A molecular epidemiological investigation of contagious caprine pleuropneumonia in goats and captive Arabian sand gazelle (Gazella marica) in Oman.

BACKGROUND: Contagious caprine pleuropneumonia (CCPP) is a fatal WOAH-listed, respiratory disease in small ruminants with goats as primary hosts that is caused by Mycoplasma capricolum subspecies capripneumoniae (Mccp). Twelve CCPP outbreaks were investigated in 11 goat herds and a herd of captive Arabian sand gazelle (Gazella marica) in four Omani governorates by clinical pathological and molecular analysis to compare disease manifestation and Mccp genetic profiles in goats and wild ungulates. RESULTS: The CCPP forms in diseased and necropsied goats varied from peracute (5.8%), acute (79.2%) and chronic (4.5%) while all of the five necropsied gazelles showed the acute form based on the clinical picture, gross and histopathological evaluation. Colonies of Mccp were recovered from cultured pleural fluid, but not from lung tissue samples of one gazelle and nine goats and all the isolates were confirmed by Mccp-specific real time PCR. Whole genome-single nucleotide polymorphism (SNP) analysis was performed on the ten isolates sequenced in this study and twenty sequences retrieved from the Genbank database. The Mccp strains from Oman clustered all in phylogroup A together with strains from East Africa and one strain from Qatar. A low variability of around 125 SNPs was seen in the investigated Omani isolates from both goats and gazelles indicating mutual transmission of the pathogen between wildlife and goats. CONCLUSION: Recent outbreaks of CCPP in Northern Oman are caused by Mccp strains of the East African Phylogroup A which can infect goats and captive gazelles likewise. Therefore, wild and captive ungulates should be considered as reservoirs and included in CCPP surveillance measures.

Multi-locus sequence analysis reveals great genetic diversity among Mycoplasma capricolum subsp. capripneumoniae strains in Asia.

Multi-Locus Sequence Analysis (MLSA) of Mycoplasma capricolum subsp. capripneumoniae (Mccp) strains from Asia revealed unforeseen diversity and a central position for genotyping groups representing strains from Central/East Asia, suggesting a possible origin of contagious caprine pleuropneumonia in this continent. A better assessment of the emergence, diversity and distribution of Mccp in Asia and Africa calls for renewed efforts to dramatically enlarge the sample of strains. Availability and affordability in the field, added to superior typeability (directly from poor samples) and high stability, discriminatory power and concordance with epidemiological and phylogenetic analyses, make MLSA an excellent tool for such investigations.

Structure of a tRNA-specific deaminase with compromised deamination activity.

Nucleotide 34 in tRNA is extensively modified to ensure translational fidelity and efficacy in cells. The deamination of adenosine at this site catalyzed by the enzyme TadA gives rise to inosine (I), which serves as a typical example of the wobble hypothesis due to its diverse basepairing capability. However, recent studies have shown that tRNAArgACG in Mycoplasma capricolum contains unmodified adenosine, in order to decode the CGG codon. The structural basis behind the poorly performing enzyme M. capricolum TadA (McTadA) is largely unclear. Here we present the structures of the WT and a mutant form of McTadA determined at high resolutions. Through structural comparison between McTadA and other active TadA enzymes as well as modeling efforts, we found that McTadA presents multiple structural conflicts with RNA substrates and thus offered support to previous studies from a structural perspective. These clashes would potentially lead to reduced substrate binding affinity of McTadA, consistent with our in vitro deamination activity and binding assays. To rescue the deamination activity of McTadA, we carried out two rounds of protein engineering through structure-guided design. The unsuccessful attempts of the activity restoration could be attributed to the altered dimer interface and stereo hindrance from the non-catalytic subunit of McTadA, which could be the inevitable outcome of the natural evolution. Our study provides structural insight into an alternative decoding and evolutionary strategy by a compromised TadA enzyme at a molecular level.

Direct Transfer of a Mycoplasma mycoides Genome to Yeast Is Enhanced by Removal of the Mycoides Glycerol Uptake Factor Gene glpF.

We previously discovered that intact bacterial chromosomes can be directly transferred to a yeast host cell where they can propagate as centromeric plasmids by fusing bacterial cells with S accharomyces cerevisiae spheroplasts. Inside the host any desired number of genetic changes can be introduced into the yeast centromeric plasmid to produce designer genomes that can be brought to life using a genome transplantation protocol. Earlier research demonstrated that the removal of restriction-systems from donor bacteria, such as Mycoplasma mycoides, Mycoplasma capricolum, or Haemophilus influenzae increased successful genome transfers. These findings suggested that other genetic factors might also impact the bacteria-to-yeast genome transfer process. In this study, we demonstrated that the removal of a particular genetic factor, the glycerol uptake facilitator protein gene glpF from M. mycoides, significantly increased direct genome transfer by up to 21-fold. Additionally, we showed that intact bacterial cells were endocytosed by yeast spheroplasts producing organelle-like structures within these yeast cells. These might lead to the possibility of creating novel synthetic organelles.

