INTERNATIONALJOURNAL OF SYSTEMATICBACTERIOLOGY, Oct. 1989, p. 413419 Vol. 39, No. 4 OO20-7713/89/O4O413-07$02.00/0 Copyright 0 1989, International Union of Microbiological Societies Pyrophosphate-Dependent Enzymes in Walled Bacteria Phylogenetically Related to the Wall-Less Bacteria of the Class Mollicutes? JAMES P. PETZEL,'S PAUL A. HARTMAN,'* AND MILTON J. ALLISON2 Department of Microbiology, Iowa State University, Ames, Iowa 5001 1-321I ,I and National Animal Disease Center, U.S. Department of Agriculture, Ames, Iowa 500102 Some of the wall-less bacteria of the class Mollicutes (mycoplasmas) have pyrophosphate (PP,)-dependent enzymic activities, including PP,-dependent phosphofructokinase (PP,-PFK), PP,-dependent nucleoside kinase, and pyruvate,orthophosphate dikinase (PPDK) activities. In most other bacteria, adenosine 5'-triphosphate (ATP), not PP,, is the cofactor of analogous enzymic reactions. Because PP,-dependent enzymes are more common among mollicutes than other bacteria, we describe here an examination of the six walled bacteria that have been reported to be phylogenetically related to the mollicutes (Clostridium innocuum, Clostridium ramosum, Erysipelothrix rhusiopathiae, Lactobacillus catenaformis, Lactobacillus vitulinus, and Streptococcus pleomorphus) for PP,-PFK, ATP-dependent PFK, phosphoenolpyruvate carboxytransphosphorylase, PPDK, and PP,- and ATP-dependent acetate kinases. Two anaerobic mollicutes, Anaeroplasma intermedium and Asteroleplasma anuerobium, were also tested. C. innocuum, E. rhusiopathiue, S. pleomorphus, and Anuero- plasma intermedium had PPi-PFK activities, whereas C. ramosum, the two lactobacilli, and Asteroleplasma anaerobium had only ATP-dependent PFK activities. Asteroleplasma anaerobium and all of the walled bacteria except E. rhusiopathiue had PPDK activities. All of the species except Asteroleplasma anaerobium and E. rhusiopathiae also had pyruvate kinase activities; the effects of allosteric activators were tested. Phosphoe- nolpyruvate carboxytransphosphorylase was detected by using two methods in C. innocuum, C. ramosum, and S. pleomorphus. All of the species tested had ATP-dependent acetate kinase activities, but none had detectable PP,-dependent acetate kinase activity. The occurrence of one or more PPi-dependent enzymes in the mollicutes and their walled relatives is a phenotypic indicator of the phylogenetic relatedness of these organisms. The distribution of these enzymes among members of this group substantiates the subgroups proposed by other workers who used 16s ribosomal ribonucleic acid analysis. The wall-less eubacteria belonging to the genera Myco- no. P114, 1988). These authors found that Clostridium in- plasma, Ureaplasma, Spiroplasma, Acholeplasma, Anaero- nocuum, Clostridium ramosum, and six species of molli- plasma, and Asteroleplasma are grouped together in the cutes (belonging to four genera) were all resistant to high class Mollicutes. Phylogenetic analyses of 5s and 16s ribo- levels of rifampin. Generally, eubacteria are susceptible to somal ribonucleic acid (rRNA) sequences have revealed that rifampin, an inhibitor of ribonucleic acid polymerase; some the mollicutes cluster with the low-guanine-plus-cytosine mollicutes, however, are resistant (10). content subdivision of gram-positive eubacteria that includes Some members of the orders Mycoplasmatales and the bacillus-lactobacillus-streptococcusbranch (31, 40, 41). Acholeplasrnatales have activities for (deoxy)ribonucleoside Among the members of the low-guanine-plus-cytosine con- kinases that are dependent on pyrophosphate (PP,) as a tent gram-positive eubacteria, the mollicutes are most cofactor (18, 38), and Acholeplasma spp. have PP,-depen- closely related to Clostridium innocuum, Clostridium ramo- dent 6-phosphofructokinase (PP,-PFK) activity (7, 23). sum, Erysipelothrix rhusiopathiae, Lactobacillus cat- Among the anaerobic mollicutes, both Anaeroplasma inter- enaformis (Lactobacillus catenaforme), Lactobacillus vitu- medium (18) and Asteroleplasma anaerobium (J. P. Petzel, linus, and Streptococcus pleomorphus (16,31,40-42; W. G. M. C. McElwain, D. DeSantis, J. Manolukas, M. V. Weisburg, J. G. Tully, D. L. Rose, J. P. Petzel, H. Oyaizu, Williams, P. A. Hartman, M. J. Allison, and J. D. Pollack, D. Yang, L. Mandelco, J. Sechrest, T. G. Lawrence, J. Van Arch, Microbiol., in press) have activities for PP,-dependent Etten, J. Maniloff, and C. R. Woese, submitted for publica- nucleoside kinases. Anaeroplasma intermedium also has tion). PP,-PFK activity, whereas Asteroleplasma anaerobium has The mollicutes and their walled phylogenetic relatives activity for a third PP,-dependent enzyme, pyruvate ,ortho- have few phenotypic similarities. The only comparative phosphate dikinase (PPDK; Petzel et al., in press). PP,- study of the phenotypes of the mollicutes and their walled dependent nucleoside kinase has not been reported in any phylogenetic relatives was conducted by Pellegrin et al. organism other than some mollicutes (18,22, 38). Adenosine (J. L. Pellegrin, J. Maugein, M. Clerc, B. Leng, and C. 5'-triphosphate (ATP) serves as the phosphate donor in the Bebear, Abstr. 