Ureaplasma urealyticum: an opportunity for combinatorial genomics.

Examination of genomic or enzymatic activity data alone neither provides a complete picture of metabolic function or potential nor confidently reveals sites amenable to inhibition. Furthermore, in some cases, gene annotation and in aqua assays disagree by describing gene annotation without enzyme activity and enzyme activity without homologous annotation. The newly sequenced genome of Ureaplasma urealyticum (parvum) is another prokaryote example of the class Mollicutes where such confounding differences are observed. The little-considered role of some proteins as multifunctional enzymes - substitutes for 'missing' genes - could both partially explain the apparent anomalies and relate to any inaccurate deductions of inhibitor function. A combinatorial analysis involving available evidence of genomic sequence, transcription, translational phenomena, structure and enzymatic activity gives the best picture of the organism's vital metabolic alternatives.

The proteome of Mycoplasma genitalium. Chaps-soluble component.

Mycoplasma genitalium is the smallest member of the class Mollicutes, with a genome size of 580 kb. It has the potential to express 480 gene products, and is therefore considered to be an excellent model to assess: (a) the minimum metabolism required by a free living cell; and (b) proteomic technologies and the information obtained by proteome analysis. Here, we report on the most complete proteome observed at 73% (expected proteome), and analysed at 33% (reported proteome). The use of four overlapping pH windows in conjunction with SDS/PAGE has allowed 427 distinct proteins to be resolved in association with the exponential growth of M. genitalium. Proof of expression for 201 proteins of sufficient abundance on silver stained two-dimensional gels was obtained using peptide mass fingerprinting (PMF) of which 158 were identified. The potential for gene product modification in even the simplest known self-replicating organism was quantified at a ratio of 1.22 : 1, more proteins than genes. A reduction in protein expression of 42% was observed for post-exponentially-grown cells. DnaK, GroEL, DNA gyrase, and a cytadherence accessory protein were significantly elevated, while some ribosomal proteins were reduced in relative abundance. The strengths and weaknesses of techniques employed were assessed with respect to the observed and predicted proteome derived from DNA sequence information. Proteomics was shown to provide a perspective into the biochemical and metabolic activities of this organism, beyond that obtainable by sequencing alone.

Mycoplasma genes: a case for reflective annotation.

Although function can be assigned to genome sequence by homology at a macroscopic level, this can be misleading in the absence of data on enzyme activities. Together, such data can reveal whether open reading frames are expressed, identify multienzyme function and point to 'orphan' function. Because of their small size and small genomes, the genome sequences of some Mycoplasma spp. are very amenable to detailed analyses.

Malate/lactate dehydrogenase in mollicutes: evidence for a multienzyme protein.

The malate (MDH) and lactate (LDH) dehydrogenases belong to the homologous class of 2-ketoacid dehydrogenases. The specificity for their respective substrates depends on residues differing at two or three regions within each molecule. Theoretical peptide-mass fingerprinting and PROSITE analysis of nine MDH and six LDH molecules were used to describe conserved sites related to function. A unique LDH is described which probably also confers MDH activity within the 580 kbp genome of Mycoplasma genitalium (class: Mollicutes). A single hydrophilic arginine residue was found in the active site of the M. genitalium LDH enzyme, differing from an hydrophobic residue normally present in these molecules. The effect of this residue may be to alter active site substrate specificity, allowing the enzyme to perform two closely related tasks. Evidence for a single gene affording dual enzymatic function is discussed in terms of genome size reduction in the simplest of free-living organisms. Since Mollicutes are thought to lack enzymes of the tricarboxylic acid cycle that would otherwise bind and interact with MDH in bacterial species possessing this pathway, active site modification of M. genitalium LDH is the sole requirement for MDH activity of this molecule. The closely related helical Mollicute, Spiroplasma melliferum, was shown to possess two distinct gene products for MDH/LDH activity.

The comparative metabolism of the mollicutes (Mycoplasmas): the utility for taxonomic classification and the relationship of putative gene annotation and phylogeny to enzymatic function in the smallest free-living cells.