23S rRNA and L22 ribosomal protein are involved in the acquisition of macrolide and lincosamide resistance in Mycoplasma capricolum subsp. capricolum.

Mycoplasma capricolum subsp. capricolum (Mcc) is one of the causative agents of contagious agalactia, and antimicrobial therapy is the most commonly applied measure to treat outbreaks of this disease. Macrolides and lincosamides bind specifically to nucleotides at domains II and V of the 23S rRNA. Furthermore, rplD and rplV genes encode ribosomal proteins L4 and L22, which are also implicated in the macrolide binding site. The aim of this work was to study the relationship between mutations in these genes and the acquisition of macrolide and lincosamide resistance in Mcc. For this purpose, in vitro selected resistant mutants and field isolates were studied. This study demonstrates the appearance of DNA point mutations at the 23S rRNA encoding genes (A2058G, A2059G and A2062C) and rplV gene (Ala89Asp) in association to high minimum inhibitory concentration values. Hence, it proves the importance of alterations in 23S rRNA domain V and ribosomal protein L22 as molecular mechanisms responsible for the acquisition of macrolide and lincosamide resistance in both field isolates and in vitro selected mutants. Moreover, these mutations enable us to provide an interpretative breakpoint of antimicrobial resistance for Mcc at MIC 0.8 µg/ml.

Resistance mechanisms against quinolones in Mycoplasma capricolum subsp. capricolum.

Quinolones interact with bacterial DNA gyrase and topoisomerase IV, the subunits of which are encoded by gyrA/gyrB and parC/parE, respectively. The aim of this study was to evaluate the relationship between changes in these genes and quinolone susceptibility of Mycoplasma capricolum subsp. capricolum (Mcc). Using in vitro selected resistant mutants and field isolates from goats, predicted amino acid changes in gyrA, gyrB and parC were associated with higher minimum inhibitory concentration values for quinolones. Alterations in parC predicted amino acid sequences were most frequently associated with quinolone resistance in Mcc.

Antimicrobial susceptibility and multilocus sequence typing of Mycoplasma capricolum subsp. capricolum.

Mycoplasma capricolum subsp. capricolum is one of the causative agents of contagious agalactia (CA). Nevertheless, there is still a lack of information about its antimicrobial susceptibility and genetic characteristics. Therefore, the aim of this work was to study the antimicrobial and genetic variability of different Mycoplasma capricolum subsp. capricolum field isolates. For this purpose, the growth inhibition effect of 18 antimicrobials and a multilocus sequence typing (MLST) scheme based on five housekeeping genes (fusA, glpQ, gyrB, lepA and rpoB) were performed on 32 selected field isolates from Italy and Spain.The results showed a wide range of growth inhibitory effects for almost all the antimicrobials studied. Macrolides presented lower efficacy inhibiting Mcc growth than in previous works performed on other CA-causative mycoplasmas. Erythromycin was not able to inhibit the growth of any of the studied strains, contrary to doxycycline, which inhibited the growth of all of them from low concentrations. On the other hand, the study of the concatenated genes revealed a high genetic variability among the different Mcc isolates. Hence, these genetic variations were greater than the ones reported in prior works on other mycoplasma species.

Contagious caprine pleuropneumonia: The first isolation and molecular characterization of Mycoplasma capricolum subsp. capripneumoniae in the Kingdom of Saudi Arabia.

Classical contagious caprine pleuropneumonia is one of the most fatal contagious disease of goats listed by World Organization for Animal Health that leads to major economic losses. It is caused by infection with Mycoplasma capricolum subspecies capripneumoniae. In order to isolate the causative agents of CCPP for the first time in the Kingdom of Saudi Arabia, fifteen flocks from Eastern region (Al Ahsa, Dammam and Hafr Albaten) and ten flocks from Riyadh and Al-Kharj regions were selected for this study. A total of 700 samples (400 nasal swabs, 300 pleural fluid samples and lung samples (from necropsied animals)) were collected from goats showing typical signs of CCPP. The clinical signs of diseased cases revealed serous to mucoid nasal discharge, coughing, dyspnea, frothy salivation, and fever (40-42°C). Necropsied animals showed fibrinous pleuropneumonia and increased pleural fluid. Of 400 nasal swabs, 190 pleural fluid, and 110 lung samples, 26 (6.5%), 31 (16.3%) and 19 (17.3%) Mycoplasma isolates were recovered, respectively. Biochemically, all isolates were sensitive to digitonin and fermented glucose. Sixty seven of Mycoplasma isolates were belonged to Mycoplasma mycoides cluster based on detection of 16S rRNA. Polymerase chain reaction screening of Mycoplasma isolates using specific primer for M. capricolum subsp. capripneumoniae confirmed 55 isolates to be M. capricolum subsp. capripneumoniae.

