Type II restriction modification systems of Prevotella bryantii TC1-1 and Prevotella ruminicola 23 strains and their effect on the efficiency of DNA introduction via electroporation.
The restriction endonucleases PbrTI and Pru2I, isoschizomers of Sau3AI and HaeIII, were partially purified and characterized from anaerobic rumen bacteria Prevotella bryantii TC1-1 and Prevotella ruminicola 23, respectively. These are the first type II restriction endonucleases discovered in strains of the genus Prevotella, and they represent one of the barriers hindering gene transfer in these microorganisms. Heterologous DNA was protected against the action of the PbrTI or Pru2I by incubation in a cell-free extract of the respective strain which contained 20 mM EDTA. This led to the development of a protocol enabling successful electrotransformation of the P. bryantii TC1-1 strain with a pRH3 Bacteroides--Escherichia coli shuttle vector containing up to 7-kb long DNA inserts. Plasmid DNA isolated from the transformed strain facilitated the transfer with further increased efficiency and made possible the introduction of ligation reaction products directly to P. bryantii TC1-1 without passing them first through E. coli.
Accetto
Tomaz
T
University of Ljubljana, Biotechnical Faculty, Zootechnical Department, Groblje 3, 1230 Domzale, Slovenia.
Peterka
Matjaz
M
Avgustin
Gorazd
G
D001483:Base Sequence; D004247:DNA / Q000378:metabolism; D015252:Deoxyribonucleases, Type II Site-Specific / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D004492:Edetic Acid / Q000494:pharmacology; D018274:Electroporation; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005822:Genetic Vectors / Q000235:genetics / Q000378:metabolism; D018720:Prevotella / Q000201:enzymology*; D013379:Substrate Specificity; D014169:Transformation, Bacterial
D004791:Enzyme Inhibitors; D004247:DNA; D004492:Edetic Acid; D015252:Deoxyribonucleases, Type II Site-Specific