Limits...
The use of nano-sized acicular material, sliding friction, and antisense DNA oligonucleotides to silence bacterial genes.

Mitsudome Y, Takahama M, Hirose J, Yoshida N - AMB Express (2014)

Bottom Line: Viable bacterial cells impaled with a single particle of a nano-sized acicular material formed when a mixture containing the cells and the material was exposed to a sliding friction field between polystyrene and agar gel; hereafter, we refer to these impaled cells as penetrons.Upon formation of Escherichia coli penetrons, β-lactamase and β-galactosidase expression was evaluated by counting the numbers of colonies formed on LB agar containing ampicillin and by measuring β-galactosidase activity respectively.This novel method of gene silencing has substantial promise for elucidation of gene function in bacterial species that have been refractory to experimental introduction of exogenous DNA.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Biochemistry and Applied Biosciences, University of Miyazaki, 1-1 Gakuen, Kibanadai-Nishi, Miyazaki 889-2192, Japan.

ABSTRACT
Viable bacterial cells impaled with a single particle of a nano-sized acicular material formed when a mixture containing the cells and the material was exposed to a sliding friction field between polystyrene and agar gel; hereafter, we refer to these impaled cells as penetrons. We have used nano-sized acicular material to establish a novel method for bacterial transformation. Here, we generated penetrons that carried antisense DNA adsorbed on nano-sized acicular material (α-sepiolite) by providing sliding friction onto the surface of agar gel; we then investigated whether penetron formation was applicable to gene silencing techniques. Antisense DNA was artificially synthesized as 15 or 90mer DNA oligonucleotides based on the sequences around the translation start codon of target mRNAs. Mixtures of bacterial cells with antisense DNA adsorbed on α-sepiolite were stimulated by sliding friction on the surface of agar gel for 60 s. Upon formation of Escherichia coli penetrons, β-lactamase and β-galactosidase expression was evaluated by counting the numbers of colonies formed on LB agar containing ampicillin and by measuring β-galactosidase activity respectively. The numbers of ampicillin resistant colonies and the β-galactosidase activity derived from penetrons bearing antisense DNA (90mer) was repressed to 15% and 25%, respectively, of that of control penetrons which lacked antisense DNA. Biphenyl metabolite, ring cleavage yellow compound produced by Pseudomonas pseudoalcaligenes penetron treated with antisense oligonucleotide DNA targeted to bphD increased higher than that lacking antisense DNA. This result indicated that expression of bphD in P. pseudoalcaligenes penetrons was repressed by antisense DNA that targeted bphD mRNA. Sporulation rates of Bacillus subtilis penetrons treated with antisense DNA (15mer) targeted to spo0A decreased to 24.4% relative to penetrons lacking antisense DNA. This novel method of gene silencing has substantial promise for elucidation of gene function in bacterial species that have been refractory to experimental introduction of exogenous DNA.

No MeSH data available.


Related in: MedlinePlus

Schematic representation of the Yoshida effect. The Yoshida effect is defined as the formation of complexes called penetrons, which are bacterial cells, each impaled by a single nano-sized acicular material in a friction field formed at a hydrogel interface. The hydrogel, interface forming material, nano-sized acicular material, bacterial cells, sliding friction, and an energy source to provide the friction force are each essential to the formation of penetrons. The hydrogel (e. g., agar, gellan gum, κ-karagenan) involved shear stress at more than 2.1 N. The interface forming material could comprise polymer material such as polystyrene, polyethylene, or acrylonitrile butanediene rubber. The optical vertical reaction force against hydrogel was around 40 gf/cm2. Multi-walled carbon nanotube, maghemite, or α-sepiolite are each nano-sized acicular materials that can generate a Yoshida effect. The sliding friction force can be represented with the following formula: F = μW, where μ and W denote the frictional coefficient and the vertical reaction force, respectively. When using 2 and 5% agar hydrogel, frictional coefficient value increased 0.038 to 0.078 and 0.081 to 0.233 respectively by given sliding stimulus for 15 seconds. The rapid increase in frictional resistance was essential for the Yoshida effect.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4230895&req=5

