Transformation of the cyanobacterium Synechocystis sp. PCC 6803 as a tool for genetic mapping: optimization of efficiency.
The cyanobacterium Synechocystis sp. PCC 6803 is transformable at high efficiency and integrates DNA by homologous double recombination. However, several genetic mapping procedures depend on the ability to generate transformants even with very small amounts of added DNA. This study is aimed at optimizing the transformation efficiency at limiting concentrations of exogenous DNA. The transformation efficiency showed little sensitivity to experimental conditions. Transformation with circular plasmid DNA was found to be no more than 30% more efficient than with linearized plasmid DNA. The efficiency of transformation remained essentially the same in the presence of competing DNA, indicating that the capacity of DNA uptake by the cells is not limiting. The incubation time of cells with DNA before plating (0-8 h) affected the transformation efficiency by up to 3-fold. Only minor changes in the efficiency were observed as a function of the presence of a membrane filter on the plate or the presence of TAE or TBE gel buffer residues in the transformation mixture. However, transformability of the host strain of Synechocystis sp. PCC 6803 was increased by two orders of magnitude if the sll1354 gene encoding the exonuclease RecJ was deleted. Therefore, the transformation efficiency of Synechocystis sp. PCC 6803 with exogenous DNA appears to be determined primarily by intracellular processes such as the efficiency of DNA processing and homologous recombination.
Kufryk
Galyna I
GI
Department of Plant Biology and Center for The Study of Early Events in Photosynthesis, Arizona State University, Tempe, 85287-1601, USA. galya@imap3.asu.edu
Sachet
Monika
M
Schmetterer
Georg
G
Vermaas
Wim F J
WF
D001426:Bacterial Proteins / Q000235:genetics; D002874:Chromosome Mapping / Q000379:methods*; D000458:Cyanobacteria / Q000235:genetics*; D005090:Exodeoxyribonucleases / Q000235:genetics; D005816:Genetic Complementation Test; D014169:Transformation, Bacterial
D001426:Bacterial Proteins; D005090:Exodeoxyribonucleases; C070765:recJ protein, Bacteria