Enzymes of Purine Metabolism in Mycoplasma mycoides subsp. mycoides

The major pathways of ribonucleotide biosynthesis in Mycoplasma mycoides subsp. mycoides were proposed previously from studies of its usage of radioactive purines and pyrimidines. To interpret more fully the pattern of purine usage, we have assayed cell-free extracts of this organism for several enzymes associated with the salvage synthesis of purine nucleotides. M. mycoides possessed phosphoribosyltransferases for adenine, guanine, and hypoxanthine, purine nucleoside phosphorylase, GMP reductase, GMP kinase, adenylosuccinate synthetase, and adenylosuccinate lyase. Purine nucleoside kinase and adenosine deaminase were not detected. Examination of kinetic properties and regulation of some of the above enzymes revealed differences between M. mycoides and Escherichia coli. Most notable of these were the greater susceptibility of the enzymes from M. mycoides to inhibition by nucleotides and the more widespread involvement of GMP as an inhibitor. Observations on enzyme activities in vitro allow an adequate explanation of the capacity of guanine to provide M. mycoides with its full requirement for purine nucleotides.     

Pathways of Nucleotide Biosynthesis in Mycoplasma mycoides subsp. mycoides

By measuring the specific activity of nucleotides isolated from ribonucleic acid after the incorporation of (14)C-labeled precursors under various conditions of growth, we have defined the major pathways of ribonucleotide synthesis in Mycoplasma mycoides subsp. mycoides. M. mycoides did not possess pathways for the de novo synthesis of nucleotides but was capable of interconversion of nucleotides. Thus, uracil provided the requirement for both pyrimidine ribonucleotides. Thymine is also required, suggesting that the methylation step is unavailable. No use was made of cytosine. Uridine was rapidly degraded to uracil. Cytidine competed effectively with uracil to provide most of the cytidine nucleotide and also provided an appreciable proportion of uridine nucleotide. In keeping with these results, there was a slow deamination of cytidine to uridine with further degradation to uracil in cultures of M. mycoides. Guanine was capable of meeting the full requirement of the organism for purine nucleotide, presumably by conversion of guanosine 5'-monophosphate to adenosine 5'-monophosphate via the intermediate inosine 5'-monophosphate. When available with guanine, adenine effectively gave a complete provision of adenine nucleotide, whereas hypoxanthine gave a partial provision. Neither adenine nor hypoxanthine was able to act as a precursor for the synthesis of guanine nucleotide. Exogenous guanosine, inosine, and adenosine underwent rapid cleavage to the corresponding bases and so show a pattern of utilization similar to that of the latter.     

Enzymes of Pyrimidine Metabolism in Mycoplasma mycoides subsp. mycoides

The major pathways of ribonucleotide biosynthesis in Mycoplasma mycoides subsp. mycoides have been proposed from studies on its use of radioactive purines and pyrimidines. To interpret more fully the observed pattern of pyrimidine usage, cell extracts of this organism have been assayed for several enzymes associated with the salvage synthesis of pyrimidine nucleotides. M. mycoides possessed uracil phosphoribosyltransferase, uridine phosphorylase, uridine (cytidine) kinase, uridine 5'-monophosphate kinase, and cytidine 5'-triphosphate synthetase. No activity for phosphorolysis of cytidine was detected, and no in vitro conditions were found to give measurable deamination of cytidine. Of the two potential pathways for incorporation of uridine, our data suggest that this precursor would largely undergo initial phosphorolysis to uracil and ribose-1-phosphate. Conversely, cytidine is phosphorylated directly to cytidine 5'-monophosphate in its major utilization, although conversion of cytidine to uracil, uridine, and uridine nucleotide has been observed in vivo, at least when uracil is provided in the growth medium. Measurements of intracellular nucleotide contents and their changes on additions of pyrimidine precursors have allowed suggestions as to the operation of regulatory mechanisms on pyrimidine nucleotide biosynthesis in M. mycoides in vivo. With uracil alone or uracil plus uridine as precursors of pyrimidine ribonucleotides, the regulation of uracil phosphoribosyltransferase and cytidine 5'-triphosphate synthetase is probably most important in determining the rate of pyrimidine nucleotide synthesis. When cytidine supplements uracil in the growth medium, control of cytidine kinase activity would also be important in this regard.     

Studies of lactate dehydrogenase of Mycoplasma mycoides var. mycoides.

Polyacrylamide gel electrophoresis and isoelectric focusing techniques have been used to compare NAD-dependent L(plus) lactate dehydrogenases (LDH) from ten different strains of Mycoplasma mycoides var. mycoides. The enzymes were not distinguished from one another, or from normal bovine LDH 1 by these methods. The kinetic behaviour of LDH form M. mycoides (T1 vaccine strain) suggested that the enzyme could readily reduce pyruvate or oxidize lactate in a manner which, in vertebrates, requires two different isoenzymes.     

