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.     

Comparative genomics analysis of Mycoplasma capricolum subsp. capripneumoniae 87001

Mycoplasma capricolum subsp. capripneumoniae (Mccp), belongs to Mycoplasma mycoides cluster and is a causal pathogen of contagious caprine pleuropneumonia (CCPP). This paper presents the complete annotated genome sequence of Mccp Strain 87001—a strain that was isolated from pneumonia affected goats on a farm in China, and comparative genomics analysis of five Mccp genomes in addition to comparative genomics within Mycoplasma mycoides cluster. The Mccp strain 87001 genome consists of a single circular chromosome 1017333 bp in length and encodes 898 open reading frames (orfs) averaging 944 bp in length. Fifty eight potential virulence genes were identified, including variable surface lipoproteins, hemolysin A, and P60 surface lipoprotein. Comparative genomic analysis revealed eight virulence genes and four extracellular genes which remained unchanged in five Mccp genomes for forty years, which can be used as potential target for drug development and vaccine design. We revealed 183 Mccp unique genes as markers to distinguish Mccp with other mycoplasma strains from goats, and different virulence factors contributing to host specificity and different syndrome of bovine pathogens and caprine pathogens.     

Genome sequence of Mycoplasma capricolum subsp. capripneumoniae strain M1601

Mycoplasma capricolum subsp. capripneumoniae is the causative agent of contagious caprine pleuropneumonia, a devastating disease of goats listed by the World Organization for Animal Health. Here we report the first complete genome sequence of this organism (strain M1601, a clinically isolated strain from China).     

Immunohistochemical and ultrastructural characteristics of Mycoplasma capricolum subsp. capricolum in caprine abortion: a case report

Described in this study are the immunohistochemical and ultrastructural findings in a case of caprine abortion due to the experimental infection of the dam with strain GM13 of Mycoplasma capricolum subsp. capricolum. Mycoplasma antigens were seen mainly in choriallantoic trophoblasts and in the lumen of blood vessels in the allantoic membrane. Examination with an electron microscope showed that the chorioallantoic trophoblasts were filled with typical mycoplasma organisms. No other bacteria were observed in any of the samples. Our results confirm by immunohistochemical and electron microscopic techniques that Mycoplasma capricolum subsp. capricolum can cause caprine abortion and that the process can occur without premonitory signs.     

Iron storage in Mycoplasma capricolum.

Considerable quantities or iron were incorporated into the Mycoplasma capricolum cell membrane. Mossbauer studies showed that the iron is in a form which becomes magnetically ordered at low temperatures. The iron-enriched cells contained membrane-bound electron-dense particles of about 6.0 nm in diameter.     

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.     

A Versatile Zero Background T-Vector System for Gene Cloning and Functional Genomics

With the recent availability of complete genomic sequences of many organisms, high-throughput and cost-efficient systems for gene cloning and functional analysis are in great demand. Although site-specific recombination-based cloning systems, such as Gateway cloning technology, are extremely useful for efficient transfer of DNA fragments into multiple destination vectors, the two-step cloning process is time consuming and expensive. Here, we report a zero background TA cloning system that provides simple and high-efficiency direct cloning of PCR-amplified DNA fragments with almost no self-ligation. The improved T-vector system takes advantage of the restriction enzyme XcmI to generate a T-overhang after digestion and the negative selection marker gene ccdB to eliminate the self-ligation background after transformation. We demonstrate the feasibility and flexibility of the technology by developing a set of transient and stable transformation vectors for constitutive gene expression, gene silencing, protein tagging, protein subcellular localization detection, and promoter fragment activity analysis in plants. Because the system can be easily adapted for developing specialized expression vectors for other organisms, zero background TA provides a general, cost-efficient, and high-throughput platform that complements the Gateway cloning system for gene cloning and functional genomics.     

Uptake of fatty acids by Mycoplasma capricolum.

The energy requirements for fatty acid uptake by Mycoplasma capricolum were studied. Fatty acid transport and esterification to phospholipid appeared to be tightly coupled, since there was little intracellular accumulation of free fatty acid. Uptake was blocked by iodoacetate, n-ethylmaleimide, and p-chloromercuribenzoate. Glucose, glycerol, and potassium ions were necessary for fatty acid uptake by whole cells. A reduction in uptake was observed in cells treated with valinomycin or dicyclohexylcarbodiimide. The effect of temperature on the rate of oleate uptake showed a discontinuity at 24 degrees C. Above 24 degrees C an energy of activation of 4.6 kcal (ca. 19.2 kJ)/mol was obtained. The data suggest that uptake of fatty acid by M. capricolum is an energy-linked, protein-mediated process. A membrane-bound enzyme activity that catalyzed the synthesis of fatty acyl-hydroxamate was demonstrated. This activity was virtually independent or only marginally dependent on coenzyme A, depending on the assay system, but was stimulated approximately twofold by ATP.     

Lipid interconversions in aging Mycoplasma capricolum cultures.

During the progression of Mycoplasma capricolum cultures from the early exponential to the stationary phase of growth, a decrease in the phospholipid-to-protein ratio and increases in both the unsaturated-to-saturated fatty acid ratio and the diphosphatidylglycerol (DPG)-to-phosphatidylglycerol (PG) ratio were found. The freedom of motion of spin-labeled fatty acids incorporated into the membrane remained unchanged throughout the growth cycle. The increase in DPG was almost stoichiometric with the decrease in PG. Furthermore, exogenous PG added to the medium was incorporated by the cells and partially converted to DPG. The DPG that was accumulated upon aging was always more unsaturated than the PG. This accumulation was enhanced in palmitic acid-poor media, but was inhibited even in aged cells when the cells were grown in palmitic acid-rich media, suggesting that the accumulation of DPG upon aging was associated with changes in the fatty acid composition of membrane lipids rather than with the transition of the cells from the exponential- to stationary-growth phase.     

