Mollicutes is a class of parasitic bacteria that have evolved from a common Firmicutes ancestor mostly by massive genome reduction. With genomes under 1 Mbp in size, most Mollicutes species retain ...the capacity to replicate and grow autonomously. The major goal of this work was to identify the minimal set of proteins that can sustain ribosome biogenesis and translation of the genetic code in these bacteria. Using the experimentally validated genes from the model bacteria Escherichia coli and Bacillus subtilis as input, genes encoding proteins of the core translation machinery were predicted in 39 distinct Mollicutes species, 33 of which are culturable. The set of 260 input genes encodes proteins involved in ribosome biogenesis, tRNA maturation and aminoacylation, as well as proteins cofactors required for mRNA translation and RNA decay. A core set of 104 of these proteins is found in all species analyzed. Genes encoding proteins involved in post-translational modifications of ribosomal proteins and translation cofactors, post-transcriptional modifications of t+rRNA, in ribosome assembly and RNA degradation are the most frequently lost. As expected, genes coding for aminoacyl-tRNA synthetases, ribosomal proteins and initiation, elongation and termination factors are the most persistent (i.e. conserved in a majority of genomes). Enzymes introducing nucleotides modifications in the anticodon loop of tRNA, in helix 44 of 16S rRNA and in helices 69 and 80 of 23S rRNA, all essential for decoding and facilitating peptidyl transfer, are maintained in all species. Reconstruction of genome evolution in Mollicutes revealed that, beside many gene losses, occasional gains by horizontal gene transfer also occurred. This analysis not only showed that slightly different solutions for preserving a functional, albeit minimal, protein synthetizing machinery have emerged in these successive rounds of reductive evolution but also has broad implications in guiding the reconstruction of a minimal cell by synthetic biology approaches.
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.
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.
Mycoplasmas are small, genome-reduced bacteria. They are obligate parasites that can be found in a wide range of host species, including the majority of livestock animals and humans. Colonization of ...the host can result in a wide spectrum of outcomes. In many cases, these successful parasites are considered commensal, as they are found in the microbiota of asymptomatic carriers. Conversely, mycoplasmas can also be pathogenic, as they are associated with a range of both acute and chronic inflammatory diseases which are problematic in veterinary and human medicine. The chronicity of mycoplasma infections and the ability of these bacteria to infect even recently vaccinated individuals clearly indicate that they are able to successfully evade their host's humoral immune response. Over the years, multiple strategies of immune evasion have been identified in mycoplasmas, with a number of them aimed at generating important antigenic diversity. More recently, mycoplasma-specific anti-immunoglobulin strategies have also been characterized. Through the expression of the immunoglobulin-binding proteins protein M or mycoplasma immunoglobulin binding (MIB), mycoplasmas have the ability to target the host's antibodies and to prevent them from interacting with their cognate antigens. In this review, we discuss how these discoveries shed new light on the relationship between mycoplasmas and their host's immune system. We also propose that these strategies should be taken into consideration for future studies, as they are key to our understanding of mycoplasma diseases' chronic and inflammatory nature and are probably a contributing factor to reduce vaccine efficacy.
Horizontal gene transfer (HGT) is a main driving force of bacterial evolution and innovation. This phenomenon was long thought to be marginal in mycoplasmas, a large group of self-replicating ...bacteria characterized by minute genomes as a result of successive gene losses during evolution. Recent comparative genomic analyses challenged this paradigm, but the occurrence of chromosomal exchanges had never been formally addressed in mycoplasmas. Here, we demonstrated the conjugal transfer of large chromosomal regions within and among ruminant mycoplasma species, with the incorporation of the incoming DNA occurring by homologous recombination into the recipient chromosome. By combining classical mating experiments with high-throughput next-generation sequencing, we documented the transfer of almost every position of the mycoplasma chromosome. Mycoplasma conjugation relies on the occurrence of an integrative conjugative element (ICE) in at least one parent cell. While ICE propagates horizontally from ICE-positive to ICE-negative cells, chromosomal transfers (CTs) occurred in the opposite direction, from ICE-negative to ICE-positive cells, independently of ICE movement. These findings challenged the classical mechanisms proposed for other bacteria in which conjugative CTs are driven by conjugative elements, bringing into the spotlight a new means for rapid mycoplasma innovation. Overall, they radically change our current views concerning the evolution of mycoplasmas, with particularly far-reaching implications given that over 50 species are human or animal pathogens.
Horizontal gene transfers (HGT) shape bacterial genomes and are key contributors to microbial diversity and innovation. One main mechanism involves conjugation, a process that allows the simultaneous transfer of significant amounts of DNA upon cell-to-cell contact. Recognizing and deciphering conjugal mechanisms are thus essential in understanding the impact of gene flux on bacterial evolution. We addressed this issue in mycoplasmas, the smallest and simplest self-replicating bacteria. In these organisms, HGT was long thought to be marginal. We showed here that nearly every position of the Mycoplasma agalactiae chromosome could be transferred via conjugation, using an unconventional mechanism. The transfer involved DNA blocks containing up to 80 genes that were incorporated into the host chromosome by homologous recombination. These findings radically change our views concerning mycoplasma evolution and adaptation with particularly far-reaching implications given that over 50 species are human or animal pathogens.
