Escherichia coli cells penetrated by chrysotile fibers are transformed to antibiotic resistance by incorporation of exogenous plasmid DNA

Our plain-English summary of Escherichia coli cells penetrated by chrysotile fibers are transformed to antibiotic resistance by incorporation of exogenous plasmid DNA, by N. Yoshida, K. Kodama, K. Nakata, et al (Applied Microbiology and Biotechnology, 2002).

The study

• Authors: N. Yoshida, K. Kodama, K. Nakata, M. Yamashita, T. Miwa
• Journal: Applied Microbiology and Biotechnology
• Published: 2002;60(4):461-8
• Cited by: 13 articles, as counted by NIH iCite
• Read it: PubMed · Publisher

Abstract

A suspension of recipient Escherichia coli cells in stationary phase, chrysotile asbestos, and pUC18 donor DNA spread over the surface of a Luria-Bertani agar plate using a streak bar several times, resulted in intracellular uptake of the plasmid DNA by the E. coli cells. The transformation efficiency was highest with a duration of cell exposure to chrysotile of more than 60 s and an agar concentration of 2%. To improve chrysotile-mediated transformation efficiency, we systematically optimized various conditions and parameters. In comparison to chrysotile exposure without cations, exposure with cations produced up to 100-fold more transformants. Optimized conditions resulted in 10(6) transformants/ micro g pUC18 DNA. The drastic physical change due to 'quick drying on the surface of the agar plate' when cells were exposed to chrysotile, was essential for chrysotile-mediated transformation. We suggest that DNA uptake mediated by chrysotile asbestos is the result of a mechanical physical transformation of E. coli, since the E. coli cells are not chemically competent. Electron microscopy of cells exposed to chrysotile suggested penetration of the E. coli membrane by chrysotile fibers. It is suggested that E. coli transformation by the plasmid DNA was the result of penetration by chrysotile fibers to which plasmid DNA is bound or adsorbed.

In plain English

This laboratory experiment tested whether chrysotile asbestos fibers could carry plasmid DNA into bacteria. Suspensions of stationary-phase Escherichia coli JM109, chrysotile fibers (95% between 0.05 and 0.12 micrometres in diameter) and pUC18 plasmid DNA were spread and rubbed on dried Luria-Bertani agar containing ampicillin, and resistant colonies were counted; efficiency was expressed as transformants per microgram of plasmid DNA. No transformants appeared with 1 s of rubbing; fewer than 1,000 per microgram at 30 s and fewer than 5,000 per microgram at more than 60 s on 1.5% agar. After 60 s, 2% agar gave 1.4 x 10^4 transformants per microgram versus 3.8 x 10^3 on 1.5% agar. Adding sodium, potassium, calcium or magnesium at their best concentrations (200, 100, 300 and 100 mM) gave 2.2, 2.1, 2.0 and 1.6 x 10^6 transformants per microgram. Chrysotile carrying only bound plasmid gave 1.6 x 10^6 per microgram. Electron microscopy suggested fibers penetrating the cell membrane. E. coli DH5a and C600 and other Gram-negative bacteria were also transformed; Bacillus subtilis was not, under the conditions employed. The authors concluded that chrysotile-bound plasmid DNA entered E. coli by mechanical fiber penetration.

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 bacterial transformation experiment with electron microscopy
• Who was studied: Bacterial strains in culture: Escherichia coli JM109, DH5a and C600, Pseudomonas sp., Klebsiella aerogenes W70 and Bacillus subtilis ISW1214; work done at Miyazaki University, Japan (paper received 2002)
• Exposure: Chrysotile asbestos fibers (purchased, size-separated; 95% of fibers 0.05 to 0.12 micrometres diameter and 0.34 to 2.30 micrometres length; about 40 to 50 micrograms per ml) mixed with cells and pUC18 plasmid DNA and rubbed onto agar plates
• Main results:
  • Efficiency was expressed throughout as transformants per microgram of plasmid DNA
  • In E. coli JM109 on 1.5% agar, no ampicillin-resistant colonies formed with 1 s of exposure; fewer than 1,000 transformants per microgram plasmid DNA at 30 s and fewer than 5,000 per microgram at more than 60 s
  • Agar concentration affected efficiency in E. coli JM109 after 60 s exposure: 1.4 x 10^4 transformants per microgram on 2% agar versus 3.8 x 10^3 on 1.5% agar, and fewer than 100 transformants per microgram pUC18 at 0.5 or 1.0% agar
  • Adding cations to the E. coli JM109 cell-chrysotile suspension raised efficiency; best concentrations of sodium, potassium, calcium and magnesium (200, 100, 300 and 100 mM) gave 2.2, 2.1, 2.0 and 1.6 x 10^6 transformants
  • When only chrysotile-bound plasmid DNA (unbound plasmid washed away) was used with E. coli JM109, 1.6 x 10^6 transformants were obtained, the same number as with untreated chrysotile-DNA solution
  • E. coli DH5a and C600 were also transformed to ampicillin resistance, with efficiencies of 1.3 x 10^6 and 1.8 x 10^6 per microgram pUC18 DNA
  • Pseudomonas sp. and Klebsiella aerogenes W70 took up pHSG298 DNA and became kanamycin resistant; Bacillus subtilis ISW1214 was not transformed with pHY300PLK DNA
  • Electron microscopy of exposed E. coli JM109 showed chrysotile fibers in the cell membrane; cells exposed for 1 s showed no penetration
  • Viable E. coli JM109 cells fell from 1.85 x 10^8 cfu/plate after 1 s exposure to about 4 x 10^7 after 60 s, and beta-galactosidase leaked from cells
• The authors' conclusion: The authors concluded that chrysotile fibers with plasmid DNA bound to them carry the DNA into E. coli by physically penetrating the cells, and suggested this gives chrysotile the potential to cause bacterial mutations
• Limitations the authors state:
  • The authors state the method worked only in Gram-negative bacteria under the conditions they used; Bacillus subtilis was not transformed, which they attribute to its thicker cell wall
  • The authors present the mutation-causing potential as a possibility and a surmise rather than a demonstrated finding
  • Some supporting data are not shown in the paper (chrysotile/cell ratio optimum; plasmid detection by gel electrophoresis)

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, Cation.

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• 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)
• Quantitative detection of asbestos fiber in gravelly sand using elastic body-exposure method — N. Yoshida, K. Takebe (2006)
• Asbestos fibers mediate transformation of monkey cells by exogenous plasmid DNA — J.D. Appel, T.M. Fasy, et al. (1988)
• Mutagenesis of Escherichia coli (CSH50) by asbestos — M.G. Cleveland (1984)

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