Regulation of a phage defence island by RptR, a novel repressor that controls restriction-modification systems in...

Bacteria encode a panoply of defence systems to overcome phage infection. In recent years, over 100 defence systems have been identified, with the majority of these found co-localized in defence islands. Although there has been much progress in understanding the mechanisms of anti-phage defence employed by bacteria, far less is known about their regulation before and during phage infection. Here, we describe RptR (RMS-proximal transcriptional regulator), a small transcriptional regulator of a defence island in enteropathogenic Escherichia coli composed of a toxin-antitoxin system, DarTG2, embedded within a Type I restriction-modification system (RMS). We determined the molecular structure of a RptR homodimer and, using transcriptional reporter and in vitro DNA binding assays, show that RptR represses the promoter of the defence island by binding to a series of three direct repeats in the promoter. Furthermore, we demonstrate, using the structural models of RptR validated with electrophoretic mobility shift assays, that the minimal RptR binding site is a 6-bp palindrome, TAGCTA. Both RptR and its binding site are highly conserved across diverse bacterial genomes with a strong genetic association with Type I RMS, highlighting the role of RptR as a novel regulatory component of an important mechanism for anti-phage defence in bacteria.