Full-length transcriptomics and proteomics reveal how genome minimization reshapes gene expression in synthetic...

JCVI-syn1.0 (Syn1.0) and JCVI-syn3A (Syn3A), genetically synthetic and genome-reduced versions of the naturally occurring bacterium Mycoplasma mycoides, are landmark platforms for defining the gene set required for life, yet how their genomes are expressed at the RNA level remains uncharacterized. We combined full-length PacBio and native long-read RNA sequencing with short-read quantification and complementary proteomics to map transcription, RNA processing, and protein abundance in both cells. In Syn1.0, full-length sequencing resolved 459 operons encompassing 911 genes, revealing pervasive RNA processing with a strong 3' bias. Analysis of the division and cell-wall cluster showed how transcriptional context explained the restoration of genes required for normal cell division in Syn3A. Most antisense and intergenic transcription in Syn1.0 reflected low-level transcriptional noise arising from inherited mis-annotation, read-through, and synthetic sequences. Much of that transcription was lost in Syn3A after genome minimization. Reducing the genome unexpectedly altered the expression of several retained genes by deleting promoters, most prominently reducing expression of the nucleoid protein HupA and central-carbon enzymes. Meanwhile, one third of the coding mRNA pool was allocated to a single 21-gene ribosomal-protein operon, while several other ribosomal proteins had reduced transcript abundance, which possibly led to imbalanced ribosome assembly. Expression of RNA polymerase and central-carbon metabolism declined at both mRNA and protein levels whereas RNase Y degradosome abundance increased. These shifts in the synthetic evolution of Syn3A suggest plausible mechanisms for its reduced chromosome contacts and slower growth. Together, these results show that genome minimization alters not only gene content but also the transcriptional context and resource allocation of retained genes. The analysis and visualization that are shared via Jupyter Notebook provide the RNA-level foundation for whole-cell modeling of the minimal cell.