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Chromosomal transfers in mycoplasmas: when minimal genomes go mobile.

ABSTRACT:

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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.

Importance

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.

SUBMITTER: Dordet-Frisoni E

PROVIDER: S-EPMC4251992 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:Mycoplasma hominis is an opportunistic human mycoplasma. Two other pathogenic human species, M. genitalium and Ureaplasma parvum, reside within the same natural niche as M. hominis: the urogenital tract. These three species have overlapping, but distinct, pathogenic roles. They have minimal genomes and, thus, reduced metabolic capabilities characterized by distinct energy-generating pathways. Analysis of the M. hominis PG21 genome sequence revealed that it is the second smallest genome among self-replicating free living organisms (665,445 bp, 537 coding sequences (CDSs)). Five clusters of genes were predicted to have undergone horizontal gene transfer (HGT) between M. hominis and the phylogenetically distant U. parvum species. We reconstructed M. hominis metabolic pathways from the predicted genes, with particular emphasis on energy-generating pathways. The Embden-Meyerhoff-Parnas pathway was incomplete, with a single enzyme absent. We identified the three proteins constituting the arginine dihydrolase pathway. This pathway was found essential to promote growth in vivo. The predicted presence of dimethylarginine dimethylaminohydrolase suggested that arginine catabolism is more complex than initially described. This enzyme may have been acquired by HGT from non-mollicute bacteria. Comparison of the three minimal mollicute genomes showed that 247 CDSs were common to all three genomes, whereas 220 CDSs were specific to M. hominis, 172 CDSs were specific to M. genitalium, and 280 CDSs were specific to U. parvum. Within these species-specific genes, two major sets of genes could be identified: one including genes involved in various energy-generating pathways, depending on the energy source used (glucose, urea, or arginine) and another involved in cytadherence and virulence. Therefore, a minimal mycoplasma cell, not including cytadherence and virulence-related genes, could be envisaged containing a core genome (247 genes), plus a set of genes required for providing energy. For M. hominis, this set would include 247+9 genes, resulting in a theoretical minimal genome of 256 genes.

Project description:Mycoplasmas are the smallest free-living organisms. These bacteria are important models for both fundamental and synthetic biology, owing to their highly reduced genomes. They are also relevant in the medical and veterinary fields, as they are pathogenic to both humans and most livestock species. Mycoplasma cells have minute sizes, often in the 300- to 800-nm range. As these dimensions are close to the diffraction limit of visible light, fluorescence imaging in mycoplasmas is often poorly informative. Recently developed superresolution imaging techniques can break this diffraction limit, improving the imaging resolution by an order of magnitude and offering a new nanoscale vision of the organization of these bacteria. These techniques have, however, not been applied to mycoplasmas before. Here, we describe an efficient and reliable protocol to perform single-molecule localization microscopy (SMLM) imaging in mycoplasmas. We provide a polyvalent transposon-based system to express the photoconvertible fluorescent protein mEos3.2, enabling photo-activated localization microscopy (PALM) in most Mycoplasma species. We also describe the application of direct stochastic optical reconstruction microscopy (dSTORM). We showcase the potential of these techniques by studying the subcellular localization of two proteins of interest. Our work highlights the benefits of state-of-the-art microscopy techniques for mycoplasmology and provides an incentive to further the development of SMLM strategies to study these organisms in the future. IMPORTANCE Mycoplasmas are important models in biology, as well as highly problematic pathogens in the medical and veterinary fields. The very small sizes of these bacteria, well below a micron, limits the usefulness of traditional fluorescence imaging methods, as their resolution limit is similar to the dimensions of the cells. Here, to bypass this issue, we established a set of state-of-the-art superresolution microscopy techniques in a wide range of Mycoplasma species. We describe two strategies: PALM, based on the expression of a specific photoconvertible fluorescent protein, and dSTORM, based on fluorophore-coupled antibody labeling. With these methods, we successfully performed single-molecule imaging of proteins of interest at the surface of the cells and in the cytoplasm, at lateral resolutions well below 50 nm. Our work paves the way toward a better understanding of mycoplasma biology through imaging of subcellular structures at the nanometer scale.

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Chromosomal transfers in mycoplasmas: when minimal genomes go mobile.

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