Mollicutes or mycoplasmas are a class of wall-less bacteria descended from low G + C% Gram-positive bacteria. Some are exceedingly small, about 0.2 micron in diameter, and are examples of the smallest free-living cells known. Their genomes are equally small; the smallest in Mycoplasma genitalium is sequenced and is 0.58 mb with 475 ORFs, compared with 4.639 mb and 4288 ORFs for Escherichia coli. Because of their size and apparently limited metabolic potential, Mollicutes are models for describing the minimal metabolism necessary to sustain independent life. Mollicutes have no cytochromes or the TCA cycle except for malate dehydrogenase activity. Some uniquely require cholesterol for growth, some require urea and some are anaerobic. They fix CO2 in anaplerotic or replenishing reactions. Some require pyrophosphate not ATP as an energy source for reactions, including the rate-limiting step of glycolysis: 6-phosphofructokinase. They scavenge for nucleic acid precursors and apparently do not synthesize pyrimidines or purines de novo. Some genera uniquely lack dUTPase activity and some species also lack uracil-DNA glycosylase. The absence of the latter two reactions that limit the incorporation of uracil or remove it from DNA may be related to the marked mutability of the Mollicutes and their tachytelic or rapid evolution. Approximately 150 cytoplasmic activities have been identified in these organisms, 225 to 250 are presumed to be present. About 100 of the core reactions are graphically linked in a metabolic map, including glycolysis, pentose phosphate pathway, arginine dihydrolase pathway, transamination, and purine, pyrimidine, and lipid metabolism. Reaction sequences or loci of particular importance are also described: phosphofructokinases, NADH oxidase, thioredoxin complex, deoxyribose-5-phosphate aldolase, and lactate, malate, and glutamate dehydrogenases. Enzymatic activities of the Mollicutes are grouped according to metabolic similarities that are taxonomically discriminating. The arrangements attempt to follow phylogenetic relationships. The relationships of putative gene assignments and enzymatic function in My. genitalium, My. pneumoniae, and My. capricolum subsp. capricolum are specially analyzed. The data are arranged in four tables. One associates gene annotations with congruent reports of the enzymatic activity in these same Mollicutes, and hence confirms the annotations. Another associates putative annotations with reports of the enzyme activity but from different Mollicutes. A third identifies the discrepancies represented by those enzymatic activities found in Mollicutes with sequenced genomes but without any similarly annotated ORF. This suggests that the gene sequence is significantly different from those already deposited in the databanks and putatively annotated with the same function. Another comparison lists those enzymatic activities that are both undetected in Mollicutes and not associated with any ORF. Evidence is presented supporting the theory that there are relatively small gene sequences that code for functional centers of multiple enzymatic activity. This property is seemingly advantageous for an organism with a small genome and perhaps under some coding restraint. The data suggest that a concept of "remnant" or "useless genes" or "useless enzymes" should be considered when examining the relationship of gene annotation and enzymatic function. It also suggests that genes in addition to representing what cells are doing or what they may do, may also identify what they once might have done and may never do again.

Reduction of benzyl viologen distinguishes genera of the class Mollicutes.

We tested the ability of 62 growing strains belonging to the class Mollicutes to reduce the redox indicator and free-radical generator 1,1'-dibenzyl-4,4'-bipyridinium dichloride (benzyl viologen [BV]) to a blue-violet-purple color. BV was reduced by 12 Acholeplasma species but not by Acholeplasma multiforme PN525T (T = type strain). BV was also reduced by five of nine Mesoplasma species and by four of six Entomoplasma species. BV was not reduced by 19 Mycoplasma species, six Spiroplasma species, five unnamed Spiroplasma strains belonging to different serogroups, three Ureaplasma species, and one unnamed Ureaplasma strain. The BV-reducing ability was localized in the membrane of Acholeplasma laidlawii B-PG9 and was dependent on NADH. Reduction of BV could be expressed in mixed cultures, and this activity may be useful for recognizing the contaminating presence of an Acholeplasma species. The reductive BV response may have phylogenetic value. We believe that the test described in this paper readily distinguishes all Acholeplasma species and some Mesoplasma and Entomoplasma species from all Mycoplasma, Spiroplasma, and Ureaplasma species tested.

