Molecular genetic studies on iron-starvation stress response of Vibrio species and its involvement in pathogenesis

1. 从不同来源分离的副溶血性弧菌菌株在铁限制条件下产生铁载体，弧菌铁蛋白（VF）的能力进行了测定。临床分离株的VF产量显著高于食物分离株和沿海分离株，这表明临床分离株更高的VF产量可能与在肠道等铁限制条件下生存和增殖的选择优势有关。采用Mn选择法分离携带fur基因突变的副溶血性弧菌菌株，以评估fur与铁载体VF和铁抑制外膜蛋白（IROMPs）坐标表达变化的关系。25个突变体中有10个产生VF并表达至少两个IROMPs。基因测序鉴定出4个不同的点突变引起氨基酸变化。Western blotting结果显示，生长培养基中铁浓度对野生型皮草细胞内丰度、多舞率无显著影响。在副溶血性弧菌中首次发现了其调控。通过核苷酸测序，从致病性弧菌中分离出多种含Fur box的铁调控基因片段。克隆并分析了副溶血性弧菌3个IROMPs的全长基因。同时，克隆并分析了该菌的有氧肌动蛋白操纵子和血红素受体。前者的遗传组织是不寻常的，除了编码生物合成酶（iucABCD）和外膜受体（iutA）的基因外，它还包含负责铁转运系统（fhuBCD）的基因。从3种弧菌中克隆出编码MnSOD的铁抑制sodA基因，并在其启动子中鉴定出Fur box。这些mnsod是为了响应铁的限制而产生的。通过自杀载体的插入诱变鉴定了一个与VF生物发生有关的基因。对上述副溶血性弧菌基因进行了插入诱变，以阐明其功能。从三种弧菌中分离到编码atp依赖性蛋白酶的基因。阐明铁调控的蛋白酶表达是否与铁饥饿反应中Fur的分解有关是一个很有意义的问题。少

1. V.parahaemolyticus strains isolated from different sources were assayed for their ability to produce a siderophore, vibrioferrin (VF), under iron-limited conditions. VF production of clinical isolates was significantly higher than those for food and coastal isolates, suggesting that greater producitivity of VF by clinical isolates may be associated with a selective advantage for survival and proliferation under conditions of iron-limitation such as in the intestine.2. V.parahaemolyticus strains carrying mutations in the fur gene were isolated by the Mn selection method to assess the function of Fur in connection with alterations in the coordinate expression of the siderophore VF and iron-repressive outer membrane proteins (IROMPs). Ten of 25 mutants constitutively produce VF and expressed at least two IROMPs. DNA sequencing of the fur genes identified 4 different point mutations causing amino acid change. Western blotting using anti-Fur antiserum revealed that the intracellular abun … More dance of the wild-type Fur was not significantly affected by the iron concentration in the growth medium. Fur regulon was identified for the first time in V.parahaemolyticus.3. Many kinds of iron-regulated gene fragments containing a putative Fur box were isolated from pathogenic vibrios by their nucleotide sequencing. The full-length genes encoding three IROMPs in V.parahaemolyticus were cloned and analyzed. Also, the aerobactin operon and heme receptor were cloned and analyzed from V.mimicus. The genetic organization of the former was unusual in that it contains the genes responsible for the iron transport system (fhuBCD) in addition to the genes encoding biosynthesis enzymes (iucABCD) and an outer membrane receptor (iutA).4. Iron-repressible sodA genes encoding MnSOD were cloned from three vibrio species, and the Fur boxes were identified in their promoters. These MnSODs were produced in response to iron-limitation.5. One of the genes responsible for VF biogenesis was identified by insertion mutagenesis using a suicide vector. The insertion mutagensis was applied to the above-described V.parahaemolyticus genes to clarify their functions.6. The genes encoding ATP-dependent protease were isolated from three vibrio species. It is of great interest to clarify whether the iron-regulated protease expression is associated with decomposition of Fur in response to iron starvation. Less