Phase variation of Ag43 in Escherichia coli: Dam-dependent methylation abrogates OxyR binding and OxyR-mediated repression of transcription

It has been shown previously that phase variation of the outer membrane pro tein Antigen43 (Ag43) of Escherichia coli requires the DNA-methylating enzy me deoxyadenosine methyltransferase (Dam) and the global regulator OxyR. In this study, we analysed the regulation of the Ag43 encoding gene (agn) usi ng isolates containing a fusion of the agn regulatory region to the reporte r gene lacZ. Our results indicate that phase variation of Ag43 is regulated at the level of transcription. Repression of agn'-lacZ transcription requi red OxyR, whereas activation required Dam. The regulatory region of agn con tains three GATC sequences, which are target sites for Dam-dependent methyl ation. In vivo, the methylation state of these GATC sequences correlated wi th the transcription state of agn'-lacZ. These GATC sequences were not prot ected from Dam-dependent methylation in an oxyR background, suggesting that OxyR binding results in Dam-dependent methylation protection in OFF cells. In vitro, both oxidized OxyR and OxyR(C199S), which is locked in the reduc ed conformation, bound to the agn regulatory region, but methylation of the three GATC sequences abrogated this binding. In vivo, OxyR(C199S) was suff icient to repress Ag43 transcription. Our data support a model in which Oxy R-mediated repression of transcription is alleviated by methylation of thre e GATC sequences in its binding site. In addition, we show that, in an oxyR background, Dam was still required for full activation, suggesting that th e model concerning the role of Dam in agn regulation is incomplete. These r esults show that Dam-dependent phase variation in E. coli is not limited to the previously identified regulatory system of the family of pap-like fimb rial operons.