In our genome-wide screens for small RNAs, we found that a number of short RNAs actually encode small proteins (1). The correct annotation of the smallest proteins is one of the biggest challenges of genome annotation, and there is little evidence that annotated short ORFs encode synthesized proteins. Although these proteins have largely been missed, the few small proteins that have been studied in detail in bacterial and mammalian cells have been shown to have important functions in signaling and in cellular defenses. We thus established a project to identify and characterize proteins of less than 50 amino acids. We used sequence conservation and ribosome binding site models to predict genes encoding small proteins of 16-50 amino acids, in the intergenic regions of the model Escherichia coli genome. We tested expression of these predicted as well as previously annotated small proteins by integrating the sequential peptide affinity tag directly upstream of the stop codon on the chromosome and assaying for synthesis using immunoblot assays. This approach confirmed that 20 previously annotated and 18 newly discovered proteins of 16-50 amino acids are synthesized. We have now initiated a complementary approach based on genome-wide ribosome profiling of ribosomes arrested in start codons to identify additional small proteins. More than half of the newly discovered proteins were predicted to consist of a single transmembrane alpha-helix and were found to be in the inner membrane in biochemical fractionation. Interestingly, assays of topology-reporter fusions and strains with defects in membrane insertion proteins, revealed that, despite their diminutive size, small membrane proteins display considerable diversity in topology and insertion pathways. Additionally, systematic assays for the accumulation of tagged versions of the proteins showed that many small proteins accumulate under specific growth conditions or after exposure to stress. We also generated and screened bar-coded null mutants and identified small proteins required for resistance to cell envelope stress and acid shock. We now are using the tagged derivatives and information about synthesis and subcellular localization and employing many of the approaches the group has used to characterize the functions of small regulatory RNAs to elucidate the functions of the small proteins. The combined approaches are beginning to give insights into how the small proteins are acting in E. coli. We found that synthesis of a 42-amino acid protein, now denoted MntS (formerly the small RNA gene rybA) is repressed by high levels of manganese through MntR. The lack of MntS leads to decreased activities of manganese-dependent enzymes under manganese-poor conditions, while overproduction of MntS leads to very high intracellular manganese and bacteriostasis under manganese-rich conditions. These and other phenotypes led us to propose that MntS modulates intracellular manganese levels, possibly by inhibiting the manganese exporter MntP. We also showed that the 31-amino acid inner membrane protein MgtS (formerly denoted YneM) whose synthesis is induced by very low Mg2+ in a PhoPQ-dependent manner, acts to increase intracellular Mg2+ levels and maintain cell integrity upon Mg2+ depletion. Upon development of a functional tagged derivative of MgtS, we found that MgtS interacts with MgtA to increase the levels of this P-type ATPase Mg2+ transporter under Mg2+-limiting conditions. Correspondingly, the effects of MgtS upon Mg2+ limitation are lost in a mgtA mutant, and MgtA overexpression can suppress the mgtS phenotype. MgtS stabilization of MgtA provides an additional layer of regulation of this tightly-controlled Mg2+ transporter (2). Most recently we found that surprisingly MgtS interacts with and modulates the activity of a second protein, the PitA cation-phosphate symporter. Finally, we discovered the 49-amino acid inner membrane protein AcrZ (formerly named YbhT) whose synthesis is increased in response to noxious compounds such as antibiotics and oxidizing agents, associates with the AcrAB-TolC multidrug efflux pump, which confers resistance to a wide variety of antibiotics and other compounds. Co-purification of AcrZ with AcrB, in the absence of both AcrA and TolC, two-hybrid assays and suppressor mutations indicate this interaction occurs through the inner membrane protein AcrB. Mutants lacking AcrZ are sensitive to many, but not all, of the antibiotics transported by AcrAB-TolC. This differential antibiotic sensitivity suggests that AcrZ enhances the ability of the AcrAB-TolC pump to export certain classes of substrates. Detailed structural and mutational studies are now giving insight into how AcrZ changes AcrB activity. This work, together with our ongoing studies of other small proteins and related findings by others in eukaryotic cells, supports our hypothesis that many small proteins are acting as regulators of larger membrane proteins.