Lu D, Boyd B, Lingwood C A
Department of Medical Genetics and Microbiology, Biochemistry, Toronto, Ontario M5G 1X8, Canada.
J Biol Chem. 1997 Nov 14;272(46):29033-8. doi: 10.1074/jbc.272.46.29033.
Very little is known about specific mechanisms for zinc accumulation and transport in bacteria. In this study a putative adhesin B in Hemophilus influenzae, the product of gene HI0119, has been identified as a periplasmic zinc-binding protein (PZP1). A pzp1-deficient mutant has been constructed which is defective for growth under aerobic conditions and grows poorly under anaerobic conditions. The growth defect is specifically rescued by supplementing the growth medium with high concentrations of zinc. Subcellular fractionation was used to localize PZP1 to the periplasmic region in a nontypeable H. influenzae strain and in a transfected recombinant Escherichia coli strain (TApzp1). Recombinant PZP1, purified from a periplasmic extract of E. coli strain TApzp1, contained approximately two zinc atoms/protein molecule as determined by neutron activation analysis and atomic absorption spectroscopy. The zinc atoms could be removed by incubation with EDTA, and, by further addition of zinc, a total of five zinc atoms/PZP1 could be bound. Direct binding of 65Zn to the recombinant protein by Western blot was demonstrated. Taken together, these results provide direct evidence that PZP1 plays a key role in zinc uptake by H. influenzae.
关于细菌中锌积累和转运的具体机制，人们了解甚少。在本研究中，已确定流感嗜血杆菌中一种假定的粘附素B（基因HI0119的产物）为周质锌结合蛋白（PZP1）。构建了一个pzp1缺陷型突变体，该突变体在有氧条件下生长有缺陷，在厌氧条件下生长也很差。通过在生长培养基中添加高浓度的锌，可特异性挽救生长缺陷。利用亚细胞分级分离技术，将PZP1定位在不可分型流感嗜血杆菌菌株和转染的重组大肠杆菌菌株（TApzp1）的周质区域。从大肠杆菌菌株TApzp1的周质提取物中纯化得到的重组PZP1，通过中子活化分析和原子吸收光谱法测定，每个蛋白质分子含有约两个锌原子。锌原子可通过与EDTA孵育去除，进一步添加锌后，每个PZP1总共可结合五个锌原子。通过蛋白质印迹法证明了65Zn与重组蛋白的直接结合。综上所述，这些结果提供了直接证据，表明PZP1在流感嗜血杆菌摄取锌的过程中起关键作用。
