De novo origin of numerous microproteins in enterobacteria

Bacterial genomes encompass numerous small open reading frames (smORFs), some of which encode functional microproteins or perform noncoding regulatory roles. The evolution of microproteins remains poorly understood, largely due to challenges in homology detection for these short sequences. To address this challenge, we constructed 36 957 orthologous groups of microproteins (microOGs) across 5668 Enterobacteriaceae genomes. Our pipeline identified dozens of novel, widely distributed microprotein families and refined conservation patterns for known ones. However, 86% of the microOGs are genus-specific and functionally uncharacterized, suggesting that enterobacteria harbor a pool of evolutionarily young, de novo-originated small genes. Nevertheless, the microprotein-encoding smORFs in the microOGs are preferentially adjacent to membrane transporter genes suggesting a role in regulating transport processes. MicroOGs formed closed pangenomes, indicative of a limited contribution to the noncore genome of enterobacteria, likely due to the limitations on the size of intergenic regions where microproteins could arise de novo and frequent loss of microprotein-encoding smORFs during bacterial evolution. Overall, we identified 4838 microOGs with clear signatures of de novo origin from noncoding sequences. Many of the microprotein-encoding smORFs overlap transcriptional regulatory signals or repetitive elements suggesting that the origin of microproteins is tied to selection for maintenance of regulatory sequences.