Isoforms of heat shock protein (Hsp) 27 were used as intracellular markers to study tumor necrosi... more Isoforms of heat shock protein (Hsp) 27 were used as intracellular markers to study tumor necrosis factor/interleukin-1 (TNF/IL-1) regulation of protein phosphatases in primary human fibroblasts. These isoforms were rapidly phosphorylated to varying degrees when fibroblasts were treated with either TNF, IL-1, okadaic acid, calyculin A, ARS, epidermal growth factor, fibroblast growth factor, H2O2, buthionine sulfoximine, N-ethylmaleimide, diethylmaleimide, or iodoacetate. However, inhibitors of protein kinases A and C, tyrosyl protein kinases, and general protein kinases had no effect on the enhanced phosphorylation of these isoforms in TNF, IL-1, okadaic acid, or calyculin A-stimulated cells, suggesting that the activation of protein kinases by itself is insufficient to produce these changes. Isoforms of 32P-labeled Hsp27 were dephosphorylated during cold-chases with excess phosphate in the absence but not in the presence of TNF/IL-1 or inhibitors of protein phosphatases suggesting that inactivation of protein phosphatase(s) plays a role in TNF/IL-1 signal transduction. Assays of phosphatase activity of cytosolic fractions from TNF or okadaic acid treated human fibroblasts showed an inactivation of protein phosphatase activity against the 32P-labeled Hsp27 protein substrates. In vitro assays of partially purified phosphatase activity from primary human fibroblasts with Hsp27 substrate also showed the protein phosphatase activity to be inhibited by ARS. Like okadaic acid, ARS mimics TNF in inducing specific patterns of cellular protein phosphorylation. Taken together these findings are consistent with the hypothesis that a SH-dependent protein phosphatase is inactivated during the early events of TNF/IL-1 signal transduction, hence inhibitors of protein phosphatases and SH modifying compounds can mimic the early effects of TNF/IL-1 on cells.
High resolution two-dimensional gel electrophoresis was used to analyze the signal transduction p... more High resolution two-dimensional gel electrophoresis was used to analyze the signal transduction pathways of tumor necrosis factor (TNF-alpha) and interleukin 1 (IL-1 alpha and -beta) in human fibroblasts. Approximately 450 discrete radioactive spots were electrophoretically resolved from cytosolic extracts of cells prelabeled with 32P. At least 63 of these polypeptides exhibited significant and concordant phosphorylation or dephosphorylation in response to TNF or IL-1, despite the fact that different receptors are involved. Most of these changes concerned serine/threonine residues although enhanced tyrosine phosphorylation of several polypeptides was also observed. Phosphorylation patterns induced by a number of other agonists were compared with the patterns induced by IL-1 and TNF. These included activators of protein kinases C and A, bradykinin (a stimulator of inositol phospholipid hydrolysis), epidermal growth factor, heatshock, and mellitin (an activator of phospholipase A2). Although each of these agonists induced changes resulting in a distinct pattern of protein phosphorylation, none of these patterns had significant homology with that induced by IL-1 and TNF. Other assays were performed to verify the involvement of specific kinases. Collectively, these data indicate that IL-1 and TNF activate multiple protein kinases viz. a kinase(s) which activates microtubule-associated protein 2 (MAP-2) kinase, a kinase that phosphorylates the cap-binding protein, and a possibly novel serine/threonine protein kinase.
Natural products have long been used as a source of antimicrobial agents against various microorg... more Natural products have long been used as a source of antimicrobial agents against various microorganisms. Actinobacteria are a group of bacteria best known to produce a wide variety of bioactive secondary metabolites, including many antimicrobial agents. In this study, four actinobacterial strains found in Singapore terrestrial soil were investigated as potential sources of new antimicrobial compounds. Large-scale cultivation, chemical, and biological investigation led to the isolation of a previously undescribed tetronomycin A (1) that demonstrated inhibitory activities against both Gram-positive bacteria Staphylococcus aureus (SA) and methicillin-resistant Staphylococcus aureus (MRSA) (i.e., MIC90 of 2–4 μM and MBC90 of 9–12 μM), and several known antimicrobial compounds, namely nonactin, monactin, dinactin, 4E-deacetylchromomycin A3, chromomycin A2, soyasaponin II, lysolipin I, tetronomycin, and naphthomevalin. Tetronomycin showed a two- to six-fold increase in antibacterial activ...