Enzymes of pyrimidine deoxyribonucleotide metabolism in Mycoplasma mycoides subsp. mycoides.

Cell-free extracts of Mycoplasma mycoides subsp. mycoides were assayed for enzymes associated with the salvage synthesis of pyrimidine deoxyribonucleotides. They possessed kinases for deoxycytidine, (d)CMP, thymidine (deoxyuridine), dTMP, and nucleoside diphosphates; dCTPase and dUTPase; dCMP deaminase; thymidine (deoxyuridine) phosphorylase; and dUMP (dTMP) phosphatase. The existence of these enzymic activities together with ribonucleoside diphosphate reductase explains the capacity of cytidine to provide M. mycoides with deoxyribose for the synthesis of thymidine nucleotides from thymine.     

Isolation of a second cryptic plasmid from Mycoplasma mycoides subsp. mycoides

Plasmids have rarely been detected in organisms constituting the genus Mycoplasma. Recently, the isolation of a cryptic plasmid from Mycoplasma mycoides subsp. mycoides has been described, and we report here the isolation of a second cryptic plasmid from this species. Restriction map and Southern blot analyses show that the second plasmid is distinct from the previously described plasmid, although a limited region of homology was detected. The availability of mycoplasmal cryptic plasmids may lead to the development of cloning vectors that replicate in these organisms.     

Pathways of pyrimidine deoxyribonucleotide biosynthesis in Mycoplasma mycoides subsp. mycoides.

By measuring the specific activity of deoxyribonucleotides isolated from DNA after the incorporation of 14C-labeled precursors with and without competition from other nucleotide precursors, we defined the major pathways of pyrimidine deoxyribonucleotide synthesis in Mycoplasma mycoides subsp. mycoides. Uracil, guanine, and thymine are required for the synthesis of nucleotides. Cytidine competed effectively with uracil to provide all of the deoxycytidine nucleotide, as well as most of the deoxyribose-1-phosphate, for the synthesis of thymidylate from thymine via thymidine phosphorylase. Each of dUMP, dCMP, and dTMP competed with cytidine for incorporation into DNA thymidylate. Appreciable incorporation of exogenous deoxyribonucleoside 5'-monophosphates into DNA without prior dephosphorylation was observed. Dephosphorylation also occurred since the added deoxyribonucleotide provided phosphate for the synthesis of the other nucleotides in DNA in competition with the 32Pi in the growth medium. Hydroxyurea inhibited cell growth and decreased the intracellular level of dATP, consistent with the action of a ribonucleoside diphosphate reductase with regulatory properties similar to those of the Escherichia coli enzyme.     

Domain analysis of lipoprotein LppQ in Mycoplasma mycoides subsp. mycoides SC

The lipoprotein LppQ is the most prominent antigen of Mycoplasma mycoides subsp. mycoides small colony type (SC) during infection of cattle. This pathogen causes contagious bovine pleuropneumonia (CBPP), a devastating disease of considerable socio-economic importance in many countries worldwide. The dominant antigenicity and high specificity for M. mycoides subsp. mycoides SC of lipoprotein LppQ have been exploited for serological diagnosis and for epidemiological investigations of CBPP. Scanning electron microscopy and immunogold labelling were used to provide ultrastructural evidence that LppQ is located to the cell membrane at the outer surface of M. mycoides subsp. mycoides SC. The selectivity and specificity of this method were demonstrated through discriminating localization of extracellular (i.e., in the zone of contact with host cells) vs. integral membrane domains of LppQ. Thus, our findings support the suggestion that the accessible N-terminal domain of LppQ is surface exposed and such surface localization may be implicated in the pathogenesis of CBPP.     

Formylation of methionyl-transfer ribonucleic acid in Mycoplasma mycoides subsp. mycoides.

Mycoplasma mycoides subsp. mycoides grows readily but does not formylate methionyl-transfer ribonucleic acid in a defined medium without an added formyl donor. Formylation occurs when the medium is supplemented with N5,N10-methenyltetrahydrofolate or N10-formyltetrahydrofolate, but not with folate.     

Uptake and utilization of deoxynucleoside 5''-monophosphates by Mycoplasma mycoides subsp. mycoides

Mycoplasma mycoides subsp. mycoides has been shown to possess an unusual capacity for the uptake and utilization of exogenous deoxyribonucleoside 5'-monophosphates intact without prior dephosphorylation. In this study, it was found that once inside the cell, deoxyribonucleoside 5'-monophosphates were rapidly phosphorylated to the triphosphate level and incorporated into DNA. Catabolism of deoxyribonucleoside 5'-monophosphates was also observed. Competition studies indicated that a single uptake system with a higher affinity for deoxyribonucleotides mediates the uptake of nucleoside 5'-monophosphates.     

