Background Information
Genetic transformation of calcium treated Escherichia coli with plasmid DNA is a common laboratory technique, yet little is known about the mechanism by which transformation occurs.1 Transformation, the uptake of naked, exogenous DNA,2 is one of three methods of horizontal gene transfer: the movement of genetic information between bacteria in a method other than descent.3 While approximately 43 bacterial species are known to be naturally transformable,4 the transformation competence of others, such as E. coli, must be induced by treatment with calcium ions.5 Natural and artificially competent bacteria use different mechanisms of DNA translocation across the cell membrane.6 While acquired chromosomal DNA would have to be homologous enough to integrate into the bacterial chromosome for expression,7 acquired plasmid DNA can replicate and be expressed independently of the bacterial genome. Plasmid expression must be activated by factors in the cell, requiring some homology of the regulatory elements in the plasmid to those of the bacteria. Transformants can be selected for by transforming an antibiotic resistance gene into the bacteria and plating the bacteria on that antibiotic.
1. Cells must be made competent
2. Plasmid DNA must remain intact in the environment until contact with a competent cell
3. DNA must be translocated across the cell membrane
4. DNA must not be degraded by restriction enzymes in the cytoplasm
5. DNA must be expressed. Some homology of regulatory elements between the new DNA and the cell’s DNA is required for this.
6. Transformants, cells expressing the new DNA, must be positively selected for
Image adapted from Access Excellence, http://www.accessexcellence.org/RC/AB/WYW/cohen/cohen_4.html
Using the calcium treatment method, the transformation efficiency ([number of transformants]/[µg of DNA])8 is dependent on the presence of divalent cations, the growth phase of the culture, and the pH of the buffer.9 One E. coli study shows the transformation frequency ([number of transformants]/[number of recipients])10 increases linearly up to at least pH 8,11 while another suggests pH 7.25-7.75 are optimal for transformation.12
Many theories exist about why extracellular pH affects transformation frequencies. E. coli intracellular pH remains relatively constant, 7.44 to 7.84, at external pH 6.00 to 10.00.13 Any impact pH has on transformation therefore occurs outside the cell or at the cell membrane, where pH or the pH gradient may vary. Most theories regarding extracellular pH and transformation involve the proton motive force (PMF), the energized state of the membrane as a result of the charge separation.14 The membrane potential (ΔΨ) and pH gradient across the cell membrane (ΔpH) comprise the PMF.15 In E. coli, the ΔpH, with the interior alkaline, is about -120mV, at pH 5.0-5.5, and decreases to zero at pH 7.5 and above.16 The ΔΨ, with the interior negative, increases in magnitude from about -80mV at pH 5.0-5.5 to –145 mV at pH 8.5,17 becoming nearly constant at pH levels greater than 8.5.18 The PMF decreases from -200mV at pH 5.5 to –140mV at pH 7.519 Findings that transformation frequency in E. coli increases up to at least pH 8.0,20 suggest that primarily increases in ΔΨ, not the ΔpH, increase transformation frequency. This supports Grinius’ proposal of an electrogenic calcium-DNA symport mechanism in artificially transformable bacteria, with the ΔΨ as part of the driving force for translocation of DNA across the cell membrane.21 Alternatively, the ΔΨ may regulate transformation by affecting properties of the proteins in the translocation system.22 The PMF has previously been found to control the redox state of redox-sensitive groups in E. coli membrane-bound proteins.23 A combination of the ΔΨ as the driving force and as a regulator of protein properties may also be possible, as has been found for the PMF in E. coli solute transport systems.24 The ΔΨ may also, or instead, be required for proper binding of competent factors to the cell, allowing the cell to take up DNA.25 That the PMF, not just ΔΨ, is involved in E. coli infection with T4 phage and in E. coli transport systems supports that the entire PMF, not just ΔΨ, is actually involved in E. coli transformation.26 In contrast, studies using various ionophores have suggested that neither ΔΨ nor ΔpH impact the transformation frequency of calcium treated E. coli.27
To clarify the role of the PMF in E. coli, E. coli were tested at various extracellular pH levels. E. coli is an ideal subject for testing transformation at a wide range of pH levels, as E. coli can survive at extracellular pH 328 to pH 10.2,29 and grow at pH 5-9.30 Plasmid DNA has been chosen for use due to the lower degree of homology required for its expression, and its relative stability in a wide range of pH conditions, including extremely alkaline ones.31 It is hypothesized that, according to the electrogenic calcium-DNA symport mechanism,32 transformation frequencies will increase from pH 5 to pH 8, as ΔΨ increases. Transformation frequency at pH 8 through 10 will be relatively constant and no smaller than at pH 8, as the ΔΨ is relatively constant above pH 8.5.