Thiopeptides are macrocyclic natural products with potent bioactivity. Nine new natural thiopepti... more Thiopeptides are macrocyclic natural products with potent bioactivity. Nine new natural thiopeptides (1–9) were obtained from a Nonomuraea jiangxiensis isolated from a terrestrial soil sample collected in Singapore. Even though some of these compounds were previously synthesized or isolated from engineered strains, herein we report the unprecedented isolation of these thiopeptides from a native Nonomuraea jiangxiensis. A comparison with the literature and a detailed analysis of the NMR and HRMS of compounds 1–9 was conducted to assign their chemical structures. The structures of all new compounds were highly related to the thiopeptide antibiotics GE2270, with variations in the substituents on the thiazole and amino acid moieties. Thiopeptides 1–9 exhibited a potent antimicrobial activity against the Gram-positive bacteria, Staphylococcus aureus with MIC90 values ranging from 2 µM to 11 µM. In addition, all compounds were investigated for their cytotoxicity against the human cancer c...
Nature has provided unique molecular scaffolds for applications including therapeutics, agricultu... more Nature has provided unique molecular scaffolds for applications including therapeutics, agriculture, and food. Due to differences in ecological environments and laboratory conditions, engineering is often necessary to uncover and utilize the chemical diversity. Although we can efficiently activate and mine these often complex 3D molecules, sufficient production of target molecules for further engineering and application remain a considerable bottleneck. An example of these bioactive scaffolds are armeniaspirols, which are potent polyketide antibiotics against gram-positive pathogens and multi-resistance gram-negative Helicobacter pylori. Here, we examine upregulation of armeniaspirols through engineering of biosynthetic pathways and primary metabolism; including perturbation of genes in biosynthetic gene clusters and regulation for triacylglycerols pool towards armeniaspirol upregulation. With either overexpression of extender unit pathway or late-stage N-methylation, or the deletio...
The present study investigated the molecular phylogeny, antimicrobial and cytotoxic activities of... more The present study investigated the molecular phylogeny, antimicrobial and cytotoxic activities of fungal endophytes obtained from the A*STAR Natural Organism Library (NOL) and previously isolated from Sungei Buloh Wetland Reserve, Singapore. Phylogenetic analysis based on ITS2 gene suggests that these isolates belong to 46 morphotypes and are affiliated to 23 different taxa in 17 genera of the Ascomycota phylum. Colletotrichum was the most dominant fungal genus accounting for 37% of all the isolates, followed by Diaporthe (13%), Phyllosticta (10.9%) and Diplodia (8.7%). Chemical elicitation using 5-azacytidine, a DNA methyltransferase inhibitor and suberoylanilide hydroxamic acid, a histone deacetylase inhibitor resulted in an increase in the number of active strains. Bioassay-guided isolation and structural elucidation yielded pestahivin and two new analogues from Bartalinia sp. F9447. Pestahivin and its related analogues did not exhibit antibacterial activity against Staphylococcu...
In recent years, CRISPR-Cas toolboxes for Streptomyces editing have rapidly accelerated natural p... more In recent years, CRISPR-Cas toolboxes for Streptomyces editing have rapidly accelerated natural product discovery and engineering. However, Cas efficiencies are also oftentimes strain dependent, subsequently a variety of Cas proteins would allow for flexibility and enable genetic manipulation within a wider range of Streptomyces strains. In this work, we have further expanded the Cas toolbox by presenting the first example of Cas12j mediated editing in Streptomyces sp. A34053. In our study, we have also observed significantly improved editing efficiencies with Acidaminococcus sp. Cas12j compared to Cas12a, Francisella tularensis subsp. novicida U112’s type V-A Cas (FnCpf1).
