Highly Dynamic Genomic Loci Drive the Synthesis of Two Types of Capsular or Secreted Polysaccharides within the Mycoplasma mycoides Cluster

Mycoplasmas of the Mycoplasma mycoides cluster are all ruminant pathogens. Mycoplasma mycoides subsp. mycoides is responsible for contagious bovine pleuropneumonia and is known to produce capsular polysaccharide (CPS) and exopolysaccharide (EPS). Previous studies have strongly suggested a role for Mycoplasma mycoides subsp. mycoides polysaccharides in pathogenicity. Mycoplasma mycoides subsp. mycoides-secreted EPS was recently characterized as a β(1→6)-galactofuranose homopolymer (galactan) identical to the capsular product. Here, we extended the characterization of secreted polysaccharides to all other members of the M. mycoides cluster: M. capricolum subsp. capripneumoniae, M. capricolum subsp. capricolum, M. leachii, and M. mycoides subsp. capri (including the LC and Capri serovars). Extracted EPS was characterized by nuclear magnetic resonance, resulting in the identification of a homopolymer of β(1→2)-glucopyranose (glucan) in M. capricolum subsp. capripneumoniae and M. leachii. Monoclonal antibodies specific for this glucan and for the Mycoplasma mycoides subsp. mycoides-secreted galactan were used to detect the two polysaccharides. While M. mycoides subsp. capri strains of serovar LC produced only capsular galactan, no polysaccharide could be detected in strains of serovar Capri. All strains of M. capricolum subsp. capripneumoniae and M. leachii produced glucan CPS and EPS, whereas glucan production and localization varied among M. capricolum subsp. capricolum strains. Genes associated with polysaccharide synthesis and forming a biosynthetic pathway were predicted in all cluster members. These genes were organized in clusters within two loci representing genetic variability hot spots. Phylogenetic analysis showed that some of these genes, notably galE and glf, were acquired via horizontal gene transfer. These findings call for a reassessment of the specificity of the serological tests based on mycoplasma polysaccharides.     

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.     

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 β-galactosidase, and the gene encoding spiralin, an abundant surface lipoprotein of the related mollicute Spiroplasma citri, were successfully expressed. Functional Escherichia coli β-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.     

Further evidence to justify reassignment of Mycoplasma mycoides subspecies mycoides Large Colony type to Mycoplasma mycoides subspecies capri

Analysis, using the polymerase chain reaction (PCR), restriction enzyme endonuclease analysis (REA), protein profile patterns, random amplification of polymorphic DNA (RAPD) fingerprinting, 16S rRNA gene sequencing and antisera growth inhibition tests, of 22 strains of Mycoplasma mycoides subsp. mycoides Large Colony type (MmmLC) and eight strains of M. mycoides subsp. capri (Mmc) are presented, along with a summary of comparative data from the literature for over 100 strains, all of which supports the reclassification of the MmmLC and Mmc strains into the single subspecies, M. mycoides subspecies capri.     

Host specificity of mollicutes oriC plasmids: functional analysis of replication origin

Recently, artificial oriC plasmids containing the chromosomal dnaA gene and surrounding DnaA box sequences were obtained for the mollicutes Spiroplasma citri and Mycoplasma pulmonis. In order to study the specificity of these plasmids among mollicutes, a set of similar oriC plasmids was developed for three mycoplasmas belonging to the mycoides cluster, Mycoplasma mycoides subsp. mycoides LC (MmmLC), M.mycoides subsp. mycoides SC (MmmSC) and Mycoplasma capricolum subsp. capricolum. Mycoplasmas from the mycoides cluster, S.citri and M.pulmonis were used as recipients for transformation experiments by homologous and heterologous oriC plasmids. All five mollicutes were successfully transformed by homologous plasmids, suggesting that the dnaA gene region represents the functional replication origin of the mollicute chromosomes. However, the ability of mollicutes to replicate heterologous oriC plasmids was found to vary noticeably with the species. For example, the oriC plasmid from M.capricolum did not replicate in the closely related species MmmSC and MmmLC. In contrast, plasmids harbouring the oriC from MmmSC, MmmLC and the more distant species S.citri were all found to replicate in M.capricolum. Our results suggest that the cis-elements present in oriC sequences are not the only determinants of this host specificity.     