Comparative metabolism of Mesoplasma, Entomoplasma, Mycoplasma, and Acholeplasma.

Cytoplasmic fractions from species of the Mollicutes genera Entomoplasma, Mesoplasma, Mycoplasma, and Acholeplasma were assayed for NADH oxidase (NADH ox), ATP- and PPi-dependent phosphofructokinase (PFK), ATP- and PPi-dependent deoxyguanosine kinase (dGUOK), thymidine kinase (TK), TMP kinase (TMPK), glucose-6-phosphate dehydrogenase (G6Pde), lactate dehydrogenase (LDH), malate dehydrogenase (MDH), phosphoenolpyruvate carboxylase, hypoxanthine-guanine phosphoribosyl transferase, dUTPase, and uracil-DNA glycosylase (UNG) activities. Membrane fractions were also examined for NADH ox activity. These activities were used as indicators of the presence and relative activities of major Mollicutes metabolic and DNA repair pathways. This was the first study to determine the presence of these enzymes in members of the genera Entomoplasma and Mesoplasma. Using the data obtained, we constructed a preliminary scheme for distinguishing genera of the class Mollicutes on the basis of the results of signature functional enzyme assays. This scheme includes phylogenetic relationships deduced from rRNA analyses, but is more informative with respect to metabolic potential. The criteria used include the presence of PPi-dependent PFK, urease, dUTPase, and dGUOK activities. Entomoplasma ellychniae ELCN-1T (T = type strain), Entomoplasma melaleucae M-1T, Mesoplasma seiffertii F7T, Mesoplasma entomophilum TACT, Mesoplasma florum L1T, Mycoplasma fermentans PG18T, and Acholeplasma multilocale PN525T were similar in most respects. NADH ox activity was localized in the cytoplasm of these organisms. These strains had ATP-dependent PFK, MDH, LDH, ATP- and PPi-dependent dGUOK, and UNG activities, but not dUTPase or G6Pde activities. In contrast, Acholeplasma equifetale C112T, Acholeplasma oculi 19LT, Acholeplasma hippikon C1T, Acholeplasma modicum PG49T, and Acholeplasma morum 72-043T had membrane-localized NADH ox activity, PPi-dependent PFK, G6Pde, and dUTPase activities, and significantly lower MDH and LDH activities and exhibited a faster rate with PPi than with ATP in the dGUOK reaction. All of the members of the Mollicutes tested had hypoxanthine-guanine phosphoribosyl transferase, phosphoenolpyruvate carboxylase, and (except for Mesoplasma entomophilum TAC(T)) UNG activities. All of the Acholeplasma strains except Acholeplasma multilocale PN525T had TK, TMPK, and UNG activities. Mesoplasma entomophilum TAC(T) was distinguished by having no detectable dUTPase, UNG, TK, and TMPK activities, indicating that there is a severe restriction in or an absence of a synthetic route to dTTP. Our data also suggest that A. multilocale PN525T is a member of an unrecognized metabolic subgroup of the genus Acholeplasma or is not an Acholeplasma strain.

Improved cultivation systems for isolation of the colorado potato beetle spiroplasma.

In North America, the Colorado potato beetle, Leptinotarsa decemlineata, is often infected with the host-specific, gut-inhabiting Colorado potato beetle spiroplasma (CPBS). CPBS is apparently a commensal, but it may be useful in biocontrol if it can be transformed to express an insect-lethal gene. Difficulty in cultivating the organism, however, has hindered the development of a suitable transformation system. In this study, we eliminated the need for coculturing CPBS with insect cells. CPBS was reliably isolated with the BBL Anaerobic GasPak Jar system (low redox, enhanced CO(inf2)), which was easier to use and less expensive than insect cell coculture methods. A further advantage is a reduction in contaminating insect cell components. Use of anaerobiosis should facilitate early-passage screening of isolates for extrachromosomal elements, for use in gene vector constructs. The unique spiral (decreasing amplitude of coils) morphology of CPBS was preserved by anaerobiosis. The use of low-pH (6.0 to 6.5) media allowed aerobic adaptation of CPBS to M1D and SP-4 broth media. These formulations permitted the first cultivation of CPBS on solid media, an accomplishment that will simplify the selection of molecular transformants. Potato beetles collected at four sites in Poland yielded CPBS strains similar to those previously obtained from populations in North America.

