Metabolomics-Driven Discovery of a Prenylated Isatin Antibiotic Produced by Streptomyces Species MBT28.

Actinomycetes are a major source of antimicrobials, anticancer compounds, and other medically important products, and their genomes harbor extensive biosynthetic potential. Major challenges in the screening of these microorganisms are to activate the expression of cryptic biosynthetic gene clusters and the development of technologies for efficient dereplication of known molecules. Here we report the identification of a previously unidentified isatin-type antibiotic produced by Streptomyces sp. MBT28, following a strategy based on NMR-based metabolomics combined with the introduction of streptomycin resistance in the producer strain. NMR-guided isolation by tracking the target proton signal resulted in the characterization of 7-prenylisatin (1) with antimicrobial activity against Bacillus subtilis. The metabolite-guided genome mining of Streptomyces sp. MBT28 combined with proteomics identified a gene cluster with an indole prenyltransferase that catalyzes the conversion of tryptophan into 7-prenylisatin. This study underlines the applicability of NMR-based metabolomics in facilitating the discovery of novel antibiotics.

The ROK family regulator Rok7B7 pleiotropically affects xylose utilization, carbon catabolite repression, and antibiotic production in streptomyces coelicolor.

Members of the ROK family of proteins are mostly transcriptional regulators and kinases that generally relate to the control of primary metabolism, whereby its member glucose kinase acts as the central control protein in carbon control in Streptomyces. Here, we show that deletion of SCO6008 (rok7B7) strongly affects carbon catabolite repression (CCR), growth, and antibiotic production in Streptomyces coelicolor. Deletion of SCO7543 also affected antibiotic production, while no major changes were observed after deletion of the rok family genes SCO0794, SCO1060, SCO2846, SCO6566, or SCO6600. Global expression profiling of the rok7B7 mutant by proteomics and microarray analysis revealed strong upregulation of the xylose transporter operon xylFGH, which lies immediately downstream of rok7B7, consistent with the improved growth and delayed development of the mutant on xylose. The enhanced CCR, which was especially obvious on rich or xylose-containing media, correlated with elevated expression of glucose kinase and of the glucose transporter GlcP. In liquid-grown cultures, expression of the biosynthetic enzymes for production of prodigionines, siderophores, and calcium-dependent antibiotic (CDA) was enhanced in the mutant, and overproduction of prodigionines was corroborated by matrix-assisted laser desorption ionization-time-of-flight analysis. These data present Rok7B7 as a pleiotropic regulator of growth, CCR, and antibiotic production in Streptomyces.

Identification of glucose kinase-dependent and -independent pathways for carbon control of primary metabolism, development and antibiotic production in Streptomyces coelicolor by quantitative proteomics.

Members of the soil-dwelling prokaryotic genus Streptomyces are indispensable for the recycling of complex polysaccharides, and produce a wide range of natural products. Nutrient availability is a major determinant for the switch to development and antibiotic production in streptomycetes. Carbon catabolite repression (CCR), a main signalling pathway underlying this phenomenon, was so far considered fully dependent on the glycolytic enzyme glucose kinase (Glk). Here we provide evidence of a novel Glk-independent pathway in Streptomyces coelicolor, using advanced proteomics that allowed the comparison of the expression of some 2000 proteins, including virtually all enzymes for central metabolism. While CCR and inducer exclusion of enzymes for primary and secondary metabolism and precursor supply for natural products is mostly mediated via Glk, enzymes for the urea cycle, as well as for biosynthesis of the γ-butyrolactone Scb1 and the responsive cryptic polyketide Cpk are subject to Glk-independent CCR. Deletion of glkA led to strong downregulation of biosynthetic proteins for prodigionins and calcium-dependent antibiotic (CDA) in mannitol-grown cultures. Repression of bldB, bldN, and its target bldM may explain the poor development of S. coelicolor on solid-grown cultures containing glucose. A new model for carbon catabolite repression in streptomycetes is presented.

In vitro incorporation of nonnatural amino acids into protein using tRNA(Cys)-derived opal, ochre, and amber suppressor tRNAs.

