Mini-Tn7 Insertion in an Artificial attTn7 Site Enables Depletion of the Essential Master Regulator CtrA in the Phytopathogen Agrobacterium tumefaciens.
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 plasmid-based 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.



Agrobacterium tumefaciens is a bacterial plant pathogen and natural genetic engineer. Thus, studies of essential processes, including cell cycle progression, DNA replication and segregation, cell growth, and division, may provide insights for limiting disease or improving biotechnology applications.
Division of Biological Sciences, University of Missouri, Columbia, Missouri, USA.
Wanda
W
Figueroa-Cuilan
Division of Biological Sciences, University of Missouri, Columbia, Missouri, USA.
Jeremy J
JJ
Daniel
Division of Biological Sciences, University of Missouri, Columbia, Missouri, USA.
Matthew
M
Howell
Division of Biological Sciences, University of Missouri, Columbia, Missouri, USA.
Aliyah
A
Sulaiman
Division of Biological Sciences, University of Missouri, Columbia, Missouri, USA brownpb@missouri.edu.
Pamela J B
PJ
Brown
D016960:Agrobacterium tumefaciens / Q000235:genetics*; D001426:Bacterial Proteins / Q000235:genetics*; D004251:DNA Transposable Elements; D015964:Gene Expression Regulation, Bacterial; D020043:Genes, Essential; D011401:Promoter Regions, Genetic
D001426:Bacterial Proteins; D004251:DNA Transposable Elements