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We studied multiple sequence alignment (MSA) consensus amino acid distributional patterns in 2844 amino acid sequences of the eight enzymes of the Kreb's oxidative tricarboxylic acid pathway (oTCA) in Archaea, Bacteria and Eukarya and 5545 sequences of 33 bacteria as geochronologically separated enzymes with MSA consensus site modal identities. The 33 bacteria were 20 presumptive examples of early-oldest (Hadean-Archaean) ('Epoch I') or 13 late-newest (contemporary) ('Epoch III') appearing enzymes on Earth. The enzyme's MSA consensus sites were identified by their modal identity, % Occupancy in one of nine-graded evolutionary-conservation zones (CZs) and the Euclidean distance (Å) from each of their consensus MSA Cɑs to the same atom (Anchor-atom) in their reported functional center. These MSA consensus sites are tetrad-data points called recovered-amino acids (RAA). Across Domains, the % Occupancies of the eight-dominant RAAs of the Kreb's cycle and the 33 bacteria were found to be similarly ranked. Compared to Trifonov's 'putative ranked temporal order of the appearance of amino acids on Earth' (TOAE), the greatest statistical concordance with tetrad-RAAs across Domains were those characterized as within the most-evolutionary conserved conservation zone (CZ9), typically nearest (Å) their enzyme's catalytic/active center. The geochronologically characterized early-oldest Hadean-Archaean Bacteria 'Epoch I' enzymes, compared to late-newest Bacteria enzymes, had greater average numbers of amino acid residues/sequence and a statistically significant larger variability in their RAA compositional-Å 3 -volumes. The late-newest 'Epoch III' enzymes had statistically significant lower volumetric values, specifically, their native Å 3 -volume, void-volume and volume change on unfolding. Our enzyme data suggest a geochronological trace of ' metabolism's progressive emergence '.Communicated by Ramaswamy H. Sarma.

The consensus of the members of the International Committee on Systematics of Prokaryotes' Subcommittee on the taxonomy of Mollicutes is that recently proposed sweeping changes to nomenclature of members of the Mycoplasmatales , specifically involving introduction of the names Malacoplasma gen. nov., Mesomycoplasma gen. nov., Metamycoplasma gen. nov., Metamycoplasmataceae fam. nov., Mycoplasmoidaceae fam. nov., Mycoplasmoidales ord. nov., Mycoplasmoides gen. nov., Mycoplasmopsis gen. nov., and all proposed species or subspecies comb. nov. placed therein, should be rejected because they violate one or more essential points of the International Code of Nomenclature of Prokaryotes.

The distributions of amino acids at most-conserved sites nearest catalytic/active centers (C/AC) in 4,645 sequences of ten enzymes of the glycolytic Embden-Meyerhof-Parnas pathway in Archaea, Bacteria and Eukaryota are similar to the proposed temporal order of their appearance on Earth. Glycine, isoleucine, leucine, valine, glutamic acid and possibly lysine often described as prebiotic, i.e., existing or occurring before the emergence of life, were localized in positional and conservational defined aggregations in all enzymes of all Domains. The distributions of all 20 biologic amino acids in most-conserved sites nearest their C/ACs were quite different either from distributions in sites less-conserved and further from their C/ACs or from all amino acids regardless of their position or conservation. The major concentrations of glycine, e.g., perhaps the earliest prebiotic amino acid, occupies ≈ 16 % of all the most-conserved sites within a volume of ≈ 7-8 Å radius from their C/ACs and decreases linearly towards the molecule's peripheries. Spatially localized major concentrations of isoleucine, leucine and valine are in the mid-conserved and mid-distant sites from their C/ACs in protein interiors. Lysine and glutamic acid comprise ≈ 25-30 % of all amino acids within an irregular volume bounded by ≈ 24-28 Å radii from their C/ACs at the most-distant least-conserved sites. The unreported characteristics of these amino acids: their spatially and conservationally identified concentrations in Archaea, Bacteria and Eukaryota, suggest some common structural organization of glycolytic enzymes that may be relevant to their evolution and that of other proteins. We discuss our data in relation to enzyme evolution, their reported prebiotic putative temporal appearances on Earth, abundances, biological "cost", neighbor-sequence preferences or "ordering" and some thermodynamic parameters.

