Defining a minimal cell: essentiality of small ORFs and nc RNAs in a genome-reduced bacterium.

Identifying all essential genomic components is critical for the assembly of minimal artificial life. In the genome-reduced bacterium Mycoplasma pneumoniae, we found that small ORFs (sm ORFs; < 100 residues), accounting for 10% of all ORFs, are the most frequently essential genomic components (53%), followed by conventional ORFs (49%). Essentiality of sm ORFs may be explained by their function as members of protein and/or DNA/ RNA complexes. In larger proteins, essentiality applied to individual domains and not entire proteins, a notion we could confirm by expression of truncated domains. The fraction of essential non-coding RNAs (nc RNAs) non-overlapping with essential genes is 5% higher than of non-transcribed regions (0.9%), pointing to the important functions of the former. We found that the minimal essential genome is comprised of 33% (269,410 bp) of the M. pneumoniae genome. Our data highlight an unexpected hidden layer of sm ORFs with essential functions, as well as non-coding regions, thus changing the focus when aiming to define the minimal essential genome. [ABSTRACT FROM AUTHOR]