Hydroxylamine assimilation by Rhodobacter capsulatus E1F1 -: Requirement of the hcp gene (hybrid cluster protein) located in the nitrate assimilation nas gene region for hydroxylamine reduction
Hydroxylamine assimilation by Rhodobacter capsulatus E1F1 -: Requirement of the hcp gene (hybrid cluster protein) located in the nitrate assimilation nas gene region for hydroxylamine reduction
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DOI：
10.1074/jbc.m404417200
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发表时间：
2004-10-29
影响因子：
4.8
通讯作者：
Moreno-Vivián, C
中科院分区：
文献类型：
--
作者：
Cabello, P;Pino, C;Moreno-Vivián, C
荚膜红细菌E1 F1是以硝酸盐为氮源的光合细菌。利用针对细菌同化硝酸还原酶中保守基序设计的引物，通过PCR扩增450-bp的DNA，并用于筛选基因组文库。一个粘粒携带的插入与四个SalI片段的2.8，4.1，4.5，和5.8 kb的分离，和DNA测序显示，它包含一个硝酸盐同化（nas）基因区，包括hcp基因编码的杂交簇蛋白（HCP）。hcp的表达可能是由相邻的hcp受体基因编码的亚硝酸盐敏感性阻遏物调节的。在大肠杆菌中过量产生并纯化了His（6）-HCP。HCP每分子含有约6个铁原子和4个不稳定的硫原子，与[2Fe-2S]和[4Fe-2S-2 O]簇的存在一致，并显示羟胺还原酶活性，在体外与甲基紫精作为还原剂形成氨。在最佳pH和温度（9.3和40 degreesC）下，NH 2 OH和甲基紫精的表观K-m值分别为1 mM和7 μ M。该酶活性对氧敏感，可被硫化物和铁试剂抑制。R.荚膜梭菌E1 F1以1 mM NH 2 OH为氮源，以光养方式生长，但不以异养方式生长，厌氧休眠细胞吸收高达10 mM NH 2 OH。铵在培养基中瞬时积累，其同化被谷氨酰胺合成酶抑制剂L-甲硫氨酸-D，L-亚砜亚胺阻止。此外，羟胺或亚硝酸盐生长的细胞表现出较高的羟胺还原酶活性。然而，R. capsulatus B10 S，一种缺乏整个hcp-nas区域的菌株，在1 mM NH 2 OH中不能生长。所以，E。过量产生HCP的大肠杆菌细胞在厌氧生长期间耐受羟胺更好。这些结果表明，HCP是参与同化NH 2 OH，一种有毒的产品，可能是在硝酸盐同化过程中形成的，可能在亚硝酸盐还原步骤。
Rhodobacter capsulatus E1F1 grows phototrophically with nitrate as nitrogen source. Using primers designed for conserved motifs in bacterial assimilatory nitrate reductases, a 450-bp DNA was amplified by PCR and used for the screening of a genomic library. A cosmid carrying an insert with four SalI fragments of 2.8, 4.1, 4.5, and 5.8 kb was isolated, and DNA sequencing revealed that it contains a nitrate assimilation (nas) gene region, including the hcp gene coding for a hybrid cluster protein (HCP). Expression of hcp is probably regulated by a nitrite-sensitive repressor encoded by the adjacent nsrR gene. A His(6)-HCP was overproduced in Escherichia coli and purified. HCP contained about 6 iron and 4 labile sulfide atoms per molecule, in agreement with the presence of both [2Fe-2S] and [4Fe-2S-2O] clusters, and showed hydroxylamine reductase activity, forming ammonia in vitro with methyl viologen as reductant. The apparent K-m values for NH2OH and methyl viologen were 1 mM and 7 muM, respectively, at the pH and temperature optima (9.3 and 40 degreesC). The activity was oxygen-sensitive and was inhibited by sulfide and iron reagents. R. capsulatus E1F1 grew phototrophically, but not heterotrophically, with 1 mM NH2OH as nitrogen source, and up to 10 mM NH2OH was taken up by anaerobic resting cells. Ammonium was transiently accumulated in the media, and its assimilation was prevented by L-methionine-D, L-sulfoximine, a glutamine synthetase inhibitor. In addition, hydroxylamine- or nitrite-grown cells showed the higher hydroxylamine reductase activities. However, R. capsulatus B10S, a strain lacking the whole hcp-nas region, did not grow with 1 mM NH2OH. Also, E. coli cells overproducing HCP tolerate hydroxylamine better during anaerobic growth. These results suggest that HCP is involved in assimilation of NH2OH, a toxic product that could be formed during nitrate assimilation, probably in the nitrite reduction step.