State Key Laboratory of Agrobiotechnology, College of Biological Sciences, China Agricultural University, Beijing, China.
School of Life Sciences, Beijing University of Chinese Medicine, Beijing, China.
Appl Environ Microbiol. 2019 Jan 9;85(2). doi: 10.1128/AEM.02394-18. Print 2019 Jan 15.
The bacterium MSR-1 forms nanosized membrane-enclosed organelles termed magnetosomes. The operon, part of the magnetosome island (MAI), includes the , , , and -like genes, which initiate gene transcription via the same promoter. We used a combination of molecular biological techniques (targeting of cross-linking reagents) and high-resolution mass spectrometry to investigate the coordinated activity of the four MamXY proteins in magnetite biomineralization. The FtsZ-like protein was shown by confocal laser scanning microscopy to be dispersed in the cytoplasm in the early stage of cell growth and then gradually polymerized along the magnetosome chain. Interactions of various pairs of MamXY proteins were observed using a bacterial two-hybrid system. We constructed a recombinant FtsZ-like-overexpressing strain, examined its growth patterns, and extracted magnetosome membrane proteins using a modified SDS/boiling method with BSG-d/d reagent, which helped stabilize interactions among MamXY proteins. In liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis, MamY expression was detected first and remained highest among the four proteins throughout all stages of cell growth. MamX and MamZ expression was detected subsequently. The four proteins displayed coordinated expression patterns during the magnetosome maturation process. Unique peptides discovered in the MamXY protein sequences appeared to constitute "hidden" interaction sites involved in the formation of MamXY complex that helped control magnetosome shape and size. operon genes play an essential role in magnetite biomineralization, participate in redox reactions, and control magnetosome shape and size. However, mechanisms whereby the four MamXY proteins function together in iron oxidoreduction and transport are poorly understood. We used a combination of targeted cross-linking techniques and high-resolution mass spectrometry to elucidate the coordinated activity patterns of the MamXY proteins during magnetite biomineralization. Our findings indicate that the FtsZ-like protein undergoes polymerization and then recruits MamY, MamX, and MamZ in turn, and that these interactions depend on unique peptides present in the protein sequences. A hypothetical model of the functionalities of these proteins is proposed that accounts for the findings and provides a basis for further studies of coordination among magnetosome island (MAI) gene clusters during the process of magnetosome formation.
该细菌 MSR-1 形成纳米大小的膜封闭的称为磁小体的细胞器。操纵子，是磁体岛（MAI）的一部分，包括 ， ， ，和 - 样基因，它们通过相同的启动子启动基因转录。我们使用了分子生物学技术（靶向交联试剂）和高分辨率质谱相结合的方法来研究四个 MamXY 蛋白在磁铁矿生物矿化中的协调活性。通过共焦激光扫描显微镜显示 FtsZ 样蛋白在细胞生长的早期在细胞质中分散，然后沿着磁小体链逐渐聚合。使用细菌双杂交系统观察了各种对 MamXY 蛋白的相互作用。我们构建了一个重组 FtsZ 样过表达菌株，检查了它的生长模式，并使用改良的 SDS/煮沸方法提取磁小体膜蛋白，其中 BSG-d/d 试剂有助于稳定 MamXY 蛋白之间的相互作用。在液相色谱 - 串联质谱（LC-MS/MS）分析中，首先检测到 MamY 表达，并且在细胞生长的所有阶段，其表达量都是四个蛋白中最高的。随后检测到 MamX 和 MamZ 的表达。在磁小体成熟过程中，这四个蛋白显示出协调的表达模式。在 MamXY 蛋白序列中发现的独特肽似乎构成了“隐藏”的相互作用位点，参与了 MamXY 复合物的形成，有助于控制磁小体的形状和大小。操纵子基因在磁铁矿生物矿化中起着至关重要的作用，参与氧化还原反应，并控制磁小体的形状和大小。然而，四个 MamXY 蛋白在铁氧化还原和运输中协同作用的机制尚不清楚。我们使用了靶向交联技术和高分辨率质谱相结合的方法来阐明在磁铁矿生物矿化过程中 MamXY 蛋白的协调活性模式。我们的发现表明 FtsZ 样蛋白进行聚合，然后依次招募 MamY、MamX 和 MamZ，并且这些相互作用依赖于蛋白序列中存在的独特肽。提出了这些蛋白质的功能的假设模型，该模型解释了这些发现，并为进一步研究磁体岛（MAI）基因簇在磁小体形成过程中的协调作用提供了基础。
