Taxonomic significance of differences in DNA methylation within the 'Mycoplasma mycoides cluster' detected with restriction endonucleases MboI and DpnI

DNA from 22 strains belonging to the ' Mycoplasma mycoides cluster' was tested for methylation of adenine in GATC sequences by its resistance or susceptibility to digestion by the restriction endonucleases MboI , which is inhibited by the methylation, or DpnI , which requires the methylation. Strains of bovine serogroup 7, M. mycoides subsp. mycoides SC, and some M. mycoides subsp. capri strains plus M. capricolum strain Cal Kid lacked the methylation, whereas other strains of M. mycoides subsp. capri and of M. capricolum , plus the F38-like and M. mycoides subsp. mycoides LC strains, possessed it. We conclude that this simple test could provide a valuable criterion in identifying these mycoplamas.     

A novel method for the determination of electrical potentials across cellular membranes. II. Membrane potentials of Acholeplasmas, Mycoplasmas, Streptococci and erythrocytes

The membrane potentials of Acholeplasma laidlawii, Mycoplasma mycoides subsp. capri, Mycoplasma gallisepticum, Streptococcus faecalis and human erythrocytes have been determined by applying a novel technique. The membrane potentials were calculated simply from potassium concentrations determined by atomic absorption spectroscopy, and gravimetry. The versatility of the new technique is demonstrated by comparing our results with data obtained by different techniques.     

Ultrastructural visualization of anionic sites on mycoplasma membranes by polycationic ferritin.

Anionic sites on mycoplasma membranes were visualized in the electron microscope by a polycationized ferritin derivative. The technique of thin sectioning was used. Staining prior to fixation led to clustering of ferritin granules on the mycoplasma cell surface. On glutaraldehyde-fixed Mycoplasma mycoides subsp. capri, M. gallisepticum, M. pneumoniae, and Acholeplasma laidlawii, the anionic sites were uniformly distributed over the entire membrane surface. M. hominis did not bind the polycationic ferritin label. Chemical and enzymatic treatments of the mycoplasmas indicated that the anionic sites may be lipid phosphate groups. Isolated M. mycoides subsp. capri membranes were labeled exclusively on only one membrane surface, presumably the outer one. Liposomes prepared from diphosphatidylglycerol and phosphatidylcholine were also labeled by the polycationic ferritin.     

Reduced nicotinamide adenine dinucleotide oxidase activity in membranes and cytoplasm of Acholeplasma laidlawii and Mycoplasma mycoides subsp. capri.

The properties of the membrane-bound reduced nicotinamide adenine dinucleotide (NADH) oxidase of Acholeplasma laidlawii were compared with those of the corresponding cytoplasmic activity of Mycoplasma mycoides subsp. capri. The striking differences in pH optima, susceptibility to inhibitors and detergents, and heat inactivation between the NADH oxidase activity, with oxygen as an electron acceptor, and the NADH oxidoreductase activity, with dichlorophenol indophenol (DCPIP) as an alternate electron acceptor, support the presence of more than one catalytic protein in both the membrane-bound and soluble enzyme systems. The detection of more than one band positive for the NADH-nitroblue tetrazolium oxidoreductase reaction on electrophoresis of either the membranes of A. laidlawii or the cytoplasm of M mycoides subsp. capri also points in the same direction. The membrane-bound enzyme system differed, however, form the soluble one because it had a lower ratio of oxidase activity to oxidoreductase activity, and because it was less susceptible to heat inactivation and more readily incorporated incorporated into reaggregated membranes. In addition, the specific activity of the membrane-bound enzyme system increased as the culture aged, whereas that of the soluble system decreased as the culture aged. It is suggested that the different location in the cell could be responsible for some of the differences between the membrane-bound NADH oxidase activity of A. laidlawii and that found in the cytoplasm of M. mycoides subsp. capri.     

