Use of cloned DNA methylase genes to increase the frequency of transfer of foreign genes into Xanthomonas campestris pv. malvacearum.
In vitro-packaged cosmid libraries of DNA from the bacterium Xanthomonas campestris pv. malvacearum were restricted 200- to 1,000-fold when introduced into Mcr+ strains of Escherichia coli compared with restriction in the Mcr- strain HB101. Restriction was predominantly associated with the mcrBC+ gene in E. coli. A plasmid (pUFR052) encoding the XmaI and XmaIII DNA methylases was isolated from an X. campestris pv. malvacearum library by a screening procedure utilizing Mcr+ and Mcr- E. coli strains. Transfer of plasmids from E. coli strains to X. campestris pv. malvacearum by conjugation was enhanced by up to five orders of magnitude when the donor cells contained pUFR052 as well as the plasmid to be transferred. Subcloning of pUFR052 revealed that at least two regions of the plasmid were required for full modification activity. Use of such modifier plasmids is a simple, novel method that may allow the efficient introduction of genes into any organism in which restriction systems provide a potent barrier to such gene transfer.
De Feyter
R
R
Plant Pathology Department, University of Florida, Gainesville 32611.
Gabriel
D W
DW
D003001:Cloning, Molecular / Q000379:methods*; D003227:Conjugation, Genetic; D015254:DNA Modification Methylases / Q000235:genetics* / Q000378:metabolism; D004262:DNA Restriction Enzymes / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D015723:Gene Library; D010957:Plasmids / Q000235:genetics; D015183:Restriction Mapping; D014162:Transfection / Q000235:genetics*; D016959:Xanthomonas campestris / Q000201:enzymology / Q000235:genetics*
D015254:DNA Modification Methylases; D004262:DNA Restriction Enzymes