In alignments of 1969 protein sequences the amino acid glycine and others were found concentrated at most-conserved sites within approximately 15 A of catalytic/active centers (C/AC) of highly conserved kinases, dehydrogenases or lyases of Archaea, Bacteria and Eukaryota. Lysine and glutamic acid were concentrated at least-conserved sites furthest from their C/ACs. Logistic-regression analyses corroborated the "movement" of glycine towards and lysine away from their C/ACs: the odds of a glycine occupying a site were decreased by 19%, while the odds for a lysine were increased by 53%, for every 10 A moving away from the C/AC. Average conservation of MSA consensus sites was highest surrounding the C/AC and directly decreased in transition toward model's peripheries. Findings held with statistical confidence using sequences restricted to individual Domains or enzyme classes or to both. Our data describe variability in the rate of mutation and likelihoods for phylogenetic trees based on protein sequence data and endorse the extension of substitution models by incorporating data on conservation and distance to C/ACs rather than only using cumulative levels. The data support the view that in the most-conserved environment immediately surrounding the C/AC of taxonomically distant and highly conserved essential enzymes of central metabolism there are amino acids whose identity and degree of occupancy is similar to a proposed amino acid set and frequency associated with prebiotic evolution.

We studied 131 protein sequences of the essentially ubiquitous glycolytic enzyme 3-phosphoglycerate kinase (3-PGK) by Bayesian analyses in three Domains: 15 Archaea, 83 Bacteria, and 33 Eukaryota. The posterior distribution of phylogenetic trees developed were based on a uniform prior, the WAG model of protein evolution, Metropolis-Hastings sampling in a Markov chain Monte Carlo analysis, and a package of diagnostics to critically evaluate the validity of the analyses. The 15 Archaea separated with high posterior probability. The archaean Phyla Euryarchaeota and the apparently Euryarchaeota derived Crenarchaeota were monophyletic. The 33 Eukaryota separated into two main groups: the non-chlorophyllous forms with coherent sub-groupings of Euglenozoa, Alveolata, Fungi, and Metazoa and all the chlorophyllous species studied: the Plantae (Viridaeplantae), chlorophyllous Stramenopiles, and the chlorophyllous Bacteria. This association supports other opinions concerning the related lineage of cyanobacteria and the Plantae. The 3-PGK sequences from 83 Bacteria in almost every instance associated by their recognized taxal group: alpha-, beta-, gamma-, epsilon-proteobacteria, Chlamydia, Actinobacteridae, and Firmicutes. Firmicutes sequences were subdivided into three apparently monophyletic groups: the anaerobic Clostridia, the spore-forming Bacillales and a group containing the Mollicutes, Lactobacillales and non-spore-forming Bacillales. The 3-PGK-gene tree assemblage was notable both for its pervasive clustering in three Domains according to recognized taxonomic groupings of Class, Order, Family, and Genus. The 3-PGK enzyme or 3-PGK-like activity may have played a central role in the metabolism of the Universal Ancestor.

The phylogenetic position of the Mollicutes has been re-examined by using phosphoglycerate kinase (Pgk) amino acid sequences. Hitherto unpublished sequences from Mycoplasma mycoides subsp. mycoides, Mycoplasma hyopneumoniae and Spiroplasma citri were included in the analysis. Phylogenetic trees based on Pgk data indicated a monophyletic origin for the Mollicutes within the Firmicutes, whereas Bacilli (Firmicutes) and Clostridia (Firmicutes) appeared to be paraphyletic. With two exceptions, i.e. Thermotoga (Thermotogae) and Fusobacterium (Fusobacteria), which clustered within the Firmicutes, comparative analyses show that at a low taxonomic level, the resolved phylogenetic relationships that were inferred from both the Pgk protein and 16S rRNA gene sequence data are congruent.