Translational Quality Control by Bacterial Threonyl-tRNA Synthetases.

Translational fidelity mediated by aminoacyl-tRNA synthetases ensures the generation of the correct aminoacyl-tRNAs, which is critical for most species. Threonyl-tRNA synthetase (ThrRS) contains multiple domains, including an N2 editing domain. Of the ThrRS domains, N1 is the last to be assigned a function. Here, we found that ThrRSs from Mycoplasma species exhibit differences in their domain composition and editing active sites compared with the canonical ThrRSs. The Mycoplasma mobile ThrRS, the first example of a ThrRS naturally lacking the N1 domain, displays efficient post-transfer editing activity. In contrast, the Mycoplasma capricolum ThrRS, which harbors an N1 domain and a degenerate N2 domain, is editing-defective. Only editing-capable ThrRSs were able to support the growth of a yeast thrS deletion strain (ScΔthrS), thus suggesting that ScΔthrS is an excellent tool for studying the in vivo editing of introduced bacterial ThrRSs. On the basis of the presence or absence of an N1 domain, we further revealed the crucial importance of the only absolutely conserved residue within the N1 domain in regulating editing by mediating an N1-N2 domain interaction in Escherichia coli ThrRS. Our results reveal the translational quality control of various ThrRSs and the role of the N1 domain in translational fidelity.

Impact of donor-recipient phylogenetic distance on bacterial genome transplantation.

Genome transplantation (GT) allows the installation of purified chromosomes into recipient cells, causing the resulting organisms to adopt the genotype and the phenotype conferred by the donor cells. This key process remains a bottleneck in synthetic biology, especially for genome engineering strategies of intractable and economically important microbial species. So far, this process has only been reported using two closely related bacteria, Mycoplasma mycoides subsp. capri (Mmc) and Mycoplasma capricolum subsp. capricolum (Mcap), and the main factors driving the compatibility between a donor genome and a recipient cell are poorly understood. Here, we investigated the impact of the evolutionary distance between donor and recipient species on the efficiency of GT. Using Mcap as the recipient cell, we successfully transplanted the genome of six bacteria belonging to the Spiroplasma phylogenetic group but including species of two distinct genera. Our results demonstrate that GT efficiency is inversely correlated with the phylogenetic distance between donor and recipient bacteria but also suggest that other species-specific barriers to GT exist. This work constitutes an important step toward understanding the cellular factors governing the GT process in order to better define and eventually extend the existing genome compatibility limit.

One-Step Multiplex RT-qPCR Assay for the Detection of Peste des petits ruminants virus, Capripoxvirus, Pasteurella multocida and Mycoplasma capricolum subspecies (ssp.) capripneumoniae.

Respiratory infections, although showing common clinical symptoms like pneumonia, are caused by bacterial, viral or parasitic agents. These are often reported in sheep and goats populations and cause huge economic losses to the animal owners in developing countries. Detection of these diseases is routinely done using ELISA or microbiological methods which are being reinforced or replaced by molecular based detection methods including multiplex assays, where detection of different pathogens is carried out in a single reaction. In the present study, a one-step multiplex RT-qPCR assay was developed for simultaneous detection of Capripoxvirus (CaPV), Peste de petits ruminants virus (PPRV), Pasteurella multocida (PM) and Mycoplasma capricolum ssp. capripneumonia (Mccp) in pathological samples collected from small ruminants with respiratory disease symptoms. The test performed efficiently without any cross-amplification. The multiplex PCR efficiency was 98.31%, 95.48%, 102.77% and 91.46% whereas the singleplex efficiency was 93.43%, 98.82%, 102.55% and 92.0% for CaPV, PPRV, PM and Mccp, respectively. The correlation coefficient was greater than 0.99 for all the targets in both multiplex and singleplex. Based on cycle threshold values, intra and inter assay variability, ranged between the limits of 2%-4%, except for lower concentrations of Mccp. The detection limits at 95% confidence interval (CI) were 12, 163, 13 and 23 copies/reaction for CaPV, PPRV, PM and Mccp, respectively. The multiplex assay was able to detect CaPVs from all genotypes, PPRV from the four lineages, PM and Mccp without amplifying the other subspecies of mycoplasmas. The discriminating power of the assay was proven by accurate detection of the targeted pathogen (s) by screening 58 viral and bacterial isolates representing all four targeted pathogens. Furthermore, by screening 81 pathological samples collected from small ruminants showing respiratory disease symptoms, CaPV was detected in 17 samples, PPRV in 45, and PM in six samples. In addition, three samples showed a co-infection of PPRV and PM. Overall, the one-step multiplex RT-qPCR assay developed will be a valuable tool for rapid detection of individual and co-infections of the targeted pathogens with high specificity and sensitivity.

