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 organisms 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°C. Optimization of the conditions for natural transformation resulted in a transformation efficiency of up to 1.7×10³ transformants per g of DNA. The exogenous DNAs were integrated stably into the targeted sites of the T. elongatus genome via homologous recombination by double crossovers.Communicated by H. Ikeda