In our recent work, we set out to answer a deceptively simple question: which parts of a bacterium’s DNA are truly essential for it to live? Not just the generally studied gene units, but also the smaller control elements that help those genes work properly, such as promoters, terminators, ribosome binding sites, and other non-coding elements.
Think of it like fixing a machine: most people would check the big gears (the genes). But a machine also depends on tiny screws and switches (the regulatory parts). Remove the wrong one, and the whole system might collapse. What we wanted was not only to identify which pieces matter, but also to measure how much they matter. If you remove a part, does the machine stop working altogether, or does it still run at 50% power?
In the study “Quantitative essentiality in a reduced genome: a functional, regulatory and structural fitness map”, recently published in Molecular Systems Biology, we present a dynamic and quantitative approach to assessing essentiality. By coupling engineered transposon libraries with temporal Tn-Seq data, we created one of the most detailed essentiality maps ever made—reaching near-single-nucleotide precision in the genome-reduced bacterium Mycoplasma pneumoniae. This allowed us to move beyond a static, binary view of essentiality and instead provide a nuanced model that captures the fine-scale contribution of each genomic element to cell fitness.
Essentiality experiments using transposon mutagenesis are not new as they have been widely applied in in bacteria and even in cancer research. We also repurposed the methodology to identify translated small proteins (also known as microproteins or SEPs, from smORF-Encoded Proteins), produce random deletions in a genome, or assess metabolic pathways that are active. What’s new here is the resolution and scope. Previous approaches typically worked at the scale of 1 in every 100 DNA bases. Ours achieved nearly base-by-base detail, requiring new computational methods to handle the complexity of the data. This unprecedented resolution provided opportunities to explore how transcription and termination locally affect fitness, to reveal essential protein domains and small non-coding elements that are critical or inaccessible to transposon insertion, and to uncover unexpected patterns of genetic flexibility.
For example, even within “essential” genes, we found structural regions that can tolerate insertions without killing the cell. Even more striking, some essential genes can be split in two and still produce functional proteins. This would be like discovering a car can still drive if part of the engine is cut in half, as long as both halves remain present.
As an application, this map gives synthetic biologists a precise guide to which DNA elements can or cannot be removed when designing or reprogramming bacteria. In the longer term, this research refines our picture of what a “minimal cell” really needs to live. That knowledge is key for building synthetic organisms or engineering bacteria that, for example, manufacture medicines. Looking ahead, we plan to apply this method to other bacteria and ask whether these fine-scale essentiality rules are universal or species-specific. We’re also intrigued by evolutionary questions: could split essential genes be remnants of smaller proteins that once functioned independently and later fused into larger proteins?
In short, essentiality isn’t always all-or-nothing. Life can be surprisingly flexible—even when it comes to the most crucial parts of the genome.