Evolution of a minimal cell.

Possessing only essential genes, a minimal cell can reveal mechanisms and processes that are critical for the persistence and stability of life1,2. Here we report on how an engineered minimal cell3,4 contends with the forces of evolution compared with the Mycoplasma mycoides non-minimal cell from which it was synthetically derived. Mutation rates were the highest among all reported bacteria, but were not affected by genome minimization. Genome streamlining was costly, leading to a decrease in fitness of greater than 50%, but this deficit was regained during 2,000 generations of evolution. Despite selection acting on distinct genetic targets, increases in the maximum growth rate of the synthetic cells were comparable. Moreover, when performance was assessed by relative fitness, the minimal cell evolved 39% faster than the non-minimal cell. The only apparent constraint involved the evolution of cell size. The size of the non-minimal cell increased by 80%, whereas the minimal cell remained the same. This pattern reflected epistatic effects of mutations in ftsZ, which encodes a tubulin-homologue protein that regulates cell division and morphology5,6. Our findings demonstrate that natural selection can rapidly increase the fitness of one of the simplest autonomously growing organisms. Understanding how species with small genomes overcome evolutionary challenges provides critical insights into the persistence of host-associated endosymbionts, the stability of streamlined chassis for biotechnology and the targeted refinement of synthetically engineered cells2,7-9.

Contribution of tRNA sequence and modifications to the decoding preferences of E. coli and M. mycoides tRNAGlyUCC for synonymous glycine codons.

tRNA superwobbling, used by certain bacteria and organelles, is an intriguing decoding concept in which a single tRNA isoacceptor is used to decode all synonymous codons of a four-fold degenerate codon box. While Escherichia coli relies on three tRNAGly isoacceptors to decode the four glycine codons (GGN), Mycoplasma mycoides requires only a single tRNAGly. Both organisms express tRNAGly with the anticodon UCC, which are remarkably similar in sequence but different in their decoding ability. By systematically introducing mutations and altering the number and type of tRNA modifications using chemically synthesized tRNAs, we elucidated the contribution of individual nucleotides and chemical groups to decoding by the E. coli and M. mycoides tRNAGly. The tRNA sequence was identified as the key factor for superwobbling, revealing the T-arm sequence as a novel pivotal element. In addition, the presence of tRNA modifications, although not essential for providing superwobbling, was shown to delicately fine-tune and balance the decoding of synonymous codons. This emphasizes that the tRNA sequence and its modifications together form an intricate system of high complexity that is indispensable for accurate and efficient decoding.

Seroprevalence of contagious bovine pleuropneumonia (CBPP) in cattle from Karamoja region, North-eastern Uganda.

BACKGROUND: Contagious bovine pleuropneumonia [CBPP] is a transboundary animal disease of cattle caused by Mycoplasma mycoides subsp. mycoides [Mmm]. CBPP causes severe economic losses to livestock producers in sub-Saharan Africa mainly due to high mortality, morbidity, reduction in productivity as well as livestock trade restrictions. This study aimed at determining seroprevalence of Mmm in cattle from Karamoja region, north-eastern Uganda; data that are required to design and implement risk based CBPP control program. METHODS: We randomly collected blood samples from 2,300 cattle spread across Karamoja region. Serum was extracted and screened for antibodies against Mycoplasma mycoides subsp. mycoides [Mmm] using the competitive enzyme linked immunosorbent assay [cELISA]. RESULTS: A quarter [25.4%; 95% CI: 23.7-27.3] of the screened cattle [n = 2,300] were sero-positive for Mmm. Amudat and Kaabong districts recorded the lowest [12.3%] and highest [30.7%] Mmm seroprevalence respectively. Increasing age, overnight stay in cattle kraals and location [certain districts, villages, herds and sub counties] of the cattle herds, the factors that promote animal commingling, were the most significant risk factors of seroconversion with Mmm. CONCLUSION: Results from this study indicated a higher seroprevalence of Mmm in Karamoja region cattle herds. This could be due to the increased frequency of CBPP outbreaks in recent years. To be effective, CBPP vaccination programs should target high risk herds along the international borders and other hotspot areas [e.g., parishes or sub counties] where cattle commingling is high.

