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Huarong Tan - One of the best experts on this subject based on the ideXlab platform.
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Specialised metabolites regulating Antibiotic Biosynthesis in Streptomyces spp.
FEMS microbiology reviews, 2016Co-Authors: Guoqing Niu, Keith F. Chater, Yuqing Tian, Jihui Zhang, Huarong TanAbstract:Streptomyces bacteria are the major source of Antibiotics and other secondary metabolites. Various environmental and physiological conditions affect the onset and level of production of each Antibiotic by influencing concentrations of the ligands for conserved global regulatory proteins. In addition, as reviewed here, well-known autoregulators such as γ-butyrolactones, themselves products of secondary metabolism, accumulate late in growth to concentrations allowing their effective interaction with cognate binding proteins, in a necessary prelude to Antibiotic Biosynthesis. Most autoregulator binding proteins target the conserved global regulatory gene adpA, and/or regulatory genes for 'cluster-situated regulators' (CSRs) linked to Antibiotic biosynthetic gene clusters. It now appears that some CSRs bind intermediates and end products of Antibiotic Biosynthesis, with regulatory effects interwoven with those of autoregulators. These ligands can exert cross-pathway effects within producers of more than one Antibiotic, and when excreted into the extracellular environment may have population-wide effects on production, and mediate interactions with neighbouring microorganisms in natural communities, influencing speciation. Greater understanding of these autoregulatory and cross-regulatory activities may aid the discovery of new signalling molecules and their use in activating cryptic Antibiotic biosynthetic pathways.
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molecular regulation of Antibiotic Biosynthesis in streptomyces
Microbiology and Molecular Biology Reviews, 2013Co-Authors: Gang Liu, Guoqing Niu, Keith F. Chater, Govind Chandra, Huarong TanAbstract:SUMMARY Streptomycetes are the most abundant source of Antibiotics. Typically, each species produces several Antibiotics, with the profile being species specific. Streptomyces coelicolor, the model species, produces at least five different Antibiotics. We review the regulation of Antibiotic Biosynthesis in S. coelicolor and other, nonmodel streptomycetes in the light of recent studies. The Biosynthesis of each Antibiotic is specified by a large gene cluster, usually including regulatory genes (cluster-situated regulators [CSRs]). These are the main point of connection with a plethora of generally conserved regulatory systems that monitor the organism9s physiology, developmental state, population density, and environment to determine the onset and level of production of each Antibiotic. Some CSRs may also be sensitive to the levels of different kinds of ligands, including products of the pathway itself, products of other Antibiotic pathways in the same organism, and specialized regulatory small molecules such as gamma-butyrolactones. These interactions can result in self-reinforcing feed-forward circuitry and complex cross talk between pathways. The physiological signals and regulatory mechanisms may be of practical importance for the activation of the many cryptic secondary metabolic gene cluster pathways revealed by recent sequencing of numerous Streptomyces genomes.
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differential regulation of Antibiotic Biosynthesis by drar k a novel two component system in streptomyces coelicolor
Molecular Microbiology, 2012Co-Authors: Hong Zhu, Huarong Tan, Fujun Dang, Weiwen Zhang, Zhongjun Qin, Sheng Yang, Weihong JiangAbstract:A novel two-component system (TCS) designated as DraR-K (sco3063/sco3062) was identified to be involved in differential regulation of Antibiotic Biosynthesis in Streptomyces coelicolor. The S. coelicolor mutants with deletion of either or both of draR and draK exhibited significantly reduced actinorhodin (ACT) but increased undecylprodigiosin (RED) production on minimal medium (MM) supplemented separately with high concentration of different nitrogen sources. These mutants also overproduced a yellow-pigmented type I polyketide (yCPK) on MM with glutamate (Glu). It was confirmed that DraR-K activates ACT but represses yCPK production directly through the pathway-specific activator genes actII-ORF4 and kasO, respectively, while its role on RED Biosynthesis was independent of pathway-specific activator genes redD/redZ. DNase I footprinting assays revealed that the DNA binding sites for DraR were at -124 to -98 nt and -24 to -1 nt relative to the respective transcription start point of actII-ORF4 and kasO. Comparison of the binding sites allowed the identification of a consensus DraR-binding sequence, 5'-AMAAWYMAKCA-3' (M: A or C; W: A or T; Y: C or T; K: G or T). By genome screening and gel-retardation assay, 11 new targets of DraR were further identified in the genome of S. coelicolor. Functional analysis of these tentative targets revealed the involvement of DraR-K in primary metabolism. DraR-K homologues are widely spread in different streptomycetes. Interestingly, deletion of draR-Ksav (sav_3481/sav_3480, homologue of draR-K) in the industrial model strain S. avermitilis NRRL-8165 led to similar abnormal Antibiotic Biosynthesis, showing higher avermectin while slightly decreased oligomycin A production, suggesting that DraR-K-mediated regulation system might be conserved in streptomycetes. This study further reveals the complexity of TCS in regulation of Antibiotic Biosynthesis in Streptomyces.
