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 (smORFs; < 100 residues), accounting for 10% of all ORFs, are the most frequently essential genomic components (53%), followed by conventional ORFs (49%). Essentiality of smORFs 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 (ncRNAs) 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 smORFs with essential functions, as well as non-coding regions, thus changing the focus when aiming to define the minimal essential genome.

Identification of small open reading frames (sm ORF s) encoding small proteins (≤ 100 amino acids; SEP s) is a challenge in the fields of genome annotation and protein discovery. Here, by combining a novel bioinformatics tool (Ran SEP s) with “‐omics” approaches, we were able to describe 109 bacterial small ORF omes. Predictions were first validated by performing an exhaustive search of SEP s present in Mycoplasma pneumoniae proteome via mass spectrometry, which illustrated the limitations of shotgun approaches. Then, Ran SEP s predictions were validated and compared with other tools using proteomic datasets from different bacterial species and SEP s from the literature. We found that up to 16 ± 9% of proteins in an organism could be classified as SEP s. Integration of Ran SEP s predictions with transcriptomics data showed that some annotated non‐coding RNA s could in fact encode for SEP s. A functional study of SEP s highlighted an enrichment in the membrane, translation, metabolism, and nucleotide‐binding categories. Additionally, 9.7% of the SEP s included a N‐terminus predicted signal peptide. We envision Ran SEP s as a tool to unmask the hidden universe of small bacterial proteins.

The PII proteins are key mediators of the cellular response to carbon and nitrogen status and are found in all domains of life. In eukaryotes, PII has only been identified in red algae and plants, and in these organisms, PII localizes to the plastid. PII proteins perform their role by assessing cellular carbon, nitrogen, and energy status and conferring this information to other proteins through proteinprotein interaction. We have used affinity chromatography and mass spectrometry to identify the PII-binding proteins of Arabidopsis thaliana. The major PII-interacting protein is the chloroplast-localized enzyme N-acetyl glutamate kinase, which catalyzes the key regulatory step in the pathway to arginine biosynthesis. The interaction of PII with N-acetyl glutamate kinase was confirmed through pull-down, gel filtration, and isothermal titration calorimetry experiments, and binding was shown to be enhanced in the presence of the downstream product, arginine. Enzyme kinetic analysis showed that PII increases N-acetyl glutamate kinase activity slightly, but the primary function of binding is to relieve inhibition of enzyme activity by the pathway product, arginine. Knowing the identity of PII-binding proteins across a spectrum of photosynthetic and non-photosynthetic organisms provides a framework for a more complete understanding of the function of this highly conserved signaling protein.In prokaryotic organisms, the PII protein is recognized as the key mediator of energy, carbon, and nitrogen interactions and is referred to as the central processing unit of carbon:nitrogen metabolism (1-4). Escherichia coli PII is a 112-amino acid protein that as a homotrimer senses the cellular status of both ATP and the carbon skeleton 2-oxoglutarate (2KG) 3 via allosteric means. Nitrogen status is assessed through glutamine levels by covalent modification (uridylylation) of PII. This metabolic information is signaled to other proteins by proteinprotein interaction and produces an appropriate response that alters gene expression and the activity of glutamine synthetase (3, 5). In terms of metabolic sensing, cyanobacterial PII plays a similar role, but in this case, covalent modification is by phosphorylation (6). To date, the processes known to be regulated by PII in cyanobacteria are: ammoniumdependent nitrate/nitrite uptake (7), high affinity bicarbonate transport (8), regulation of the global transcriptional activation by NtcA (9, 10), and arginine biosynthesis (11).In eukaryotes, PII has only been identified in plants and red algae (12), and its sequence is highly conserved when compared with prokaryotic PIIs, with Arabidopsis thaliana PII being 50 and 55% identical to E. coli and Synechococcus elongatus PII, respectively. Plant PII proteins have a conserved N-terminal extension that functions as a chloroplast transit peptide, which is consistent with biochemical data indicating that PII resides in this compartment. We have previously shown that the plant PII protein is not regulated by phosphorylation (13). Like the bacterial...

Fluorescence resonance energy transfer (FRET)-based detection of protein interactions is limited by the very narrow range of FRET-permitting distances. We show two different strategies for the rational design of weak helper interactions that co-recruit donor and acceptor fluorophores for a more robust detection of bimolecular FRET: (i) in silico design of electrostatically driven encounter complexes and (ii) fusion of tunable domain-peptide interaction modules based on WW or SH3 domains. We tested each strategy for optimization of FRET between (m)Citrine and mCherry, which do not natively interact. Both approaches yielded comparable and large increases in FRET efficiencies with little or no background. Helper-interaction modules can be fused to any pair of fluorescent proteins and could, we found, enhance FRET between mTFP1 and mCherry as well as between mTurquoise2 and mCitrine. We applied enhanced helper-interaction FRET (hiFRET) probes to study the binding between full-length H-Ras and Raf1 as well as the drug-induced interaction between Raf1 and B-Raf.