Multilocus sequence typing of pathogenic Mycoplasma mycoides subsp. capri reveals the predominance of a novel clonal complex among isolates from goats in India.

Mycoplasma mycoides subsp. capri (Mmc) typically causes pneumonia, mastitis, arthritis, keratitis and septicaemia in goats. Mortality associated with Mmc in goat flocks is lower compared to Mycoplasma capricolum subsp. capripneumoniae-associated respiratory infections. Case fatality rates associated with Mmc ranged from 9.8 to 26.8% among several states in India. Molecular epidemiology approaches aimed at genotyping help to identify the diversity of isolates involved in a disease. Ten clinical pathogenic Mmc isolates were analysed by multilocus sequence typing (MLST) for studying genotypic relationships with 50 isolates available from public databases. The MLST analysis indicates high genetic diversity among Mmc isolates. From a total number of 60 isolates, 43 six sequence types (STs) were recognized comprising of six STs from India and 37 STs from other geographical regions. MLST profiles of isolates revealed none of the STs observed in Indian isolates were shared with global isolates. Some of the STs representing Indian isolates (four STs) were clustered into a novel clonal complex 1 (CC1). Maintenance of genetically related STs forming CCs among the goat population in India for longer periods indicates disease causing potentiality of these isolates. Based on various recombination analysis, weak clonal relationship among Mmc isolates were identified. The present study has enlightened further steps in disease investigations and to design future control measures by employing prevalent genotypes as vaccine candidates against Mmc infections.

A Method for High-Throughput Measurements of Viscosity in Sub-micrometer-Sized Membrane Systems.

To unravel the underlying principles of membrane adaptation in small systems like bacterial cells, robust approaches to characterize membrane fluidity are needed. Currently available relevant methods require advanced instrumentation and are not suitable for high-throughput settings needed to elucidate the biochemical pathways involved in adaptation. We developed a fast, robust, and financially accessible quantitative method to measure the microviscosity of lipid membranes in bulk suspension using a commercially available plate reader. Our approach, which is suitable for high-throughput screening, is based on the simultaneous measurements of absorbance and fluorescence emission of a viscosity-sensitive fluorescent dye, 9-(2,2-dicyanovinyl)julolidine (DCVJ), incorporated into a lipid membrane. We validated our method using artificial membranes with various lipid compositions over a range of temperatures and observed values that were in good agreement with previously published results. Using our approach, we were able to detect a lipid phase transition in the ruminant pathogen Mycoplasma mycoides.

Diatom centromeres suggest a mechanism for nuclear DNA acquisition.

Centromeres are essential for cell division and growth in all eukaryotes, and knowledge of their sequence and structure guides the development of artificial chromosomes for functional cellular biology studies. Centromeric proteins are conserved among eukaryotes; however, centromeric DNA sequences are highly variable. We combined forward and reverse genetic approaches with chromatin immunoprecipitation to identify centromeres of the model diatom Phaeodactylum tricornutum We observed 25 unique centromere sequences typically occurring once per chromosome, a finding that helps to resolve nuclear genome organization and indicates monocentric regional centromeres. Diatom centromere sequences contain low-GC content regions but lack repeats or other conserved sequence features. Native and foreign sequences with similar GC content to P. tricornutum centromeres can maintain episomes and recruit the diatom centromeric histone protein CENH3, suggesting nonnative sequences can also function as diatom centromeres. Thus, simple sequence requirements may enable DNA from foreign sources to persist in the nucleus as extrachromosomal episomes, revealing a potential mechanism for organellar and foreign DNA acquisition.

Seroprevalence and determinants of contagious bovine pleuropneumonia in cattle in Burkina Faso.