Experimental Contagious Caprine Pleuropneumonia: A Long Term Study on the Course of Infection and Pathology in a Flock of Goats Infected with <Emphasis Type="Italic">Mycoplasma capricolum</Emphasis> subsp. <Emphasis Type="Italic">capripneumoniae</Emphasis>

Contagious caprine pleuropneumonia (CCPP) is a major threat to goat farming in parts of Africa and Asia. It classically causes acute high morbidity and mortality early in infection, but little is known of its long term epizootiology and course. In this study, 10 goats were inoculated with Mycoplasma capricolum subsp. capripneumoniae (M. capripneumoniae) and then mixed with 15 goats for contact transmission. The disease course was monitored in each goat for 56–105 days, whereafter the goats were killed and necropsied. Varying features signifying infection occurred in altogether 17 goats (7 inoculated, 10 in-contact). Clinical signs were severe in 8 goats but no fatalities occurred. Only 6 goats had serum antibody titres against M. capripneumoniae in ELISA. Fourteen goats (5 inoculated, 9 in-contact) had chronic pleuropulmonary lesions compatible with CCPP at necropsy and 7 of those showed M. capripneumoniae antigen in the lung by immunohistochemistry. Neither cultivation nor PCR tests were positive for the agent in any goat. The results indicate that the clinical course of CCPP in a flock may be comparatively mild, M. capripneumoniae- associated lung lesions may be present at a late stage of infection, and chronic infection may occur without a significant serological response.     

Caprine mycoplasmal mastitis in Nigeria

In a study to investigate the current status of intramammary mycoplasmosis in caprine udders in Nigeria, a total of 57 and 24 milk samples were collected from udders of goats affected by mastitis and from apparently normal goats’ udders, respectively. Acute and chronic mastitis were more commonly observed in goats between 1 and 3 years old. Mycoplasma agalactiae and Mycoplasma capricolum occurred at a significantly higher rate (p<0.05) in udders affected by mastitis than in normal healthy udders. Other mycoplasma occurring in low prevalence include Mycoplasma bovis and Mycoplasma mycoides subsp. mycoides LC. It is concluded that cultural (microbiological) surveillance is necessary for effective treatment and control of the disease in Nigeria.     

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.     

Disruption of the S41 Peptidase Gene in Mycoplasma mycoides capri Impacts Proteome Profile,H2O2 Production,and Sensitivity to Heat Shock

Members of the Mycoplasma mycoides cluster are among the most virulent of the mycoplasmas, causing worldwide economically significant diseases of cattle and goats. A distinguishing phenotype among the members of the cluster is the ability to degrade casein. The MMCAP2_0241 gene, an S41 peptidase, confers the proteolytic phenotype in Mycoplasma mycoides subsp. capri GM12. In order to determine the impact of disruption of the gene, we used differential proteome profiling to compare the M. mycoides subsp. capri wild type with a mutant lacking the proteolytic phenotype. Disruption of MMCAP2_0241 resulted in altered phenotypes reminiscent of M. mycoides subsp. mycoides SC and had significant impacts on the proteome profile of the microbe. The mutant exhibited increased production of hydrogen peroxide, decreased lactate dehydrogenase activity, and increased sensitivity to heat shock.     

Nucleotide sequence of the rrnB 16S ribosomal RNA gene from Mycoplasma capricolum

Summary The nucleotide sequences of the rrnB 16S ribosomal RNA gene and its 5-and 3-flanking regions from Mycoplasma capricolum have been determined. The coding sequence is 1521 base pairs long, being 21 base pairs shorter than that of the Scherichia coli 16S rRNA gene. The 16S rRNA sequence of M. capricolum reveals 74% and 76% identity with that of E. coli and Anacystis nidulans, respectively. The secondary structure model constructed from the M. capricolum 16S rRNA.gene sequence resembles that proposed for E. coli 16S rRNA. A large stem structure can be constructed between the 5- and 3-flanking sequences of the 16S rRNA gene. The flanking regions are extremely rich in AT.     

The number of ribosomal RNA genes in Mycoplasma capricolum

Summary We have examined the number of rRNA genes in Mycoplasma capricolum (KID) by hybridization of BglII-, EcoRI- and XbaI-digests of DNA to [3-³²P] 16S, 23S and 5S rRNAs according to the Southern procedure (1975). All the restriction gels gave two radioactive bands with three kinds of rRNA. Furthermore, band positions were indistinguishable from one another when 16S, 23S and 5S rRNAs were used as probes, indicating that each band contains sequences corresponding to the 3-termini of 16S, 23S and 5S rRNAs. It is thus concluded that Mycoplasma capricolum chromosome carries at least two sets of genes for 16S, 23S and 5S rRNAs.     

