Initial Characterization of the FlgE Hook High Molecular Weight Complex of <i>Borrelia burgdorferi</i>

The spirochete periplasmic flagellum has many unique attributes. One unusual characteristic is the flagellar hook. This structure serves as a universal joint coupling rotation of the membrane-bound motor to the flagellar filament. The hook is comprised of about 120 FlgE monomers, and in most bacteria these structures readily dissociate to monomers (∼ 50 kDa) when treated with heat and detergent. However, in spirochetes the FlgE monomers form a large mass of over 250 kDa [referred to as a high molecular weight complex (HMWC)] that is stable to these and other denaturing conditions. In this communication, we examined specific aspects with respect to the formation and structure of this complex. We found that the Lyme disease spirochete Borrelia burgdorferi synthesized the HMWC throughout the in vitro growth cycle, and also in vivo when implanted in dialysis membrane chambers in rats. The HMWC was stable to formic acid, which supports the concept that the stability of the HMWC is dependent on covalent cross-linking of individual FlgE subunits. Mass spectrometry analysis of the HMWC from both wild type periplasmic flagella and polyhooks from a newly constructed ΔfliK mutant indicated that other proteins besides FlgE were not covalently joined to the complex, and that FlgE was the sole component of the complex. In addition, mass spectrometry analysis also indicated that the HMWC was composed of a polymer of the FlgE protein with both the N- and C-terminal regions remaining intact. These initial studies set the stage for a detailed characterization of the HMWC. Covalent cross-linking of FlgE with the accompanying formation of the HMWC we propose strengthens the hook structure for optimal spirochete motility.螺旋体（spirochete）周质鞭毛（periplasmic flagellum）具备诸多独特属性，其中一项非同寻常的特征为鞭毛钩（flagellar hook）。该结构充当万向节，将膜结合马达（membrane-bound motor）的旋转运动与鞭毛丝（flagellar filament）偶联起来。鞭毛钩由约120个FlgE单体（FlgE monomer）构成；在多数细菌中，这类结构经热与去污剂处理后可轻易解离为单体（约50千道尔顿）。然而，螺旋体中的FlgE单体会形成分子量超250千道尔顿的大型聚集体[称之为高分子量复合物（high molecular weight complex，HMWC）]，该复合物对热、去污剂及其他变性条件均保持稳定。 本研究针对该复合物的形成与结构展开了特定维度的考察。我们发现，莱姆病螺旋体伯氏疏螺旋体（Borrelia burgdorferi）可在整个体外生长周期中合成HMWC，当被植入大鼠体内的透析膜腔室（dialysis membrane chambers）时同样可完成该合成过程。HMWC对甲酸（formic acid）保持稳定，这为“HMWC的稳定性依赖于单个FlgE亚基间的共价交联（covalent cross-linking）”这一观点提供了支撑。 对野生型周质鞭毛与新构建的ΔfliK突变体（ΔfliK mutant）所产生的多聚钩（polyhooks）中的HMWC进行质谱（mass spectrometry）分析后结果显示：除FlgE外，并无其他蛋白质与该复合物发生共价结合，FlgE是该复合物的唯一组成成分。此外，质谱分析还表明，HMWC由FlgE蛋白的聚合物构成，其N端与C端区域均保持完整。 这些初步研究为HMWC的详细表征奠定了基础。我们推测，FlgE发生共价交联并伴随HMWC形成的过程，可强化鞭毛钩结构，以保障螺旋体获得最优的运动能力（spirochete motility）。