Amber suppressor tRNAs are widely used to incorporate nonnatural amino acids into proteins to serve as probes of structure, environment, and function. The utility of this approach would be greatly enhanced if multiple probes could be simultaneously incorporated at different locations in the same protein without other modifications. Toward this end, we have developed amber, opal, and ochre suppressor tRNAs derived from Escherichia coli, and yeast tRNA(Cys) that incorporate a chemically modified cysteine residue with high selectivity at the cognate UAG, UGA, and UAA stop codons in an in vitro translation system. These synthetic tRNAs were aminoacylated in vitro, and the labile aminoacyl bond was stabilized by covalently attaching a fluorescent dye to the cysteine sulfhydryl group. Readthrough efficiency (amber > opal > ochre) was substantially improved by eRF1/eRF3 inhibition with an RNA aptamer, thus overcoming an intrinsic hierarchy in stop codon selection that limits UGA and UAA termination suppression in higher eukaryotic translation systems. This approach now allows concurrent incorporation of two different modified amino acids at amber and opal codons with a combined apparent readthrough efficiency of up to 25% when compared with the parent protein lacking a stop codon. As such, it significantly expands the possibilities for incorporating nonnative amino acids for protein structure/function studies.

Analysis of two distinct mycelial populations in liquid-grown Streptomyces cultures using a flow cytometry-based proteomics approach.

Streptomycetes are proficient producers of enzymes and antibiotics. When grown in bioreactors, these filamentous microorganisms form mycelial pellets that consist of interconnected hyphae. We here employed a flow cytometry approach designed for large particles (COPAS) and demonstrate that liquid-grown Streptomyces cultures consist of two distinct populations of pellets. One population consists of mycelia with a constant mean diameter of approximately 260 µm, whereas the other population contains larger mycelia whose diameter depends on the strain, the age of the culture, and medium composition. Quantitative proteomics analysis revealed that 37 proteins differed in abundance between the two populations of pellets. Stress-related proteins and biosynthetic proteins for production of the calcium-dependent antibiotic were more abundant in the population of large mycelia, while proteins involved in DNA topology, modification, or degradation were overrepresented in the population of small mycelia. Deletion of genes for the cellulose synthase-like protein CslA and the chaplins affected the average size of the population of large pellets but not that of small pellets. Considering the fact that the production of enzymes and metabolites depends on pellet size, these results provide new leads toward rational strain design of Streptomyces strains tailored for industrial fermentations.

Protein complexes in bacterial and yeast mitochondrial membranes differ in their sensitivity towards dissociation by SDS.

Previously, a 2D gel electrophoresis approach was developed for the Escherichia coli inner membrane, which detects membrane protein complexes that are stable in sodium dodecyl sulfate (SDS) at room temperature, and dissociate under the influence of trifluoroethanol [R. E. Spelbrink et al., J. Biol. Chem. 280 (2005), 28742-8]. Here, the method was applied to the evolutionarily related mitochondrial inner membrane that was isolated from the yeast Saccharomyces cerevisiae. Surprisingly, only very few proteins were found to be dissociated by trifluoroethanol of which Lpd1p, a component of multiple protein complexes localized in the mitochondrial matrix, is the most prominent. Usage of either milder or more stringent conditions did not yield any additional proteins that were released by fluorinated alcohols. This strongly suggests that membrane protein complexes in yeast are less stable in SDS solution than their E. coli counterparts, which might be due to the overall reduced hydrophobicity of mitochondrial transmembrane proteins.

Depletion of phosphatidylcholine in yeast induces shortening and increased saturation of the lipid acyl chains: evidence for regulation of intrinsic membrane curvature in a eukaryote.

To study the consequences of depleting the major membrane phospholipid phosphatidylcholine (PC), exponentially growing cells of a yeast cho2opi3 double deletion mutant were transferred from medium containing choline to choline-free medium. Cell growth did not cease until the PC level had dropped below 2% of total phospholipids after four to five generations. Increasing contents of phosphatidylethanolamine (PE) and phosphatidylinositol made up for the loss of PC. During PC depletion, the remaining PC was subject to acyl chain remodeling with monounsaturated species replacing diunsaturated species, as shown by mass spectrometry. The remodeling of PC did not require turnover by the SPO14-encoded phospholipase D. The changes in the PC species profile were found to reflect an overall shift in the cellular acyl chain composition that exhibited a 40% increase in the ratio of C16 over C18 acyl chains, and a 10% increase in the degree of saturation. The shift was stronger in the phospholipid than in the neutral lipid fraction and strongest in the species profile of PE. The shortening and increased saturation of the PE acyl chains were shown to decrease the nonbilayer propensity of PE. The results point to a regulatory mechanism in yeast that maintains intrinsic membrane curvature in an optimal range.

