Quantitative Proteome and Phosphoproteome Analyses of Streptomyces coelicolor Reveal Proteins and Phosphoproteins Modulating Differentiation and Secondary Metabolism

Abstract

Streptomycetes are multicellular bacteria with complex developmental cycles. They are of biotechnological importance as they produce most bioactive compounds used in biomedicine, antibiotic, antitumoral and immunosupressor compounds. genomes encode many Ser/Thr/Tyr kinases, making this genus an outstanding model for the study of bacterial protein phosphorylation events. We used mass spectrometry based quantitative proteomics and phosphoproteomics to characterize bacterial differentiation and activation of secondary metabolism of We identified and quantified 3461 proteins corresponding to 44.3% of the proteome across three developmental stages: vegetative hypha (first mycelium); secondary metabolite producing hyphae (second mycelium); and sporulating hyphae. A total of 1350 proteins exhibited more than 2-fold expression changes during the bacterial differentiation process. These proteins include 136 regulators (transcriptional regulators, transducers, Ser/Thr/Tyr kinases, signaling proteins), as well as 542 putative proteins with no clear homology to known proteins which are likely to play a role in differentiation and secondary metabolism. Phosphoproteomics revealed 85 unique protein phosphorylation sites, 58 of them differentially phosphorylated during differentiation. Computational analysis suggested that these regulated protein phosphorylation events are implicated in important cellular processes, including cell division, differentiation, regulation of secondary metabolism, transcription, protein synthesis, protein folding and stress responses. We discovered a novel regulated phosphorylation site in the key bacterial cell division protein FtsZ (pSer319) that modulates sporulation and regulates actinorhodin antibiotic production. We conclude that manipulation of distinct protein phosphorylation events may improve secondary metabolite production in industrial streptomycetes, including the activation of cryptic pathways during the screening for new secondary metabolites from streptomycetes.

Mycelium Differentiation and Development ofStreptomycesin Liquid Nonsporulating Cultures: Programmed Cell Death, Differentiation, and Lysis Condition Secondary Metabolite Production

“…This was the first time that secondary metabolism was associated with a specific mycelial stage (MII) [10]. Transcriptomic [11] and proteomic [12,13] analyses performed by our group corroborated that MI is the vegetative mycelium expressing/translating primary metabolism genes/proteins, while MII is the reproductive mycelium expressing/translating secondary metabolism and sporulation genes/proteins.…”

sRNA scr5239 Involved in Feedback Loop Regulation of Streptomyces coelicolor Central Metabolism

“…After protein extraction and fragmentation, the peptides where labeled with a different TMT labels for each strain and analyzed using LC-MS/MS ( Figure 1A). This method has already been successfully applied in S. coelicolor to characterize differentiation and activation of the secondary metabolism (Rioseras et al, 2018). A total of 1669 proteins were found to be expressed in all three strains ( Supplementary Table S3).…”

Regulation of Protein Post-Translational Modifications on Metabolism of Actinomycetes

“…Phosphorylation is an effective type of modification, changing the spatial structure of proteins. It is vital for coordinate gene functions for cell growth [ 11 ], virulence [ 36 ], biofilms [ 11 ] and quorum sensing [ 37 ]. After modification with a negatively charged phosphate group, proteins can be activated by exposing or stabilizing the active domain, thus incorporating the signal into protein sequence for subsequent transcriptional regulation.…”

“…These processes are called protein post-translational modifications (PTMs), which highly expand the chemical composition and information normally encoded in the amino acid sequence of the proteins [ 8 , 9 ]. In prokaryotic cells, although the number and degree of protein PTMs are much lower than eukaryotes and the level of modifications fluctuate greatly with environmental changes [ 8 ], there are increasing evidences that PTMs play a vital role in various cellular processes, such as protein stability [ 10 ], protein synthesis [ 11 ], spore formation [ 11 ], dormancy [ 12 ] and virulence [ 13 ]. The addition of modifications ranges from small chemical groups, such as acetyl groups (about 42 Da) or phosphate groups (about 80 Da), to more complex structures, including N -glycosylation (2–3 kDa) and polypeptide chains such as the prokaryotic ubiquitin-like protein (Pup) (about 8 kDa) ( Table 1 ).…”

“…The addition of modifications ranges from small chemical groups, such as acetyl groups (about 42 Da) or phosphate groups (about 80 Da), to more complex structures, including N -glycosylation (2–3 kDa) and polypeptide chains such as the prokaryotic ubiquitin-like protein (Pup) (about 8 kDa) ( Table 1 ). While the functions of PTMs in primary metabolism are well-established, for example, their functions in cell division [ 11 ] and glucose assimilation [ 14 ], their roles in secondary metabolism remain elusive. With the development of two-dimensional gel electrophoresis and matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) mass spectrometry, the concept that PTMs regulate secondary metabolism was first proposed in 2002 [ 9 ].…”