Bacteria of the class Mollicutes have no cell wall. One species, Mycoplasma genitalium is the personification of the simplest form of independent cell-free life. Its small genome (580 kbp) is the smallest of any cell. Mollicutes have unique metabolic properties, perhaps because of their limited coding space and high mutability. Based on 16S rRNA analyses the Mollicutes Mycoplasma gallisepticum is thought to be the most mutable bacteria. Enzyme activities found in most Bacteria are absent from Mollicutes. The functions of apparently absent genes and enzymes can apparently be fulfilled by other genes and their expression products that have multiple capabilities. Because of these and other properties predictions of their metabolism based only on, e.g., either annotation, enzymatic assay, proteomic studies or structural analyses is problematic. To obtain a more confident appraisal of the functional capabilities of these simplest cells genomic and enzymatic data were combined to obtain a "metabolic consensus". The consensus is represented by a biochemical circuit for central metabolism involving purine and pyrimidine interconversions and their linkages to amino acid metabolism, glycolysis and the pentose phosphate pathway in three human Mollicutes pathogens: Mycoplasma pneumoniae, Mycoplasma genitalium and Ureaplasma urealyticum.

The small genome Mollicutes whose DNAs are completely sequenced (Mycoplasma genitalium, Mycoplasma pneumoniae, Mycoplasma pulmonis, and Ureaplasma urealyticum [parvum]) lack a gene (ndk) for the presumably essential nucleoside diphosphate kinase (NDPK). We hypothesized that other activities might replace NDPK activity. We found in M. genitalium G37(T), Mycoplasma pneumoniae FH(T), Mycoplasma fermentans PG18(T), and Mycoplasma capricolum subsp. capricolum Kid(T) that their 6-phosphofructokinases (6-PFKs), phosphoglycerate kinases (PGKs), pyruvate kinases (PKs), and acetate kinases (AKs), besides reactant ADP/ATP, could use other ribo- and deoxyribo-purine and pyrimidine NDPs and NTPs. These activities could compensate for the absence of an orthologous ndk gene in the Mycoplasmataceae. They suggest a metabolically varied and consequential role for unrelated and perhaps unsuspected "replacement" or compensatory enzymes that may confound metabolic prediction. We partially purified and biochemically characterized the PKs, 6-PFKs, PGKs, and AKs from M. capricolum subsp. capricolum Kid(T) and M. fermentans PG18(T).

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

Extracts of Acholeplasma laidlawii B-PG9 were examined for the enzymes associated with the interconversion of the pyrimidine deoxyribonucleotides and the biosynthesis of thymidine nucleotides. A. laidlawii B-PG9 possessed deaminases for deoxycytidine and dCMP, pyrophosphatases for dUTP, phosphorylases for thymidine and uridine, and a membrane-associated pyrimidine deoxyribonucleoside monophosphate phosphatase activity. The role these enzyme activities have in the generation of deoxyribose-1-phosphate during growth may explain the ability of A. laidlawii B-PG9 to utilize either thymine or thymidine for biosynthesis.

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.

