TITLE:
Characterization of Avirulent TnphoA Mutants in Agrobacterium tumefaciens to Enhance Transformation Efficiency
AUTHORS:
Dilip K. Das, Eugene W. Nester
KEYWORDS:
Transposon, Mutagenesis, Agrobacterium tumefaciense, Bradyrhizobium japonicum
JOURNAL NAME:
Advances in Microbiology,
Vol.4 No.9,
July
28,
2014
ABSTRACT:
Protein fusion with
the Escherichia coli alkaline
phosphatase is used extensively for the analysis of the topology of membrane
protein. Agrobacterium strain A6007
was mutagenized with E. coli strain
mm294A plasmid pRK609 having TnphoA to obtain mutants defective in virulence.
Because alkaline phosphatase activity is only detected when the PhoA gene
product from the transposon is secreted out of the protoplasm, the virulence
mutants are located in genes that code for transmembrane or periplasmic
proteins. Attempts were made to obtain the sequences adjacent to the TnphoA
inserts through several different approaches including Inverse PCR, Cloning,
and Tail PCR. Transposon-adjacent sequence was obtained from one membrane
anchor subunit in Bradyrhizobium
japonicum i.e. succinate dehydrogenase which has enhanced transformation efficiency.