An Epigenetic Switch Involving Overlapping Fur and DNA Methylation Optimizes Expression of a Type VI Secretion Gene Cluster

摘要

Type VI secretion systems (T6SS) are macromolecular machines of the cell envelope of Gram-negative bacteria responsible for bacterial killing and/or virulence towards different host cells. Here, we characterized the regulatory mechanism underlying expression of the enteroagregative Escherichia coli sci1 T6SS gene cluster. We identified Fur as the main regulator of the sci1 cluster. A detailed analysis of the promoter region showed the presence of three GATC motifs, which are target of the DNA adenine methylase Dam. Using a combination of reporter fusion, gel shift, and in vivo and in vitro Dam methylation assays, we dissected the regulatory role of Fur and Dam-dependent methylation. We showed that the sci1 gene cluster expression is under the control of an epigenetic switch depending on methylation: fur binding prevents methylation of a GATC motif, whereas methylation at this specific site decreases the affinity of Fur for its binding box. A model is proposed in which the sci1 promoter is regulated by iron availability, adenine methylation, and DNA replication. Author Summary DNA methylation plays an important role in the regulation of genes involved in assembly of cell surface adhesins or appendages. Methylation at a GATC motif by the Dam methylase influences binding of transcriptional regulators, leading to variation in the gene expression pattern. In several cases, this may lead to different cell subpopulations allowing a rapid adaptation to varying environments. In this work, we uncover the regulatory mechanism controlling expression of the sci1 Type VI secretion gene cluster in entero-aggregative Escherichia coli , which encodes a structure required for inter-bacterial interaction. We showed that this gene cluster is repressed by Fur in iron-replete conditions and that Fur binding on the promoter prevents methylation of a GATC motif. In iron-limited conditions, Fur is relieved from the promoter allowing expression of the gene cluster and methylation of the GATC motif. Methylation prevents de novo Fur binding allowing constitutive expression. Our findings support a model in which the expression of the Type VI secretion gene cluster is regulated by a non-stochastic epigenetic switch: switch from the OFF to ON phases depends on iron availability whereas the ON to OFF switch depends on DNA replication and competition between Dam-dependent methylation and Fur binding.