In many bacteria, there is a genome-wide bias towards co-orientation of replication and transcription, with essential and/or highly-expressed genes further enriched co-directionally. We previously found that reversing this bias in the bacterium Bacillus subtilis slows replication elongation, and we proposed that this effect contributes to the evolutionary pressure selecting the transcription-replication co-orientation bias. This selection might have been based purely on selection for speedy replication; alternatively, the slowed replication might actually represent an average of individual replication-disruption events, each of which is counter-selected independently because genome integrity is selected. To differentiate these possibilities and define the precise forces driving this aspect of genome organization, we generated new strains with inversions either over ∼1/4 of the chromosome or at ribosomal RNA (rRNA) operons. Applying mathematical analysis to genomic microarray snapshots, we found that replication rates vary dramatically within the inverted genome. Replication is moderately impeded throughout the inverted region, which results in a small but significant competitive disadvantage in minimal medium. Importantly, replication is strongly obstructed at inverted rRNA loci in rich medium. This obstruction results in disruption of DNA replication, activation of DNA damage responses, loss of genome integrity, and cell death. Our results strongly suggest that preservation of genome integrity drives the evolution of co-orientation of replication and transcription, a conserved feature of genome organization.
An important feature of genome organization is that transcription and replication are selectively co-oriented. This feature helps to avoid conflicts between head-on replication and transcription. The precise consequences of the conflict and how it affects genome organization remain to be understood. We previously found that reversing the transcription bias slows replication in the Bacillus subtilis genome. Here we engineered new inversions to avoid changes in other aspects of genome organization. We found that the reversed transcription bias is sufficient to decrease replication speed, and it results in lowered fitness of the inversion strains and a competitive disadvantage relative to wild-type cells in minimal medium. Further, by analyzing genomic copy-number snapshots to obtain replication speed as a function of genome position, we found that inversion of the strongly-transcribed rRNA genes obstructs replication during growth in rich medium. This confers a strong growth disadvantage to cells in rich medium, turns on DNA damage responses, and leads to cell death in a subpopulation of cells, while the surviving cells are more sensitive to genotoxic agents. Our results strongly support the hypothesis that evolution has favored co-orientation of transcription with replication, mainly to avoid these effects.