A PPi-dependent phosphofructotransferase (PPi-fructose 6-phosphate 1-phosphotransferase, EC 2.7.1.90) which catalyzes the conversion of fructose 6 phosphate (F-6-P) to fructose 1,6-bisphosphate (F-1, 6-P2) was isolated from a cytoplasmic fraction of Acholeplasma laidlawii B-PG9 and partially purified (430-fold). PPi was required as the phosphate donor. ATP, dATP, CTP, dCTP, GTP, dGTP, UTP, dUTP, ITP, TTP, ADP, or Pi could not substitute for PPi. The PPi-dependent reaction (2.0 mM PPi) was not altered in the presence of any of these nucleotides (2.0 mM) or in the presence of smaller (less than or equal to 300 microM) amounts of fructose 2,6-bisphosphate, (NH4)2SO4, AMP, citrate, GDP, or phosphoenolpyruvate. Mg2+ and a pH of 7.4 were required for maximum activity. The partially purified enzyme in sucrose density gradient experiments had an approximate molecular weight of 74,000 and a sedimentation coefficient of 6.7. A second form of the enzyme (molecular weight, 37,000) was detected, although in relatively smaller amounts, by using Blue Sepharose matrix when performing electrophoresis experiments. The back reaction, F-1, 6-P2 to F-6-P, required Pi; arsenate could substitute for Pi, but not PPi or any other nucleotide tested. The computer-derived kinetic constants (+/- standard deviation) for the reaction in the PPi-driven direction of F-1, 6-P2 were as follows: v, 38.9 +/- 0.48 mM min-1; Ka(PPi), 0.11 +/- 0.04 mM; Kb(F-6-P), 0.65 +/- 0.15 mM; and Kia(PPi), 0.39 +/- 0.11 mM. A. laidlawii B-PG9 required PPi not only for the PPi-phosphofructotransferase reaction which we describe but also for purine nucleoside kinase activity. a dependency unknown in any other organism. In A. laidlawii B-PG9, the PPi requirement may be met by reactions in this organism already known to synthesize PPi (e.g., dUTPase and purine nucleobase phosphoribosyltransferases). In almost all other cells, the conversion of F-6-P to F-1,6-P2 is ATP dependent, and the reaction is generally considered to be the rate-limiting step of glycolysis. The ability of A. laidlawii B-PG9 and one other acholeplasma to use PPi instead of ATP as an energy source may offer these cytochrome-deficient organisms some metabolic advantage and may represent a conserved metabolic remnant of an earlier evolutionary process.

Extracts of the Mollicutes Acholeplasma equifetale, Acholeplasma laidlawii B, Mycoplasma arthritidis. Mycoplasma pulmonis, and Mycoplasma pneumoniae had DNase and endonuclease activity. A. laidlawii B had at least two peaks of DNase activity in sucrose gradients with sedimentation coefficients of 3.1S and 4.3S. These fractions also had endonuclease activity with different substrate specificities. A. laidlawii B may have more than two peaks of endonuclease activity in sucrose gradients.

The metabolism of the Mollicutes Acholeplasma and Mycoplasma may be characterized as restricted, for example, by virtue of the apparent absence of cytochrome pigments. Some Mollicutes have lowered ECA values during their logarithmic growth phase, which we speculate may be related to insufficient substrate phosphorylation or insufficient ATP synthesis linked to glycolysis. We found that PEP is carboxylated by preparations of A. laidlawii, but not by other Mollicutes; thus in this organism oxaloacetate from PEP may be a link to other pathways. We found phosphoribosylpyrophosphate in A. laidlawii, which suggests that ribosylation of purines and pyrimidines occurs in Mollicutes other than M. mycoides.

Adenosine 5'-triphosphate, adenosine 5'-diphosphate, and adenosine 5'-monophosphate were produced by Acholeplasma laidlawii B-PG9 growing in modified Edward medium. The adenylate energy charge was calculated to be 0.84 +/- 0.07 and ranged from 0.91 to 0.78 during exponential growth (12 to 24 h). During exponential growth, A. laidlawii contained, at 17.5 h, 2.3 X 10(-17) mol of adenosine 5'-triphosphate per colony-forming unit and, at 16 h, 27.3 nmol of adenosine 5'-triphosphate per mg (dry weight). The medium supported a doubling time of 0.95 h. The molar growth yields (Yglucose = grams [dry weight] per mole of glucose used) were 40.2 +/- 3.4 (16 h) and 57.1 +/- 9.7 (20 h) during midexponential growth. A maximum yield of 8.3 X 10(9) colony-forming units was reached at 24 h, when 56% of the initial concentration of glucose had been used. At 40 h, during the stationary phase, 14.95 +/- 3.75 mumol of glucose per ml of medium had been used. At this time, the culture fluids contained 21.86 +/0 mumol of lactate per ml and 3.14 +/- 0.13 mumol of pyruvate per ml.

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.

An apparatus to trap and quantitate 14CO2 is described. When used to measure antibiotic effects on Histoplasma capsulatum and Mycobacterium bovis (BCG), there was an inverse quantitative relationship between 14CO2 evolved and antibiotic concentration. The technique should prove useful for analyses that require trapping and quantitation of 14CO2, including antimicrobial sensitivities of slow-growing organisms.