Contagious bovine pleuropneumonia (CBPP) is a bacterial disease caused by Mycoplasma mycoides subsp. Mycoides. This disease affects ruminants mainly cattle with respiratory disorders as predominant symptoms. In Burkina Faso, this condition has been considered as enzootic since several years but data on its seroprevalence remains scares. This study aimed to establish the serological prevalence and determinants of CBPP in Burkina Faso in 2017. For this purpose, 3969 serum samples have been collected following a stratified sampling plan based on vaccination coverage in 12 regions, 84 communes, and 210 villages and analyzed using c-ELISA test. Individual seroprevalence was 16.91% (95% CI: 15.74-18.07%), while 84.5% (95% CI: 60.46-80.02%) of communes, chosen as epidemiological units were found positive. The individual prevalence was found to be associated with agro-ecological area (p < 0.05) and a prevalence of 18.70% (95% CI: 16.74-20.66%) was noted in Sahelian areas, while 15.79% (95% CI: 14.34-17.23%) was found in Soudanian areas. The prevalence was also significantly associated with vaccination coverage (p < 0.05) with a prevalence of 13.92% (95% CI: 11.66-16.18%), 19.21% (95% CI: 16.66-20.75%) and 11.61%(95% CI: 9.00-14.23%) for high, moderate, and low vaccination coverage respectively. The individual prevalence was respectively 16.97 (95% CI: 15.56-18.39%) and 17.13% (95% CI: 15.93-18.33%) for female and animals more than 2 years old. According to regions, the highest seroprevalence was found in Plateau Central region (38.18%, 95% CI: 29.1-47.26%), while the lowest was found in Centre-Est Region (7%, 95% CI: 4.5-9.5%). These prevalence data will allow us to adapt the ongoing strategy to control CBPP in Burkina Faso.

In vivo role of capsular polysaccharide in Mycoplasma mycoides.

Capsular polysaccharides have been confirmed to be an important virulence trait in many gram-positive and gram-negative bacteria. Similarly, they are proposed to be virulence traits in minimal Mycoplasma that cause disease in humans and animals. In the current study, goats were infected with the caprine pathogen Mycoplasma mycoides subsp. capri or an engineered mutant lacking the capsular polysaccharide, galactofuranose. Goats infected with the mutant strain showed only transient fever. In contrast, 5 of 8 goats infected with the parental strain reached end-point criteria after infection. These findings confirm that galactofuranose is a virulence factor in M. mycoides.

Evaluation of new generation macrolides for the treatment and metaphylaxis of contagious bovine pleuropneumonia (CBPP) in cattle experimentally infected with Mycoplasma mycoides subspecies mycoides.

BACKGROUND: Contagious bovine pleuropneumonia (CBPP) caused by Mycoplasma mycoides subspecies mycoides (Mmm) is an important disease of cattle that causes serious economic losses. With the known effectiveness of new generation macrolides, tulathromycin and gamithromycin were assessed in comparison with oxytetracycline as a positive control and saline as a negative control for effectiveness in inhibiting lung lesion development, promoting resolution, preventing spread and bacteriological clearance in susceptible local cattle breeds in two separate studies in Kenya and Zambia. Animals were monitored for clinical signs, sero-conversion as well as detailed post-mortem examination for CBPP lesions. RESULTS: Using the Hudson and Turner score for lesion type and size, tulathromycin protected 90%, gamithromycin 80%, and oxytetracycline 88% of treated animals in Kenya. In Zambia, all animals (100%) treated with macrolides were free of lung lesions, while oxytetracycline protected 77.5%. Using the mean adapted Hudson and Turner score, which includes clinical signs, post-mortem findings and serology, tulathromycin protected 82%, gamithromycin 56% and oxytetracycline 80% of the animals in Kenya whereas in Zambia, tulathromycin protected 98%, gamithromycin 94% and oxytetracycline 80%. The saline-treated groups had 93 and 92% lesions in Kenya and Zambia respectively, with Mmm recovered from 5/14 in Kenya and 10/13 animals in Zambia. Whereas the groups treated with macrolides were free from lesions in Zambia, in Kenya 5/15 tulathromycin-treated animals and 6/15 gamithromycin-treated animals showed lesions. Oxytetracycline-treated animals showed similarities with 3/14 and 4/15 showing lesions in Zambia and Kenya respectively and Mmm recovery from one animal in Kenya and six in Zambia. In both studies, lesion scores of saline-treated groups were significantly higher than those of the antibiotic treated groups (p < 0.001). In sentinel animals, CBPP lesions were detected and Mmm recovered from one and two animals mixed with the saline-treated groups in Kenya and Zambia respectively. CONCLUSIONS: This study demonstrated that tulathromycin, a mycoplasmacidal, can achieve metaphylactic protection of up to 80%, while non-recovery of Mmm from sentinels suggests macrolides effectiveness in preventing spread of Mmm. It is recommended that further studies are conducted to evaluate strategies comparing vaccination alone or combining vaccination and antibiotics to control or eradicate CBPP.

