Correa Nidia E, Klose Karl E
Department of Biology, University of Texas at San Antonio, TX 78249, USA.
J Bacteriol. 2005 May;187(9):3158-70. doi: 10.1128/JB.187.9.3158-3170.2005.
The human pathogen Vibrio cholerae is a highly motile organism by virtue of a polar flagellum, and motility has been inferred to be an important aspect of virulence. It has previously been demonstrated that the sigma(54)-dependent activator FlrC is necessary for both flagellar synthesis and for enhanced intestinal colonization. In order to characterize FlrC binding, we analyzed two FlrC-dependent promoters, the highly transcribed flaA promoter and the weakly transcribed flgK promoter, utilizing transcriptional lacZ fusions, mobility shift assays, and DNase I footprinting. Promoter fusion studies showed that the smallest fragment with wild-type transcriptional activity for flaAp was from positions -54 to +137 with respect to the start site, and from -63 to +144 for flgKp. Gel mobility shift assays indicated that FlrC binds to a fragment containing the region from positions +24 to +95 in the flaAp, and DNase I footprinting identified a protected region between positions +24 and +85. Mobility shift and DNase I footprinting indicated weak binding of FlrC to a region downstream of the flgKp transcription start site. These results demonstrate a relatively novel sigma(54)-dependent promoter architecture, with the activator FlrC binding downstream of the sigma(54)-dependent transcription start sites. When the FlrC binding site(s) in the flaA promoter was moved a large distance (285 bp) upstream of the transcription start site of either flaAp or flgKp, high levels of FlrC-dependent transcription resulted, indicating that this binding region functions as an enhancer element. In contrast, the relatively weak FlrC binding site(s) in the flgK promoter failed to function as an enhancer element at either promoter, suggesting that FlrC binding strength contributes to enhancer activity. Our results suggest that the differences in FlrC binding to various flagellar promoters results in the differences in transcription levels that mirror the relative requirement for the flagellar components within the flagellum.
人类病原体霍乱弧菌凭借一根极鞭毛成为高度能动的生物体，并且能动性已被推断为毒力的一个重要方面。先前已经证明，σ⁵⁴依赖性激活因子FlrC对于鞭毛合成和增强肠道定殖都是必需的。为了表征FlrC的结合情况，我们利用转录lacZ融合、迁移率变动分析和DNase I足迹法，分析了两个FlrC依赖性启动子，即高转录的flaA启动子和低转录的flgK启动子。启动子融合研究表明，flaAp具有野生型转录活性的最小片段相对于起始位点为-54至+137位，flgKp为-63至+144位。凝胶迁移率变动分析表明，FlrC与flaAp中包含+24至+95位区域的片段结合，DNase I足迹法确定了+24至+85位之间的一个受保护区域。迁移率变动和DNase I足迹法表明FlrC与flgKp转录起始位点下游区域的结合较弱。这些结果证明了一种相对新颖的σ⁵⁴依赖性启动子结构，激活因子FlrC在σ⁵⁴依赖性转录起始位点下游结合。当flaA启动子中的FlrC结合位点向上游移动到flaAp或flgKp转录起始位点的285 bp处时，会产生高水平的FlrC依赖性转录，这表明该结合区域起到增强子元件的作用。相比之下，flgK启动子中相对较弱的FlrC结合位点在两个启动子中均未能起到增强子元件的作用，这表明FlrC的结合强度有助于增强子活性。我们的结果表明，FlrC与各种鞭毛启动子结合的差异导致了转录水平的差异，这反映了鞭毛内鞭毛成分的相对需求。
