Electrotransformation of Thiobacillus ferrooxidans with plasmids containing a mer determinant.
The mer operon from a strain of Thiobacillus ferrooxidans (C. Inoue, K. Sugawara, and T. Kusano, Mol. Microbiol. 5:2707-2718, 1991) consists of the regulatory gene merR and an operator-promoter region followed by merC and merA structural genes and differs from other known gram-negative mer operons. We have constructed four potential shuttle plasmids composed of a T. ferrooxidans-borne cryptic plasmid, a pUC18 plasmid, and the above-mentioned mer determinant as a selectable marker. Mercury ion-sensitive T. ferrooxidans strains were electroporated with constructed plasmids, and one strain, Y4-3 (of 30 independent strains tested), was found to have a transformation efficiency of 120 to 200 mercury-resistant colonies per microgram of plasmid DNA. This recipient strain was confirmed to be T. ferrooxidans by physiological, morphological, and chemotaxonomical data. The transformants carried a plasmid with no physical rearrangements through 25 passages under no selective pressure. Cell extracts showed mercury ion-dependent NADPH oxidation activity.
Kusano
T
T
Laboratory of Plant Genetic Engineering, Akita Prefectural College of Agriculture, Japan.
Sugawara
K
K
Inoue
C
C
Takeshima
T
T
Numata
M
M
Shiratori
T
T
D015139:Blotting, Southern; D003001:Cloning, Molecular; D004352:Drug Resistance, Microbial / Q000235:genetics; D004926:Escherichia coli / Q000187:drug effects / Q000235:genetics; D005798:Genes, Bacterial / Q000235:genetics*; D005819:Genetic Markers / Q000235:genetics; D005822:Genetic Vectors / Q000235:genetics*; D008628:Mercury / Q000494:pharmacology; D010957:Plasmids / Q000235:genetics*; D015183:Restriction Mapping; D013855:Thiobacillus / Q000187:drug effects / Q000235:genetics*; D014169:Transformation, Bacterial / Q000235:genetics*
D005819:Genetic Markers; D008628:Mercury