The metabolism of AIDS-associated mycoplasmas.

Mycoplasma fermentans and Mycoplasma pirum have been recovered from human immunodeficiency virus (HIV)-positive persons. M. fermentans has been isolated with much higher frequency from HIV-positive than from HIV-negative persons. Mycoplasma genitalium has been detected by polymerase chain reaction in the blood of a patient with AIDS. Little is known about the biology of these mycoplasmas, especially their physiology, biochemistry, and growth response to inhibitors of essential metabolic loci or transport. Metabolically, they resemble other Mycoplasma species. Those studied lack cytochromes, the tricarboxylic acid cycle, and portions of the hexose monophosphate shunt. According to limited data, they fix CO2, use ATP to phosphorylate fructose-6-phosphate, have substrate phosphorylation and transaminase(s), and interconvert most purines and pyrimidines. The synthesis of thymidine may be limited. They may require a variety of essential small molecules for optimal growth (e.g., pyridoxal phosphate, ribose-1-phosphate). Their pathogenic potential and cultural lability may involve the production of the superoxide anion and the hydroxyl radical. We hypothesize that the mycoplasmas generate toxic oxygenated products that damage the host cell, probably membrane, permitting the mycoplasmas to gain easier access to the interior of the cell. The mycoplasma-damaged host cell membrane may also effect the maturation or release of HIV particles from the cell.

Early changes in the permeability of the blood-brain barrier produced by toxins associated with liver failure.

Our study was designed to determine whether substances that appear in the serum during the course of liver failure have a detrimental effect on the passive permeability of the blood-brain [blood-cerebrospinal fluid (CSF)] barrier. Lactic acid, octanoic acid, and ammonia were infused into rabbits for 4 h. The permeability changes of the blood-brain barrier were quantified by infusing polyethylene glycol 400 (PEG 400) and measuring the quantity and average mol wt of the PEG 400 that entered the CSF. The lipid solubility and effective diffusional radius of the PEG molecules were also quantified to provide greater precision for measurements using this probe. None of the animals receiving toxic infusions became seriously ill during the infusions. Low dose infusions of lactic acid, octanoic acid, and ammonia increased the effective pore diameter of the blood-brain barrier from 7.3 A to an average of 8.5 A. The amount of PEG entering the CSF increased from 1.7 to 4.0 (p less than 0.025), 4.7 (p less than 0.025), and 6.7 (p less than 0.001) mmol/L, respectively. Rabbits with galactosamine-induced liver failure had 10.1 mmol/L PEG 400 in the CSF (P less than 0.001) before any evidence of cerebral edema. These changes occur soon after these toxins accumulate in the plasma and may alone or together with other toxins account for the permeability changes that allow neurotoxic substances to enter the brain during hepatic disease and encephalopathies such as Reye's syndrome.

A mollicute (mycoplasma) DNA repair enzyme: purification and characterization of uracil-DNA glycosylase.

