Natural transformation of the thermophilic cyanobacterium Thermosynechococcus elongatus BP-1: a simple and efficient method for gene transfer.
Proteins derived from the thermophilic cyanobacterium Thermosynechococcus elongatus BP-1, which performs plant-type oxygenic photosynthesis, are suitable for biochemical, biophysical and X-ray crystallographic studies. We found that T. elongatus displays natural transformation, and we established a simple and efficient protocol for transferring exogenous DNAs into the organism's genome. We obtained transformants directly on selective agar plates without having to amplify them prior to plating. We constructed several targeting vectors that enabled us to insert exogenous DNAs into specific sites without disrupting endogenous genes and operons. We also developed a new selectable marker gene for T. elongatus by optimizing the codons of the gene encoding a kanamycin nucleotidyltransferase derived from the thermophilic bacterium Bacillus stearothermophilus. This synthetic gene enabled us to select transformants as kanamycin-resistant colonies on agar plates at 52 degrees C. Optimization of the conditions for natural transformation resulted in a transformation efficiency of up to 1.7 x 10(3) transformants per microg of DNA. The exogenous DNAs were integrated stably into the targeted sites of the T. elongatus genome via homologous recombination by double crossovers.
Onai
K
K
Center for Gene Research, Nagoya University, Furo, Chikusa, 464-8602 Nagoya, Japan.
Morishita
M
M
Kaneko
T
T
Tabata
S
S
Ishiura
M
M
D000458:Cyanobacteria / Q000235:genetics*; D004269:DNA, Bacterial / Q000235:genetics; D018274:Electroporation; D018014:Gene Transfer Techniques; D005798:Genes, Bacterial; D005822:Genetic Vectors; D006358:Hot Temperature; D016133:Polymerase Chain Reaction; D014170:Transformation, Genetic
D004269:DNA, Bacterial