Intertwined Precursor Supply during Biosynthesis of the Catecholate-Hydroxamate Siderophores Qinichelins in Streptomyces sp. MBT76.

The explosive increase in genome sequencing and the advances in bioinformatic tools have revolutionized the rationale for natural product discovery from actinomycetes. In particular, this has revealed that actinomycete genomes contain numerous orphan gene clusters that have the potential to specify many yet unknown bioactive specialized metabolites, representing a huge unexploited pool of chemical diversity. Here, we describe the discovery of a novel group of catecholate-hydroxamate siderophores termed qinichelins (2-5) from Streptomyces sp. MBT76. Correlation between the metabolite levels and the protein expression profiles identified the biosynthetic gene cluster (named qch) most likely responsible for qinichelin biosynthesis. The structure of the molecules was elucidated by bioinformatics, mass spectrometry, and NMR. The genome of Streptomyces sp. MBT76 contains three gene clusters for the production of catecholate-peptide siderophores, including a separate cluster for the production of a shared catecholate precursor. In addition, an operon in the qch cluster was identified for the production of the ornithine precursor for qinichelins, independent of primary metabolism. This biosynthetic complexity provides new insights into the challenges scientists face when applying synthetic biology approaches for natural product discovery.

Genome-wide analysis of in vivo binding of the master regulator DasR in Streptomyces coelicolor identifies novel non-canonical targets.

Streptomycetes produce a wealth of natural products, including over half of all known antibiotics. It was previously demonstrated that N-acetylglucosamine and secondary metabolism are closely entwined in streptomycetes. Here we show that DNA recognition by the N-acetylglucosamine-responsive regulator DasR is growth-phase dependent, and that DasR can bind to sites in the S. coelicolor genome that have no obvious resemblance to previously identified DasR-responsive elements. Thus, the regulon of DasR extends well beyond what was previously predicted and includes a large number of genes with functions far removed from N-acetylglucosamine metabolism, such as genes for small RNAs and DNA transposases. Conversely, the DasR regulon during vegetative growth largely correlates to the presence of canonical DasR-responsive elements. The changes in DasR binding in vivo following N-acetylglucosamine induction were studied in detail and a possible molecular mechanism by which the influence of DasR is extended is discussed. Discussion of DasR binding was further informed by a parallel transcriptome analysis of the respective cultures. Evidence is provided that DasR binds directly to the promoters of all genes encoding pathway-specific regulators of antibiotic production in S. coelicolor, thereby providing an exquisitely simple link between nutritional control and secondary metabolism.

Natural product proteomining, a quantitative proteomics platform, allows rapid discovery of biosynthetic gene clusters for different classes of natural products.

Information on gene clusters for natural product biosynthesis is accumulating rapidly because of the current boom of available genome sequencing data. However, linking a natural product to a specific gene cluster remains challenging. Here, we present a widely applicable strategy for the identification of gene clusters for specific natural products, which we name natural product proteomining. The method is based on using fluctuating growth conditions that ensure differential biosynthesis of the bioactivity of interest. Subsequent combination of metabolomics and quantitative proteomics establishes correlations between abundance of natural products and concomitant changes in the protein pool, which allows identification of the relevant biosynthetic gene cluster. We used this approach to elucidate gene clusters for different natural products in Bacillus and Streptomyces, including a novel juglomycin-type antibiotic. Natural product proteomining does not require prior knowledge of the gene cluster or secondary metabolite and therefore represents a general strategy for identification of all types of gene clusters.

Relative quantification of proteasome activity by activity-based protein profiling and LC-MS/MS.

Activity-based protein profiling (ABPP) is a functional proteomics technique for directly monitoring the expression of active enzymes in cell extracts and living cells. The technique relies on irreversible inhibitors equipped with reactive groups (warheads) that covalently attach to the active site of enzymes and fluorescent or affinity tags for imaging and purification purposes, respectively. Here, a high-throughput and robust protocol for high-resolution quantitative activity-based proteasome profiling is described. We use both panreactive and subunit-specific fluorescent activity-based probes (ABPs) to quantify the proteasome activity in living cells, in the presence or absence of the potent proteasome inhibitor bortezomib. Active proteasome subunits from cell lysates are affinity-purified via a biotinylated ABP. Purification from live cells involves a two-step ABP approach using a reagent with a cell-permeable azide-warhead and postlysis installation of biotin. By means of liquid chromatography-mass spectrometry (LC-MS)-based proteomics, we can accurately identify the enriched proteins and the active site peptides of the enzymes, and relatively quantify all the proteasome activities in one experiment. The fluorescence ABPP protocols takes 2-3 d, and approximately 8-10 d are needed to complete the entire protocol.

