Chestnut bur-shaped aggregates of chrysotile particles enable inoculation of Escherichia coli cells with plasmid DNA

Our plain-English summary of Chestnut bur-shaped aggregates of chrysotile particles enable inoculation of Escherichia coli cells with plasmid DNA, by N. Yoshida, Y. Saeki (Applied Microbiology and Biotechnology, 2004).

The study

• Authors: N. Yoshida, Y. Saeki
• Journal: Applied Microbiology and Biotechnology
• Published: 2004;65(5):566-75
• Cited by: 8 articles, as counted by NIH iCite
• Read it: PubMed · Publisher

Abstract

In the present study, Escherichia coli cells exhibited antibiotic resistance after transformation with exogenous plasmid DNA adsorbed onto chrysotile particles during agar-exposure. We previously demonstrated penetration of E. coli by chrysotile particles during agar-exposure. To further investigate the mechanism by which transformation of E. coli is achieved through the use of chrysotile fibers, the interaction between E. coli cells and chrysotile was examined during agar-exposure. Dispersion of chrysotile particles within the chrysotile solution was analyzed by flow cytometry. A suspension containing E. coli cells expressing blue fluorescence protein and chrysotile particles was exposed to agar using stirring apparatus, which allowed a constant vertical reaction force to be applied to the surface of the gel. Fluorescence microscopy was then used to illustrate the adsorption of fluorescein isothiocyanate-conjugated DNA oligomers to chrysotile. Larger aggregates were observed when increasing concentrations of chrysotile were added to the solution. With prolonged exposure, during which surface moisture diffused into the agar gel, greater concentrations of chrysotile were observed on the agar surface. In addition, chrysotile aggregates exceeding 50 microm developed on the agar surface. They were shaped like a chestnut bur. The chrysotile aggregates penetrated the cell membranes of adherent E. coli cells during agar-exposure due to sliding friction forces generated at the interface of the agar and the stirring stick. E. coli cells thus acquired plasmid DNA and antibiotic resistance, since the plasmid DNA had been adsorbed onto the chrysotile particles. The inoculation of plasmid DNA into E. coli cells demonstrates the usefulness of chrysotile for E. coli transformation.

In plain English

This was a laboratory study of how chrysotile asbestos particles carry plasmid DNA into Escherichia coli during a procedure the authors call "agar-exposure", in which a cell-chrysotile suspension is spread on an agar plate with a stirring stick under a fixed vertical reaction force of 40 g. Chrysotile particles were characterised by flow cytometry, scanning electron microscopy and fluorescence microscopy using FITC-tagged DNA oligomers, and E. coli expressing blue fluorescent protein were used to show where cells attached. Flow cytometry indicated 95% of particles in solution were less than 2.76 micrometres; scanning electron microscopy gave fibre dimensions of 0.05-0.12 micrometres in diameter and 0.34-2.30 micrometres in length. Chrysotile solution filtered through 0.2-5.0 micrometre mesh produced no transformants, while 10-30 micrometre mesh and unfiltered solution produced about 1.1 to 2.3 times 10 to the sixth transformants per microgram pUC18. Aggregates grew with concentration, and aggregates exceeding 50 micrometres, shaped like a chestnut bur, formed on the agar surface with adherent E. coli. The authors concluded that these aggregates penetrated cell membranes through sliding friction, allowing uptake of adsorbed plasmid DNA.

We wrote this plain-English summary from the full article with the help of AI, and a second AI model checked each sentence against the article. It is not the authors' wording; the abstract and the article are their own account.

What the study did and found

• Design: laboratory in vitro experimental study of E. coli transformation during "agar-exposure" (stirring stick on agar at a fixed 40 g vertical reaction force), with flow cytometry, electron microscopy and fluorescence microscopy
• Who was studied: Escherichia coli strains JM109 (recipient for pUC18 transformation) and BL21 (DE3) carrying pQBI67 expressing blue fluorescent protein; laboratory work at the University of Miyazaki, Japan (article published 2004)
• Exposure: chrysotile asbestos fibres in aqueous suspension (stock 50 micrograms per millilitre, concentrated up to 1,000 micrograms per millilitre), with pUC18 plasmid DNA or FITC-conjugated DNA oligomers adsorbed onto the fibres, applied to bacteria during agar-exposure
• Main results:
  • Flow cytometry of the chrysotile solution indicated that 95% of particles were less than 2.76 micrometres, with asymmetrical light scatter taken to indicate needle-shaped particles
  • Chrysotile solution filtered through 0.2, 0.5, 0.8, 3.0 and 5.0 micrometre mesh gave no detectable transformants, while 10.0, 20.0 and 30.0 micrometre mesh gave 1.2x10^6, 2.3x10^6 and 1.1x10^6 colony-forming units per microgram pUC18 and unfiltered solution gave 2.0x10^6
  • Aggregate size in solution increased with chrysotile concentration: 7-8 micrometres at 200 micrograms per millilitre, 10 micrometres at 250, and about 70 micrometres at 1,000 micrograms per millilitre
  • By DAPI flow cytometry, aggregates larger than 2.6 micrometres were 16.1% of DAPI-positive particles at 500 micrograms per millilitre and 2.52% at 50 micrograms per millilitre
  • Scanning electron microscopy gave chrysotile fibres of 0.05-0.12 micrometres in diameter and 0.34-2.30 micrometres in length, compared with E. coli cells 0.65 micrometres in diameter and 1.6-2.7 micrometres long
  • After agar-exposure, chrysotile aggregates larger than 50 micrometres, resembling a chestnut bur, formed on the agar surface and blue-fluorescent E. coli cells were adsorbed onto them
• The authors' conclusion: The authors concluded that chestnut bur-shaped chrysotile aggregates penetrated the E. coli cell membrane through sliding friction during agar-exposure, so that surviving cells took up the adsorbed plasmid DNA and became antibiotic resistant, a mechanical rather than chemical transformation
• Limitations the authors state:
  • The authors stated that the mechanism by which chrysotile fibres disrupt cell integrity had not been examined, and that the toxic or mutagenic potential of physical cell disruption by these aggregates still needs study
  • The authors noted a result they did not expect: flow cytometry showed most particles were smaller than 2.76 micrometres, yet filtration at 3.0-5.0 micrometres abolished transformation

Topics

The National Library of Medicine indexes this study under subjects our site covers: Chrysotile.

Medical terms in this study, explained in our Lexicon: Chrysotile.

Related studies

• Escherichia coli cells penetrated by chrysotile fibers are transformed to antibiotic resistance by incorporation of exogenous plasmid DNA — N. Yoshida, K. Kodama, et al. (2002)
• Chrysotile asbestos fibers mediate transformation of Escherichia coli by exogenous plasmid DNA — N. Yoshida, T. Ikeda, et al. (2001)
• Quantitative detection of asbestos fiber in gravelly sand using elastic body-exposure method — N. Yoshida, K. Takebe (2006)
• Discovery and application of the Yoshida effect: nano-sized acicular materials enable penetration of bacterial cells by sliding friction force — N. Yoshida (2007)
• Plasmid DNA is released from nanosized acicular material surface by low molecular weight oligonucleotides: exogenous plasmid acquisition mechanism for penetration intermediates based on the Yoshida effect — N. Yoshida, K. Ide (2008)
• Mutagenesis of Escherichia coli (CSH50) by asbestos — M.G. Cleveland (1984)

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