The DNA repair enzyme uracil-DNA glycosylase from Mycoplasma lactucae (831-C4) was purified 1,657-fold by using affinity chromatography and chromatofocusing techniques. The only substrate for the enzyme was DNA that contained uracil residues, and the Km of the enzyme was 1.05 +/- 0.12 microM for dUMP containing DNA. The product of the reaction was uracil, and it acted as a noncompetitive inhibitor of the uracil-DNA glycosylase with a Ki of 5.2 mM. The activity of the enzyme was insensitive to Mg2+, Mn2+, Zn2+, Ca2+, and Co2+ over the concentration range tested, and the activity was not inhibited by EDTA. The enzyme activity exhibited a biphasic response to monovalent cations and to polyamines. The enzyme had a pI of 6.4 and existed as a nonspherical monomeric protein with a molecular weight of 28,500 +/- 1,200. The uracil-DNA glycosylase from M. lactucae was inhibited by the uracil-DNA glycosylase inhibitor from bacteriophage PBS-2, but the amount of inhibitor required for 50% inhibition of the mycoplasmal enzyme was 2.2 and 8 times greater than that required to cause 50% inhibition of the uracil-DNA glycosylases from Escherichia coli and Bacillus subtilis, respectively. Previous studies have reported that some mollicutes lack uracil-DNA glycosylase activity, and the results of this study demonstrate that the uracil-DNA glycosylase from M. lactucae has a higher Km for uracil-containing DNA than those of the glycosylases of other procaryotic organisms. Thus, the low G + C content of the DNA from some mollicutes and the A.T-biased mutation pressure observed in these organisms may be related to their decreased capacity to remove uracil residues from DNA.

The anaplerotic phosphoenolpyruvate carboxylase of the tricarboxylic acid cycle deficient Acholeplasma laidlawii B-PG9.

Phosphoenolpyruvate carboxylase (EC 4.1.1.31) (PEP-C) was purified approximately 770-fold from the mollicute Acholeplasma laidlawii B-PG9. The partially purified PEP-C required phosphoenolpyruvate (PEP) and MnCl2 at pH 7.4 or MgCl2 at pH 8.6 for optimal activity. The product is oxaloacetate as detected by a malate dehydrogenase indicator system. The KmA (PEP variable) was 0.66 mM and the KmB (bicarbonate variable) was 1.02 mM. At low bicarbonate concentrations (0.5 mM), PEP-C activity was stimulated approximately 240% by fructose 1,6-bisphosphate. Aspartate was a non-competitive inhibitor of PEP-C activity. The KiA (PEP variable) for aspartate was 0.69 mM and the KiB (bicarbonate variable) was 0.99 mM. Malate, citrate, isocitrate, 2-oxoglutarate, acetyl-CoA, CMP, CDP, GDP, GTP, ADP and ATP had no effect on the PEP-C reaction. The Hill interaction coefficient was 0.98-1.11. The molecular mass by sucrose density gradient analysis was 353 kDa; by gel filtration chromatography it was 384 kDa. The Stokes radius was about 7.4 nm. PEP-C activity and its inhibition by aspartate in Acholeplasma laidlawii B-PG-9 extracts may reflect an involvement of this enzyme in the interdependent regulation of protein, lipid and nucleic acid precursor metabolism of this TCA-cycle-deficient and cytochrome-less mollicute.

Enzymic activities of carbohydrate, purine, and pyrimidine metabolism in the Anaeroplasmataceae (class Mollicutes).

Cell-free extracts of two strictly anaerobic mollicutes, Anaeroplasma intermedium 5LA and Asteroleplasma anaerobium 161T, were tested for enzymic activities of intracellular carbohydrate metabolism. Asteroleplasma anaerobium was also tested for enzymes of purine and pyrimidine metabolism. Both organisms had enzymic activities associated with the nonoxidative portion of the pentose phosphate pathway, and with the Embden-Meyerhoff-Parnas pathway. The 6-phosphofructokinase (PFK) of Asteroleplasma anaerobium was ATP-dependent, whereas the PFK of Anaeroplasma intermedium was PPi-dependent. The two anaerobic mollicutes also differed with respect to the enzymes that converted phosphoenolpyruvate (PEP) to pyruvate; Anaeroplasma intermedium had pyruvate kinase activity, but Asteroleplasma anaerobium had pyruvate, orthophosphate dikinase activity (PPi-dependent). Both organisms had lactate dehydrogenase activity which was activated by fructose 1,6-bisphosphate (Fru-1,6-P2). Anaeroplasma intermedium had activity for PEP carboxykinase (activated by Fru-1,6-P2), but Asteroleplasma anaerobium did not. PEP carboxytransphosphorylase activity was not detected in either organism. Anaeroplasma intermedium had malate dehydrogenase and isocitrate dehydrogenase activities, but it had no activities for the three other tricarboxylic acid cycle enzymes examined; Asteroleplasma anaerobium had malate dehydrogenase activity only. Asteroleplasma anaerobium had enzymic activities for the interconversion of purine nucleobases, (deoxy)ribonucleosides, and (deoxy)ribomononucleotides, including PPi-dependent nucleoside kinase, reported heretofore only in some other mollicutes. Asteroleplasma anaerobium could synthesize dTDP by the thymine salvage pathway if deoxyribose 1-phosphate was provided, and it had dUTPase, ATPase, and dCMP kinase activities. It lacked (deoxy)cytidine deaminase, dCMP deaminase, and deoxycytidine kinase activities.