MIB-MIP is a mycoplasma system that captures and cleaves immunoglobulin G.

Mycoplasmas are "minimal" bacteria able to infect humans, wildlife, and a large number of economically important livestock species. Mycoplasma infections include a spectrum of clinical manifestations ranging from simple fever to fulminant inflammatory diseases with high mortality rates. These infections are mostly chronic, suggesting that mycoplasmas have developed means to evade the host immune response. Here we present and functionally characterize a two-protein system from Mycoplasma mycoides subspecies capri that is involved in the capture and cleavage of IgG. The first component, Mycoplasma Ig binding protein (MIB), is an 83-kDa protein that is able to tightly bind to the Fv region of a wide range of IgG. The second component, Mycoplasma Ig protease (MIP), is a 97-kDa serine protease that is able to cleave off the VH domain of IgG. We demonstrate that MIB is necessary for the proteolytic activity of MIP. Cleavage of IgG requires a sequential interaction of the different partners of the system: first MIB captures the IgG, and then MIP is recruited to the MIB-IgG complex, enabling protease activity. MIB and MIP are encoded by two genes organized in tandem, with homologs found in the majority of pathogenic mycoplasmas and often in multiple copies. Phylogenetic studies suggest that genes encoding the MIB-MIP system are specific to mycoplasmas and have been disseminated by horizontal gene transfer. These results highlight an original and complex system targeting the host immunoglobulins, playing a potentially key role in the immunity evasion by mycoplasmas.

Respiratory explants as a model to investigate early events of contagious bovine pleuropneumonia infection.

Contagious bovine pleuropneumonia (CBPP) is a severe disease caused by Mycoplasma mycoides subsp. mycoides (Mmm). Knowledge on CBPP pathogenesis is fragmented and hampered by the limited availability of laboratory animal and in vitro models of investigation. The purpose of the present study is to assess respiratory explants as useful tools to study the early stages of CBPP. Explants were obtained from trachea, bronchi and lungs of slaughtered cattle, tested negative for Mycoplasma spp. and for the major bacterial and viral respiratory pathogens. The interaction of Mmm with explant cells was studied by immunohistochemistry (IHC), double-labelling indirect immunofluorescence (DLIIF) and laser scanning confocal microscopy (LSCM). Mmm capability to survive and proliferate within the explants was evaluated by standard microbiological procedures. Finally, the putative cellular internalization of Mmm was further investigated by the gentamicin invasion assay. IHC and DLIIF indicated that Mmm can colonize explants, showing a marked tropism for lower airways. Specifically, Mmm was detected on/inside the bronchiolar and alveolar epithelial cells, the alveolar macrophages and the endothelial cells. The interaction between Mmm and explant cells was abolished by the pre-incubation of the pathogen with bovine anti-Mmm immune sera. Mmm was able to survive and proliferate in all tracheal, bronchial and lung explants, during the entire time course of the experiments. LSCM and gentamicin invasion assay both confirmed that Mmm can enter non-phagocytic host cells. Taken together, our data supports bovine respiratory explants as a promising tool to investigate CBPP, alternative to cattle experimental infection.

