Lin Min, Dan Hanhong, Li Yijing
Canadian Food Inspection Agency, Animal Diseases Research Institute, 3851 Fallowfield Road, Nepean, Ontario, Canada.
Curr Microbiol. 2004 Feb;48(2):145-52. doi: 10.1007/s00284-003-1089-1.
Leptospira borgpetersenii, one of the causative agents of leptospirosis in both animals and humans, is a bacterial pathogen with characteristic motility that is mediated by the rotation of two periplasmic flagella (PF). The flaB gene coding for a core polypeptide subunit of PF was previously characterized by sequence analysis of its open reading frame (ORF) (M. Lin, J Biochem Mol Biol Biophys 2:181-187, 1999). The present study was undertaken to isolate and clone the uncharacterized sequence upstream of the flaB gene by using a PCR-based genome walking procedure. This has resulted in a 1470-bp genomic DNA sequence in which an 846-bp ORF coding for a 281-amino acid polypeptide (31.3 kDa) is identified 455 bp upstream from the flaB start codon. The encoded protein exhibits 72% amino acid identity to the deduced FlaB protein sequence of L. borgpetersenii and a high degree of sequence homology to the FlaB proteins of other spirochaetes. This has demonstrated for the first time that a second flaB gene homolog is present in a Leptospira species. The newly identified gene is designated flaB1, and the previously cloned flaB renamed flaB2. Within the intergenic sequence between flaB1 and flaB2, a potential stem-loop structure (12-bp inverted repeats) was identified 25 bp downstream of the flaB1 stop codon; this could serve as a transcription terminator for the flaB1 mRNA. Three E. coli-like promoter regions (I, II, and III) for binding Esigma(70), a regulatory sequence uncommonly found in flagellar genes, were predicted upstream of the flaB2 ORF. Only promoter region II contains a promoter that is functional in E. coli, as revealed at phenotypic and transcriptional levels by its capability of directing the expression of the chloramphenicol acetyltransferase (CAT) gene in the promoter probe vector pKK232-8. These observations may suggest that flaB1 and flaB2 are transcribed separately and do not form a transcriptional operon controlled by a single promoter.
波氏钩端螺旋体是人和动物钩端螺旋体病的病原体之一，是一种细菌病原体，具有由两个周质鞭毛（PF）旋转介导的特征性运动性。编码PF核心多肽亚基的flaB基因先前已通过其开放阅读框（ORF）的序列分析进行了表征（M. Lin，《生物化学与分子生物学与生物物理学杂志》2：181 - 187，1999）。本研究旨在通过基于PCR的基因组步移程序分离和克隆flaB基因上游未表征的序列。这产生了一个1470 bp的基因组DNA序列，其中在flaB起始密码子上游455 bp处鉴定出一个编码281个氨基酸多肽（31.3 kDa）的846 bp ORF。编码的蛋白质与波氏钩端螺旋体推导的FlaB蛋白序列具有72%的氨基酸同一性，并且与其他螺旋体的FlaB蛋白具有高度的序列同源性。这首次证明在钩端螺旋体物种中存在第二个flaB基因同源物。新鉴定的基因命名为flaB1，先前克隆的flaB重新命名为flaB2。在flaB1和flaB2之间的基因间隔序列中，在flaB1终止密码子下游25 bp处鉴定出一个潜在的茎环结构（12 bp反向重复序列）；这可能作为flaB1 mRNA的转录终止子。在flaB2 ORF上游预测到三个类似大肠杆菌的启动子区域（I、II和III），用于结合Esigma(70)，这是一种在鞭毛基因中不常见的调控序列。只有启动子区域II包含一个在大肠杆菌中具有功能的启动子，如通过其在启动子探针载体pKK232 - 8中指导氯霉素乙酰转移酶（CAT）基因表达的能力在表型和转录水平上所揭示的那样。这些观察结果可能表明flaB1和flaB2是分别转录的，并且不形成由单个启动子控制的转录操纵子。
