The impact of O(2) on the Fe-S cluster biogenesis requirements of Escherichia coli FNR.
The impact of O(2) on the Fe-S cluster biogenesis requirements of Escherichia coli FNR.
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O(2) 对大肠杆菌 FNR 的 Fe-S 簇生物合成需求的影响。
DOI：
10.1016/j.jmb.2008.09.080
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发表时间：
2008
影响因子：
5.6
通讯作者：
Kiley,PatriciaJ
中科院分区：
文献类型：
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作者：
Mettert,ErinL;Outten,FWayne;Wanta,Brendan;Kiley,PatriciaJ
在这项研究中，功能的两个已建立的铁-硫簇生物合成途径，Isc（铁-硫簇）和Suf（硫动员），在有氧和厌氧生长条件下，通过测量大肠杆菌的全球厌氧调节FNR的活性进行比较。在厌氧条件下，FNR活性需要[4Fe-4S]簇。在含有各种缺失的iscSUAhscBAfdx操纵子的菌株中FNR依赖性启动子的表达的测定显示，在厌氧条件下，在不存在Isc途径的情况下，FNR活性降低了60%。相反，缺乏整个Suf途径的突变体具有正常的FNR活性，尽管Suf操纵子的过表达完全挽救了缺乏Isc途径的菌株中FNR活性的厌氧缺陷。SufA启动子的表达和SufD蛋白的水平在厌氧条件下的Isc−菌株中上调了2倍至3倍，这表明Suf途径的表达增加可能是缺乏Isc途径的菌株中剩余FNR活性的部分原因。相比之下，使用O2稳定的[4Fe-4S]簇FNR变体FNR-L28 H表明，在有氧条件下，suf操纵子的过表达不能恢复缺乏Isc途径的菌株的FNR活性。此外，FNR-L28 H活性在有氧条件下比在厌氧条件下受损更严重。如55 Fe标记实验所示，在有氧条件下对Isc途径的更大需求不是由于FNR-L28 H在有氧和厌氧条件下Fe-S簇的获取速率的变化。使用[35 S]蛋氨酸脉冲追踪测定，我们观察到Isc途径，而不是Suf途径，是在厌氧生长条件下开始时将O2失活的apo-FNR转化为[4Fe-4S]FNR所需的主要途径。综上所述，这些研究结果表明，在有氧和厌氧条件下的FNR Fe-S簇生物合成的Isc途径的主要作用。
In this study, the functions of two established Fe–S cluster biogenesis pathways, Isc (iron–sulfur cluster) and Suf (sulfur mobilization), under aerobic and anaerobic growth conditions were compared by measuring the activity of the Escherichia coli global anaerobic regulator FNR. A [4Fe–4S] cluster is required for FNR activity under anaerobic conditions. An assay of the expression of FNR-dependent promoters in strains containing various deletions of the iscSUAhscBAfdx operon revealed that, under anaerobic conditions, FNR activity was reduced by 60% in the absence of the Isc pathway. In contrast, a mutant lacking the entire Suf pathway had normal FNR activity, although overexpression of the suf operon fully rescued the anaerobic defect in FNR activity in strains lacking the Isc pathway. Expression of the sufA promoter and levels of SufD protein were upregulated by twofold to threefold in Isc−strains under anaerobic conditions, suggesting that increased expression of the Suf pathway may be partially responsible for the FNR activity remaining in strains lacking the Isc pathway. In contrast, use of the O2-stable [4Fe–4S] cluster FNR variant FNR-L28H showed that overexpression of the suf operon did not restore FNR activity to strains lacking the Isc pathway under aerobic conditions. In addition, FNR-L28H activity was more impaired under aerobic conditions than under anaerobic conditions. The greater requirement for the Isc pathway under aerobic conditions was not due to a change in the rate of Fe–S cluster acquisition by FNR-L28H under aerobic and anaerobic conditions, as shown by55Fe-labeling experiments. Using [35S]methionine pulse-chase assays, we observed that the Isc pathway, but not the Suf pathway, is the major pathway required for conversion of O2-inactivated apo-FNR into [4Fe–4S]FNR upon the onset of anaerobic growth conditions. Taken together, these findings indicate a major role for the Isc pathway in FNR Fe–S cluster biogenesis under both aerobic and anaerobic conditions.