Post Graduate Department of Biotechnology, T. M. Bhagalpur University, Bhagalpur, India. Department of Microbiology, School of Medicine, University of Washington, Seattle, USA.
TnphoA construct was derived from the transposon Tn5, which contained a kanamycin resistance gene flanked by two IS50 elements. This construct contains a version of the alkaline phosphatase gene with its signal sequence and promoter deleted, which will result in a blue colony phenotype on X-phos (5-bromo-4-chloro-3-indolyl phosphate toluidine salt) containing media when secreted into the periplasm. This secretion can occur only if the TnphoA has been inserted into a gene with a signal sequence which directs that gene-product to leave the cytosol. Additionally, the transposon must be inserted into the correct orientation and the same reading frame as the gene has been inserted into. TnphoA’s transposition activity derives from that of this Tn5 transposon by a conservative mechanism in a relatively rare but highly regulated process. The phoA portion of the TnphoA construct came from Escherichia coli K12 where it coded for the periplasmic protein alkaline phosphatase. This enzyme must be secreted from the cytoplasm in order to demonstrate activity and its secretion is determined by the presence of a signal sequence at its amino-terminal end. TnphoA mutagenesis is the identification of new genes that code for transmembrane or secreted proteins. TnphoA is an extremely useful construct, which along with other derivatives of the transposon Tn5 is used extensively in transposon mutagenesis based genetic analysis. TnphoA will ensure its continued significance and prominence in the area of transposon mutagenesis. TnphoA has been used to isolate new chromosomal genes, whose products are secreted, as in the case of some virulence genes in Agrobacterium tumefaciens and symbiotic genes in Rhizobium meliloti.
D. Das and E. Nester, "TnphoA and Its Use in Transposon Mutagenesis," Advances in Microbiology, Vol. 4 No. 1, 2014, pp. 15-19. doi: 10.4236/aim.2014.41004.
[1] C. Manoil and J. Beckwith, “TnphoA: A Transposon Probe for Protein Export Signals,” Proceedings of the National Academy of Sciences of the USA, Vol. 82, No. 23, 1985, pp. 8129-8133. http://dx.doi.org/10.1073/pnas.82.23.8129 [2] W. S. Reznikoff, “The Tn5 Transposon,” Annual Review of Microbiology, Vol. 47, 1993, pp. 945-963.
http://dx.doi.org/10.1146/annurev.mi.47.100193.004501 [3] I. Y. Goryshin and W. S. Reznikoff, “Tn5 in Vitro Transposition,” Journal of Biological Chemistry, Vol. 273, 1998, pp. 7367-7374. http://dx.doi.org/10.1074/jbc.273.13.7367 [4] C. S. Hoffman and A. Wright, “Fusions of Secreted Proteins to Alkaline Phosphatase: An Approach for Studying Protein Secretion,” Proceedings of the National Academy of Sciences of the USA, Vol. 82, No. 15, 1985, pp. 5107-5111. http://dx.doi.org/10.1073/pnas.82.15.5107 [5] J. Kadonga, A. Gautier, D. Straus, A. Charles, M. Edge and J. Knowles, Journal of Biological Chemistry, Vol. 259, 1984, pp. 2149-2154. [6] J. S. Kroll, B. Loynds, L. N. Brophy and E. R. Moxon, “The Bex Locus in Encapsulated Haemophilus influenze: A Chromosomal Region Involved in Capsule Polysaccharide Export,” Molecular Microbiology, Vol. 4, No. 11, 1990, pp. 1853-1862.
http://dx.doi.org/10.1111/j.1365-2958.1990.tb02034.x [7] C. Manoil, “Analysis of Protein Localization by Use of Gene Fusions with Complementary Properties,” Journal of Bacteriology, Vol. 172, 1990, pp. 1035-1042. [8] G. A. Cangelosi, E. A. Best, G. Martinetti and E. W. Nester, “Genetic Analysis of Agrobacterium,” Methods in Enzymology, Vol. 204, 1991, pp. 384-397. [9] S. Long, S. McCune and G. C. Walker, “Symbiotic Loci of Rhizobium meliloti Identified by Random TnphoA Mutagenesis,” Journal of Bacteriology, Vol. 170, 1988, pp. 4257-4265. [10] V. de Lorenzo, M. Herrero, U. Jakubzik and K. N. Timmis, “Mini-Tn5 Transposon Derivatives for Insertion Mutagenesis, Promoter Probing, and Chromosomal Insertion of Cloned DNA in Gram-Negative Eubacteria,” Journal of Bacteriology, Vol. 172, 1990, pp. 6568-6572. [11] J. J. Dennis and G. J. Zylstra, “Plasposons: Modular SelfCloning Minitransposon Derivatives for Rapid Genetic Analysis of Gram-negative Bacterial Genomes,” Applied and Environmental Microbiology, Vol. 64, 1998, pp. 2710-2715. [12] R. Biovin, F. P. Chalifour and P. Dion, “Construction of a Tn5 Derivative Encoding Bioluminescence and Its Introduction in Pseudomonas, Agrobacterium and Rhizobium,” Molecular and General Genetics, Vol. 213, 1988, pp. 50-55. http://dx.doi.org/10.1007/BF00333397