MOLECULAR CHARACTERIZATION OF THE SALMONELLA-TYPHIMURIUM FLHB OPERON AND ITS PROTEIN PRODUCTS
MOLECULAR CHARACTERIZATION OF THE SALMONELLA-TYPHIMURIUM FLHB OPERON AND ITS PROTEIN PRODUCTS
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DOI：
10.1128/jb.176.24.7630-7637.1994
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发表时间：
1994-12-01
影响因子：
3.2
通讯作者：
KUTSUKAKE, K
中科院分区：
文献类型：
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作者：
MINAMINO, T;IINO, T;KUTSUKAKE, K
flhB和flhA基因在鼠伤寒沙门氏菌染色体上构成称为flhB操纵子的操纵子。它们的基因产物是鞭毛器杆状结构形成所必需的。此外，一些证据表明，它们与FliI和FliH一起构成鞭毛蛋白的输出装置，鞭毛蛋白是鞭毛丝的组成蛋白。在这项研究中，我们确定了整个flhB操纵子的核苷酸序列从S。鼠伤寒。结果表明，flhB和flhA基因编码高度疏水的多肽，计算分子量分别为42，322和74，848 Da。这两种蛋白质都有几个潜在的跨膜片段，表明它们可能是整合的膜蛋白。发现flhB操纵子含有一个额外的开放阅读框架，能够编码一个计算分子量为14，073 Da的多肽，我们将这个开放阅读框架命名为flhE。FlhE的N-末端16个氨基酸显示出典型信号序列的特征。一个大细胞标记实验使我们能够识别的前体和成熟形式的flhE基因产物。将卡那霉素抗性基因盒插入染色体flhE基因中并不影响细胞的运动性，这表明flhE基因对于鞭毛的形成和功能并不是必需的。我们已经过量生产和纯化了N-末端截短的FlhB和FlhA蛋白，并提出了针对它们的抗体。通过使用这些抗体，用分级分离的细胞提取物通过Western印迹（免疫印迹）分析FlhB和FlhA蛋白的定位。结果表明，这两种蛋白质都定位于细胞质膜。
The flhB and flhA genes constitute an operon called flhB operon on the Salmonella typhimurium chromosome. Their gene products are required for formation of the rod structure of flagellar apparatus. Furthermore, several lines of evidence suggest that they, together with FliI and FliH, mag constitute the export apparatus of flagellin, the component protein of flagellar filament. In this study, we determined the nucleotide sequence of the entire flhB operon from S. typhimurium. It was shown that the flhB and flhA genes encode highly hydrophobic polypeptides with calculated molecular masses of 42,322 and 74,848 Da, respectively. Both proteins have several potential membrane-spanning segments, suggesting that they may be integral membrane proteins. The flhB operon was found to contain an additional open reading frame capable of encoding a polypeptide with a calculated molecular mass of 14,073 Da. We designated this open reading frame flhE. The N-terminal 16 amino acids of FlhE displays a feature of a typical signal sequence. A maxicell labeling experiment enabled us to identify the precursor and mature forms of the flhE gene products. Insertion of a kanamycin-resistant gene cartridge into the chromosomal flhE gene did not affect the motility of the cells, indicating that the flhE gene is not essential for flagellar formation and function. We have overproduced and purified N-terminally truncated FlhB and FlhA proteins and raised antibodies against them. By use of these antibodies, localization of the FlhB and FlhA proteins was analyzed by Western blotting (immunoblotting) with the fractionated cell extracts. The results obtained indicated that both proteins are localized in the cytoplasmic membrane.