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.

The experience of contagious bovine pleuropneumonia ring trials in Botswana.

The Botswana National Veterinary Laboratory (BNVL) has conducted ring trials (proficiency testing) on an annual basis since 2010. Proficiency testing is carried out to evaluate the ability of veterinary laboratories to perform serological complement fixation tests (CFTs) and molecular polymerase chain reaction (PCR) tests for the diagnosis of contagious bovine pleuropneumonia (CBPP). In this paper, the authors discuss the experience gained and the lessons learned in coordinating these ring trials over a period of six years, from 2010 to 2015.The number of participating laboratories increased from five in 2010 to 11 in2015. Their performance also improved over this period. The proportion of unsatisfactory results decreased from 40% to 10% for serological testing, while questionable results decreased from 60% to 10%. The proportion of unsatisfactory results for the molecular test decreased from 33% to 0%. Systematic errors (i.e. technical errors or imperfect experimental design) were the principal causes of questionable and unsatisfactory results. An analysis of responses from customer satisfaction surveys conducted annually since 2013 provided valuable information that enabled BNVL to redesign the programme in 2014 and 2015 to improve the overall quality of the proficiency testing programme. Among the changes made were sending freeze-dried sera for CFTs and DNA for PCR instead of sera and liquid cultures.

Le Laboratoire national vétérinaire du Botswana (BNVL) effectue depuis 2010 des essais comparatifs inter-laboratoires à intervalles d'un an. Ces essais d'aptitude inter-laboratoires visent à évaluer la capacité des laboratoires vétérinaires du pays à réaliser efficacement le test de fixation du complément (FC) ainsi qu'un test moléculaire (l'amplification en chaîne par polymérase ou PCR) en vue du diagnostic de la péripneumonie contagieuse bovine (PPCB). Les auteurs décrivent l'expérience acquise et les enseignements tirés lors de la coordination d'essais comparatifs inter-laboratoires sur une période de six ans (de 2010 à2015). Le nombre de laboratoires participants est passé de cinq en 2010 à onze en 2015. Les performances des laboratoires se sont également améliorées au cours de cette période. Le pourcentage de résultats sérologiques non concluants est passé de 40 % à 10 % tandis que la part des résultats douteux est passée de60 % à 10 %. Pour ce qui concerne la technique moléculaire, la proportion de résultats non concluants est passée de 33 % à 0 %. Les résultats douteux et non concluants étaient essentiellement dus à des erreurs systématiques (c'est-à-dire à des erreurs techniques ou imputables à une conception imparfaite du protocole de test). Une analyse des réponses fournies aux enquêtes de satisfaction réalisées chaque année depuis 2013 a fourni des informations précieuses qui ont permis au BNVL de corriger le protocole du programme en 2014 et en 2015 afin d'améliorer la qualité globale du programme d'essais d'aptitude. Les principales modifications introduites ont été le recours à des sérums lyophilisés pour le test de FC et l'utilisation d'ADN lyophilisé pour les PCR au lieu des sérums et des cultures en milieu liquide.

El Laboratorio Veterinario Nacional de Botsuana (BNVL) lleva realizando pruebas interlaboratorios anuales (pruebas de competencia) desde 2010. Las pruebas de competencia sirven para evaluar la aptitud de un laboratorio veterinario para efectuar pruebas serológicas (la de fijación del complemento, FdC) y moleculares (la de reacción en cadena de la polimerasa, PCR) de diagnóstico de la perineumonía contagiosa bovina. Los autores examinan la experiencia adquirida y las enseñanzas extraídas con la coordinación de estas pruebas interlaboratorios durante el sexenio que va de 2010 a 2015. El número de laboratorios participantes pasó de 5 en 2010 a 11 en 2015. Su eficacia también mejoró durante esos años: en el caso de la técnica serológica, el porcentaje de resultados insatisfactorios pasó de un 40% a un 10 y el de resultados cuestionables de un 60% a un 10%. En el caso de la técnica molecular, el porcentaje de resultados insatisfactorios pasó de un 33% a un 0%. Los resultados insatisfactorios o cuestionables se deben principalmente a errores sistemáticos (esto es, errores técnicos o concepción imperfecta de la prueba). Al analizar las respuestas a los cuestionarios de satisfacción del cliente realizados anualmente desde 2013 se obtuvo información de gran interés, que sirvió al BNVL para remodelar el programa de pruebas de competencia en 2014 y 2015 a fin de mejorar su calidad general. Entre los cambios introducidos está el envío de sueros liofilizados para la prueba de FdC y de ADN liofilizado para la PCR, en lugar de sueros y cultivos líquidos.

