TITLE:
TnphoA and Its Use in Transposon Mutagenesis
AUTHORS:
D. K. Das, E. W. Nester
KEYWORDS:
TnphoA Mutagenesis; Agrobacterium tumefaciens; Transposition; Transmembrane Proteins; Fusion Proteins; Tranposase Protein
JOURNAL NAME:
Advances in Microbiology,
Vol.4 No.1,
January
16,
2014
ABSTRACT:
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.