The shufflon of IncI1 plasmids is rearranged constantly during different growth conditions

One of the factors that can affect conjugation of IncI1 plasmids, amongst others, is the genetic region known as the shufflon. This multiple inversion system modifiesthe pilus tip proteins used during conjugation, thus affecting the affinity for different recipient cells. Although recombination is known to occur inin vitroconditions,little is known about the regulation and the extent of recombination that occurs. To measure the recombination of the shufflon, we have amplified the entire shufflonregion and sequenced the amplicons using nanopore long-read sequencing. This method was effective to determine the order of the segments of the shufflon andallow for the analysis of the shufflon variants that are present in a heterogeneous pool of templates. Analysis was performed over different growth phases and afteraddition of cefotaxime. Furthermore, analysis was performed in differentE. colihost cells to determine if recombination is likely to be influenced. Recombination ofthe shufflon was constantly ongoing in all conditions that were measured, although no differences in the amount of different shufflon variants or the rate at whichnovel variants were formed could be found. As previously reported, some variants were abundant in the population while others were scarce. This leads to theconclusion that the shufflon is continuously recombining at a constant rate, or that the method used here was not sensitive enough to detect differences in this rate.For one of the plasmids, the host cell appeared to have an effect on the specific shufflon variants that were formed which were not predominant in another host,indicating that host factors may be involved. As previously reported, thepilV-AandpilV-A'ORFs are formed at higher frequencies than otherpilVORFs. These resultsdemonstrate that the recombination that occurs within the shufflon is not random. While any regulation of the shufflon affected by thesein vitroconditions could notbe revealed, the method of amplifying large regions for long-read sequencing for the analysis of multiple inversion systems proved effective.