TnphoA and Its Use in Transposon Mutagenesis

Abstract

TnphoA construct was derived from the transposon Tn5, which contain ed 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 insert ed 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 code d 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 .