DNA Replication in Engineered <i>Escherichia coli</i> Genomes with Extra Replication Origins

The standard outline of bacterial genomes is a single circular chromosome with a single replication origin. From the bioengineering perspective, it appears attractive to extend this basic setup. Bacteria with split chromosomes or multiple replication origins have been successfully constructed in the last few years. The characteristics of these engineered strains will largely depend on the respective DNA replication patterns. However, the DNA replication has not been investigated systematically in engineered bacteria with multiple origins or split replicons. Here we fill this gap by studying a set of strains consisting of (i) <i>E. coli</i> strains with an extra copy of the native replication origin (<i>oriC</i>), (ii) <i>E. coli</i> strains with an extra copy of the replication origin from the secondary chromosome of <i>Vibrio cholerae</i> (<i>oriII</i>), and (iii) a strain in which the <i>E. coli</i> chromosome is split into two linear replicons. A combination of flow cytometry, microarray-based comparative genomic hybridization (CGH), and modeling revealed silencing of extra <i>oriC</i> copies and differential timing of ectopic <i>oriII</i> copies compared to the native <i>oriC</i>. The results were used to derive construction rules for future multiorigin and multireplicon projects