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Quantitative proteomics analysis of Streptomyces coelicolor development demonstrates that onset of secondary metabolism coincides with hypha differentiation.

ABSTRACT: Streptomyces species produce many clinically important secondary metabolites, including antibiotics and antitumorals. They have a complex developmental cycle, including programmed cell death phenomena, that makes this bacterium a multicellular prokaryotic model. There are two differentiated mycelial stages: an early compartmentalized vegetative mycelium (first mycelium) and a multinucleated reproductive mycelium (second mycelium) arising after programmed cell death processes. In the present study, we made a detailed proteomics analysis of the distinct developmental stages of solid confluent Streptomyces coelicolor cultures using iTRAQ (isobaric tags for relative and absolute quantitation) labeling and LC-MS/MS. A new experimental approach was developed to obtain homogeneous samples at each developmental stage (temporal protein analysis) and also to obtain membrane and cytosolic protein fractions (spatial protein analysis). A total of 345 proteins were quantified in two biological replicates. Comparative bioinformatics analyses revealed the switch from primary to secondary metabolism between the initial compartmentalized mycelium and the multinucleated hyphae.

SUBMITTER: Manteca A

PROVIDER: S-EPMC2938082 | biostudies-other | 2010 Jul

REPOSITORIES: biostudies-other

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Quantitative proteomics analysis of Streptomyces coelicolor development demonstrates that onset of secondary metabolism coincides with hypha differentiation.

Manteca Angel A Sanchez Jesus J Jung Hye R HR Schwämmle Veit V Jensen Ole N ON

Molecular & cellular proteomics : MCP 20100311 7

Streptomyces species produce many clinically important secondary metabolites, including antibiotics and antitumorals. They have a complex developmental cycle, including programmed cell death phenomena, that makes this bacterium a multicellular prokaryotic model. There are two differentiated mycelial stages: an early compartmentalized vegetative mycelium (first mycelium) and a multinucleated reproductive mycelium (second mycelium) arising after programmed cell death processes. In the present stud ...[more]

PMID: 20224110

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Project description:Streptomycetes are multicellular bacteria with complex developmental cycles. They are of biotechnological importance as they produce most bioactive compounds used in biomedicine, e.g. antibiotic, antitumoral and immunosupressor compounds. Streptomyces 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 Streptomyces coelicolor We identified and quantified 3461 proteins corresponding to 44.3% of the S. coelicolor 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.

Project description:We report here the physiological and genetic characterization of an orphan histidine kinase (HK) (OhkA, SCO1596) in Streptomyces coelicolor and its homolog (OhkAsav, SAV_6741) in Streptomyces avermitilis. The physiological analysis showed that the ohkA mutant of S. coelicolor exhibits impaired aerial mycelium formation and sporulation and overproduction of multiple antibiotics on mannitol-soy flour (MS) medium, especially actinorhodin (ACT) and calcium-dependent antibiotic (CDA), and disruption of ohkAsav in S. avermitilis also led to the similar phenotypes of impaired morphological differentiation and significantly increased oligomycin A production. DNA microarray analysis combined with real-time reverse transcription-PCR (RT-PCR) and RNA dot blot assay in the S. coelicolor ohkA deletion mutant confirmed the physiological results by showing the upregulation of genes involved in the biosynthesis of ACT, CDA, undecylprodigiosin (RED), a yellow type I polyketide (CPK, SCO6273-6289), and a sesquiterpene antibiotic, albaflavenone (SCO5222-5223). The results also suggested that the increased production of ACT and RED in the mutant could be partly ascribed to the enhanced precursor malonyl coenzyme A (malonyl-CoA) supply through increased transcription of genes encoding acetyl-CoA carboxylase (ACCase). Interestingly, DNA microarray analysis also showed that deletion of ohkA greatly downregulated the transcription of chpABCDEFGH genes essential for aerial mycelium formation by S. coelicolor on MS medium but significantly increased transcription of ramS/C/R, which is responsible for SapB formation and regulation and is normally absent on MS medium. Moreover, many other genes involved in development, such as bldM/N, whiG/H/I, ssgA/B/E/G/R, and whiE, were also significantly downregulated upon ohkA deletion. The results clearly demonstrated that OhkA is an important global regulator for both morphological differentiation and secondary metabolism in S. coelicolor and S. avermitilis.

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Quantitative proteomics analysis of Streptomyces coelicolor development demonstrates that onset of secondary metabolism coincides with hypha differentiation.

Publications

Quantitative proteomics analysis of Streptomyces coelicolor development demonstrates that onset of secondary metabolism coincides with hypha differentiation.

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