Isolation and characterization of Mycoplasma mycoides subsp. mycoides mutants deficient in nucleoside monophosphate transport.

To select mutants lacking dAMP uptake, log-phase cells of Mycoplasma mycoides subsp. mycoides Y were incubated with high-specific-activity [32P]dAMP and then stored several weeks at -20 degrees C to allow 32P decay before plating out. Mutants were screened for lack of labeling by [32P]dAMP. Two mutants were studied further by uptake and growth experiments with other nucleotides.     

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.     

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 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.     

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.     

Isolation of Mycoplasma mycoides subsp. mycoides (LC variant, Y-Goat) from naturally aborted bovine fetuses

Seven strains of Mycoplasma mycoides subsp. mycoides (LC, Y-Goat) were isolated from 4 of 85 (4.7%) cases of bovine abortion. The biochemical characters of all the isolates were homogenous (except for one sucrose negative isolate), but variation in sensitivity to neomycin, kanamycin and streptomycin were noted. The only gross lesions of internal organs in aborted fetuses were congestion of liver and lungs, and hemorrhagic patches on the heart surface. Significant microscopic lesions were encountered in lungs (edema of the interlobular septae, thickened alveolar wall, lymphocytic infiltration, septal hyperplasia and alveolar infiltration with neutrophils and macrophages), liver (mild lymphocytic infiltration in the hepatic triad, hypertrophy of Von-kupffer cells), kidney (degenerative necrosis and desquamation of tubular epithelium, and the marked lymphocytic infiltration in the interstitium), spleen (depleted lymphoid tissues, infiltration of lymphocytes, macrophage and plasma cells in the red pulp) and heart (lymphocytic myocarditis). The observations of this study focus on the role of M. mycoides subsp. mycoides (LC variant) in bovine abortions.     

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.     

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.     

Isolation and preliminary characterisation of twenty-five temperature-sensitive mutants of mouse cytomegalovirus

Abstract To study the pathogenicity of mouse cytomegalovirus (MCMV) and to identify virulence determinants, we have isolated and phenotypically characterised 25 temperature-sensitive ( ts ) mutants. Six of these ( tsm 9, tsm 13, tsm 20, tsm 22, tsm 28 and tsm 30) failed to replicate in mice and were avirulent. Five mutants ( tsm 14, tsm 18, tsm 19, tsm 25 and tsm27 ) were to similar virulence to the parenthal wild-type ( wt ) virus, five ( tsm 7, tsm 15, tsm 24, tsm31 ) were 12–100 fold less virulent, five ( tsm 8, tsm 12, tsm 16, tsm 23 and tsm 29) were 150–1500 fold less virulent and four ( tsm 10, tsm 11, tsm 17 and tsm 21) were between 2,000 and 85,000 fold less virulent than wt . One mutant ( tsm 28) did not plaque or replicate at 39°C while 5 other mutants ( tsm 7, tsm 9, tsm 23, tsm 24 and tsm 27) also failed to plaque at 39°C but only failed to replicate or replicated poorly at 40°C. A further two mutants ( tsm 10 and tsm 13) were able to plaque and replicate at 39°C but not 40°C. Six other mutants ( tsm 14, tsm 15, tsm 16, tsm21 , tsm 22 and tsm 30) failed to form plaques at 40°C and were severely restricted in their replication at 40°C. The remaining 11 mutants exhibited varying degrees of restriction in ability to plaque and/or replicate at non-permissive temperatures. These 25 mutants, together with 6 isolated previously, comprise at least 24 complementation groups.     

One Test Microbial Diagnostic Microarray for Identification of Mycoplasma mycoides subsp. mycoides and Other Mycoplasma Species

The present study describes the use of microarray technology for rapid identification and differentiation of Mycoplasma mycoides subsp. mycoides from other mycoplasmas that may be pathogenic to ruminants, including those of the Mycoplasma mycoides cluster, genetically and antigenically strictly correlated with Mycoplasma mycoides subsp. mycoides. A microarray containing genetic sequences of 55 different bacterial species from Acholeplasma, Mycoplasma, Spiroplasma and Ureaplasma genera was constructed. Sequences to genes of interest were collected in FASTA format from NCBI. The collected sequences were processed with OligoPicker software. Oligonucleotides were then checked for their selectivity with BLAST searches in GenBank. The microarray was tested with ATCC/NCTC strains of Mycoplasma spp. of veterinary importance in ruminants including Mycoplasma belonging to the mycoides cluster as well as Mycoplasma mycoides subsp. mycoides and Mycoplasma mycoides subsp. capri field strains. The results showed that but one ATCC/NCTC reference strains hybridized with their species-specific sequences showed a profile/signature different and distinct from each other. The heat-map of the hybridization results for the nine genes interrogated for Mycoplasma mycoides subsp. mycoides demonstrated that the reference strain Mycoplasma mycoides subsp mycoides PG1 was positive for all of the gene sequences spotted on the microarray. CBPP field, vaccine and reference strains were all typed to be M. mycoides subsp. mycoides, and seven of the nine strains gave positive hybridization results for all of the nine genes. Two Italian strains were negative for some of the genes. Comparison with non-Mycoplasma mycoides subsp. mycoides reference strains showed some positive signals or considerable homology to Mycoplasma mycoides subsp. mycoides genes. As expected, some correlations were observed between the strictly genetically and antigenically correlated Mycoplasma mycoides subsp. mycoides and Mycoplasma mycoides subsp. capri strains. Specifically, we observed that some Italian Mycoplasma mycoides subsp. mycoides strains were positive for two out of the three Mycoplasma mycoides subsp. capri genes, differently from what has been observed for other European or African Mycoplasma mycoides subsp. mycoides strains. This study highlighted the use of microarray technology as a simple and effective method for a single-step identification and differentiation of Mycoplasma mycoides subsp. mycoides from other mycoplasmas that may be pathogenic to ruminants, including those of the Mycoplasma mycoides cluster, genetically and antigenically strictly correlated with Mycoplasma mycoides subsp. mycoides. The opportunity to discriminate several mycoplasmas in a single analysis enhances diagnostic rapidity and may represent a useful tool to screen occasionally mycoplasmas affecting animal farming in territories where diagnostic laboratory support is limited. The heat-map of the hybridization results of the comparative genomic hybridizations DNA-designed chip clearly indicates that the microarray performs well for the identification of the tested Mycoplasma mycoides subsp. mycoides reference and field strains, discriminating them from other mycoplasmas.     

