IDENTIFICATION AND CHARACTERIZATION OF PHON-SF, A GENE ON THE LARGE PLASMID OF SHIGELLA-FLEXNERI 2A ENCODING A NONSPECIFIC PHOSPHATASE

A gene encoding a nonspecific phosphatase, named PhoN-Sf, was identifi ed on the large virulence plasmid (pMYSH6000) of Shigella flexneri 2a YSH6000. The phosphatase activity in YSH6000 was observed under high-p hosphate conditions. However, it was found that low-phosphate conditio ns induced a slightly higher level of activity. The nucleotide sequenc e of the phoN-Sf region cloned from pMYSH6000 possessing the phoN-Sf g ene encoded 249 amino acids with a typical signal sequence at the N te rminus, The deduced amino acid sequence of the PhoN-Sf protein reveale d significant homology to sequences of nonspecific acid phosphatases o f other bacteria, such as Providencia stuartii (PhoN, 83.2%), Morganel la morganii (PhoC, 80.6%), Salmonella typhimurium (PhoN, 47.8%), and Z ymomonas mobilis (PhoC, 34.8%). The PhoN-Sf protein was purified, and its biochemical properties were characterized. The apparent molecular mass of the protein on sodium dodecyl sulfate-polyacrylamide gel elect rophoresis was calculated to be 27 kDa, The 20 amino acids at the N te rminus corresponded to the 20 amino acid residues following the putati ve signal sequence of PhoN-Sf protein deduced from the nucleotide sequ ence. The PhoN-Sf activity had a pH optimum of 6.6, and the optimum te mperature was 37 degrees C. The enzymatic activity was inhibited by di isopropyl fluorophosphate, N-bromosuccinimide, or dithiothreitol but n ot by EDTA. The subcellular localization of the PhoN-Sf protein in YSH 6000 revealed that the protein was found predominantly in the periplas m. Examination of Shigella and enteroinvasive Escherichia coli strains for PhoN-Sf production by immunoblotting with the PhoN-specific antib ody and for the presence of phoN-Sf DNA by using a phoN-Sf probe indic ated that approximately one-half of the strains possessed the phoN-Sf gene on the large plasmid and expressed the PhoN-Sf protein. The Tn5 i nsertion mutants of YSH6000 possessing phoN-Sf::Tn5 still retained wil d-type levels of invasiveness, as well as the subsequent spreading cap acity in MK2 epithelial cell monolayers, thus suggesting that the PhoN -Sf activity is not involved in expression of the virulence phenotypes of Shigella strains under in vitro conditions.