Proteome-wide detection of phospholipid-protein interactions in mitochondria by photocrosslinking and click chemistry.

Photoactivatable lipid analogues are uniquely suited for the detection of lipid-protein interactions in biological membranes. Based on photocrosslinking, new methodology has been developed for the proteome-wide detection of lipid-protein interactions. Bifunctional lipid analogues containing a tag for click chemistry in addition to the photoactivatable moiety enable the enrichment of the crosslinked proteins that is required for subsequent identification by mass spectrometry. In principle the phospholipid interaction-based membrane protein proteomics approach is applicable to any biomembrane and any lipid. Here, we review the background and the development of the new methodology. Results obtained with photocrosslinking in purified mitochondrial membranes from the yeast Saccharomyces cerevisiae are summarized and future perspectives discussed.

Photocrosslinking and click chemistry enable the specific detection of proteins interacting with phospholipids at the membrane interface.

New lipid analogs mimicking the abundant membrane phospholipid phosphatidylcholine were developed to photocrosslink proteins interacting with phospholipid headgroups at the membrane interface. In addition to either a phenylazide or benzophenone photoactivatable moiety attached to the headgroup, the lipid analogs contained azides attached as baits to the acyl chains. After photocrosslinking in situ in the biomembrane, these baits were used for the attachment of a fluorescent tetramethylrhodamine-alkyne conjugate or a biotin-alkyne conjugate using click chemistry, allowing for the selective detection and purification of crosslink products, respectively. Proteins crosslinked to the lipid analogs in inner mitochondrial membranes from Saccharomyces cerevisiae were detected and subsequently identified by mass spectrometry. Established interaction partners of phosphatidylcholine were found, as well as known integral and peripheral inner membrane proteins, and proteins that were not previously considered mitochondrial inner membrane proteins.

Probing the membrane interface-interacting proteome using photoactivatable lipid cross-linkers.

To analyze proteins interacting at the membrane interface, a phospholipid analogue was used with a photoactivatable headgroup (ASA-DLPE, N-(4-azidosalicylamidyl)-1,2-dilauroyl-sn-glycero-3-phosphoethanolamine) for selective cross-linking. The peripheral membrane protein cytochrome c from the inner mitochondrial membrane was rendered carbonate wash-resistant by cross-linking to ASA-DLPE in a model membrane system, validating our approach. Cross-link products of cytochrome c and its precursor apocytochrome c were demonstrated by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) and were specifically detected by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), taking advantage of the intrinsic UV absorbance of the cross-linker. Application of the method to inner mitochondrial membranes from Saccharomyces cerevisae revealed cross-link products of both exogenously added apocytochrome c and endogenous proteins with molecular weights around 34 and 72 kDa. Liquid chromatograpy (LC)-MS/MS was performed to identify these proteins, resulting in a list of candidate proteins potentially cross-linked at the membrane interface. The approach described here provides methodology for capturing phospholipid-protein interactions in their native environment of the biomembrane using modern proteomics techniques.

Tissue-specific expression and dimerization of the endoplasmic reticulum oxidoreductase Ero1beta.

Endoplasmic reticulum oxidoreductases (Eros) are essential for the formation of disulfide bonds. Understanding disulfide bond catalysis in mammals is important because of the involvement of protein misfolding in conditions such as diabetes, arthritis, cancer, and aging. Mammals express two related Ero proteins, Ero1alpha and Ero1beta. Ero1beta is incompletely characterized but is of physiological interest because it is induced by the unfolded protein response. Here, we show that Ero1beta can form homodimers and mixed heterodimers with Ero1alpha, in addition to Ero-PDI dimers. Ero-Ero dimers require the Ero active site, occur in vivo, and can be modeled onto the Ero1p crystal structure. Our data indicate that the Ero1beta protein is constitutively strongly expressed in the stomach and the pancreas, but in a cell-specific fashion. In the stomach, selective expression of Ero1beta occurs in the enzyme-producing chief cells. In pancreatic islets, Ero1beta expression is high, but is inversely correlated with PDI and PDIp levels, demonstrating that cell-specific differences exist in the regulation of oxidative protein folding in vivo.