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.     

Mycoplasma bovis shares insertion sequences with Mycoplasma agalactiae and Mycoplasma mycoides subsp. mycoides SC: Evolutionary and developmental aspects

Three new insertion elements, ISMbov1, ISMbov2 and ISMbov3, which are closely related to ISMag1 (Mycoplasma agalactiae), ISMmy1 and IS1634 (both Mycoplasma mycoides subsp. mycoides SC), respectively, have been discovered in Mycoplasma bovis, an important pathogen of cattle. Southern blotting showed that the genome of M. bovis harbours 6-12 copies of ISMbov1, 11-15 copies of ISMbov2 and 4-10 copies of ISMbov3, depending on the strain. A fourth insertion element, the IS30-like element, is present in 4-8 copies. This high number of IS elements in M. bovis, which represent a substantial part of its genome, and their relatedness with IS elements of both M. agalactiae and M. mycoides subsp. mycoides SC suggest the occurrence of two evolutionary events: (i) a divergent evolution into M. agalactiae and M. bovis upon infection of different hosts; (ii) a horizontal transfer of IS elements during co-infection with M. mycoides subsp. mycoides SC and M. bovis of a same bovine host.     

Surveillance of Mycoplasma agalactiae and Mycoplasma mycoides subsp. capri in dairy goat herds

This study was designed to monitor the presence of Mycoplasma agalactiae and Mycoplasma mycoides subsp. capri (Mmc) in 66 dairy goat herds of a genetic improvement programme in a region of Spain where contagious agalactia is endemic. Over a whole lactation period, 300 bulk tank milk and 381 milk samples from goats with clinical mastitis were subjected to polymerase chain reaction (PCR) to detect the two mycoplasma species. The presence of mycoplasmas (either species or both) was detected in 66.7% of the herds and M. agalactiae was identified in 95.45% of these positives herds. In a given infected herd, mycoplasmas were not continuously detected over the whole study period. Our findings indicate that in an endemic area, M. agalactiae and Mmc can be monitored through PCR analysis of mastitic milk and bulk tank milk (BTM) samples. Over a lactation period we recommend testing multiple BTM samples on a herd. No relationship was observed between the use of inactivated mycoplasma vaccines and the PCR detection of both mycoplasmas.     

Selection and study of mutants of Dekkera intermedia and Candida wickerhamii derepressed for β-glucosidase production

Abstract Mutants of Candida wickerhamii and Dekkera intermedia , derepressed for β-glucosidase biosynthesis, were isolated. These mutants were also shown to hyperproduce this enzyme. In anaerobic culture, the C. wickerhamii mutant still hyperproduced β-glucosidase and was derepressed. Glucose-cellobiose anaerobic fermentation by this strain was thus improved. On the other hand, the D. intermedia mutant did not show any difference from the wild-type strain in anaerobic culture.     

Rapid photometric assay of growth of Mycoplasma mycoides subsp. capri

A new spectrophotometric technique for evaluation of early growth in liquid culture of Mycoplasma mycoides subsp. capri has been developed. As turbidity does not appear until after incubation to 18 h the method utilizes the change in absorbance of the medium at 550 nm to monitor growth. The change in absorbance of the medium (which contains phenol red) occurs when the pH changes due to microbial growth. For measurement of growth at later stages when turbidity is proportional to number of colony forming units, two other wavelengths (450 nm and 700 nm) have been suggested.     

Isolation and preliminary characterization of β-lactamase excretory mutants of Escherichia coli K-12

Abstract Escherichia coli exc mutants able to release the plasmid pBR322-encoded β-lactamase (EC 3.5.2.6) into the extracellular medium have been isolated using a new in situ plate assay.
A preliminary characterization of the exc mutants was carried out: the presence of exc mutations was associated with a specific or pleiotropic pattern of excretion of periplasmic enzymes, an increased sensitivity to different growth inhibitors (EDTA, chloramphenicol, cholic acid) and a poor growth on various carbon sources.
After quantitative analysis, three groups of exc mutants were identified on the basis of their temperature-dependent or -independent pattern of growth and β-lactamase synthesis and excretion.