The origin of the 'Mycoplasma mycoides cluster' coincides with domestication of ruminants

The 'Mycoplasma mycoides cluster' comprises the ruminant pathogens Mycoplasma mycoides subsp. mycoides the causative agent of contagious bovine pleuropneumonia (CBPP), Mycoplasma capricolum subsp. capripneumoniae the agent of contagious caprine pleuropneumonia (CCPP), Mycoplasma capricolum subsp. capricolum, Mycoplasma leachii and Mycoplasma mycoides subsp. capri. CBPP and CCPP are major livestock diseases and impact the agricultural sector especially in developing countries through reduced food-supply and international trade restrictions. In addition, these diseases are a threat to disease-free countries. We used a multilocus sequence typing (MLST) approach to gain insights into the demographic history of and phylogenetic relationships among the members of the 'M. mycoides cluster'. We collected partial sequences from seven housekeeping genes representing a total of 3,816 base pairs from 118 strains within this cluster, and five strains isolated from wild Caprinae. Strikingly, the origin of the 'M. mycoides cluster' dates to about 10,000 years ago, suggesting that the establishment and spread of the cluster coincided with livestock domestication. In addition, we show that hybridization and recombination may be important factors in the evolutionary history of the cluster.     

Classification and Identification of Ovine and Caprine Mycoplasmas

The purpose of this investigation was to give a survey of the classification of ovine/caprine mycoplasmas as a basis for the identification of strains isolated from sheep and goats. A total of 13 strains representing 13 species and/or serogroups were biochemically examined, and serological cross-titrations were performed using metabolism inhibition, growth inhibition and immunofluorescence. Serogroup 6 (Al-Aubaidi) was found to be identical with Mycoplasma capricolum. The results of identification of 57 isolates, sent to the reference centre from different countries, are given. On the basis of the above investigations and a comparison of some of the classification systems described in the literature, it is concluded that the following species have been isolated from goats and/or sheep: M. agalactiae, M. arginini, M. bovis, M. capricolum, M. conjunctivae, M. mycoides subsp. capri, M. mycoides subsp. mycoides, M. ovipneumoniae, M. putrefaciens, Acholeplasma granularum, A. laidlawii and A. oculi. In addition, both Ureaplasmas and strains representing 6 serogroups (groups 5, 7, 10 and 11 of Al-Aubaidi and groups 17 and 18 of Cottew) have been isolated. These serogroups ought to be finally species-classified as soon as possible. kw|Keywords|k]ovine/caprine mycoplasmas; k]classification; k]identification     

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.     

A rapid biochemical test to aid identification of Mycoplasma mycoides subsp. mycoides small colony (SC) strains

The ability to utilize maltose, as determined by measurement of oxygen uptake, is used to differentiate Mycoplasma mycoides subsp. mycoides small colony (SC) and M. capricolum subsp. capripneumoniae (all strains negative) from other members of the M. mycoides cluster (M. mycoides subsp. capri, M. mycoides subsp. mycoides large colony (LC), M. capricolum subsp. capricolum; and bovine serogroup 7; 94% of strains positive). Rapid tests for maltose utilizing ability were developed, based on hydrolysis of a chromogenic alpha-glucosidase (maltase) substrate (p-nitrophenyl-alpha-D-glucopyranoside, colourless) to give a brightly coloured product (p-nitrophenol, yellow). On agar plates, colonies of maltose-utilizing strains became coloured within 40 min.     

Comparison of the conserved region in the dnaA gene from three mollicute species

Abstract Polymerase chain reaction was carried out to amplify the conserved region (789 bp in the case of Mycoplasma capricolum ) of the dnaA gene (1350 bp in the case of M. capricolum ) of 15 representatives of the class Mollicutes using degenerate oligonucleotide primers. The dnaA gene fragments were amplified from M. mycoides subsp. capri, Spiroplasma apis and S. citri . The amino acid sequences deduced from the nucleotide sequences of the amplified fragments showed very low similarities to those of the corresponding regions of four walled bacteria. The values of similarity between any two of the three mollicute species were lower than those between any two of the four walled bacteria.     

