TnphoA and Its Use in Transposon Mutagenesis

Abstract

TnphoA construct was derived from the transposon Tn5,\rwhich contained a kanamycin resistance gene flanked by two IS50 elements. This\rconstruct contains a version of the alkaline phosphatase gene with its signal\rsequence and promoter deleted, which will result in a blue colony phenotype on\rX-phos (5-bromo-4-chloro-3-indolyl phosphate toluidine salt) containing media\rwhen secreted into the periplasm. This secretion can occur only if the TnphoA\rhas been inserted into a gene with a signal sequence which directs that\rgene-product to leave the cytosol. Additionally, the transposon must be inserted into\rthe correct orientation and the same reading frame as the gene has been inserted\rinto. TnphoA’s transposition activity derives from that of this Tn5 transposon\rby a conservative mechanism in a relatively rare but highly regulated process.\rThe phoA portion of the TnphoA construct came from Escherichia coli K12 where it coded for the periplasmic\rprotein alkaline phosphatase. This enzyme must be secreted from the cytoplasm\rin order to demonstrate activity and its secretion is determined by the\rpresence of a signal sequence at its amino-terminal end. TnphoA mutagenesis is\rthe identification of new genes that code for transmembrane or secreted\rproteins. TnphoA is an extremely useful construct, which along with other\rderivatives of the transposon Tn5 is used extensively in transposon mutagenesis\rbased genetic analysis. TnphoA will ensure its continued significance and\rprominence in the area of transposon mutagenesis. TnphoA has been used to\risolate new chromosomal genes, whose products are secreted, as in the case of\rsome virulence genes in Agrobacterium\rtumefaciens and symbiotic genes in Rhizobium\rmeliloti.