Mycoplasma mycoides, from "mycoides Small Colony" to "capri". A microevolutionary perspective

<p>Abstract</p> <p>Background</p> <p>The <it>Mycoplasma mycoides </it>cluster consists of five species or subspecies that are ruminant pathogens. One subspecies, <it>Mycoplasma mycoides </it>subspecies <it>mycoides </it>Small Colony (MmmSC), is the causative agent of contagious bovine pleuropneumonia. Its very close relative, <it>Mycoplasma mycoides </it>subsp. <it>capri </it>(Mmc), is a more ubiquitous pathogen in small ruminants causing mastitis, arthritis, keratitis, pneumonia and septicaemia and is also found as saprophyte in the ear canal. To understand the genetics underlying these phenotypic differences, we compared the MmmSC PG1 type strain genome, which was already available, with the genome of an Mmc field strain (95010) that was sequenced in this study. We also compared the 95010 genome with the recently published genome of another Mmc strain (GM12) to evaluate Mmc strain diversity.</p> <p>Results</p> <p>The MmmSC PG1 genome is 1,212 kbp and that of Mmc 95010 is ca. 58 kbp shorter. Most of the sequences present in PG1 but not 95010 are highly repeated Insertion Sequences (three types of IS) and large duplicated DNA fragments. The 95010 genome contains five types of IS, present in fewer copies than in PG1, and two copies of an integrative conjugative element. These mobile genetic elements have played a key role in genome plasticity, leading to inversions of large DNA fragments. Comparison of the two genomes suggested a marked decay of the PG1 genome that seems to be correlated with a greater number of IS. The repertoire of gene families encoding surface proteins is smaller in PG1. Several genes involved in polysaccharide metabolism and protein degradation are also absent from, or degraded in, PG1.</p> <p>Conclusions</p> <p>The genome of MmmSC PG1 is larger than that of Mmc 95010, its very close relative, but has less coding capacity. This is the result of large genetic rearrangements due to mobile elements that have also led to marked gene decay. This is consistent with a non-adaptative genomic complexity theory, allowing duplications or pseudogenes to be maintained in the absence of adaptive selection that would lead to purifying selection and genome streamlining over longer evolutionary times. These findings also suggest that MmmSC only recently adapted to its bovine host.</p

Purification process of recombinant monoclonal antibodies with mixed mode chromatography

An innovative process to purify mAb from CHO cell culture supernatant was developed. This three-step process involved two mixed mode resins and an anion exchange membrane. We used a human IgG mixture to determine the optimal conditions for each purification step. Thereafter, the whole process was evaluated and improved for the purification of a recombinant mAb produced in the supernatant of CHO cells. Once optimized, yield and purity of 88% and 99.9%, respectively were comparable to those obtained in a conventional process based on a capture step using protein A. In addition, aggregates, HCPs and DNA levels in the purified fraction were below regulatory specifications. Then we used mass spectrometry to identify contaminating proteins in the antibody fraction in order to highlight the behavior of HCPs. (C) 2015 Elsevier B.V. All rights reserved

A proteomic study of water deficit-responsive proteins in poplar roots

In order to analyze root proteins involved in drought tolerance in poplar, a proteomic study was conducted on the roots of two Populus deltoïdes × Populus nigra cultivars, Carpaccio and Soligo. These cultivars were selected based on contrasted physiological responses under water stress. Plants were exposed to different water stress levels (Control, Early, Medium and Severe), and root tips collected. Technical optimization included protein extraction and resolubilization, as well as the finetuning of electrophoretic conditions. The best results were obtained upon using phenol extraction, a resolubilization solution containing two reduction reagents, DTT and 2-ME, and 100 μg protein load on both acidic (pH 4-7) and basic (pH 7-11NL) IPG strips. A total of 48 2-D gels were then produced and quantitatively analyzed using SameSpot Progenesis software. Statistical analyses were completed using normalized spot volumes. Multivariate analyses (correlation matrix, PCA and HCA) and twoway ANOVAs showed that protein expression was first regulated by genotype effect and second by treatment effect. Differentially regulated proteins are being identified based on tandem mass spectrometry. This functional information should shed some lights on the molecular players involved in drought-stress response and potentially underlying adaptation to this abiotic constraint

A proteomic study of water deficit-responsive proteins in poplar roots

In order to analyze root proteins involved in drought tolerance in poplar, a proteomic study was conducted on the roots of two Populus deltoïdes × Populus nigra cultivars, Carpaccio and Soligo. These cultivars were selected based on contrasted physiological responses under water stress. Plants were exposed to different water stress levels (Control, Early, Medium and Severe), and root tips collected. Technical optimization included protein extraction and resolubilization, as well as the finetuning of electrophoretic conditions. The best results were obtained upon using phenol extraction, a resolubilization solution containing two reduction reagents, DTT and 2-ME, and 100 μg protein load on both acidic (pH 4-7) and basic (pH 7-11NL) IPG strips. A total of 48 2-D gels were then produced and quantitatively analyzed using SameSpot Progenesis software. Statistical analyses were completed using normalized spot volumes. Multivariate analyses (correlation matrix, PCA and HCA) and twoway ANOVAs showed that protein expression was first regulated by genotype effect and second by treatment effect. Differentially regulated proteins are being identified based on tandem mass spectrometry. This functional information should shed some lights on the molecular players involved in drought-stress response and potentially underlying adaptation to this abiotic constraint

Leaf proteome analysis of eight Populus xeuramericana genotypes : genetic variation in drought response and in water-use efficiency involves photosynthesis-related proteins

