Fur-Dam Regulatory Interplay at an Internal Promoter of the Enteroaggregative Escherichia coli Type VI Secretion sci1 Gene Cluster
Fur-Dam Regulatory Interplay at an Internal Promoter of the Enteroaggregative Escherichia coli Type VI Secretion sci1 Gene Cluster
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DOI：
10.1128/jb.00075-20
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发表时间：
2020-05-01
影响因子：
3.2
通讯作者：
Cascales, Eric
中科院分区：
文献类型：
--
作者：
Brunet, Yannick R.;Bernard, Christophe S.;Cascales, Eric
VI型分泌系统（T6 SS）是用于将效应物递送到革兰氏阴性细菌中广泛存在的靶细胞中的武器。T6 SS是一种高度通用的机器，因为它可以靶向真核和原核细胞，并且已经提出T6 SS适应每种细菌的特定需求。因此，T6 SS基因簇的表达和分泌装置的激活受到严格控制。在肠聚集性大肠杆菌（EAEC）中，sci 1 T6 SS基因簇受到涉及铁摄取调节剂（Fur）和DNA腺嘌呤甲基化酶（Dam）依赖的DNA甲基化的复杂调节。在这项研究中，一个额外的，内部，启动子被确定在sci 1基因簇内使用+1转录作图。进一步的分析表明，这个内部启动子是由一个机制严格相同的主启动子。Fur结合盒与-10转录元件和Dam甲基化位点GATC-32重叠。因此，在富铁条件下，远端snit基因的表达被抑制，GATC-32位点被保护免于甲基化。通过体外甲基化测定证实了GATC-32的毛皮依赖性保护作用。此外，GATC-32的甲基化对Fur结合产生负面影响。因此sci 1内部启动子的表达是通过Fur调节铁的可用性来控制的，而Dam依赖的甲基化在铁限制的条件下保持稳定的ON表达。其中一种武器，VI型分泌系统（T6 SS），组装了一个可收缩的尾巴，作为弹簧来推动装载毒素的针头。因此，它的表达和激活需要严格调控。在此，我们在肠聚集性大肠杆菌sci 1 T6 SS基因簇中鉴定了一个内部启动子。杆菌我们发现，这个内部启动子是由毛皮和Dam-dependent甲基化控制。我们进一步证明了Fur和Dam在-10转录元件处竞争以精细地调节T6 SS基因的表达。我们认为，这种优雅的调节机制允许在肠聚集性E.大肠杆菌遇到竞争物种。
The type VI secretion system (T6SS) is a weapon for delivering effectors into target cells that is widespread in Gram-negative bacteria. The T6SS is a highly versatile machine, as it can target both eukaryotic and prokaryotic cells, and it has been proposed that T6SSs are adapted to the specific needs of each bacterium. The expression of T6SS gene clusters and the activation of the secretion apparatus are therefore tightly controlled. In enteroaggregative Escherichia coli (EAEC), the sci1 T6SS gene cluster is subject to a complex regulation involving both the ferric uptake regulator (Fur) and DNA adenine methylase (Dam)-dependent DNA methylation. In this study, an additional, internal, promoter was identified within the sci1 gene cluster using +1 transcriptional mapping. Further analyses demonstrated that this internal promoter is controlled by a mechanism strictly identical to that of the main promoter. The Fur binding box overlaps the -10 transcriptional element and a Dam methylation site, GATC-32. Hence, the expression of the distal snit genes is repressed and the GATC-32 site is protected from methylation in iron-rich conditions. The Fur-dependent protection of GATC-32 was confirmed by an in vitro methylation assay. In addition, the methylation of GATC-32 negatively impacted Fur binding. The expression of the sci1 internal promoter is therefore controlled by iron availability through Fur regulation, whereas Dam-dependent methylation maintains a stable ON expression in iron-limited conditions.IMPORTANCE Bacteria use weapons to deliver effectors into target cells. One of these weapons, the type VI secretion system (T6SS), assembles a contractile tail acting as a spring to propel a toxin-loaded needle. Its expression and activation therefore need to be tightly regulated. Here, we identified an internal promoter within the sci1 T6SS gene cluster in enteroaggregative E. coli. We show that this internal promoter is controlled by Fur and Dam-dependent methylation. We further demonstrate that Fur and Dam compete at the -10 transcriptional element to finely tune the expression of T6SS genes. We propose that this elegant regulatory mechanism allows the optimum production of the T6SS in conditions where enteroaggregative E. coli encounters competing species.