A large-scale genomic approach affords unprecedented resolution for the molecular epidemiology and evolutionary history of contagious caprine pleuropneumonia.

Contagious caprine pleuropneumonia (CCPP), caused by Mycoplasma capricolum subsp. capripneumoniae (Mccp), is a devastating disease of domestic goats and of some wild ungulate species. The disease is currently spreading in Africa and Asia and poses a serious threat to disease-free areas. A comprehensive view of the evolutionary history and dynamics of Mccp is essential to understand the epidemiology of CCPP. Yet, analysing the diversity of genetically monomorphic pathogens, such as Mccp, is complicated due to their low variability. In this study, the molecular epidemiology and evolution of CCPP was investigated using a large-scale genomic approach based on next-generation sequencing technologies, applied to a sample of strains representing the global distribution of this disease. A highly discriminatory multigene typing system was developed, allowing the differentiation of 24 haplotypes among 25 Mccp strains distributed in six genotyping groups, which showed some correlation with geographic origin. A Bayesian approach was used to infer the first robust phylogeny of the species and to date the principal events of its evolutionary history. The emergence of Mccp was estimated only at about 270 years ago, which explains the low genetic diversity of this species despite its high mutation rate, evaluated at 1.3 × 10(-6) substitutions per site per year. Finally, plausible scenarios were proposed to elucidate the evolution and dynamics of CCPP in Asia and Africa, though limited by the paucity of Mccp strains, particularly in Asia. This study shows how combining large-scale genomic data with spatial and temporal data makes it possible to obtain a comprehensive view of the epidemiology of CCPP, a precondition for the development of improved disease surveillance and control measures.

Nucleotide composition bias and codon usage trends of gene populations in Mycoplasma capricolum subsp. capricolum and M. Agalactiae.

Because of the low GC content of the gene population, amino acids of the two mycoplasmas tend to be encoded by synonymous codons with an A or T end. Compared with the codon usage of ovine, Mycoplasma capricolum and M. agalactiae tend to select optimal codons, which are rare codons in ovine. Due to codon usage pattern caused by genes with key biological functions, the overall codon usage trends represent a certain evolutionary direction in the life cycle of the two mycoplasmas. The overall codon usage trends of a gene population of M. capricolum subsp. capricolum can be obviously separated from other mycoplasmas, and the overall codon usage trends of M. agalactiae are highly similar to those of M. bovis. These results partly indicate the independent evolution of the two mycoplasmas without the limits of the host cell's environment. The GC and AT skews estimate nucleotide composition bias at different positions of nucleotide triplets and the protein consideration caused by the nucleotide composition bias at codon positions 1 and 2 largely take part in synonymous codon usage patterns of the two mycoplasmas. The correlation between the codon adaptation index and codon usage variation indicates that the effect of codon usage on gene expression in M. capricolum subsp. capricolum is opposite to that of M. agalactiae, further suggesting independence of the evolutionary process influencing the overall codon usage trends of gene populations of mycoplasmas.

Comparative analysis of the Mycoplasma capricolum subsp. capricolum GM508D genome reveals subrogation of phase-variable contingency genes and a novel integrated genetic element.

Mycoplasma capricolum subspecies capricolum is both a pathogen of small ruminants and a model recipient organism for gene transplantation and synthetic biology. With the availability of the complete genome of the type strain California kid (released in 2005), a draft genome of strain GM508D was determined to investigate genomic variation in this subspecies. Differences in mobile genetic element location and complement, catabolic pathway genes, contingency loci, surface antigen genes and type II restriction-modification systems highlight the plasticity and diversity within this taxon.

Discrimination between Mycoplasma mycoides subsp. capri and Mycoplasma capricolum subsp. capricolum using PCR-RFLP and PCR.