Genetic variation of leaf proteome in drought response was investigated among eight Populus ×euramericana genotypes contrasting for their leaf carbon isotope discrimination (Δ), an estimate of intrinsic water-use efficiency. Plants were grown in open field on two similar plots. Drought was induced by an 86-day irrigation cessation on one plot, whereas a second plot remained regularly irrigated. Using 2-DE, 863 reproducible spots were detected; about 60% presented at least one significant effect i.e. treatment, genotype and/or genotype by treatment interaction effect. A significant genotype by treatment interaction was detected for 62 reliably identified proteins among which, about 65% consisted in chloroplast-associated proteins either involved in the Calvin cycle or in the electron-transport chains. The other proteins were involved in oxidative stress, amino acid or protein metabolisms. Correlations between protein abundance and Δ variations were found for 45 reliably identified proteins. The abundance of ribulose-1,5-bisphosphate carboxylase/oxygenase activase isoforms scaled negatively with Δ regardless of the treatment, suggesting that a large intrinsic water-use efficiency could be due to higher abundance of ribulose-1,5-bisphosphate carboxylase/oxygenase activase. Under control condition, abundance of enzymes involved in carbon fixation was also negatively correlated with Δ, whereas abundance of enzymes involved in photorespiration or respiration was positively correlated with Δ

High gellan gum concentration and secondary somatic embryogenesis: two key factors to improve somatic embryo development in Pseudotsuga menziesii [Mirb.]

Douglas-fir is a conifer species of major economic importance worldwide, including Western Europe and New Zealand. Herein we describe some characterization and significant refinement of somatic embryogenesis in Douglas-fir, with focus on maturation. The most typical structures observed in the embryonal masses were large polyembryogenic centres (up to 800-1500 μm) with a broad meristem, creating a compact ce ll “package” with suspensor cells. Singulated somatic embryos composed of both a embryonal head (300-400 μm) and long, tightly arranged suspensor were also frequent. Embryo development was enhanced following embryonal mass dispersion on filter paper discs at low density (50-100 mg fresh mass). Moreover, increasing gellan gum concentration in maturation medium (up to 10 g L-1) improved both the quantity and quality of cotyledonary somatic embryos (SEs), which were subsequently able to germinate and develop into plantlets at high frequency. Embryogenic yield was highly variable among the seven embryogenic lines tested (27-1544 SE g-1 fresh mass). Interestingly secondary somatic embryogenesis could be induced from cotyledonary SEs of both low- and highly-productive lines with some useful practical outcomes: secondary lines from low-performance lines (30-39 478 SE g-1) displayed significantly higher embryogenic yield (148-1343 SE g-1). In our best conditions, the total protein content in cotyledonary SEs increased significantly with maturation 41 duration (up to 150 μg mg-1 fresh mass after 7 weeks) but remained below that of mature zygotic embryos (300 μg mg-1). The protein pattern was similar in both somatic and zygotic embryos, with major storage proteins identified as 7S-vicilin- and legumin-like proteins

Repetitive somatic embryogenesis induced cytological and proteomic changes in embryogenic lines of Pseudotsuga menziesii

International audienc

Development of a method for the extraction and analysis of grape skin proteins strongly bound to cell walls

Aim: To better understand the protein composition of grape skin cell walls, we have developed a method to analyse the strongly bound cell wall proteins. Methods and results: The protocol was developed with grape skins at full maturity. The critical steps of this protocol were : (i) the elimination of cellular aggregates, (ii) the elimination of soluble proteins, and (iii) the localization of the identified proteins within the cell wall. To verify whether these three conditions were met, the decrease in the quantity of cellular aggregates was followed by optical microscopy, the removal of soluble proteins was measured by chemical assay, and the presence of proteins located in cell walls was demonstrated by extensive bioinformatic analysis. The process made it possible to obtain a four-fold reduction in the amount of cellular aggregates, a reduction in the concentration of soluble proteins below the method detection limit, and a high proportion of proteins predicted to be secreted (79 %). Conclusion: The protocol described in this paper constitutes the first method to analyse proteins strongly bound to cell walls in grape skins. However, this method excludes the identification of labile proteins or proteins weakly bound to the cell wall. Significance and impact of the study: This protocol can be used for studying the role that strongly bound cell wall proteins play in development and defense processes in grape skins

The extracellular matrix of the oleolytic biofilms of Marinobacter hydrocarbonoclasticus comprises cytoplasmic proteins and T2SS effectors that promote growth on hydrocarbons and lipids.

International audienceSummary : The assimilation of the nearly water insol. substrates hydrocarbons and lipids by bacteria entails specific adaptations such as the formation of oleolytic biofilms. The present article reports that the extracellular matrix of an oleolytic biofilm formed by Marinobacter hydrocarbonoclasticus at n-hexadecane-water interfaces is largely composed of proteins typically cytoplasmic such as translation factors and chaperones, and a lesser amt. of proteins of unknown function that are predicted extra-cytoplasmic. Matrix proteins appear to form a structured film on hydrophobic interfaces and were found mandatory for the development of biofilms on lipids, alkanes and polystyrene. Exo-proteins secreted through the type-2 secretion system (T2SS) were shown to be essential for the formation of oleolytic biofilms on both alkanes and triglycerides. The T2SS effector involved in biofilm formation on triglycerides was identified as a lipase. In the case of biofilm formation on n-hexadecane, the T2SS effector is likely involved in the mass transfer, capture or transport of alkanes. We propose that M. hydrocarbonoclasticus uses cytoplasmic proteins released by cell lysis to form a proteinaceous matrix and dedicated proteins secreted through the T2SS to act specifically in the assimilation pathways of hydrophobic substrates