCES 1. Barile, M. F., and B. G. Levinthal. 1968. Possible mechanism for mycoplasma inhibition of lymphocyte transformation induced by phytohemagglutinin. Nature (London) 219:751-752. 2. Cole, B. C., Y. Naot, E. J. Stanbridge, and K. S. Wise. 1985. Interactions of mycoplasmas and their products with lymphoid cell in vitro, p. 203-257. In S. Razin and M. F. Barile (ed.), The mycoplasmas, vol. 4. Academic Press, Inc., New York. 3. Copperman, R., and H. E. Morton. 1966. Reversible inhibition of mitosis in lymphocyte cultures by non-viable mycoplasma. Proc. Soc. Exp. Biol. Med. 123:790-795. 4. Del Giudice, R. A., J. G. Tully, D. L. Rose, and R. M. Cole. 1985. Mycoplasma pirum sp. nov., a terminal structured mollicute from cell cultures. Int. J. Syst. Bacteriol. 35:285-291. 5. Erickson, B. Z., R. F. Ross, D. L. Rose, J. G. Tully, and J. M. Bove. 1986. Mycoplasma hyopharyngis, a new species from swine. Int. J. Syst. Bacteriol. 36:55-59. 6. Fenske, J. D., and G. E. Kenny. 1976. Role of arginine deiminase in growth of Mycoplasma hominis. J. Bacteriol. 126:501-510. 7. Hill, A. C. 1984. Mycoplasma cavipharyngis, a new species isolated from the nasophaynx of guinea-pigs. J. Gen. Microbiol. 130:3183-3188. 8. Kahane, I., A. Muhlrad, and S. Razin. 1978. Possible role of
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