Abstract
How gene order along chromosomes affects cellular homeostasis and genome evolution remains poorly understood. Bacterial chromosomes are organized along the replication origin (oriC)-terminus (ter) axis. The spatial arrangement of genes within this axis may influence cellular physiology, genome evolution, and transcriptional regulation. We tested the importance of the universally conserved rplKAJL-rpoBC locus, which encodes the β/β' subunits of the sole bacterial RNA polymerase (RNAP), by relocating it to different genomic positions in the fast-growing pathogen Vibrio cholerae. Relocation close from locus native site was neutral but relocating it near either chromosomal terminus reduced exponential growth and competitive fitness specifically in nutrient-rich media. Marker-frequency analysis showed that distal positioning lowered locus copy number from ~3 to ~1 per cell, causing a 20%-25% depletion in cellular RNAP without altering its subcellular distribution. Introducing an additional oriC-proximal copy restored wild-type phenotypes, whereas two terminus copies rescued growth solely through increased dosage. Deleting the oriC-proximal RNAP genes reproduced all defects, identifying them as the primary drivers. Selection keeps RNAP genes close to oriC to harness replication-associated dosage increment during exponential growth, ensuring adequate transcription capacity for rapid proliferation. Gene order is a key but overlooked layer of bacterial genome evolution and ecological adaptation.