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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.

The heritable natural competency trait of Burkholderia pseudomallei in other Burkholderia species through comE and crp