Natural products are a family of diverse compounds with multiple impactful applications, especial... more Natural products are a family of diverse compounds with multiple impactful applications, especially in therapeutics. Recent advances in genomics and bioinformatics have also hinted at vast untapped chemical potential within Nature. However, despite the many strategies available for activation and upregulation of natural product biosyntheses in native and heterologous microbial strains, there is yet to be a generalizable and efficient approach for interrogating diverse native strain collections. Here, we describe and demonstrate a flexible and robust one-step integrase-mediated genetic- and cultivation-based approach to perturb and activate antibiotics production in a set of 54 actinobacterial strains. Our multi-pronged strategy significantly increases accessible metabolite space by two-fold, resulting in the discovery of the first example of Gram-negative bioactivity in new tetramic acid analogs. We envision these results to serve as the first step toward a more streamlined, acceler...
Adaptation to a wide variety of habitats allows fungi to develop unique abilities to produce dive... more Adaptation to a wide variety of habitats allows fungi to develop unique abilities to produce diverse secondary metabolites with diverse bioactivities. In this study, 30 Ascomycetes fungi isolated from St. John’s Island, Singapore were investigated for their general biosynthetic potential and their ability to produce antimicrobial secondary metabolites (SMs). All the 30 fungal isolates belong to the Phylum Ascomycota and are distributed into 6 orders and 18 genera with Order Hypocreales having the highest number of representative (37%). Screening for polyketide synthase (PKS) and nonribosomal peptide synthetase (NRPS) genes using degenerate PCR led to the identification of 23 polyketide synthases (PKSs) and 5 nonribosomal peptide synthetases (NRPSs) grouped into nine distinct clades based on their reduction capabilities. Some of the identified PKSs genes share high similarities between species and known reference genes, suggesting the possibility of conserved biosynthesis of closely ...
Isoforms of heat shock protein (Hsp) 27 were used as intracellular markers to study tumor necrosi... more Isoforms of heat shock protein (Hsp) 27 were used as intracellular markers to study tumor necrosis factor/interleukin-1 (TNF/IL-1) regulation of protein phosphatases in primary human fibroblasts. These isoforms were rapidly phosphorylated to varying degrees when fibroblasts were treated with either TNF, IL-1, okadaic acid, calyculin A, ARS, epidermal growth factor, fibroblast growth factor, H2O2, buthionine sulfoximine, N-ethylmaleimide, diethylmaleimide, or iodoacetate. However, inhibitors of protein kinases A and C, tyrosyl protein kinases, and general protein kinases had no effect on the enhanced phosphorylation of these isoforms in TNF, IL-1, okadaic acid, or calyculin A-stimulated cells, suggesting that the activation of protein kinases by itself is insufficient to produce these changes. Isoforms of 32P-labeled Hsp27 were dephosphorylated during cold-chases with excess phosphate in the absence but not in the presence of TNF/IL-1 or inhibitors of protein phosphatases suggesting that inactivation of protein phosphatase(s) plays a role in TNF/IL-1 signal transduction. Assays of phosphatase activity of cytosolic fractions from TNF or okadaic acid treated human fibroblasts showed an inactivation of protein phosphatase activity against the 32P-labeled Hsp27 protein substrates. In vitro assays of partially purified phosphatase activity from primary human fibroblasts with Hsp27 substrate also showed the protein phosphatase activity to be inhibited by ARS. Like okadaic acid, ARS mimics TNF in inducing specific patterns of cellular protein phosphorylation. Taken together these findings are consistent with the hypothesis that a SH-dependent protein phosphatase is inactivated during the early events of TNF/IL-1 signal transduction, hence inhibitors of protein phosphatases and SH modifying compounds can mimic the early effects of TNF/IL-1 on cells.
