Limberger R J, Slivienski L L, Samsonoff W A
David Axelrod Institute for Public Health, Wadsworth Center for Laboratories and Research, New York State Department of Health, Albany 12201-0509.
J Bacteriol. 1994 Jun;176(12):3631-7. doi: 10.1128/jb.176.12.3631-3637.1994.
The periplasmic flagellum of Treponema phagedenis consists of the flagellar filament and hook-basal body. We report here a characterization of the hook gene and flagellar hook of T. phagedenis, and in the process of this analysis we found evidence that the hook polypeptide is likely cross-linked in situ. A T. phagedenis genomic library was screened with a Treponema pallidum antiserum, and the DNA segments from several positive plaques were subcloned and sequenced. DNA sequencing of two overlapping segments revealed a 1,389-nucleotide (nt) open reading frame (ORF) with a deduced amino acid sequence that was 36% identical to that of FlgE, the hook polypeptide of Salmonella typhimurium. This gene was designated T. phagedenis flgE. Beginning at 312 nt downstream from flgE was a partial ORF of 486 nt with a deduced amino acid sequence that was 33% identical to that of MotA of Bacillus subtilis, a polypeptide that enables flagellar rotation. Upstream of flgE, separated by 39 nt, was a partial (291-nt) ORF with a deduced amino acid sequence that was homologous to that of ORF8, a polypeptide of unknown function located in an operon encoding polypeptides involved in motility of B. subtilis. The T. phagedenis flgE gene was cloned into an Escherichia coli protein expression plasmid, and the purified recombinant protein was used to prepare a FlgE antiserum. Western blots (immunoblots) of whole-cell lysates probed with this antiserum revealed a 55-kDa polypeptide and a ladder of polypeptide bands with increasing molecular masses. T. phagedenis hooks were then isolated and purified, and electron microscopic analysis revealed that the morphology of the hooks resembled that in other bacteria. The hooks were slightly curved and had an average length of 69 +/- 8 nm and a diameter of 23 +/- 1 nm. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blots of purified hook preparations using the FlgE antiserum also revealed a polypeptide ladder, suggesting that the hooks are composed of a covalently cross-linked polypeptide.
溶牙密螺旋体的周质鞭毛由鞭毛丝和钩形基体组成。我们在此报告了溶牙密螺旋体钩基因和鞭毛钩的特征，并且在该分析过程中，我们发现有证据表明钩多肽可能在原位发生交联。用梅毒螺旋体抗血清筛选溶牙密螺旋体基因组文库，将来自几个阳性噬菌斑的DNA片段进行亚克隆并测序。对两个重叠片段的DNA测序揭示了一个1389个核苷酸（nt）的开放阅读框（ORF），其推导的氨基酸序列与鼠伤寒沙门氏菌的钩多肽FlgE的氨基酸序列有36%的同一性。该基因被命名为溶牙密螺旋体flgE。在flgE下游312 nt处开始是一个486 nt的部分ORF，其推导的氨基酸序列与枯草芽孢杆菌的MotA有33%的同一性，MotA是一种能使鞭毛旋转的多肽。在flgE上游，相隔39 nt，是一个部分（291 nt）ORF，其推导的氨基酸序列与ORF8同源，ORF8是位于编码参与枯草芽孢杆菌运动性的多肽的操纵子中的一个功能未知的多肽。将溶牙密螺旋体flgE基因克隆到大肠杆菌蛋白表达质粒中，用纯化的重组蛋白制备FlgE抗血清。用该抗血清探测全细胞裂解物的蛋白质免疫印迹（免疫印迹）显示出一条55 kDa的多肽以及分子量递增的多肽条带梯。然后分离并纯化溶牙密螺旋体的钩，电子显微镜分析显示钩的形态与其他细菌中的相似。钩略微弯曲，平均长度为69±8 nm，直径为23±1 nm。使用FlgE抗血清对纯化的钩制剂进行十二烷基硫酸钠-聚丙烯酰胺凝胶电泳和蛋白质免疫印迹也显示出一条多肽条带梯，表明钩由共价交联的多肽组成。
