FIG. 3.

Construction of inducible NixA-PhoA fusions. A 1,417-bp PCR fragment encoding the mature PhoA polypeptide of TnphoA was amplified from plasmid pRT733 and subcloned into the EcoRV site of pBluescript II SK(+). Two EcoRI sites within the phoA sequence were eliminated by PCR mutagenesis (G717C and A1050G). The phoA sequence was then excised at primer-encoded PstI-NruI sites and ligated into these sites in pLKC480 to create pAPF1. The phoA sequence was reamplified from pAPF1 by using primers encoding XhoI and KpnI restriction sites and ligated into pBluescript II SK(+), yielding the IPTG-inducible PhoA fusion vector pBAF. Black boxes indicate the convergence of the TnphoA sequence and the sequence encoding the mature E. coli PhoA polypeptide. The last 8 of 16 amino acids of the in-frame TnphoA insertion sequence and their nucleotide sequence are shown, along with the corresponding sequences encoded by restriction sites in pBAF. nixA truncates were ligated in EcoRI-SalI fragments.