DNA replication in Escherichia coli is normally initiated at a single origin, 
oriC, dependent on initiation protein DnaA. However, replication can be 
initiated elsewhere on the chromosome at multiple ectopic oriK sites. Genetic 
evidence indicates that initiation from oriK depends on RNA-DNA hybrids 
(R-loops), which are normally removed by enzymes such as RNase HI to prevent 
oriK from misfiring during normal growth. Initiation from oriK sites occurs in 
RNase HI-deficient mutants, and possibly in wild-type cells under certain 
unusual conditions. Despite previous work, the locations of oriK and their 
impact on genome stability remain unclear. We combined 2D gel electrophoresis 
and whole genome approaches to map genome-wide oriK locations. The DNA copy 
number profiles of various RNase HI-deficient strains contained multiple peaks, 
often in consistent locations, identifying candidate oriK sites. Removal of 
RNase HI protein also leads to global alterations of replication fork migration 
patterns, often opposite to normal replication directions, and presumably 
eukaryote-like replication fork merging. Our results have implications for 
genome stability, offering a new understanding of how RNase HI deficiency 
results in R-loop-mediated transcription-replication conflict, as well as 
inappropriate replication stalling or blockage at Ter sites outside of the 
terminus trap region and at ribosomal operons.