In this study, the dihydrolipoyl dehydrogenase (lpdA) gene was used to distinguish Mycoplasma mycoides subsp. capri (Mmc) from Mycoplasma capricolum subsp. capricolum (Mcc), two of four Mycoplasma species that cause contagious agalactia in sheep and goats. After alignment of nucleotide sequences of both species, specific primer sets were designed from unchanging and variable gene segments. The first primer set LPD-C1-F/LPD-C1-R was used to amplify a 911 bp fragment that was subsequently co-digested with FastDigest PstI, SspI, EcoRI and ClaI enzymes. The PCR-RFLP profiles differentiated the two mycoplasma species. The second primer set was used to distinguish Mmc from Mcc by single tube PCR. Both methods were further applied to identify 54 isolates collected from dairy herds from different provinces in Sardinia. The results of this study showed that PCR-RFLP and PCR could be used in routine diagnosis for rapid and specific simultaneous discrimination of Mmc and Mcc.

The 20th anniversary of proteomics and some of its origins.

The term "proteome" was first introduced into the scientific literature in July 1995. Almost 20 years ago attempts to characterize the "total protein complement able to be encoded by a given genome" only became possible due to privileged access to what were then the world's most complete sets of genomic data. Today, proteomics has become an important pillar in the fields of disease diagnosis and drug research and development, while also playing a critical role in the much larger field of Healthcare Analytics and Biomarker Discovery and Detection. It is important to note that this industry originated mostly from building blocks in analytical science that predated the term "proteomics" by many decades. However, proteomics, as a discipline, has allowed protein scientists to more favorably compete in the face of highly fashionable Big Science and, more specifically, genomics.

Fatal transmission of contagious caprine pleuropneumonia to an Arabian oryx (Oryx leucoryx).

Contagious caprine pleuropneumonia (CCPP) is an infectious respiratory disease mainly affecting domestic goats. As CCPP has never been documented in grazing antelopes (subfamily hippotraginae), they were not considered susceptible. Mycoplasma capricolum subspecies capripneumoniae (Mccp) was isolated from pleural liquid collected during the necropsy of a severely emaciated Arabian oryx with mild nasal discharge. The Mccp isolate was then genotyped using a multilocus sequence scheme; the sequence type was identical to the Mccp strain previously identified in a sand gazelle from a nearby enclosure. This case shows for the first time that members of the hippotraginae subfamily, here the Arabian oryx, can be affected by CCPP. In addition, genotyping shows that the oryx was most probably infected, at a distance, by sand gazelles.

The flavoprotein Mcap0476 (RlmFO) catalyzes m5U1939 modification in Mycoplasma capricolum 23S rRNA.

Efficient protein synthesis in all organisms requires the post-transcriptional methylation of specific ribosomal ribonucleic acid (rRNA) and transfer RNA (tRNA) nucleotides. The methylation reactions are almost invariably catalyzed by enzymes that use S-adenosylmethionine (AdoMet) as the methyl group donor. One noteworthy exception is seen in some bacteria, where the conserved tRNA methylation at m5U54 is added by the enzyme TrmFO using flavin adenine dinucleotide together with N5,N10-methylenetetrahydrofolate as the one-carbon donor. The minimalist bacterium Mycoplasma capricolum possesses two homologs of trmFO, but surprisingly lacks the m5U54 tRNA modification. We created single and dual deletions of the trmFO homologs using a novel synthetic biology approach. Subsequent analysis of the M. capricolum RNAs by mass spectrometry shows that the TrmFO homolog encoded by Mcap0476 specifically modifies m5U1939 in 23S rRNA, a conserved methylation catalyzed by AdoMet-dependent enzymes in all other characterized bacteria. The Mcap0476 methyltransferase (renamed RlmFO) represents the first folate-dependent flavoprotein seen to modify ribosomal RNA.

Transfer RNA misidentification scrambles sense codon recoding.

Sense codon recoding is the basis for genetic code expansion with more than two different noncanonical amino acids. It requires an unused (or rarely used) codon, and an orthogonal tRNA synthetase:tRNA pair with the complementary anticodon. The Mycoplasma capricolum genome contains just six CGG arginine codons, without a dedicated tRNA(Arg). We wanted to reassign this codon to pyrrolysine by providing M. capricolum with pyrrolysyl-tRNA synthetase, a synthetic tRNA with a CCG anticodon (tRNA(Pyl)(CCG)), and the genes for pyrrolysine biosynthesis. Here we show that tRNA(Pyl)(CCG) is efficiently recognized by the endogenous arginyl-tRNA synthetase, presumably at the anticodon. Mass spectrometry revealed that in the presence of tRNA(Pyl)(CCG), CGG codons are translated as arginine. This result is not unexpected as most tRNA synthetases use the anticodon as a recognition element. The data suggest that tRNA misidentification by endogenous aminoacyl-tRNA synthetases needs to be overcome for sense codon recoding.