摘要：Natural competency requires uptake of exogenous DNA from the environment and the integration of that DNA into recipient bacteria can be used for DNA-repair or genetic diversification. The Burkholderia genus is unique in that only some of the species and strains are naturally competent. We identified and characterized two genes, comE and crp , from naturally competent B . pseudomallei 1026b that play a role in DNA uptake and catabolism. Single-copies of rhamnose-inducible comE and crp genes were integrated into a Tn7 attachment-site in non-naturally competent Burkholderia including pathogens B . pseudomallei K96243, B . cenocepacia K56-2, and B . mallei ATCC23344. Strains expressing comE or crp were assayed for their ability to uptake and catabolize DNA. ComE and Crp allowed non-naturally competent Burkholderia species to catabolize DNA, uptake exogenous gfp DNA and express GFP. Furthermore, we used synthetic comE and crp to expand the utility of the λ-red recombineering system for genetic manipulation of non-competent Burkholderia species. A newly constructed vector, pKaKa4, was used to mutate the aspartate semialdehyde dehydrogenase ( asd ) gene in four B . mallei strains, leading to the complete attenuation of these tier-1 select-agents. These strains have been excluded from select-agent regulations and will be of great interest to the field.
