Comprehensive subcellular topologies of polypeptides in Streptomyces

Abstract

BackgroundMembers of the genus Streptomyces are Gram-positive bacteria that are used as important cell factories to produce secondary metabolites and secrete heterologous proteins. They possess some of the largest bacterial genomes and thus proteomes. Understanding their complex proteomes and metabolic regulation will improve any genetic engineering approach.ResultsHere, we performed a comprehensive annotation of the subcellular localization of the proteome of Streptomyces lividans TK24 and developed the Subcellular Topology of Polypeptides in Streptomyces database (SToPSdb) to make this information widely accessible. We first introduced a uniform, improved nomenclature that re-annotated the names of ~ 4000 proteins based on functional and structural information. Then protein localization was assigned de novo using prediction tools and edited by manual curation for 7494 proteins, including information for 183 proteins that resulted from a recent genome re-annotation and are not available in current databases. The S. lividans proteome was also linked with those of other model bacterial strains including Streptomyces coelicolor A3(2) and Escherichia coli K-12, based on protein homology, and can be accessed through an open web interface. Finally, experimental data derived from proteomics experiments have been incorporated and provide validation for protein existence or topology for 579 proteins. Proteomics also reveals proteins released from vesicles that bleb off the membrane. All export systems known in S. lividans are also presented and exported proteins assigned export routes, where known.ConclusionsSToPSdb provides an updated and comprehensive protein localization annotation resource for S. lividans and other streptomycetes. It forms the basis for future linking to databases containing experimental data of proteomics, genomics and metabolomics studies for this organism.Electronic supplementary materialThe online version of this article (10.1186/s12934-018-0892-0) contains supplementary material, which is available to authorized users.

Multi-Omics and Targeted Approaches to Determine the Role of Cellular Proteases in Streptomyces Protein Secretion

“…Moreover, upon loading of secretome material derived from the same volume of culture, TK24Δ ftsH was also found to be a profuse secretor of polypeptides (not shown). As seen in other studies ( Hamed et al, 2017 ; Tsolis et al, 2018 ), there appears to be a good correlation between suppressed growth and improved secretion as seen by comparison of the total secretome expressed per unit cell biomass ( Figure 3B ).…”

“…Streptomyces lividans TK24 contains 190 protease-encoding genes (Supplementary Table S1 ; SToPSdb; Tsolis et al, 2018 ) 1 . Of these, 50% are secreted and another 10% are membrane-embedded.…”

“…In addition, TK24 secretes 3 proteins that act as protease inhibitors. To determine whether proteases play a role in the secretory processes of TK24, we first classified potential proteases based on several parameters, as follows: (I) the presence of a Sec or Tat secretion signal using multiple bioinformatics tools ( Tsolis et al, 2018 ), (II) whether the protein in question belongs to the set of core genes of S. lividans TK24, based on the comparison to the core genome of 13 Streptomyces species, and (III) the maximal transcript level, determined by RNAseq. For the latter, TK24 was grown under two media regimes (minimal medium supplemented with glucose and minimal medium with glucose supplemented with casamino acids) and cells were harvested at three different growth phases (early and late logarithmic, and stationary).…”

“…Thresholds for the analysis were set to adjusted p -value < 0.05 and fold difference > 2. Functional characterization of proteomics results was performed after filtering the dataset only to secreted proteins, excluding cytoplasmic contamination, using proteome annotation as described in the SToPSdb ( Tsolis et al, 2018 ) 2 . The percentage of differentially abundant proteins that match a specific term over the total differentially abundant proteins for each condition was plotted.…”

The Cellular Mechanisms that Ensure an Efficient Secretion in Streptomyces

“…Streptomyces lividans contains three Tat components (TatA, TatB, and TatC) with two different tatA genes [ 54 ]. Recognition of the Tat pre-secretory proteins in S. lividans is carried out by the heterodimeric complex formed by TatA and TatB, which theoretically target the pre-proteins to the membrane [ 55 ].…”