High resolution two-dimensional gel electrophoresis was used to analyze the signal transduction p... more High resolution two-dimensional gel electrophoresis was used to analyze the signal transduction pathways of tumor necrosis factor (TNF-alpha) and interleukin 1 (IL-1 alpha and -beta) in human fibroblasts. Approximately 450 discrete radioactive spots were electrophoretically resolved from cytosolic extracts of cells prelabeled with 32P. At least 63 of these polypeptides exhibited significant and concordant phosphorylation or dephosphorylation in response to TNF or IL-1, despite the fact that different receptors are involved. Most of these changes concerned serine/threonine residues although enhanced tyrosine phosphorylation of several polypeptides was also observed. Phosphorylation patterns induced by a number of other agonists were compared with the patterns induced by IL-1 and TNF. These included activators of protein kinases C and A, bradykinin (a stimulator of inositol phospholipid hydrolysis), epidermal growth factor, heatshock, and mellitin (an activator of phospholipase A2). Although each of these agonists induced changes resulting in a distinct pattern of protein phosphorylation, none of these patterns had significant homology with that induced by IL-1 and TNF. Other assays were performed to verify the involvement of specific kinases. Collectively, these data indicate that IL-1 and TNF activate multiple protein kinases viz. a kinase(s) which activates microtubule-associated protein 2 (MAP-2) kinase, a kinase that phosphorylates the cap-binding protein, and a possibly novel serine/threonine protein kinase.
Natural products have long been used as a source of antimicrobial agents against various microorg... more Natural products have long been used as a source of antimicrobial agents against various microorganisms. Actinobacteria are a group of bacteria best known to produce a wide variety of bioactive secondary metabolites, including many antimicrobial agents. In this study, four actinobacterial strains found in Singapore terrestrial soil were investigated as potential sources of new antimicrobial compounds. Large-scale cultivation, chemical, and biological investigation led to the isolation of a previously undescribed tetronomycin A (1) that demonstrated inhibitory activities against both Gram-positive bacteria Staphylococcus aureus (SA) and methicillin-resistant Staphylococcus aureus (MRSA) (i.e., MIC90 of 2–4 μM and MBC90 of 9–12 μM), and several known antimicrobial compounds, namely nonactin, monactin, dinactin, 4E-deacetylchromomycin A3, chromomycin A2, soyasaponin II, lysolipin I, tetronomycin, and naphthomevalin. Tetronomycin showed a two- to six-fold increase in antibacterial activ...
Thiopeptides are macrocyclic natural products with potent bioactivity. Nine new natural thiopepti... more Thiopeptides are macrocyclic natural products with potent bioactivity. Nine new natural thiopeptides (1–9) were obtained from a Nonomuraea jiangxiensis isolated from a terrestrial soil sample collected in Singapore. Even though some of these compounds were previously synthesized or isolated from engineered strains, herein we report the unprecedented isolation of these thiopeptides from a native Nonomuraea jiangxiensis. A comparison with the literature and a detailed analysis of the NMR and HRMS of compounds 1–9 was conducted to assign their chemical structures. The structures of all new compounds were highly related to the thiopeptide antibiotics GE2270, with variations in the substituents on the thiazole and amino acid moieties. Thiopeptides 1–9 exhibited a potent antimicrobial activity against the Gram-positive bacteria, Staphylococcus aureus with MIC90 values ranging from 2 µM to 11 µM. In addition, all compounds were investigated for their cytotoxicity against the human cancer c...
Nature has provided unique molecular scaffolds for applications including therapeutics, agricultu... more Nature has provided unique molecular scaffolds for applications including therapeutics, agriculture, and food. Due to differences in ecological environments and laboratory conditions, engineering is often necessary to uncover and utilize the chemical diversity. Although we can efficiently activate and mine these often complex 3D molecules, sufficient production of target molecules for further engineering and application remain a considerable bottleneck. An example of these bioactive scaffolds are armeniaspirols, which are potent polyketide antibiotics against gram-positive pathogens and multi-resistance gram-negative Helicobacter pylori. Here, we examine upregulation of armeniaspirols through engineering of biosynthetic pathways and primary metabolism; including perturbation of genes in biosynthetic gene clusters and regulation for triacylglycerols pool towards armeniaspirol upregulation. With either overexpression of extender unit pathway or late-stage N-methylation, or the deletio...
