Abstract
BackgroundDespite their versatile functions in multimeric protein complexes, in the modification of enzymatic activities, intercellular communication or regulatory processes, proteins shorter than 80 amino acids (μ-proteins) are a systematically underestimated class of gene products in bacteria. Photosynthetic cyanobacteria provide a paradigm for small protein functions due to extensive work on the photosynthetic apparatus that led to the functional characterization of 19 small proteins of less than 50 amino acids. In analogy, previously unstudied small ORFs with similar degrees of conservation might encode small proteins of high relevance also in other functional contexts.ResultsHere we used comparative transcriptomic information available for two model cyanobacteria, Synechocystis sp. PCC 6803 and Synechocystis sp. PCC 6714 for the prediction of small ORFs. We found 293 transcriptional units containing candidate small ORFs ≤80 codons in Synechocystis sp. PCC 6803, also including the known mRNAs encoding small proteins of the photosynthetic apparatus. From these transcriptional units, 146 are shared between the two strains, 42 are shared with the higher plant Arabidopsis thaliana and 25 with E. coli. To verify the existence of the respective μ-proteins in vivo, we selected five genes as examples to which a FLAG tag sequence was added and re-introduced them into Synechocystis sp. PCC 6803. These were the previously annotated gene ssr1169, two newly defined genes norf1 and norf4, as well as nsiR6 (nitrogen stress-induced RNA 6) and hliR1(high light-inducible RNA 1) , which originally were considered non-coding. Upon activation of expression via the Cu2+.responsive petE promoter or from the native promoters, all five proteins were detected in Western blot experiments.ConclusionsThe distribution and conservation of these five genes as well as their regulation of expression and the physico-chemical properties of the encoded proteins underline the likely great bandwidth of small protein functions in bacteria and makes them attractive candidates for functional studies.
Highlights
Despite their versatile functions in multimeric protein complexes, in the modification of enzymatic activities, intercellular communication or regulatory processes, proteins shorter than 80 amino acids (μ-proteins) are a systematically underestimated class of gene products in bacteria
Photosynthetic cyanobacteria provide a paradigm for small protein functions due to extensive work on the photosynthetic apparatus that led to the functional characterization of 19 μ-proteins of less than 50 amino acids, that play a role in photosystem II (genes psbM, psbT, psbI, psbL, psbJ, psbY, psbX, psb30, psbN, psbF, psbK [12, 13]), in photosystem I, photosynthetic electron transport (Cytb6f complex; petL, petN, petM, petG [15,16,17]), or have accessory functions (hliC [18])
We found 293 transcriptional units (TU) containing candidate small ORFs ≤80 codons in Synechocystis 6803, including all known mRNAs encoding small proteins of the photosynthetic apparatus
Summary
Despite their versatile functions in multimeric protein complexes, in the modification of enzymatic activities, intercellular communication or regulatory processes, proteins shorter than 80 amino acids (μ-proteins) are a systematically underestimated class of gene products in bacteria. Photosynthetic cyanobacteria provide a paradigm for small protein functions due to extensive work on the photosynthetic apparatus that led to the functional characterization of 19 small proteins of less than 50 amino acids. During standard genome annotation these short protein-coding genes are frequently neglected and proteomics-based analyses fail to detect this class of peptides routinely. The functional characterization of selected examples of μ-proteins has revealed their critical involvement in processes such as quorum sensing or interspecies communication [2], regulatory functions [3,4,5,6] and in the formation of multi-subunit protein complexes. The Baumgartner et al BMC Microbiology (2016) 16:285 likely diverse functions of short proteins are largely unknown, even for simple unicellular bacteria
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