Transformation of Acidiphilium by electroporation and conjugation

Abstract

Two techniques, electroporation and conjugation, have been used to introduce the RK2-based broad-host-range plasmids pRK415 and pLAFR3 into strains of the bacterial genus Acidiphilium. Using electroporation, cells were also transformed with a series of chimeric plasmids constructed by cloning cryptic Acidiphilium plasmids into the Escherichia coli vector pBR328. Various parameters affecting electroporation were investigated. Transformation efficiency varied widely with different recipient strains. Growth at an elevated temperature (37 degrees C) prior to electroporation increased transformation efficiency 10-fold compared with growth at 32 degrees C. For three strains tested, optimum transformation efficiency was obtained with field strengths of 10-15 kV/cm. Transformation efficiency increased linearly with increasing DNA concentration up to 10 micrograms/mL. Transformation efficiencies in these experiments ranged up to 10(4) transformants/micrograms DNA. Mobilization of pRK415 and pLAFR3 from E. coli strain S17.1 into several Acidiphilium strains was achieved following incubation for 3 h on nutrient agar medium (pH 7.0). Conjugation frequencies in the range of 10(-5)-10(-9) per recipient cell were obtained. Conjugation frequency was also dependent on recipient strain.

Genetic improvement of acidophilic bacteria for biohydrometallurgical applications

“…Taken as a whole, the cloning and analysis of T. ferrooxidans genes supports the concept that foreign genes can be introduced into acidophilic bacteria and controlled by elements which function in a wide variety of gram-negative bacteria, including E. coli. Expression of antibiotic resistances introduced into Acidiphilium also support this conclusion (Glenn et al, 1992).…”

“…So-called "shuttle" vectors have been constructed, which contain two or more separate replication origins, one native to the organism under study, and another E. coli origin to allow production of large amounts of the recombinant plasmids in E. coli, rather than in the study organism. A variety of these have been constructed for T. ferrooxidans and Acidiphilium (Rawlings et al, 1984Holmes et al, 1984Holmes et al, ,1986Shiratori et al, 1991;Glenn et al, 1992). Broad-host-range plasmids (with a single replication origin capable of function in a wide range of bacteria) may also be employed successfully (Glenn et al, 1992;Jin et al, 1992), and eliminate the need to clone plasmids from the desired host.…”

“…A variety of these have been constructed for T. ferrooxidans and Acidiphilium (Rawlings et al, 1984Holmes et al, 1984Holmes et al, ,1986Shiratori et al, 1991;Glenn et al, 1992). Broad-host-range plasmids (with a single replication origin capable of function in a wide range of bacteria) may also be employed successfully (Glenn et al, 1992;Jin et al, 1992), and eliminate the need to clone plasmids from the desired host. (4) Genetic control elements, or promoters (initiation sites for mRNA synthesis), which allow regulation of gene expression.…”

General Principles of Bacteria Electrotransformation: Key Steps

“…For example, an improved efficiency was obtained on S. aureus by the use of a yeast extract containing medium rather than the conventional SOC medium (Schenk and Laddaga, 1992), and the same effect was observed for E. coli C (Taketo, 1989). The effect of the growth temperature has been studied but results are not apparent in the same way, for E. coli Blue an increase of the transformation efficiency was obtained bygrowth at 18° instead ofthe classical37° (Chuang, et al, 1995), but the same effect was obtained by using a growth temperature of 44 oc for E. coli C. In fact it seems that in all studies the classical growth temperature (in terms of cellular growth rate) is not the optimal growth temperature for preparation of electrocompetent cells (Glenn, et al, 1992).…”

Characterization of a new bacteriophage which infects bacteria of the genus Acidiphilium

“…The genetic properties of these bacteria have not been extensively studied. We are investigating various techniques for transferring genetic information into such cells and conditions for transformation of Acidiphilium with plasmid DNA have recently been reported (Glenn et al, 1992). A transducing bacteriophage derived from an endogenous temperate phage would constitute an alternative mechanism for introducing DNA, which might result in higher efficiency.…”