Presence of anaplerotic reactions and transamination, and the absence of the tricarboxylic acid cycle in mollicutes.

Cell extracts of the fermentative Mollicutes Acholeplasma laidlawii B-PG9, Acholeplasma morum S2, Mycoplasma capricolum 14, Mycoplasma gallisepticum S6, Mycoplasma pneumoniae FH, Mycoplasma hyopneumoniae J and M. genitalium G-37, and the non-fermentative Mycoplasma hominis PG-21, Mycoplasma hominis 1620 and Mycoplasma bovigenitalium PG-11 were examined for 39 cytoplasmic enzyme activities associated with the tricarboxylic acid (TCA) cycle, transamination, anaplerotic reactions and other enzyme activities at the pyruvate locus. Malate dehydrogenase (EC 4.2.1.2) was the only TCA-cycle-associated enzyme activity detected and it was found only in the eight Mycoplasma species. Aspartate aminotransferase (EC 2.6.1.1) activity was detected in all Mollicutes tested except M. gallisepticum S6. Malate synthetase (EC 4.1.3.2) activity, in the direction of malate formation, was found in the eight Mycoplasma species, but not in any of the Acholeplasma species. Phosphoenolpyruvate (PEP) carboxylase (EC 4.1.1.31) was detected in the direction of oxaloacetate (OAA) formation in both Acholeplasma species, but not in any of the Mycoplasma species. Pyruvate carboxylase (EC 6.4.1.1), pyruvate kinase (EC 2.7.1.40), pyruvate dehydrogenase (EC 1.2.4.1) and lactate dehydrogenase (EC 1.1.1.27) activities were found in all ten Mollicutes tested. No activities were detected in any of the ten Mollicutes for aspartase (EC 4.3.1.1), malic enzyme (EC 1.1.1.40), PEP carboxytransphosphorylase (EC 4.1.1.38), PEP carboxykinase (EC 4.1.1.32) or pyruvate orthophosphate dikinase (EC 2.7.9.1). In these TCA-cycle-deficient Mollicutes the pyruvate-OAA locus may be a point of linkage for the carbons of glycolysis, lipid synthesis, nucleic acid synthesis and certain amino acids. CO2 fixation appears obligatory in the Acholeplasma species and either CO2 fixation or malate synthesis appears obligatory in the Mycoplasma species.

Acholeplasma laidlawii B-PG9 adenine-specific purine nucleoside phosphorylase that accepts ribose-1-phosphate, deoxyribose-1-phosphate, and xylose-1-phosphate.