Electrophoretic analysis of proteins from Mycoplasma capricolum and related serotypes using extracts from intact cells and from minicells containing cloned mycoplasma DNA

The acidic proteins of six different mycoplasma serotypes causing bovine or caprine pleuropneumonia were compared by two-dimensional gel electrophoresis of extracts of 35S-labelled cells. The organisms investigated were Mycoplasma mycoides subsp. mycoides (PG1), M. mycoides subsp. mycoides (Y-goat), M. mycoides subsp. capri (PG3), M. capricolum (California kid), the unclassified bovine serogroup 7 of Leach (PG50) and the F38-like group (F38). The results suggested a close relationship between M. capricolum and F38 and a similarly close relationship between the different M. mycoides subspecies, whereas the two M. mycoides subspecies appeared to be quite distant from M. capricolum and F38. The representative strain of the bovine serogroup 7 of Leach was equally distant from F38, M. capricolum and the three strains of M. mycoides. Strikingly, all six mycoplasma strains apparently shared six proteins in the two-dimensional gels. In Escherichia coli minicells, DNA from strain PG50 cloned in the vector pBR325 gave rise to incorporation of radioactive label into proteins which were identified as mycoplasma proteins by two-dimensional electrophoresis and immunoprecipitation.     

Methylation patterns of mycoplasma transfer and ribosomal ribonucleic acid.

The methylation patterns of transfer and ribosomal ribonucleic acid (RNA) from two mycoplasmas, Mycoplasma capricolum and Acholeplasma laidlawii, have been examined. The transfer RNA from the two mycoplasmas resembled that of other procaryotes in degree of methylation and general diversity of methylated nucleotides, and bore particular resemblance to Bacillus subtilis transfer RNA. The only unusual feature was the absence of m5U from M. capricolum transfer RNA. The methylation patterns of the mycoplasma 16S RNAs were also typically procaryotic, retaining the methylated residues previously shown to be highly conserved among eubacterial 16S RNAs. The mycoplasma 23S RNA methylation patterns were, on the other hand, quite unusual. M. capricolum 23S RNA contained only four methylated residues in stoichiometric amounts, all of which were ribose methylated. A. laidlawii 23S RNA contained the same ribose-methylated residues, plus in addition approximately six m5U residues. These findings are discussed in relation to the phylogenetic status of mycoplasma, as well as the possible role of RNA methylation.     

Relationships between members of the Mycoplasma mycoides cluster as shown by DNA probes and sequence analysis.

A gene probe, CAP-21, which demonstrated interrelationships between the members of the Mycoplasma mycoides cluster was developed. The probe easily differentiated mycoplasmas in this cluster by clear and predictable hybridization patterns in Southern blots and separated the cluster into four groups. Strains of M. mycoides subsp. mycoides which were capable of causing contagious bovine pleuropneumonia composed one group. Strains of M. mycoides subsp. mycoides which did not cause contagious bovine pleuropneumonia together with strains of M. mycoides subsp. capri composed the second group. Mycoplasma capricolum and the F38 mycoplasmas formed a third group, while the bovine group 7 mycoplasmas composed a separate, fourth group. Further support for the above grouping of the cluster was obtained when amplified DNA analogous to the probe from one representative strain of each of the cluster members was sequenced and these data were used to construct a phylogenic tree. Contagious caprine pleuropneumonia is recognized as an important disease, and the etiological agent of this disease is now known to be the F38 mycoplasma. The CAP-21 probe did not differentiate between M. capricolum and the closely related F38 mycoplasma. A second probe, F38-12, which was capable of distinguishing these two mycoplasmas was made.     

The nucleotide sequence of the 5S rRNA from Spiroplasma species BC3 and Mycoplasma mycoides sp. capri PG3

Using in vitro labelling techniques, the complete nucleotide sequence of the 5S ribosomal RNAs isolated from the honeybee pathogen, Spiroplasma species BC3 and Mycoplasma mycoides sp. capri PG3, have been determined. The latter shows only 3 differences from the reported sequence of M. capricolum 5S rRNA, indicating that these two species are very closely related. The Spiroplasma sequence is also 107 nucleotides long and a comparative analysis of the sequence confirms that this Spiroplasma species is closely related to the Mycoplasma species and that they and the Gram-positive eubacteria have descended from a common ancestor and in the process the cell wall-less organisms have lost a large percentage of their genome.     