No cytochrome pigments were detected by difference (reduced minus oxidized) spectroscopy at liquid nitrogen temperature in whole-cell preparations or membrane fractions of Acholeplasma axanthum S273, Acholeplasma equifetale N93, Acholeplasma granularum BTS39, Acholeplasma laidlawii B-PG9, Acholeplasma modicum PG-49, Acholeplasma oculi 19L, Mycoplasma arginini G230, Mycoplasma arthritidis 07, Mycoplasma pneumoniae FH, and Mycoplasma pulmonis JB. All ten Mollicutes species examined contained iron of unknown function (3.0 to 15.3 nmol of iron per mg of protein). Relatively small amounts of acid-labile sulfide were found in all fractions (0.10 to 1.07 nmol of acid-labile sulfide per mg of protein). The data suggest that, as Mollicutes lack cytochrome pigments, they would synthesize most if not all adenosine triphosphate at the substrate level.

By using ethylene glycol extraction of whole submerged cultures followed by Sephadex G-200 and diethylaminoethyl-Sephadex chromatography, we isolated four distinct glycopeptides from Trichophyton mentagrophytes, T. rubrum, and Microsporum canis. Chemical analyses revealed that these glycopeptides contained mostly carbohydrate (42.5 to 81.6%) and protein (4.3 to 11.3%), with lesser amounts of phosphorus (0.4 to 6.0%) and hexosamines (0.3 to 0.6%). Based upon total carbohydrate and monosaccharide content, these dermatophyte glycopeptides could be divided into two chemical groups: glucopeptides (DSI1) and mannopeptides (DSI2, DSII1, and DSII2). The mannopeptides and glucopeptides of each species of dermatophyte were not significantly different chemically from those derived from the other two dermatophyte species studied. Skin testing of DSI1-glycopeptides or DSI2-mannopeptides in immunized guinea pigs indicated that only the DSI2-mannopeptides elicited a delayed hypersensitivity reaction. Skin testing T. mentagrophytes 62-infected guinea pigs with the four purified DS-glycopeptides, as well as earlier fractions from the purification scheme, derived from T. mentagrophytes, T. rubrum, and M. canis, again indicated that only the DSI2-mannopeptides of the two Trichophyton species elicited a delayed hypersensitivity reaction. The number of infections or duration of infection had no effect on the size of the skin test response. DSI2-mannopeptides were non-cross-reactive between genera when tested in Trichophyton-immunized or -infected guinea pigs and Microsporum-immunized guinea pigs.

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.

dUTP was purified 120-fold from extracts of Acholeplasma laidlawii B-PG9 by Blue-Sepharose, Phenyl-Sepharose, hydroxyapatite, and DEAE-Sephacel chromatography techniques. The only substrate for the enzyme was dUTP with an apparent Km of 4.5 microM. The only reaction products were dUMP and PPi. The dUTPase did not exhibit any specific divalent cation requirement, but it was inhibited by EDTA. The enzyme was not inhibited by Pi or p-hydroxymercuribenzoate. The molecular weight of the enzyme was estimated by gel filtration chromatography to be 48,000, and its isoelectric point was 5.3. The enzyme was thermostable at 55 degrees C for 1 h. A. laidlawii dUTPase was distinguishable from KB (human epidermoid carcinoma) dUTPase by differences in electrophoretic migration, isoelectric point, and thermostability. The enzyme is important in preventing dUTP from being incorporated into DNA and may have a significant role in both the synthesis of thymidine- and PPi-dependent phosphorylations.

We examined the lipid content of bronchoalveolar (BA) washes from both mice and rats infected with Mycoplasma pulmonis, an etiological agent of murine pneumonia. During a 30-day period after intranasal inoculation, the total lipid content from infected and control rats (in milligrams per animal) remained relatively equal and unchanged. The saturated, unsaturated, and total lecithin contents in infected rats (in milligrams per animal) all increased. The maximum lecithin values were detected at 7 to 10 days after infection; later, the levels fell to control values. There was essentially no change in any lecithin value from uninfected animals. Although in BA washes from infected animals the mass of disaturated lecithins increased, the percentage of this fraction in the total lecithin pool decreased. The fatty acids of the lecithins from BA washes of infected mice had significantly less palmitic and significantly more oleic and linoleic acids than the lecithins isolated from the BA washes of control animals. Both the relative decrease in the mass of disaturated lecithins in the BA washes and the increase in the percentage of esterified unsaturated fatty acids in the lecithins may be directly related to the reduced lung function reported to occur during the course of murine M. pulmonis pneumonia.