Genomic variations of Mycoplasma capricolum subsp. capripneumoniae detected by amplified fragment length polymorphism (AFLP) analysis

The genetic diversity of Mycoplasma capricolum subsp. capripneumoniae strains based on determination of amplified fragment length polymorphisms (AFLP) is described. AFLP fingerprints of 38 strains derived from different countries in Africa and the Middle East consisted of over 100 bands in the size range of 40-500 bp. The similarity between individual AFLP profiles, calculated by Jaccard's coefficient, ranged from 0.92 to 1.0. On the basis of the polymorphisms detected, the analysed strains can explicitly be grouped into two major clusters, equivalent to two evolutionary lines of the organism found by 16S rDNA analysis. The present data support previous observations regarding genetic homogeneity of M. capricolum subsp. capripneumoniae, and confirm the two evolutionary lines of descent found by analysis of 16S rRNA genes.     

Molecular Evolution of Mycoplasma capricolum subsp. capripneumoniae Strains, Based on Polymorphisms in the 16S rRNA Genes

Mycoplasma capricolum subsp. capripneumoniae belongs to the so-called Mycoplasma mycoides cluster and is the causal agent of contagious caprine pleuropneumonia (CCPP). All members of the M. mycoides cluster have two rRNA operons. The sequences of the 16S rRNA genes of both rRNA operons from 20 strains of M. capricolum subsp. capripneumoniae of different geographical origins in Africa and Asia were determined. Nucleotide differences which were present in only one of the two operons (polymorphisms) were detected in 24 positions. The polymorphisms were not randomly distributed in the 16S rRNA genes, and some of them were found in regions of low evolutionary variability. Interestingly, 11 polymorphisms were found in all the M. capricolum subsp. capripneumoniae strains, thus defining a putative ancestor. A sequence length difference between the 16S rRNA genes in a poly(A) region and 12 additional polymorphisms were found in only one or some of the strains. A phylogenetic tree was constructed by comparative analysis of the polymorphisms, and this tree revealed two distinct lines of descent. The nucleotide substitution rate of strains within line II was up to 50% higher than within line I. A tree was also constructed from individual operonal 16S rRNA sequences, and the sequences of the two operons were found to form two distinct clades. The topologies of both clades were strikingly similar, which supports the use of 16S rRNA sequence data from homologous operons for phylogenetic studies. The strain-specific polymorphism patterns of the 16S rRNA genes of M. capricolum subsp. capripneumoniae may be used as epidemiological markers for CCPP.     

The ribosomal genes of Mycoplasma capricolum

The nucleotide sequence of 5S rRNA from Mycoplasma capricolum is more similar to that of the gram-positive bacteria than that of the gram-negative bacteria. The presence of two copies of rRNA genes in M. capricolum genome has been demonstrated. The two different rRNA gene clusters have been cloned in E. coli plasmid vectors and analyzed for the rRNA gene organizations, demonstrating that the gene arrangement is in the order of 16S, 23S, and 5S rDNA. The ribosomes of M. capricolum contain about 30 species of proteins in 50S and 20 in 30S subunits. The number and size of the ribosomal proteins are not significantly different from those of other eubacterial ribosomes.     

Physical mapping of the Mycoplasma capricolum genome

A physical map of Mycoplasma capricolum ATCC 27343 genome was constructed, based on estimation of the restriction fragment sizes by pulse-field electrophoresis. The linkage order of restriction fragments was determined by two-dimensional electrophoresis of partial and complete single digests and complete double digests and by Southern hybridization analysis. The genome size was established at 1155.5 kb, and 26 cleavage sites for 7 endonucleases were assigned to the map.     

Novel Small Stable RNAs of Mycoplasma capricolum

Two stable RNA species and their genes have been isolated fromMycoplasma capricolum, and the nucleotide sequences have beendetermined by partial RNA sequencing and sequencing of the genes.The RNAs are 92 and 105 nucleotides in length, respectively.The two RNAs reveal no sequence similarity to any stable RNAso far reported, indicating that these are novel RNA species.The RNAs, designated MCS2 and MCS3 RNA, exist in small amountsin the soluble fraction of the cell extract.     

Physical mapping of the ribosomal RNA genes of Mycoplasma capricolum.

Physical mapping of the rRNA genes of Mycoplasma capricolum was done by digestion of the mycoplasmal DNA with EcoRI, PstI and BglII and hybridization with nick-translated probes consisting of defined portions of the rrnB ribosomal RNA operon of Escherichia coli. The results indicate that the rRNA genes in the chromosome of M. capricolum are arranged in two clusters, each organized in the order 5'-16S-23S-5S-3', resembling the order of the genes in the rrnB operon, with no large spacer regions separating the genes in each cluster.     

Stable RNA synthesis and its control in Mycoplasma capricolum.

The synthesis of stable RNA in Mycoplasma capricolum was studied by [32P] labeling of cellular RNA of cells grown in a partially-defined medium in the presence or absence of an amino acid mixture supplement. The results indicate that M. capricolum employs the same stringent control mechanism used by E. coli cells, as judged by a decreased synthesis of stable RNA and accumulation of 5'-triphosphoguanosine-3'-diphosphate (pppGpp) and 5'-diphosphoguanosine-3'-diphosphate (ppGpp) in response to amino acid starvation. In addition, the results suggest that precursors of stable RNA accumulate and an intracellular pool of the precursors exists at all times under the growth conditions used by us. These findings may be interpreted to reflect a slow rate of RNA processing in M. capricolum.