Enzymes of intermediary carbohydrate metabolism in Ureaplasma urealyticum and Mycoplasma mycoides subsp. mycoides

Cell extracts of Ureaplasma urealyticum and Mycoplasma mycoides were examined for enzymes of intermediary carbohydrate metabolism using a sensitive radiochemical assay procedure. For M. mycoides, the enzyme activities detected were supporting evidence for the existence of a glycolytic pathway giving lactate anaerobically and acetate aerobically. U. urealyticum also had activities of many glycolytic enzymes. Enzymes of the pentose phosphate pathway occurred in both M. mycoides and U. urealyticum. Their presence allowed the proposal of a sequence for the synthesis from glycolytic pathway intermediates of ribose 5-phosphate, and hence phosphoribosyl diphosphate, for the synthesis of nucleotides. Pathways for the further metabolism of deoxyribose 1-phosphate and ribose 1-phosphate produced from nucleoside phosphorylase reactions operated in extracts from both organisms.     

Isolation and microcalorimetric characterisation of glucose-negative and pyruvate-negative mutants of Mycoplasma mycoides subsp. mycoides

Abstract A glucose-negative and a pyruvate-negative strain of Mycoplasma mycoides ssp. mycoides were isolated by their resistance to 3-deoxy-3-fluoro- d -glucose and β-fluoropyruvate, respectively. The ability of the mutants to metabolise various substrates was investigated microcalorimetrically. Results suggest that both mutants are transport mutants. The pyruvate-negative mutant was unable to metabolise exogenous lactate. The kinetics of N -acetylglucosamine and fructose metabolism by the glucose-negative mutant were similar to those of the parent strain; glucosamine and mannose, however, were not metabolised, and it is suggested that their transport in the parent strain involves glucose-specific uptake component(s).     

Plasmid transformation of Mycoplasma mycoides subspecies mycoides is promoted by high concentrations of polyethylene glycol.

The recent isolation and characterization of two plasmids from Mycoplasma mycoides subspecies mycoides has opened up new possibilities for studying mycoplasmal genetics. In order to facilitate the development of a genetic system in M. mycoides subsp. mycoides, parameters of polyethylene glycol (PEG)-mediated transformation were examined, as existing protocols prove very inefficient in this organism. The effects of PEG concentration, DNA concentration, presence of Ca2+ ions, and choice of buffers on the transformation of the Tn916-containing plasmid pAM120 into M. mycoides subsp. mycoides were examined. The stability of Tn916 in the M. mycoides subsp. mycoides chromosome was also evaluated. The optimal PEG concentration (53-62% (w/v)) in the transformation mixture was substantially higher than the PEG concentration reported to be optimal for transformation of other mycoplasmas (36% (w/v)). The PEG concentrations used here were also higher than the concentration used to promote transformation or fusion of gram-positive bacterial protoplasts. A necessity for the presence of Ca2+ ions for optimal transformation was shown, as was the possible involvement of cell culture growth stage. Our results demonstrate the need for expanding current transformation techniques for mycoplasmas. Studies also indicate that once Tn916 inserts into the M. mycoides subsp. mycoides chromosome, it can transpose to other sites at a relatively high frequency.     

Isolation of Mycoplasma mycoides, mycoides (LC variant), from two naturally aborted caprine fetuses

Described in this study are 2 cases of caprine abortion associated with the LC type of Mycoplasma mycoides , subsp. mycoides . This mycoplasma and Mycoplasma mycoides , subsp. capri had been previously reported in adult goats in this herd. The abortion took place in the latter part of gestation. Samples from heart blood, lung and pleural exudate were collected for the isolation of mycoplasmas and other bacterials in both fetuses. Two strains of Mycoplasma mycoides , subsp. mycoides (LC variant) were isolated. The only gross lesion of the internal organs in the aborted fetuses was congestion of the lungs. Microscopic lesions were encountered in the lungs, and these were characterized by patchy to diffuse pneumonia. Exfoliated cells, many alveolar macrophages, scattered neutrophils and lymphocytes were seen in the lumen of the terminal airways and alveolar spaces. This report appears to be the first isolation of Mycoplasma mycoides , subsp. mycoides (LC variant) from aborted caprine fetuses.     

ISMmy1, a novel insertion sequence of Mycoplasma mycoides subsp. mycoides small colony type

A new insertion sequence, ISMmy1, has been identified in the bovine pathogen Mycoplasma mycoides subsp. mycoides biotype small colony (MmymySC). The occurrence of ISMmy1 in 15 MmymySC strains and 12 other mycoplasmas was examined by Southern blotting. All MmymySC strains showed identical hybridisation patterns except for the type strain PG1(T), the vaccine strain T1Sr49, and the strain Afadé, which all had unique patterns. ISMmy1-like sequences were also found in the bovine pathogen Mycoplasma bovis strain Donetta(T) while mycoplasmas that are phylogenetically closer to MmymySC lack ISMmy1. This observation suggests horizontal transfer between MmymySC and M. bovis.