Sera from 103 fasting individuals 3 to 76 years of age and free of clinical infectious disease and sera from 183 patients with infectious disease were assayed for serum total non-esterfied fatty acids (tNEFA) and compared. Data were also separated into five groups according to age of donor: 3--7, 8--19, 20--35, 36--60, and 61--76 years. The mean group serum levels of tNEFA increased with age. Among patients with infectious diseases sixty-five were diagnosed as having hepatitis, 41 with infectious mononucleosis, 18 with cellulitis, 12 with pulmonary tuberculosis, 11 with non-pneumococcal pneumonia, 9 with pneumococcal pneumonia, 8 with pharyngitis, 6 with pyelonephritis, 6 with aseptic meningitis, 4 with Gram-negative sepsis, and 3 with encephalitis. The sera from 23 non-fasting patients with gonorrhea were also tested. The serum tNEFA levels were found to be altered, in fact depressed from normal group values, only in patients with pneumonia or tuberculosis. This depression may be related to aberrant pulmonary metabolism during pneumonia.

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.

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.

Liver mitochondria from octanoate-treated rabbits showed an impaired ability to synthesize citrulline. Two methods were used to evaluate citrulline synthesis in rat liver mitochondria. Under these conditions octanoate inhibited citrulline synthesis by over 50%. When ATP was included in the assay medium the inhibitory effect of octanoate was prevented. In the absence of ATP in the suspending medium, octanoate did not significantly lower total adenine nucleotides in rat liver mitochondria. However, under these conditions octanoate caused a change in the adenine nucleotide profile such that ATP content was decreased and AMP content was increased. When ATP was present in the assay medium, octanoate caused a similar increase in AMP content. However, ATP decreased only slightly. The alterations in mitochondrial adenine nucleotide profile by octanoate and the reversal of the effect by exogenous ATP suggests that octanoate inhibits citrulline synthesis via reduced intramitochondrial ATP levels. The ability of octanoate to lower mitochondrial ATP and elevate mitochondrial AMP may be related to its intramitochondrial activation by the medium chain fatty acid activating enzyme.

Pollack, J. D. (University of Connecticut, Storrs), Shmuel Razin, and Robert C. Cleverdon. Localization of enzymes in Mycoplasma. J. Bacteriol. 90:617-622. 1965.-Cells of eight parasitic and two saprophytic Mycoplasma strains were lysed by use of osmotic shock, and the membranes were separated from the soluble fraction by use of differential centrifugation. Cell fractions were tested for reduced nicotinamide adenine dinucleotide (NADH(2)) oxidase, reduced nicotinamide adenine dinucleotide phosphate (NADPH(2)) oxidase, glucose-6-phosphate dehydrogenase, adenosine triphosphatase, ribonuclease, and deoxyribonuclease activities. Adenosine triphosphatase was confined to the membrane fraction of all Mycoplasma strains. The NADH(2) oxidase activity was associated with the membranes of the saprophytic M. laidlawii and with the soluble fraction of the parasitic Mycoplasma strains. NADPH(2) oxidase activity was detected only in the soluble fraction of the parasitic strains. Glusose-6-phosphate dehydrogenase was demonstrated only in the soluble fraction of M. laidlawii. Ribonuclease activity was found usually in both membrane and soluble fractions, but was generally higher in the membrane fraction. In the human and bovine Mycoplasma strains, deoxyribonuclease activity could not be demonstrated in the soluble fraction; in the remaining strains, activity was highest in the soluble fraction. Dissolution of M. laidlawii strain B membranes by sodium deoxycholate significantly increased membrane-NADH(2) oxidase and adenosine triphosphatase activities.