Lung lesion score system in cattle: proposal for contagious bovine pleuropneumonia.

Contagious bovine pleuropneumonia (CBPP) is a severe infectious disease caused by Mycoplasma mycoides subsp. mycoides. The peculiar pathological features of CBPP make desirable the assessment of ad hoc score methods to grade the disease in the affected animals. Thus, the present work aims to assess a new lung score system for CBPP. Our results indicate that the present score system strongly correlates with that previously published by Turner and could be effectively used in CBPP-affected animals.

Galactofuranose in Mycoplasma mycoides is important for membrane integrity and conceals adhesins but does not contribute to serum resistance.

Mycoplasma mycoides subsp. capri (Mmc) and subsp. mycoides (Mmm) are important ruminant pathogens worldwide causing diseases such as pleuropneumonia, mastitis and septicaemia. They express galactofuranose residues on their surface, but their role in pathogenesis has not yet been determined. The M. mycoides genomes contain up to several copies of the glf gene, which encodes an enzyme catalysing the last step in the synthesis of galactofuranose. We generated a deletion of the glf gene in a strain of Mmc using genome transplantation and tandem repeat endonuclease coupled cleavage (TREC) with yeast as an intermediary host for the genome editing. As expected, the resulting YCp1.1-Δglf strain did not produce the galactofuranose-containing glycans as shown by immunoblots and immuno-electronmicroscopy employing a galactofuranose specific monoclonal antibody. The mutant lacking galactofuranose exhibited a decreased growth rate and a significantly enhanced adhesion to small ruminant cells. The mutant was also 'leaking' as revealed by a ß-galactosidase-based assay employing a membrane impermeable substrate. These findings indicate that galactofuranose-containing polysaccharides conceal adhesins and are important for membrane integrity. Unexpectedly, the mutant strain showed increased serum resistance.

Development of a Novel Cocktail Enzyme-Linked Immunosorbent Assay and a Field-Applicable Lateral-Flow Rapid Test for Diagnosis of Contagious Bovine Pleuropneumonia.

Contagious bovine pleuropneumonia (CBPP) is a severe respiratory disease that is widespread in sub-Saharan Africa. It is caused by Mycoplasma mycoides subsp. mycoides, a bacterium belonging to the Mycoplasma mycoides cluster. In the absence of an efficient CBPP vaccine, improved and easy-to-use diagnostic assays for recurrent testing combined with isolation and treatment of positive animals represent an option for CBPP control in Africa. Here we describe the comprehensive screening of 17 immunogenic Mycoplasma mycoides subsp. mycoides proteins using well-characterized bovine sera for the development of a novel cocktail enzyme-linked immunosorbent assay (ELISA) for laboratory use. Two recombinant Mycoplasma immunogens, MSC_0136 and MSC_0636, were used to set up a standardized cocktail ELISA protocol. According to the results from more than 100 serum samples tested, the sensitivity and specificity of the novel cocktail ELISA were 85.6% and 96.4%, respectively, with an overall diagnostic accuracy comparable to that of the Office International des Epizooties (OIE)-prescribed serological assays. In addition, we provide a proof of principle for a field-applicable, easy-to-use commercially produced prototype lateral-flow test for rapid (<30-min) diagnosis of CBPP.