Organization of ribosomal RNA genes in Mycoplasma capricolum

Summary DNA segments carrying rRNA genes of Mycoplasma capricolum have been cloned and characterized by restriction endonuclease mapping, DNA-RNA hybridization and nucleotide sequencing. The M. capricolum genome has two sets of rRNA gene clusters, where the arrangement is in the order of (5)16S-23S-5S(3). The spacer region between 16S and 23S rDNA is extremely rich in AT and does not carry any tRNA genes. Present address: Division of Hematology and Immunology of Internal Medicine, Kanazawa Medical University, Uchinada-Cho, Kahoku-Gun Ishikawa Pref. 920-02, Japan     

Transformation of Mycoplasma capricolum and Examination of DNA Restriction Modification in M. capricolum and Mycoplasma mycoides subsp. mycoides

Plasmids pIKΔ and pIKΔ-erm have recently been developed as mycoplasmal cloning vectors. In this report, we demonstrate that these plasmids can replicate in Mycoplasma capricolum, a mycoplasmal species for which transformation had not previously been characterized. Both plasmids are stably maintained at a higher copy number than in their parental species, Mycoplasma mycoides subsp. mycoides. We have also examined the possibility of one or more restriction-modification systems affecting transformation frequencies in both species.     

Detection of Mycoplasma mycoides subsp. mycoides SC in bronchoalveolar lavage fluids of cows based on a TaqMan real-time PCR discriminating wild type strains from an lppQ mutant vaccine strain used for DIVA-strategies

Contagious bovine pleuropneumonia (CBPP) is the most serious cattle disease in Africa, caused by Mycoplasma mycoides subsp. mycoides small-colony type (SC). CBPP control strategies currently rely on vaccination with a vaccine based on live attenuated strains of the organism. Recently, an lppQ⁻ mutant of the existing vaccine strain T1/44 has been developed (Janis et al., 2008). This T1lppQ⁻ mutant strain is devoid of lipoprotein LppQ, a potential virulence attribute of M. mycoides subsp. mycoides SC. It is designated as a potential live DIVA (Differentiating Infected from Vaccinated Animals) vaccine strain allowing both serological and etiological differentiation. The present paper reports on the validation of a control strategy for CBPP in cattle, whereby a TaqMan real-time PCR based on the lppQ gene has been developed for the direct detection of M. mycoides subsp. mycoides SC in ex vivo bronchoalveolar lavage fluids of cows and for the discrimination of wild type strains from the lppQ⁻ mutant vaccine strain.     

First report on the molecular prevalence of Mycoplasma capricolum subspecies capripneumoniae (Mccp) in goats the cause of contagious caprine pleuropneumonia (CCPP) in Balochistan province of Pakistan

Contagious caprine pleuropneumonia (CCPP) caused by Mycoplasma capricolum subspecies capripneumoniae (Mccp) is a disease of goats which causes high morbidity and mortality and is reported in many countries of the world. There are probably no reports on the molecular prevalence of Mccp, Mycoplasma capricolum subsp. capricolum (Mcc) and Mycoplasma putrefaciens (Mp) in Balochistan and any other part of Pakistan. Thirty goats (n = 30) with marked respiratory symptoms were selected and procured from forty goat flocks in Pishin district of Balochistan in 2008. The genomic deoxyribonucleic acid (DNA) from the lung samples (n = 30) of the slaughtered goats was purified and subjected to polymerase chain reaction (PCR) assays for the presence of Mycoplasma mycoides cluster members and Mp. The PCR-RFLP (restriction fragment length polymorphism) was also used to further confirm the Mccp. Of the thirty lung samples 17 (56.67%) were positive for the molecular prevalence of Mcc, Mccp and Mp. In total the molecular prevalence was observed as 17.65% for Mccp (n = 3), 70.59% for Mcc (n = 12) and 11.76% for Mp (n = 2). The RFLP profile has also validated the PCR results of Mccp by yielding two bands of 190 and 126 bp. The results of PCR-RFLP coupled with the presence of fibrinous pleuropneumonia and pleurisy during postmortem of goats (n = 3) strongly indicated the prevalence of CCPP in this part of world. Moreover the prevalence of Mcc and Mp is also alarming in the study area. We report for the very first time the molecular prevalence of Mcc, Mccp, and Mp in the lung tissues of goats in the Pishin district of Balochistan, Pakistan.     

