Abstract
Actinomycetes undergo a dramatic reorganization of metabolic and cellular machinery during a brief period of growth arrest ("metabolic switch") preceding mycelia differentiation and the onset of secondary metabolite biosynthesis. This study explores the role of phosphorylation in coordinating the metabolic switch in the industrial actinomycete Saccharopolyspora erythraea. A total of 109 phosphopeptides from 88 proteins were detected across a 150-h fermentation using open-profile two-dimensional LC-MS proteomics and TiO(2) enrichment. Quantitative analysis of the phosphopeptides and their unphosphorylated cognates was possible for 20 pairs that also displayed constant total protein expression. Enzymes from central carbon metabolism such as putative acetyl-coenzyme A carboxylase, isocitrate lyase, and 2-oxoglutarate dehydrogenase changed dramatically in the degree of phosphorylation during the stationary phase, suggesting metabolic rearrangement for the reutilization of substrates and the production of polyketide precursors. In addition, an enzyme involved in cellular response to environmental stress, trypsin-like serine protease (SACE_6340/NC_009142_6216), decreased in phosphorylation during the growth arrest stage. More important, enzymes related to the regulation of protein synthesis underwent rapid phosphorylation changes during this stage. Whereas the degree of phosphorylation of ribonuclease Rne/Rng (SACE_1406/NC_009142_1388) increased during the metabolic switch, that of two ribosomal proteins, S6 (SACE_7351/NC_009142_7233) and S32 (SACE_6101/NC_009142_5981), dramatically decreased during this stage of the fermentation, supporting the hypothesis that ribosome subpopulations differentially regulate translation before and after the metabolic switch. Overall, we show the great potential of phosphoproteomic studies to explain microbial physiology and specifically provide evidence of dynamic protein phosphorylation events across the developmental cycle of actinomycetes.
Highlights
From the §Australian Institute for Bioengineering and Nanotechnology (AIBN), The University of Queensland, St Lucia, QLD 4072, Australia
In vivo phosphorylation controlling enzyme functionality was studied in E. coli and Streptomyces coelicolor developmental cycles [16, 17], showing the yet poorly explored effect of dynamic protein phosphorylation in microbial physiology
Understanding the regulatory mechanisms underpinning the reorganization of the metabolic and cellular machinery at the metabolic switch [20] is of both fundamental and practical importance, as efficient induction is essential for high-level production
Summary
Strain and Culture Conditions—S. erythraea strain NRRL23338 was purchased from the American Type Culture Collection (ATCC number 11635TM). Sample elutions were collected in 16 2-ml fractions using a 0%– 30% gradient of solvent B (5 mM KH2PO4, 30% acetonitrile, 350 mM KCl, 0.1% trifluoroacetic acid, pH 2.7) over 30 min. All quantitative experiments were performed from total protein extracts (without TiO2 enrichment or chromatographic fractionation) using a triple quadrupole mass spectrometer (QTRAPTM 4000, Applied Biosystems) with an electrospray ion source configured in positive mode. The total protein amount was estimated from the logged sum of peak areas for the phosphorylated and nonphosphorylated peptide cognates. The concentration of glucose was determined via high-performance liquid chromatography (Agilent 1200 HPLC system) (as described in Ref. 34) using a Phenomenex Rezex RHM Monosaccharide Hϩ column (300 ϫ 7.8 mm; 8 m) and detected by Refractive index. Erythromycin production was determined via LC-MS using a Dionex UltiMate 3000 liquid chromatography system (Dionex, Sunnyvale, CA) coupled with an AB Sciex 4000 QTRAP mass spectrometer (AB Sciex, Ontario, Canada) as described in Ref. 35
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