An adenylate-specific purine nucleoside phosphorylase (purine nucleoside:orthophosphate ribosyltransferase, EC12.4.2.1) (PNP) was isolated from a cytoplasmic fraction of Acholeplasma laidlawii B-PG9 and partially purified (820-fold). This partially purified PNP could only ribosylate adenine and deribosylate adenosine and deoxyadenosine. The A. laidlawii partially purified PNP could not use hypoxanthine, guanine, uracil, guanosine, deoxyguanosine, or inosine as substrates, but could use ribose-1-phosphate, deoxyribose-1-phosphate, or xylose-1-phosphate as the pentose donor. Mg2+ and a pH of 7.6 were required for maximum activity for each of the pentoses. The partially purified enzyme in sucrose density gradient experiments had an approximate molecular weight of 108,000 and a sedimentation coefficient of 6.9, and in gel filtration experiments it had an approximate molecular weight of 102,000 and a Stoke's radius of 4.1 nm. Nondenaturing polyacrylamide tube gels of the enzyme preparation produced one major and one minor band. The major band (Rf, 0.57) corresponded to all enzyme activity. The Kms for the partially purified PNP with ribose-1-phosphate, deoxyribose-1-phosphate, and xylose-1-phosphate were 0.80, 0.82, and 0.81 mM, respectively. The corresponding Vmaxs were 12.5, 14.3, and 12.0 microM min-1, respectively. The Hill or interaction coefficients (n) for all three pentose phosphates were close to unity. The characterization data suggest the possibility of one active site on the enzyme which is equally reactive toward each of the three pentoses. This is the first report of an apparently adenine-specific PNP activity.

Synthesis of deoxyribomononucleotides in Mollicutes: dependence on deoxyribose-1-phosphate and PPi.

Cell extracts of Acholeplasma laidlawii B-PG9, Acholeplasma morum S2, Mycoplasma capricolum 14, and Mycoplasma gallisepticum S6 were examined for 37 cytoplasmic enzyme activities involved in the salvage and biosynthesis of purines. All of these organisms had adenine phosphoribosyltransferase activity (EC 2.4.2.7) and hypoxanthine phosphoribosyltransferase activity (EC 2.4.2.8). All of these organisms had purine-nucleoside phosphorylase activity (EC 2.4.2.1) in the synthetic direction using ribose-1-phosphate (R-1-P) or deoxyribose-1-phosphate (dR-1-P); this activity generated ribonucleosides or deoxyribonucleosides, respectively. The pyrimidine nucleobase uracil could also be ribosylated by using either R-1-P or dR-1-P as a donor. The synthesis of deoxyribonucleosides from nucleobases and dR-1-P has been reported from only one other procaryote, Escherichia coli (L. A. Mason and J. O. Lampen, J. Biol. Chem. 193:539-547, 1951). The reverse of this phosphorylase reaction is more widely known, and we found such activity in all mollicutes studied. Some Acholeplasma species but not the Mycoplasma species can phosphorylate deoxyribonucleosides to deoxyribomononucleotides by a PPi-dependent deoxyribonucleoside kinase activity, which was first reported in this group for the ribose analogs (V. V. Tryon and J. D. Pollack, Int. J. Syst. Bacteriol. 35:497-501, 1985). This is the first report of PPi-dependent purine deoxyribonucleoside kinase activity. An ATP-dependent purine deoxyribonucleoside kinase activity is known only in salmon milt extracts (H. L. A. Tarr, Can. J. Biochem. 42:1535-1545, 1964). Deoxyribomononucleotidase activity was also found in cytoplasmic extracts of these mollicutes. This is the first report of deoxyribomononucleotidase activity.

Metabolic distinctiveness of ureaplasmas.

Enzymes of the Embden-Meyerhof-Parnas pathway and hexose monophosphate shunt were examined in cytoplasmic extracts of three serovars of Ureaplasma urealyticum. We found no glucose-6-phosphate or 6-phosphogluconate dehydrogenase, hexokinase, phosphoglucose isomerase, aldolase, or lactic dehydrogenase activities. We failed to find cytochrome pigments in extracts and found no significant production of 14CO2 from [U-14C]glucose, nor did we find oxygen-dependent reduced nicotinamide adenine dinucleotide oxidase activity. Lactic acid was found only at trace levels in spent culture fluids. Ureaplasmas are apparently nonfermentative and are unlike all other mollicutes in that they have no detectable oxygen-dependent reduced nicotinamide adenine dinucleotide oxidase activity.