ORGANIZATION OF TCP, ACF, AND TOXT GENES WITHIN A TOXT-DEPENDENT OPERON
ORGANIZATION OF TCP, ACF, AND TOXT GENES WITHIN A TOXT-DEPENDENT OPERON
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DOI：
10.1111/j.1365-2958.1995.tb02408.x
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发表时间：
1995-05-01
影响因子：
3.6
通讯作者：
TAYLOR, RK
中科院分区：
文献类型：
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作者：
BROWN, RC;TAYLOR, RK
毒素共调节菌毛（ICP）是霍乱弧菌定植人体肠道所必需的，菌毛蛋白基因tcpA的表达依赖于ToxR和ToxT，最近，ToxT基因在TCP生物发生基因簇中被定位，并在大肠杆菌中克隆时显示能够激活tcpA::TnphoA融合。在本研究中，我们确定了ToxR/ToxT激活发生在tcpA转录水平，ToxT在E中表达。大肠杆菌可以激活顶部操纵子融合，而ToxR、ToxR与ToxS或ToxR- phoa融合不能激活顶部操纵子融合，并且在DNA迁移转移实验中，我们无法证明ToxR提取物与tcpA启动子区域的结合。通过引物延伸，该调控启动子的起始位点位于tcpA第一个密码子上游75bp处。Northern分析发现了一个800碱基的tcpA信息，其大小与转录起始点和tcpA与tcpB之间的发夹环序列之间的距离有关。RNase保护分析更敏感的试验表明，一个受调控的转录本可能延伸到下游顶端基因的其余部分，包括toxT和邻近的辅助定植因子（acf）基因，一个帧内tcpA缺失，但不包括极性tcpA::TnphoA融合。可以通过在trans中提供tcpA来补充菌毛表面表达。这一证据表明，包括toxT在内的顶端基因组织在一个由toxT直接激活的操纵子中，以依赖于toxr的方式。在诱导条件下，大多数toxT的表达需要转录tcpA启动子，进一步研究tcp::TnphoA极性插入对toxT表达的调控，发现在toxR和tcp::TnphoA菌株中存在较低的基础水平的toxT表达。总的来说，这些观察结果支持了ToxR/ ToxT级联调控fcp，一旦诱导，ToxT的表达通过顶部启动子进行自我调节，将顶部表达与其他定植因子、外毒素产生和霍乱发病机制中功能未知的基因联系起来。
The toxin coregulated pilus (ICP) is required for Vibrio cholerae to colonize the human intestine, The expression of the pilin gene, tcpA, is dependent upon ToxR and upon ToxT, The toxT gene was recently mapped within the TCP biogenesis gene cluster and shown to be capable of activating a tcpA::TnphoA fusion when cloned in Escherichia coli, In this study, we determined that ToxR/ToxT activation occurs at the level of tcpA transcription, ToxT expressed in E, coli could activate a top operon fusion, while ToxR, ToxR with ToxS, or a ToxR-PhoA fusion failed to activate the top operon fusion and we could not demonstrate binding of a ToxR extract to the tcpA promoter region in DNA mobility-shift assays, The start site for the regulated promoter was shown by primer extension to lie 75bp upstream of the first codon of tcpA. An 800-base tcpA message was identified, by Northern analysis, that correlates by size to the distance between the transcriptional start and a hairpin-loop sequence between tcpA and tcpB, The more-sensitive assay of RNase protection analysis demonstrated that a regulated transcript probably extends through the rest of the downstream top genes, including toxT and the adjacent accessory colonization factor (acf) genes, An in-frame tcpA deletion, but not a polar tcpA::TnphoA fusion, could be complemented for pilus surface expression by providing tcpA in trans. This evidence suggests that the top genes, including toxT, are organized in an operon directly activated by ToxT in a ToxR-dependent manner. Most of the toxT expression under induced conditions requires transcription of the tcpA promoter, Further investigation of how tcp::TnphoA insertions that are polar on toxT expression retain regulation showed that a low basal level of toxT expression is present in toxR and tcp::TnphoA strains. Overall, these observations support the ToxR/ ToxT cascade of regulation for fcp,Once induced, toxT expression becomes autoregulatory via the top promoter, linking top expression to that of additional colonization factors, exotoxin production, and genes of unknown function in cholera pathogenesis.