Mycoplasmas are commonly described as the simplest self-replicating organisms, whose evolution was mainly characterized by genome downsizing with a proposed evolutionary scenario similar to that of ...obligate intracellular bacteria such as insect endosymbionts. Thus far, analysis of mycoplasma genomes indicates a low level of horizontal gene transfer (HGT) implying that DNA acquisition is strongly limited in these minimal bacteria. In this study, the genome of the ruminant pathogen Mycoplasma agalactiae was sequenced. Comparative genomic data and phylogenetic tree reconstruction revealed that approximately 18% of its small genome (877,438 bp) has undergone HGT with the phylogenetically distinct mycoides cluster, which is composed of significant ruminant pathogens. HGT involves genes often found as clusters, several of which encode lipoproteins that usually play an important role in mycoplasma-host interaction. A decayed form of a conjugative element also described in a member of the mycoides cluster was found in the M. agalactiae genome, suggesting that HGT may have occurred by mobilizing a related genetic element. The possibility of HGT events among other mycoplasmas was evaluated with the available sequenced genomes. Our data indicate marginal levels of HGT among Mycoplasma species except for those described above and, to a lesser extent, for those observed in between the two bird pathogens, M. gallisepticum and M. synoviae. This first description of large-scale HGT among mycoplasmas sharing the same ecological niche challenges the generally accepted evolutionary scenario in which gene loss is the main driving force of mycoplasma evolution. The latter clearly differs from that of other bacteria with small genomes, particularly obligate intracellular bacteria that are isolated within host cells. Consequently, mycoplasmas are not only able to subvert complex hosts but presumably have retained sexual competence, a trait that may prevent them from genome stasis and contribute to adaptation to new hosts.
One remarkable achievement in synthetic biology was the reconstruction of mycoplasma genomes and their cloning in yeast where they can be modified using available genetic tools. Recently, CRISPR/Cas9 ...editing tools were developed for yeast mutagenesis. Here, we report their adaptation for the engineering of bacterial genomes cloned in yeast. A seamless deletion of the mycoplasma glycerol-3-phosphate oxidase-encoding gene (glpO) was achieved without selection in one step, using 90 nt paired oligonucleotides as templates to drive recombination. Screening of the resulting clones revealed that more than 20% contained the desired deletion. After manipulation, the overall integrity of the cloned mycoplasma genome was verified by multiplex PCR and PFGE. Finally, the edited genome was back-transplanted into a mycoplasma recipient cell. In accordance with the deletion of glpO, the mutant mycoplasma was affected in the production of H2O2. This work paves the way to high-throughput manipulation of natural or synthetic genomes in yeast.
Summary
Horizontal gene transfer (HGT) is a major force of microbial evolution but was long thought to be marginal in mycoplasmas. In silico detection of exchanged regions and of loci encoding ...putative Integrative Conjugative Elements (ICE) in several mycoplasma genomes challenged this view, raising the prospect of these simple bacteria being able to conjugate. Using the model pathogen Mycoplasma agalactiae, we demonstrated for the first time that one of these elements, ICEA, is indeed self‐transmissible. As a hallmark of conjugative processes, ICEA transfers were DNase resistant and required viable cells. ICEA acquisition conferred ICE‐negative strains with the new ability to conjugate, allowing the spread of ICEA. Analysis of transfer‐deficient mutants indicated that this process requires an ICEA‐encoded lipoprotein of unknown function, CDS14. Formation of a circular extrachromosomal intermediate and the subsequent chromosomal integration of ICEA involved CDS22, an ICEA‐encoded product distantly related to the ISLre2 transposase family. Remarkably, ICEA has no specific or no preferential integration site, often resulting in gene disruptions. Occurrence of functional mycoplasma ICE offers these bacteria with a means for HGT, a phenomenon with far‐reaching implications given their minute‐size genome and the number of species that are pathogenic for a broad host‐range.
Vaccination against CCPP in East Africa Salt, Jeremy; Jores, Joerg; Labroussaa, Fabien ...
Veterinary record,
September 2019, Letnik:
185, Številka:
9
Journal Article
Among challenges that hamper reaping the benefits of genome assembly are both unfinished assemblies and the ensuing experimental costs. First, numerous software solutions for genome de novo assembly ...are available, each having its advantages and drawbacks, without clear guidelines as to how to choose among them. Second, these solutions produce draft assemblies that often require a resource intensive finishing phase.
In this paper we address these two aspects by developing Mix , a tool that mixes two or more draft assemblies, without relying on a reference genome and having the goal to reduce contig fragmentation and thus speed-up genome finishing. The proposed algorithm builds an extension graph where vertices represent extremities of contigs and edges represent existing alignments between these extremities. These alignment edges are used for contig extension. The resulting output assembly corresponds to a set of paths in the extension graph that maximizes the cumulative contig length.
We evaluate the performance of Mix on bacterial NGS data from the GAGE-B study and apply it to newly sequenced Mycoplasma genomes. Resulting final assemblies demonstrate a significant improvement in the overall assembly quality. In particular, Mix is consistent by providing better overall quality results even when the choice is guided solely by standard assembly statistics, as is the case for de novo projects.
Mix is implemented in Python and is available at https://github.com/cbib/MIX, novel data for our Mycoplasma study is available at http://services.cbib.u-bordeaux2.fr/mix/.
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