DNA adenine methyltransferase (Dam) controls the expression of the cytotoxic enterotoxin (act) gene of Aeromonas hydrophila via tRNA modifying enzyme-glucose-inhibited division protein (GidA).
DNA adenine methyltransferase (Dam) controls the expression of the cytotoxic enterotoxin (act) gene of Aeromonas hydrophila via tRNA modifying enzyme-glucose-inhibited division protein (GidA).
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DOI：
10.1016/j.gene.2012.02.024
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发表时间：
2012-05-01
期刊：
影响因子：
3.5
通讯作者：
Chopra AK
中科院分区：
文献类型：
--
作者：
Erova TE;Kosykh VG;Sha J;Chopra AK
嗜水气单胞菌是一种人类和动物的病原体，细胞毒性肠毒素（Act）是这种细菌的一个关键毒力因子，因为它相关的溶血，细胞毒性和肠毒性活动。以前，为了确定一些调控基因在调节Act产生中的作用，我们发现，编码tRNA甲基化酶的葡萄糖抑制分裂基因（gidA）的缺失降低了Act水平，而DNA腺嘌呤甲基转移酶（Dam）的过量产生导致了A.你好重要的是，在gidA基因的上游序列内存在Dam的多个GATC结合位点，并且在act基因上游区域中存在一个这样的靶位点。我们发现dam基因对A.因此，为了更好地了解Dam和GidA编码基因在act基因调控中的相互作用，我们构建了大肠杆菌GM 28（dam+）和GM 33（Δdam）gidA读框内缺失突变株。然后，我们通过使用含有报告绿色荧光蛋白（GFP）的无启动子的pGlow-TOPO载体来测试act和gidA基因的表达活性。我们的数据表明，在GidA+菌株中，E.大肠杆菌中，通过Dam负调节act和gidA基因表达的GATC位点的组成型甲基化，如通过GFP产生所测量的。然而，在ΔgidA菌株中，无论是否存在组成型活性Dam，我们都没有观察到act基因表达的任何改变，这表明GidA在正调节Act产生中的作用。为了确定Dam和GidA如何影响Act的确切机制，进行实时定量PCR（RT-qPCR）测定。分析表明，在A.这些数据与E. coli GM 28菌株。因此，由Dam的组成型与过量生产引起的DNA甲基化程度以及DNA的可能构象影响A.我是SSU。我们的研究结果表明，act基因是在Dam和GidA修饰甲基化酶的控制下，Dam通过GidA调节Act的产生。
Aeromonas hydrophila is both a human and animal pathogen, and the cytotoxic enterotoxin (Act) is a crucial virulence factor of this bacterium because of its associated hemolytic, cytotoxic, and enterotoxic activities. Previously, to define the role of some regulatory genes in modulating Act production, we showed that deletion of a glucose-inhibited division gene (gidA) encoding tRNA methylase reduced Act levels, while overproduction of DNA adenine methyltransferase (Dam) led to a concomitant increase in Act-associated biological activities of a diarrheal isolate SSU of A. hydrophila. Importantly, there are multiple GATC binding sites for Dam within an upstream sequence of the gidA gene and one such target site in the act gene upstream region. We showed the dam gene to be essential for the viability of A. hydrophila SSU, and, therefore, to better understand the interaction of the encoding genes, Dam and GidA, in act gene regulation, we constructed a gidA in-frame deletion mutant of Escherichia coli GM28 (dam+) and GM33 (Δdam) strains. We then tested the expressional activity of the act and gidA genes by using a promoterless pGlow-TOPO vector containing a reporter green fluorescent protein (GFP). Our data indicated that in GidA+ strains of E. coli, constitutive methylation of the GATC site(s) by Dam negatively regulated act and gidA gene expression as measured by GFP production. However, in the ΔgidA strains, irrespective of the presence or absence of constitutively active Dam, we did not observe any alteration in the expression of the act gene signifying the role of GidA in positively regulating Act production. To determine the exact mechanism of how Dam and GidA influence Act, a real-time quantitative PCR (RT-qPCR) assay was performed. The analysis indicated an increase in gidA and act gene expression in the A. hydrophila Dam-overproducing strain, and these data matched with Act production in the E. coli GM28 strain. Thus, the extent of DNA methylation caused by constitutive versus overproduction of Dam, as well as possible conformation of DNA influence the expression of act and gidA genes in A. hydrophila SSU. Our results indicate that the act gene is under the control of both Dam and GidA modification methylases, and Dam regulates Act production via GidA.
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