Genome Sequence, Proteome Profile, and Identification of a Multiprotein Reductive Dehalogenase Complex in Dehalogenimonas alkenigignens Strain BRE15M

Bacteria of the genus Dehalogenimonas respire with vicinally halogenated alkanes via dihaloelimination. We aimed to describe involved proteins and their supermolecular organization. Metagenomic sequencing of a Dehalogenimonas-containing culture resulted in a 1.65 Mbp draft genome of Dehalogenimonas alkenigignens strain BRE15M. It contained 31 full-length reductive dehalogenase homologous genes (rdhA), but only eight had cognate rdhB gene coding for membrane-anchoring proteins. Shotgun proteomics of cells grown with 1,2-dichloropropane as an electron acceptor identified 1152 proteins representing more than 60% of the total proteome. Ten RdhA proteins were detected, including a DcpA ortholog, which was the strongest expressed RdhA. Blue native gel electrophoresis (BNE) demonstrating maximum activity was localized in a protein complex of 146–242 kDa. Protein mass spectrometry revealed the presence of DcpA, its membrane-anchoring protein DcpB, two hydrogen uptake hydrogenase subunits (HupL and HupS), an iron–sulfur protein (HupX), and subunits of a redox protein with a molybdopterin-binding motif (OmeA and OmeB) in the complex. BNE after protein solubilization with different detergent concentrations revealed no evidence for an interaction between the putative respiratory electron input module (HupLS) and the OmeA/OmeB/HupX module. All detected RdhAs comigrated with the organohalide respiration complex. Based on genomic and proteomic analysis, we propose quinone-independent respiration in Dehalogenimonas.脱卤单胞菌属（Dehalogenimonas）细菌通过二卤消除反应，以邻位卤代烷烃为呼吸底物进行能量代谢。本研究旨在阐明其参与呼吸过程的蛋白质及其超分子组装机制。对含脱卤单胞菌的培养物进行宏基因组测序，获得了产烯脱卤单胞菌（Dehalogenimonas alkenigignens）菌株BRE15M的1.65 Mbp草图基因组。该基因组包含31个完整的还原脱卤酶同源基因（rdhA），但仅8个带有编码膜锚定蛋白的同源rdhB基因。以1,2-二氯丙烷作为电子受体培养菌体后，通过鸟枪法蛋白质组学（shotgun proteomics）鉴定到1152种蛋白质，覆盖了总蛋白质组的60%以上。共检测到10种RdhA蛋白，其中包括DcpA同源蛋白，其表达量为所有RdhA蛋白中最高。蓝色非变性凝胶电泳（Blue Native Electrophoresis, BNE）显示，该酶的最大活性位于146–242 kDa的蛋白质复合物中。蛋白质质谱分析表明，该复合物中包含DcpA、其膜锚定蛋白DcpB、两个摄氢氢化酶亚基（HupL和HupS）、一种铁硫蛋白（HupX），以及带有钼蝶呤结合基序的氧化还原蛋白亚基（OmeA和OmeB）。使用不同浓度去污剂溶解蛋白质后进行BNE分析，未发现推定的呼吸电子输入模块（HupLS）与OmeA/OmeB/HupX模块之间存在相互作用的证据。所有检测到的RdhA蛋白均与有机卤呼吸复合物共迁移。基于基因组和蛋白质组学分析，我们提出脱卤单胞菌存在不依赖醌的呼吸途径。