Proposal to reclassify the Streptomyces albidoflavus clade on the basis of multilocus sequence analysis and DNA–DNA hybridization,and taxonomic elucidation of Streptomyces griseus subsp. solvifaciens

The Streptomyces albidoflavus 16S rRNA gene clade contains 10 species and subspecies with identical 16S rRNA gene sequences and very similar numerical taxonomic data, including Streptomyces griseus subsp. solvifaciens. Type strains of this clade, as well as three CGMCC strains which were received as Streptomyces galilaeus, Streptomyces sioyaensis and Streptomyces vinaceus, respectively, that shared the same 16S rRNA gene sequences with the clade, were subjected to multilocus sequence analysis (MLSA), DNA–DNA hybridization (DDH) and phenotypic characterization for a comprehensive reevaluation. The 13 strains still formed a distinct, albeit loosely related, clade in the phylogenetic tree based on concatenated sequences of aptD, gyrB, recA, rpoB and trpB genes, supported by a high bootstrap value and different tree-making algorithms, with MLSA evolutionary distances ranging from 0 to 0.003. DDH values among these strains were well above the 70% cut-off point for species delineation. Based on the genotypic data of MLSA and DDH, combined with key phenotypic properties in common, it is proposed that the 10 species and subspecies of the S. albidoflavus clade, namely S. albidoflavus, S. canescens, S. champavatii, S. coelicolor, S. felleus, S. globisporus subsp. caucasicus, S. griseus subsp. solvifaciens, S. limosus, S. odorifer and S. sampsonii, should be merged into a single genomic species, for which the name S. albidoflavus is retained, and that the three strains S. galilaeus CGMCC 4.1320, S. sioyaensis CGMCC 4.1306 and S. vinaceus CGMCC 4.1305 should be assigned to S. albidoflavus as well. The results also indicated that MLSA could be the procedure of choice for distinguishing between species within Streptomyces 16S rRNA gene clades.     

Presence of contagious agalactia causing mycoplasmas in Spanish goat artificial insemination centres

Male goats admitted to artificial insemination centres come from herds that have shown no clinical symptoms of contagious agalactia (CA) for the last 6 mo. However, prior reports suggest that this control measure may not be completely effective. This study was designed to detect the presence of CA-causing mycoplasmas in 9 Spanish centres, comprising 159 goats (147 males and 12 teaser does) of 8 different breeds. A microbiological study was conducted during 8 mo on 448 samples (318 ear swabs, 119 semen samples and 11 milk samples). In 86 samples (84 swabs, 1 semen sample and 1 milk sample), CA-causative mycoplasmas were detected by PCR or culture, and 52 animals (49 goat males and 3 teaser does) tested positive. Most of these positive animals were auricular carriers (n = 50), mainly of Mycoplasma mycoides subsp. capri (Mmc), although some M. agalactiae (Ma) and, interestingly, M. capricolum subsp. capricolum (Mcc) carriers were also identified. At least 1 animal infected by CA-causing mycoplasmas was detected in 8 of the 9 centres (88.8%) although in most (66.7%) no infected animals or only 1 or 2 positive animals were identified. Our results indicate the presence of CA carriers as asymptomatic animals in reproductive programmes. These findings have already prompted efficient measures to detect and avoid the entry of these carriers in Spanish centres. We recommend similar measures for all centres in areas where CA is endemic.     

Structure and rearrangements of rRNA genes in chloroplast DNA in two strains of Euglena gracilis

Summary The organisation of the rRNA genes in the chloroplast genomes of two strains of Euglena gracilis were analyzed and compared. It was previously shown that the bacillaris strain contains three complete rrn (rRNA) operons (7) and that the Z-S strain contains one operon (21). Using heteroduplex analysis it was found that the bacillaris strain contains, apart from the three complete rrn operons, an extra 16S rRNA gene, an extra partial 23S rRNA gene sequence and an inverted duplication of a stretch within the 5S–16S spacer. In addition a short (<100 bp) inverted repeat sequence (13) which forms a stem/loop structure in single-stranded cpDNA was located between the 3-end of the extra 16S rRNA gene and the partial 23 S rRNA sequence.The Z-S strain differs from the bacillaris strain by a deletion of two units of the complete rrn operons. The region upstream of the single complete rrn operon, including the inverted repeats, the partial 23S and the extra 16S rRNA sequences is identical with the bacillaris strain.The only non-homology found in heteroduplexes between the SalI fragments of B of the two strains is the deletion-insertion loop which represents the two rrn operons. A small deletion loop was found occasionally in hetero-and in homoduplexes of both strands in the region of variable size. Apart from the deletion/insertion of two rrn operons the two genomes appear to be colinear as can be seen from partial denaturation mapping. The organisation of the rRNA genes of the two strains is compared with those of the Z strain and the bacillaris-ATCC strain.