Uchiya K I, Tohsuji M, Nikai T, Sugihara H, Sasakawa C
Department of Microbiology, Faculty of Pharmacy, Meijo University, Nagoya, Japan.
J Bacteriol. 1996 Aug;178(15):4548-54. doi: 10.1128/jb.178.15.4548-4554.1996.
A gene encoding a nonspecific phosphatase, named PhoN-Sf, was identified on the large virulence plasmid (pMYSH6000) of Shigella flexneri 2a YSH6000. The phosphatase activity in YSH6000 was observed under high-phosphate conditions. However, it was found that low-phosphate conditions induced a slightly higher level of activity. The nucleotide sequence of the phoN-Sf region cloned from pMYSH6000 possessing the phoN-Sf gene encoded 249 amino acids with a typical signal sequence at the N terminus. The deduced amino acid sequence of the PhoN-Sf protein revealed significant homology to sequences of nonspecific acid phosphatases of other bacteria, such as Providencia stuartii (PhoN, 83.2%), Morganella morganii (PhoC, 80.6%), Salmonella typhimurium (PhoN, 47.8%), and Zymomonas 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 electrophoresis was calculated to be 27 kDa. The 20 amino acids at the N terminus corresponded to the 20 amino acid residues following the putative signal sequence of PhoN-Sf protein deduced from the nucleotide sequence. The PhoN-Sf activity had a pH optimum of 6.6, and the optimum temperature was 37 degrees C. The enzymatic activity was inhibited by diisopropyl fluorophosphate, N-bromosuccinimide, or dithiothreitol but not by EDTA. The subcellular localization of the PhoN-Sf protein in YSH6000 revealed that the protein was found predominantly in the periplasm. Examination of Shigella and enteroinvasive Escherichia coli strains for PhoN-Sf production by immunoblotting with the PhoN-specific antibody and for the presence of phoN-Sf DNA by using a phoN-Sf probe indicated that approximately one-half of the strains possessed the phoN-Sf gene on the large plasmid and expressed the PhoN-Sf protein. The Tn5 insertion mutants of YSH6000 possessing phoN-Sf::Tn5 still retained wild-type levels of invasiveness, as well as the subsequent spreading capacity 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.
在福氏志贺菌2a YSH6000的大毒力质粒（pMYSH6000）上鉴定出一个编码非特异性磷酸酶的基因，命名为PhoN-Sf。在高磷酸盐条件下观察到YSH6000中的磷酸酶活性。然而，发现低磷酸盐条件诱导的活性水平略高。从含有phoN-Sf基因的pMYSH6000克隆的phoN-Sf区域的核苷酸序列编码249个氨基酸，在N端具有典型的信号序列。PhoN-Sf蛋白的推导氨基酸序列与其他细菌的非特异性酸性磷酸酶序列具有显著同源性，如普罗威登斯菌（PhoN，83.2%）、摩根摩根菌（PhoC，80.6%）、鼠伤寒沙门氏菌（PhoN，47.8%）和运动发酵单胞菌（PhoC，34.8%）。纯化了PhoN-Sf蛋白并对其生化特性进行了表征。该蛋白在十二烷基硫酸钠-聚丙烯酰胺凝胶电泳上的表观分子量经计算为27 kDa。N端的20个氨基酸对应于从核苷酸序列推导的PhoN-Sf蛋白假定信号序列之后的20个氨基酸残基。PhoN-Sf活性的最适pH为6.6，最适温度为37℃。酶活性受到二异丙基氟磷酸酯、N-溴代琥珀酰亚胺或二硫苏糖醇的抑制，但不受EDTA的抑制。PhoN-Sf蛋白在YSH6000中的亚细胞定位显示该蛋白主要存在于周质中。用PhoN特异性抗体进行免疫印迹检测志贺菌和侵袭性大肠杆菌菌株的PhoN-Sf产生情况，并用phoN-Sf探针检测phoN-Sf DNA的存在情况，结果表明约一半的菌株在大质粒上拥有phoN-Sf基因并表达PhoN-Sf蛋白。具有phoN-Sf::Tn5的YSH6000的Tn5插入突变体仍保留野生型水平的侵袭性以及随后在MK2上皮细胞单层中的扩散能力，因此表明在体外条件下PhoN-Sf活性不参与志贺菌菌株毒力表型的表达。