The present study investigated the molecular phylogeny, antimicrobial and cytotoxic activities of... more The present study investigated the molecular phylogeny, antimicrobial and cytotoxic activities of fungal endophytes obtained from the A*STAR Natural Organism Library (NOL) and previously isolated from Sungei Buloh Wetland Reserve, Singapore. Phylogenetic analysis based on ITS2 gene suggests that these isolates belong to 46 morphotypes and are affiliated to 23 different taxa in 17 genera of the Ascomycota phylum. Colletotrichum was the most dominant fungal genus accounting for 37% of all the isolates, followed by Diaporthe (13%), Phyllosticta (10.9%) and Diplodia (8.7%). Chemical elicitation using 5-azacytidine, a DNA methyltransferase inhibitor and suberoylanilide hydroxamic acid, a histone deacetylase inhibitor resulted in an increase in the number of active strains. Bioassay-guided isolation and structural elucidation yielded pestahivin and two new analogues from Bartalinia sp. F9447. Pestahivin and its related analogues did not exhibit antibacterial activity against Staphylococcu...
In recent years, CRISPR-Cas toolboxes for Streptomyces editing have rapidly accelerated natural p... more In recent years, CRISPR-Cas toolboxes for Streptomyces editing have rapidly accelerated natural product discovery and engineering. However, Cas efficiencies are also oftentimes strain dependent, subsequently a variety of Cas proteins would allow for flexibility and enable genetic manipulation within a wider range of Streptomyces strains. In this work, we have further expanded the Cas toolbox by presenting the first example of Cas12j mediated editing in Streptomyces sp. A34053. In our study, we have also observed significantly improved editing efficiencies with Acidaminococcus sp. Cas12j compared to Cas12a, Francisella tularensis subsp. novicida U112’s type V-A Cas (FnCpf1).
Natural products are a family of diverse compounds with multiple impactful applications, especial... more Natural products are a family of diverse compounds with multiple impactful applications, especially in therapeutics. Recent advances in genomics and bioinformatics have also hinted at vast untapped chemical potential within Nature. However, despite the many strategies available for activation and upregulation of natural product biosyntheses in native and heterologous microbial strains, there is yet to be a generalizable and efficient approach for interrogating diverse native strain collections. Here, we describe and demonstrate a flexible and robust one-step integrase-mediated genetic- and cultivation-based approach to perturb and activate antibiotics production in a set of 54 actinobacterial strains. Our multi-pronged strategy significantly increases accessible metabolite space by two-fold, resulting in the discovery of the first example of Gram-negative bioactivity in new tetramic acid analogs. We envision these results to serve as the first step toward a more streamlined, acceler...
Adaptation to a wide variety of habitats allows fungi to develop unique abilities to produce dive... more Adaptation to a wide variety of habitats allows fungi to develop unique abilities to produce diverse secondary metabolites with diverse bioactivities. In this study, 30 Ascomycetes fungi isolated from St. John’s Island, Singapore were investigated for their general biosynthetic potential and their ability to produce antimicrobial secondary metabolites (SMs). All the 30 fungal isolates belong to the Phylum Ascomycota and are distributed into 6 orders and 18 genera with Order Hypocreales having the highest number of representative (37%). Screening for polyketide synthase (PKS) and nonribosomal peptide synthetase (NRPS) genes using degenerate PCR led to the identification of 23 polyketide synthases (PKSs) and 5 nonribosomal peptide synthetases (NRPSs) grouped into nine distinct clades based on their reduction capabilities. Some of the identified PKSs genes share high similarities between species and known reference genes, suggesting the possibility of conserved biosynthesis of closely ...
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