<span class="paragraphSection"><div class="boxTitle">Abstract</div>How gene order along chromosomes affects cellular homeostasis and genome evolution remains poorly understood. Bacterial chromosomes are organized along the replication origin (<span style="font-style: italic;">oriC</span>)–terminus (<span style="font-style: italic;">ter</span>) 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 <span style="font-style: italic;">rplKAJL-rpoBC</span> 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 <span style="font-style: italic;">Vibrio cholerae</span>. 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 <span style="font-style: italic;">oriC</span>-proximal RNAP genes reproduced all defects, identifying them as the primary drivers. Selection keeps RNAP genes close to <span style="font-style: italic;">oriC</span> 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 an