Random transposon insertion in the Mycoplasma hominis minimal genome

Rideau, Fabien; Roy, Chloé Le; Sagné, Eveline; Renaudin, H.; Pereyre, Sabine; Henrich, Birgit; Dordet‐Frisoni, Emilie; Citti, Christine; Lartigue, Carole; Bebear, Cécile

doi:10.1038/s41598-019-49919-y

Cited by 13 publications

(25 citation statements)

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“…mycoides strain PG1 as the reference. This coding sequence was subsequently cloned in the plasmid pMT85 ( 43 , 56 – 61 ). The plasmid backbone carries a transposon derived from Tn 4001 ( 62 ), the insertion of which can be selected through the gentamicin resistance gene aacA-aphD placed under the control of its natural promoter.…”

Section: Resultsmentioning

confidence: 99%

Imaging Minimal Bacteria at the Nanoscale: a Reliable and Versatile Process to Perform Single-Molecule Localization Microscopy in Mycoplasmas

et al. 2022

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Section: Resultsmentioning

confidence: 99%

Imaging Minimal Bacteria at the Nanoscale: a Reliable and Versatile Process to Perform Single-Molecule Localization Microscopy in Mycoplasmas

et al. 2022

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“…Furthermore, Meygret et al also reported that there were no antimicrobial resistance genes associated with these reported MICE. In related research, minitransposons have been used, albeit artificially, to deliver tetracycline resistance genes (30). This is further backed by the frequent occurrence of HGT that occurs in mollicutes and prokaryotes in general (31,32) and the phenomenon's ability to confer antimicrobial resistance (27,33), as well as the presence of genes in the ICE region of M. hominis, such as the aforementioned serine recombinase and Tn916 integrase, and including ArdA, a known facilitator of gene mobility (34).…”

Section: Discussionmentioning

confidence: 99%

Tetracycline Resistance Mediated by tet (M) Has Variable Integrative Conjugative Element Composition in Mycoplasma hominis Strains Isolated in the United Kingdom from 2005 to 2015

Chalker

Sharratt

Rees

et al. 2021

Antimicrob Agents Chemother

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A minimal genome and absent bacterial cell wall renders Mycoplasma hominis inherently resistant to most antimicrobials except lincosamides, tetracyclines and fluoroquinionlones. Often dismissed as a commensal (except where linked to preterm birth), it causes septic arthritis in immunodeficient patients and is increasingly associated with transplant failure (particularly lung) accompanying immunosuppression. We examined antimicrobial susceptibility (AST) on strains archived between 2005-2015 submitted to the Public Health England reference laboratory and determined the underlying mechanism of resistance by whole genome sequencing (WGS). Archived M. hominis strains included 32/115 from invasive infection (sepsis, CSF, peritoneal and pleural fluid) over the 10-year period (6.4% of all samples submitted between 2010-2015 were positive). No clindamycin resistance was detected, while two strains were resistant to moxifloxacin and levofloxacin (resistance mutations: S83L or E87G in gyrA and S81I or E84V in parC). One of these strains and 11 additional strains were tetracycline resistant, mediated by tet(M) carried within an integrative conjugative element (ICE) consistently integrated at the somatic rumA gene; however, the ICEs varied widely in 5-19 associated accessory genes. WGS analysis showed tet(M)-carrying strains were not clonal, refuting previous speculation that the ICE was broken and immobile. We found tet(M)-positive and -negative strains (including the multi-resistant 2015 strain) to be equally susceptible to tigecycline and josamycin, however, the British National Formulary does not include guidance for these. Continued M. hominis investigation and AST surveillance (especially immunocompromised patients) is warranted, and expansion of the limited therapeutics needs to be expanded in the UK.

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“…mycoides strain PG1 as reference. This coding sequence was subsequently cloned in the plasmid pMT85 (42,58,(70)(71)(72)(73)(74). The plasmid backbone carries a transposon derived from Tn4001 (75), the insertion of which can be selected through the gentamicin resistance gene aacA-aphD placed under the control of its natural promoter.…”

Section: Establishing a Polyvalent Meos32 Expression System For Palm In Multiple Mycoplasmasmentioning

confidence: 99%

Imaging minimal bacteria at the nanoscale: a reliable and versatile process to perform Single Molecule Localization Microscopy in mycoplasmas

Rideau

Villa

Belzanne

et al. 2022

Preprint

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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 of both humans and most livestock species. Mycoplasma cells have minute sizes, often in the 300-800 nanometers range. As these dimensions are close to the diffraction limit of visible light, fluorescence imaging in mycoplasmas is often poorly informative. Recently developed Super-Resolution 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 photo-convertible 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 SMLM strategies to study these organisms in the future.

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Random transposon insertion in the Mycoplasma hominis minimal genome

Cited by 13 publications

References 53 publications

Imaging Minimal Bacteria at the Nanoscale: a Reliable and Versatile Process to Perform Single-Molecule Localization Microscopy in Mycoplasmas

Imaging Minimal Bacteria at the Nanoscale: a Reliable and Versatile Process to Perform Single-Molecule Localization Microscopy in Mycoplasmas

Tetracycline Resistance Mediated by tet (M) Has Variable Integrative Conjugative Element Composition in Mycoplasma hominis Strains Isolated in the United Kingdom from 2005 to 2015

Imaging minimal bacteria at the nanoscale: a reliable and versatile process to perform Single Molecule Localization Microscopy in mycoplasmas

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