Relationship between Mycoplasma mycoides subsp. mycoides ('large-colony' strains) and M. mycoides subsp. capri, as indicated by numerical analysis of one-dimensional SDS-PAGE protein patterns

Twenty-five strains classified as Mycoplasma mycoides subsp. mycoides LC or subsp. capri have been compared by one-dimensional SDS-PAGE of their cellular proteins. A computerized numerical analysis revealed that the protein patterns of all but two aberrant strains formed one large phenon that separated clearly from representatives of the four other members of the 'M. mycoides cluster' at a similarity level (S) of 66% and which remained undivided at up to 78% S. At higher similarity levels, these strains fell heterogeneously into mixed sub-phenons containing strains of both subspecies. Serological comparisons by immunofluorescence largely confirmed the subspecies designations of the test strains, but also showed that some were serologically intermediate between subsp. mycoides and subsp. capri, being cross-reactive with both. These results confirm and enlarge upon those of our earlier studies indicating the protein-pattern inseparability of subsp. capri and subsp. mycoides LC strains and their distinctiveness from the classical M. mycoides subsp. mycoides SC strains and other members of the 'M. mycoides cluster'. As also recognized by other workers, subsp. mycoides LC and subsp. capri strains appear to comprise one large group, wherein those most readily identifiable as either type lie at either end of a serological spectrum that also contains serologically cross-reactive strains. Our observations therefore suggest the lines along which the three groups classified at present within the species M. mycoides (SC and LC strains of subsp. mycoides; subsp. capri) might eventually be reclassified, subject to direct genomic comparisons.     

Versatile use of oriC plasmids for functional genomics of Mycoplasma capricolum subsp. capricolum

Replicative oriC plasmids were recently developed for several mollicutes, including three Mycoplasma species belonging to the mycoides cluster that are responsible for bovine and caprine diseases: Mycoplasma mycoides subsp. mycoides small-colony type, Mycoplasma mycoides subsp. mycoides large-colony type, and Mycoplasma capricolum subsp. capricolum. In this study, oriC plasmids were evaluated in M. capricolum subsp. capricolum as genetic tools for (i) expression of heterologous proteins and (ii) gene inactivation by homologous recombination. The reporter gene lacZ, encoding beta-galactosidase, and the gene encoding spiralin, an abundant surface lipoprotein of the related mollicute Spiroplasma citri, were successfully expressed. Functional Escherichia coli beta-galactosidase was detected in transformed Mycoplasma capricolum subsp. capricolum cells despite noticeable codon usage differences. The expression of spiralin in M. capricolum subsp. capricolum was assessed by colony and Western blotting. Accessibility of this protein at the cell surface and its partition into the Triton X-114 detergent phase suggest a correct maturation of the spiralin precursor. The expression of a heterologous lipoprotein in a mycoplasma raises potentially interesting applications, e.g., the use of these bacteria as live vaccines. Targeted inactivation of gene lppA encoding lipoprotein A was achieved in M. capricolum subsp. capricolum with plasmids harboring a replication origin derived from S. citri. Our results suggest that the selection of the infrequent events of homologous recombination could be enhanced by the use of oriC plasmids derived from related mollicute species. Mycoplasma gene inactivation opens the way to functional genomics in a group of bacteria for which a large wealth of genome data are already available and steadily growing.     

Mycoplasmas (Mollicutes) have a low number of rRNA genes.

DNA from Mycoplasma, Ureaplasma, Acholeplasma, and Spiroplasma species digested by restriction endonucleases was hybridized with probes consisting of portions of the rrnB rRNA operon of Escherichia coli and the rRNA operon of Mycoplasma capricolum. The results indicate the presence of only one or two sets of rRNA genes in the genome of Mollicutes linked in the procaryotic fashion, 16S-23S-5S.     