Figure 1: Schematic representation of the Yoshida effect. The Yoshida effect is defined as the formation of complexes called penetrons, which are bacterial cells, each impaled by a single nano-sized acicular material in a friction field formed at a hydrogel interface. The hydrogel, interface forming material, nano-sized acicular material, bacterial cells, sliding friction, and an energy source to provide the friction force are each essential to the formation of penetrons. The hydrogel (e. g., agar, gellan gum, κ-karagenan) involved shear stress at more than 2.1 N. The interface forming material could comprise polymer material such as polystyrene, polyethylene, or acrylonitrile butanediene rubber. The optical vertical reaction force against hydrogel was around 40 gf/cm2. Multi-walled carbon nanotube, maghemite, or α-sepiolite are each nano-sized acicular materials that can generate a Yoshida effect. The sliding friction force can be represented with the following formula: F = μW, where μ and W denote the frictional coefficient and the vertical reaction force, respectively. When using 2 and 5% agar hydrogel, frictional coefficient value increased 0.038 to 0.078 and 0.081 to 0.233 respectively by given sliding stimulus for 15 seconds. The rapid increase in frictional resistance was essential for the Yoshida effect.

Mentions: As shown in Figure 1, a bacterial cell impaled by a nano-sized acicular material (penetron) was formed when bacterial cells and nano-sized acicular materials were exposed to a friction field in hydrogel. This phenomenon is called the Yoshida effect (Yoshida and Sato [2009]). Penetrons readily take up exogenous DNA; consequently, penetrons are applicable to a genetic transformation technique called tribos transformation (Yoshida and Sato [2009]). Transformation efficiency of E. coli penetrons is 107 colony forming units (cfu) per 1 μg of plasmid DNA (Yoshida et al. [2007]). Nano-sized acicular materials, e.g., chrysotile and sepiolite, have high affinity for nucleic acid. Notably, DNA adsorbed on nano-sized acicular materials is protected from enzyme-mediated nucleolytic degradation and remains stable (Somiya et al. [2012]).


The use of nano-sized acicular material, sliding friction, and antisense DNA oligonucleotides to silence bacterial genes.

Mitsudome Y, Takahama M, Hirose J, Yoshida N - AMB Express (2014)

Schematic representation of the Yoshida effect. The Yoshida effect is defined as the formation of complexes called penetrons, which are bacterial cells, each impaled by a single nano-sized acicular material in a friction field formed at a hydrogel interface. The hydrogel, interface forming material, nano-sized acicular material, bacterial cells, sliding friction, and an energy source to provide the friction force are each essential to the formation of penetrons. The hydrogel (e. g., agar, gellan gum, κ-karagenan) involved shear stress at more than 2.1 N. The interface forming material could comprise polymer material such as polystyrene, polyethylene, or acrylonitrile butanediene rubber. The optical vertical reaction force against hydrogel was around 40 gf/cm2. Multi-walled carbon nanotube, maghemite, or α-sepiolite are each nano-sized acicular materials that can generate a Yoshida effect. The sliding friction force can be represented with the following formula: F = μW, where μ and W denote the frictional coefficient and the vertical reaction force, respectively. When using 2 and 5% agar hydrogel, frictional coefficient value increased 0.038 to 0.078 and 0.081 to 0.233 respectively by given sliding stimulus for 15 seconds. The rapid increase in frictional resistance was essential for the Yoshida effect.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4230895&req=5

