Mini-Tn7 insertion in an artificial attTn7 site enables depletion of the essential master regulator ctrA in the phytopathogen agrobacterium tumefaciens

Abstract

Mechanistic studies of many processes in Agrobacterium tumefaciens have been hampered by a lack of genetic tools for characterization of essential genes. In this study, we used a Tn7-based method for inducible control of transcription from an engineered site on the chromosome. We demonstrate that this method enables tighter control of inducible promoters than plasmidbased systems and can be used for depletion studies. The method enables the construction of depletion strains to characterize the roles of essential genes in A. tumefaciens. Here, we used the strategy to deplete the alphaproteobacterial master regulator CtrA and found that depletion of this essential gene results in dramatic rounding of cells, which become nonviable.