Distinctive repertoire of contingency genes conferring mutation- based phase variation and combinatorial expression of surface lipoproteins in Mycoplasma capricolum subsp. capricolum of the Mycoplasma mycoides phylogenetic cluster

The generation of surface variation among many divergent species of Mollicutes (mycoplasmas) occurs through stochastic expression patterns of diverse lipoprotein genes. The size and wide distribution of such variable gene sets in minimal (approximately 0.6- to 1.4-Mb) mycoplasmal genomes suggest their key role in the adaptation and survival of these wall-less monoderms. Diversity through variable genes is less clearly established among phylogenetically similar mycoplasmas, such as the Mycoplasma mycoides cluster of ruminant pathogens, which vary widely in host range and pathobiology. Using (i) genome sequences from two members of this clade, Mycoplasma capricolum subsp. capricolum and M. mycoides subsp. mycoides small colony biotype (SC), (ii) antibodies to specific peptide determinants of predicted M. capricolum subsp. capricolum gene products, and (iii) analysis of the membrane-associated proteome of M. capricolum subsp. capricolum, a novel set of six genes (vmcA to vmcF) expressing distinct Vmc (variable M. capricolum subsp. capricolum) lipoproteins is demonstrated. These occur at two separate loci in the M. capricolum subsp. capricolum genome, which shares striking overall similarity and gene synteny with the M. mycoides subsp. mycoides SC genome. Collectively, Vmc expression is noncoordinate and combinatorial, subject to a single-unit insertion/deletion in a 5' flanking dinucleotide repeat that governs expression of each vmc gene. All vmc genes share modular regions affecting expression and membrane translocation. In contrast, vmcA to vmcD genes at one locus express surface proteins with highly structured size-variable repeating domains, whereas vmcE to vmcF genes express products with short repeats devoid of predicted structure. These genes confer a distinctive, dynamic surface architecture that may represent adaptive differences within this important group of pathogens as well as exploitable diagnostic targets.     

Numerical analysis of PAGE protein patterns and the taxonomic relationships within the 'Mycoplasma mycoides cluster'

Twenty-six isolates belonging to the 'Mycoplasma mycoides cluster' have been characterized by one-dimensional SDS-PAGE of their cellular proteins. A numerical classification based on the resulting patterns and using a correlation coefficient revealed four distinct phenons at a similarity (S) level of 70%, comprising: (a) bovine group 7 strains; (b) M. capricolum and F38-like strains; (c) M. mycoides subsp. capri and LC strains ('subsp. mycoides'); (d) M. mycoides subsp. mycoides (SC). At the 75% S level, they could be divided further to give eight phenons. The composition of the clusters at both levels was in good agreement with their previous classification, except for M. mycoides subsp. mycoides LC and M. mycoides subsp. capri, which were clustered in a single phenon at 70% S and could not be clearly separated at 75% S. We conclude that high-resolution SDS-PAGE, combined with computerized analysis of protein patterns, provides an extremely effective approach to the investigation of taxonomic relationships within this group of mycoplasmas.     

A novel method for the determination of electrical potentials across cellular membranes. II. Membrane potentials of acholeplasmas, mycoplasmas, streptococci and erythrocytes

The membrane potentials of Acholeplasma laidlawii, Mycoplasma mycoides subsp. capri, Mycoplasma gallisepticum, Streptococcus faecalis and human erythrocytes have been determined by applying a novel technique. The membrane potentials were calculated simply from potassium concentrations determined by atomic absorption spectroscopy, and gravimetry. The versatility of the new technique is demonstrated by comparing our results with data obtained by different techniques.     

Experimental pathogenicity of Mollicutes of bovine udder origin in hamster tracheal ring organ culture.

Hamster tracheal-ring organ culture was employed to examine pathogenic effects of 8 isolates of Mollicutes of bovine udder origin. The tested Mollicutes could be categorized into two groups: (i) Mycoplasma F-38, M. mycoides var. capri, M. bovigenitalium mixed with M. bovirhinis, and M. bovigenitalium mixed with M. bovirhinis and Mycoplasma F-38 produced significant ciliostatic effect and infiltration of neutrophils and lymphocytes in lamina propria/subepithelium, hyperplasia and desquamation of epithelial lining cells and loss of cilia; and (ii) A. laidlawii, A. axanthum, an unidentified Acholeplasma and a mixed isolate of M. bovis, M. bovigenitalium, Mycoplasma F-38 and A. laidlawii showed insignificant ciliostatic effects and produced mild histopathological lesions. This correlates with the disease causing potentials of the strains.