Figure 1: Schematic representation of the Yoshida effect. The Yoshida effect is defined as the formation of complexes called penetrons, which are bacterial cells, each impaled by a single nano-sized acicular material in a friction field formed at a hydrogel interface. The hydrogel, interface forming material, nano-sized acicular material, bacterial cells, sliding friction, and an energy source to provide the friction force are each essential to the formation of penetrons. The hydrogel (e. g., agar, gellan gum, κ-karagenan) involved shear stress at more than 2.1 N. The interface forming material could comprise polymer material such as polystyrene, polyethylene, or acrylonitrile butanediene rubber. The optical vertical reaction force against hydrogel was around 40 gf/cm2. Multi-walled carbon nanotube, maghemite, or α-sepiolite are each nano-sized acicular materials that can generate a Yoshida effect. The sliding friction force can be represented with the following formula: F = μW, where μ and W denote the frictional coefficient and the vertical reaction force, respectively. When using 2 and 5% agar hydrogel, frictional coefficient value increased 0.038 to 0.078 and 0.081 to 0.233 respectively by given sliding stimulus for 15 seconds. The rapid increase in frictional resistance was essential for the Yoshida effect.
Mentions: As shown in Figure 1, a bacterial cell impaled by a nano-sized acicular material (penetron) was formed when bacterial cells and nano-sized acicular materials were exposed to a friction field in hydrogel. This phenomenon is called the Yoshida effect (Yoshida and Sato [2009]). Penetrons readily take up exogenous DNA; consequently, penetrons are applicable to a genetic transformation technique called tribos transformation (Yoshida and Sato [2009]). Transformation efficiency of E. coli penetrons is 107 colony forming units (cfu) per 1 μg of plasmid DNA (Yoshida et al. [2007]). Nano-sized acicular materials, e.g., chrysotile and sepiolite, have high affinity for nucleic acid. Notably, DNA adsorbed on nano-sized acicular materials is protected from enzyme-mediated nucleolytic degradation and remains stable (Somiya et al. [2012]).

Bottom Line: Viable bacterial cells impaled with a single particle of a nano-sized acicular material formed when a mixture containing the cells and the material was exposed to a sliding friction field between polystyrene and agar gel; hereafter, we refer to these impaled cells as penetrons.Upon formation of Escherichia coli penetrons, β-lactamase and β-galactosidase expression was evaluated by counting the numbers of colonies formed on LB agar containing ampicillin and by measuring β-galactosidase activity respectively.This novel method of gene silencing has substantial promise for elucidation of gene function in bacterial species that have been refractory to experimental introduction of exogenous DNA.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Biochemistry and Applied Biosciences, University of Miyazaki, 1-1 Gakuen, Kibanadai-Nishi, Miyazaki 889-2192, Japan.

ABSTRACT
Viable bacterial cells impaled with a single particle of a nano-sized acicular material formed when a mixture containing the cells and the material was exposed to a sliding friction field between polystyrene and agar gel; hereafter, we refer to these impaled cells as penetrons. We have used nano-sized acicular material to establish a novel method for bacterial transformation. Here, we generated penetrons that carried antisense DNA adsorbed on nano-sized acicular material (α-sepiolite) by providing sliding friction onto the surface of agar gel; we then investigated whether penetron formation was applicable to gene silencing techniques. Antisense DNA was artificially synthesized as 15 or 90mer DNA oligonucleotides based on the sequences around the translation start codon of target mRNAs. Mixtures of bacterial cells with antisense DNA adsorbed on α-sepiolite were stimulated by sliding friction on the surface of agar gel for 60 s. Upon formation of Escherichia coli penetrons, β-lactamase and β-galactosidase expression was evaluated by counting the numbers of colonies formed on LB agar containing ampicillin and by measuring β-galactosidase activity respectively. The numbers of ampicillin resistant colonies and the β-galactosidase activity derived from penetrons bearing antisense DNA (90mer) was repressed to 15% and 25%, respectively, of that of control penetrons which lacked antisense DNA. Biphenyl metabolite, ring cleavage yellow compound produced by Pseudomonas pseudoalcaligenes penetron treated with antisense oligonucleotide DNA targeted to bphD increased higher than that lacking antisense DNA. This result indicated that expression of bphD in P. pseudoalcaligenes penetrons was repressed by antisense DNA that targeted bphD mRNA. Sporulation rates of Bacillus subtilis penetrons treated with antisense DNA (15mer) targeted to spo0A decreased to 24.4% relative to penetrons lacking antisense DNA. This novel method of gene silencing has substantial promise for elucidation of gene function in bacterial species that have been refractory to experimental introduction of exogenous DNA.

No MeSH data available.


Related in: MedlinePlus