7th Congr. Int. Organ. Mycoplasmol., abstr. phosphofructokinase (PFK) reaction instead of PP, in the great majority of bacteria. PPDK, which converts phospho- enolpyruvate (PEP) to pyruvate, has been reported in only a * Corresponding author. T Dedicated to Isadore M. Robinson. Journal Paper 5-13458 of the few procaryotes (43). Iowa Agriculture and Home Economics Experiment Station, Ames Because PP,-dependent enzymes are generally rare among (project 2678). procaryotes but occur more frequently among the molli- $ Present address: Department of Food Science and Nutrition, cutes, and because PP, may be an evolutionary precursor of University of Minnesota, Saint Paul, MN 55108. ATP (14,43), we examined the walled phylogenetic relatives 413 414 PETZEL ET AL. INT.J. SYST. BACTERIOL. of the mollicutes and some members of the family Anaero- tion of the method of Wood et al. (44). The mixture of plasmataceae for the following four PP,-dependent en- KHC03, MgCl,, and potassium phosphate buffer in a serum zymes: PP,-PFK, PPDK, PEP carboxytransphosphorylase, bottle was sparged with CO, for 30 min, the bottle was and PP,-dependent acetate kinase. We also assayed these sealed with a septum, and samples were withdrawn with a bacteria for some analogous ATP-dependent enzyme activ- syringe as needed. In addition, the concentration of CoCl, ities. was reduced to 10 FM, and dithiothreitol was substituted for p-mercaptoethanol. PEP carboxytransphosphorylase was MATERIALS AND METHODS also assayed by determining the appearance of PP,; the assay mixture used for the oxaloacetate assay was modified by Strains. Clostridium innocuum ATCC 14501T (T = type deleting reduced nicotinamide adenine dinucleotide and strain), Lactobacillus catenaformis ATCC 25536T (= DSM malate dehydrogenase and increasing the concentration of 20559T), Lactobacillus vitulinus ATCC 27783T, and Strepto- CoCl, to 100 pM. The reaction mixtures were incubated coccus pleomorphus ATCC 29734T (= DSM 20574T) were under CO, for 30 min at 37°C; mixtures without PEP served obtained from the American Type Culture Collection, Rock- as controls. PP, concentrations were determined with a ville, Md. Anaeroplasma intermedium 5LA and 7LAT (= commercial assay kit (Sigma) according to the directions of ATCC 43166T) (28, 30) and Asteroleplasma anaerobium the manufacturer. Control values were subtracted from the 161T (= ATCC 27880T) (30) were obtained from I. M. values obtained by using complete assay mixtures. ATP- Robinson, National Animal Disease Center, U.S. Depart- dependent acetate kinase (EC 2.7.2.1) reactions in the direc- ment of Agriculture, Ames, Iowa. Erysipelothrix rhusio- tion of acetyl phosphate formation were examined by using pathiae AVR-9 was obtained from R. L. Wood, National the method of Bowman et al. (3), with potassium acetate Animal Disease Center. Clostridium innocuum 7207 and added to a concentration of 50 mM and sodium succinate (a Clostridium rarnosum 8546 were received from the Anaer- positive effector in Escherichia coli [35]) added to a concen- obe Laboratory, Virginia Polytechnic Institute and State tration of 10 mM. For each strain, a second assay mixture University, Blacksburg. containing boiled cell-free extract was used as a blank for the Cultivation and extract preparation. The mollicutes were spectrophotometric determination of acetyl hydroxamate by harvested from 1.2 liters of S-2 broth (10.5% inocula; the method of Skarstedt and Silverstein (35). PP,-dependent incubation for 4 to 6 days at 37°C) as described elsewhere acetate kinase (EC 2.7.2.12) was determined similarly, ex- (Petzel et al., in press). Erysipelothrix rhusiopathiae was cept that the concentration of MgC1, was reduced to 0.75 grown (0.8% inoculum; incubation for 2 days at 37°C) in 1.2 mM to prevent precipitation with PP,. Alternatively, to liters of S-2 broth with the following modifications: the circumvent potential inhibition by PP, of color formation in medium was supplemented with 1.7% (voVvo1) heat-inacti- the hydroxylamine assay for acetyl phosphate in the PP,- vated horse serum and 10 mM MES [2-(N-morpholino)eth- dependent acetate kinase reaction, the method of Reeves anesulfonic acid]; the lipids and Na,CO, were deleted, and and Guthrie (25) was used. Sodium dithionite, which report- the culture was incubated without anaerobic precautions. All edly stimulates the reaction in DesuEfotomaculum ruminis