Core elements of the vegetative replication control of the Inc1 plasmid pO104_90 of Escherichia coli O104:H4 also regulate its transfer frequency

The highly virulent Escherichia coli O104:H4 isolate that caused a large outbreak in 2011 carries three plasmids. Out of these, only one, the IncI plasmid pO104_90 that encodes two extended spectrum beta-lactamases, can transfer itself by conjugation. Considering its potential contribution to the emergence and virulence of the outbreak strain, we aimed to get a closer insight into pO104_90 transfer efficiency and control. We tested the host spectrum of the plasmid and observed transmission into Enterobactericeae including clinically relevant enterobacterial pathogens like Salmonella typhimurium and Shigella flexneri. However, we found that this plasmid did not transfer into E. coli strains that kill the donor strain due to bacteriocin production, e.g. the probiotic E. coli Nissle 1917. Under the same conditions, the highly transmittable control plasmid RP4 was efficiently transferred into all these recipients. Therefore we hypothesized that the failure of transfer of pO104_90 was simply due to the generally much lower transmission rates of this IncI plasmid and we decided to screen for factors that negatively affect the transfer of the plasmid by an in vivo deletion analysis. Our attempts to delete larger regions of the plasmid resulted in cells containing both a truncated plasmid (Δ50 kb and Δ75 kb) and a wild type copy of pO104_90. When used as donors in conjugation experiments, these cells transferred the wild type plasmid at dramatically increased rates. This indicated that the relatively limited region shared by both plasmids contained an activator of transfer. We therefore analyzed its transcriptional organization, dissected the candidate region by subcloning and showed that additional copies of repY/INC were sufficient to increase the transfer frequency of pO104_90 to the observed level. To our knowledge, this is the first evidence for a direct regulatory cross talk between core control elements of the vegetative replication and the transfer functions of an IncI1 plasmid.