AiM  Vol.4 No.9 , July 2014
Characterization of Avirulent TnphoA Mutants in Agrobacterium tumefaciens to Enhance Transformation Efficiency
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.
Cite this paper: Das, D. and Nester, E. (2014) Characterization of Avirulent TnphoA Mutants in Agrobacterium tumefaciens to Enhance Transformation Efficiency. Advances in Microbiology, 4, 579-593. doi: 10.4236/aim.2014.49064.

[1]   Cangelosi, G.A., Huang, L., Puvanesarajah, V., Stacey, G., Ozga, D.A., Leigh, J.A. and Nester, E.W. (1987) Common Loci for Agrobacterium tumefaciens and Rhizobium meliloti Exoplysaccharide Synthesis and Their Roles in Plant Interactions. Journal of Bacteriology, 169, 2086-2091.

[2]   Douglas, C.J., Halperin, W. and Nester, E.W. (1982) Agrobacterium tumefaciens Mutants Affected in Attachment in Plant Cells. Journal of Bacteriology, 152, 1265-1275.

[3]   Manoil, C. and Beckwith, J. (1985) A Transposon Probe for Protein Export Signals. Proceedings of the National Academy of Sciences USA, 87, 6684-6688.

[4]   Long, S., McCune, S. and Walker, G.C. (1988) Symbiotic Loci of Rhizobium meliloti Identified by Random TnphoA Mutagenesis. Journal of Bacteriology, 170, 4257-4265.

[5]   Sarthy, A., Michaelis, S. and Beckwith, J. (1981) Deletion Map of the Escherichia coli Structural Gene for Alkaline Phosphatase, PhoA. Journal of Bacteriology, 145, 288-292.

[6]   Backwith, J. and Silhavy, T.J. (1983) Genetic Analysis of Protein Export in Escherichia coli. Methods in Enzymology, 97, 3-11.

[7]   Cangelosi, G.A., Best, E.A., Martinetti, G. and Nester, E.W. (1991) Genetic Analysis of Agrobacterium. Methods in Enzymology, 204, 584-397.

[8]   Cangelosi, G.A., Ankenbauer, R.G. and Nester, E.W. (1990) Sugars Induce the Agrobacterium tumefaciens Genes through a Periplasmic Binding Protein and a Transmembrane Signal Protein. Proceedings of the National Academy of Sciences USA, 87, 6708-6712.

[9]   Huang, M.L., Cangelosi, G., Halperin, W. and Nester, E.W. (1990) A Chromosomal Agrobacterium tumefaciens Gene Required for Effective Plant Signal Transduction. Journal of Bacteriology, 172, 1814-1822.

[10]   Shimoda, N., Toyoda-Yamamoto, A., Aoki, S. and Machinda, Y. (1990) Genetic Evidence for an Interaction between the VirA Sensor Protein and the ChvE Sugar Binding Protein of Agrobacterium tumefaciens. Journal of Biological Chemistry, 268, 26552-26558.

[11]   Gardiol, A., Arias, A., Cervenansky, C. and Martinez-Drets, G. (1982) Succinate Dehydrogenase Mutant of Rhizobium meliloti. Journal of Bacteriology, 151, 1621-1623.

[12]   Gardiol, A. Georges, E., Truchet, L. and Dazzo, F.B. (1987) Requirement of Succinate Dehydrogenase Activity for Symbiotic Differentiation of Rhizobium meliloti in Alfalfa Nodules. Applied and Environmen
tal Microbiology, 53, 1947-1950.

[13]   Cohen, S.N., Chang, A.C.Y. and Hsu, L. (1972) Non Chromosomal Antibiotic Resistance in Bacteria: Genetic Transformation of E. coli by R-Factor DNA. Proceedings of the National Academy of Sciences USA, 69, 2110.

[14]   Chun, K.T., Edenberg, H.J., Kelley, M.R. and Goebl, M.G. (1997) Rapid Amplification of Uncharacterized Transposon-Tagged DNA Sequence from Genomic DNA. Yeast, 13, 233-240.<233::AID-YEA88>3.0.CO;2-E