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Differential regulation of Antibiotic Biosynthesis by DraR‐K, a novel two‐component system in Streptomyces coelicolor
Molecular microbiology, 2012Co-Authors: Hong Zhu, Huarong Tan, Fujun Dang, Weiwen Zhang, Zhongjun Qin, Sheng Yang, Weihong JiangAbstract:A novel two-component system (TCS) designated as DraR-K (sco3063/sco3062) was identified to be involved in differential regulation of Antibiotic Biosynthesis in Streptomyces coelicolor. The S. coelicolor mutants with deletion of either or both of draR and draK exhibited significantly reduced actinorhodin (ACT) but increased undecylprodigiosin (RED) production on minimal medium (MM) supplemented separately with high concentration of different nitrogen sources. These mutants also overproduced a yellow-pigmented type I polyketide (yCPK) on MM with glutamate (Glu). It was confirmed that DraR-K activates ACT but represses yCPK production directly through the pathway-specific activator genes actII-ORF4 and kasO, respectively, while its role on RED Biosynthesis was independent of pathway-specific activator genes redD/redZ. DNase I footprinting assays revealed that the DNA binding sites for DraR were at -124 to -98 nt and -24 to -1 nt relative to the respective transcription start point of actII-ORF4 and kasO. Comparison of the binding sites allowed the identification of a consensus DraR-binding sequence, 5'-AMAAWYMAKCA-3' (M: A or C; W: A or T; Y: C or T; K: G or T). By genome screening and gel-retardation assay, 11 new targets of DraR were further identified in the genome of S. coelicolor. Functional analysis of these tentative targets revealed the involvement of DraR-K in primary metabolism. DraR-K homologues are widely spread in different streptomycetes. Interestingly, deletion of draR-Ksav (sav_3481/sav_3480, homologue of draR-K) in the industrial model strain S. avermitilis NRRL-8165 led to similar abnormal Antibiotic Biosynthesis, showing higher avermectin while slightly decreased oligomycin A production, suggesting that DraR-K-mediated regulation system might be conserved in streptomycetes. This study further reveals the complexity of TCS in regulation of Antibiotic Biosynthesis in Streptomyces.
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Pseudo γ-Butyrolactone Receptors Respond to Antibiotic Signals to Coordinate Antibiotic Biosynthesis
The Journal of biological chemistry, 2010Co-Authors: Juan Wang, Linqi Wang, Xiuyun Tian, Haihua Yang, Keqiang Fan, Keqian Yang, Huarong TanAbstract:In actinomycetes, the onset of secondary metabolite Biosynthesis is often triggered by the quorum-sensing signal gamma-butyrolactones (GBLs) via specific binding to their cognate receptors. However, the presence of multiple putative GBL receptor homologues in the genome suggests the existence of an alternative regulatory mechanism. Here, in the model streptomycete Streptomyces coelicolor, ScbR2 (SCO6286, a homologue of GBL receptor) is shown not to bind the endogenous GBL molecule SCB1, hence designated "pseudo" GBL receptor. Intriguingly, it could bind the endogenous Antibiotics actinorhodin and undecylprodigiosin as ligands, leading to the derepression of KasO, an activator of a cryptic type I polyketide synthase gene cluster. Likewise, JadR2 is also a putative GBL receptor homologue in Streptomyces venezuelae, the producer of chloramphenicol and cryptic Antibiotic jadomycin. It is shown to coordinate their Biosynthesis via direct repression of JadR1, which activates jadomycin Biosynthesis while repressing chloramphenicol Biosynthesis directly. Like ScbR2, JadR2 could also bind these two disparate Antibiotics, and the interactions lead to the derepression of jadR1. The Antibiotic responding activities of these pseudo GBL receptors were further demonstrated in vivo using the lux reporter system. Overall, these results suggest that pseudo GBL receptors play a novel role to coordinate Antibiotic Biosynthesis by binding and responding to Antibiotics signals. Such an Antibiotic-mediated regulatory mechanism could be a general strategy to coordinate Antibiotic Biosynthesis in the producing bacteria.
Weihong Jiang - One of the best experts on this subject based on the ideXlab platform.
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The orphan histidine kinase PdtaS-p regulates both morphological differentiation and Antibiotic Biosynthesis together with the orphan response regulator PdtaR-p in Streptomyces.
Microbiological research, 2020Co-Authors: Yawei Zhao, Guosong Zheng, Hengnuo Tao, Jun Chen, Weihong JiangAbstract:In Streptomyces pristinaespiralis, the orphan histidine kinase (HK) PdtaS-p (encoded by SSDG_02492), which belongs to proteins of two-component systems (TCSs), plays an important role in both morphological differentiation and Antibiotic Biosynthesis. Owing to the isolated genetic organization of pdtaS-p, it is a challenge to identify its cognate response regulator (RR) and hampers the efforts to elucidate the regulation mechanism of PdtaS-p. In this study, based on bioinformatics analysis, we identify the cognate RR PdtaR-p (encoded by SSDG_04087) of PdtaS-p by phenotype similarity of gene deletion mutants as well as in vitro phosphor-transfer assay. We show that the mutants (ΔpdtaR-p and ΔpdtaS-p) exhibit almost the same phenotypical changes, showing a bald phenotype on MS agar and reduced pristinamycin Biosynthesis. Further phosphor-transfer assay indicates that the phosphoryl group of HK PdtaS-p can be specifically transferred to RR PdtaR-p. Compared with the majority of RRs that harbor DNA-binding domains, PdtaR-p contains a putative ANTAR RNA-binding domain involved in controlling gene expression at the post-transcription level. Finally, we demonstrate that their ortholog from the model strain Streptomyces coelicolor, PdtaS-c/PdtaR-c, also regulates both morphological differentiation and Antibiotics Biosynthesis, suggesting that PdtaS-p/PdtaR-p-mediated molecular regulation may be conserved in the genus Streptomyces. To our knowledge, this is the first report describing the functional identification of ANTAR RNA-binding regulators in Streptomyces.
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Overexpression of the diguanylate cyclase CdgD blocks developmental transitions and Antibiotic Biosynthesis in Streptomyces coelicolor.
Science China. Life sciences, 2019Co-Authors: Xiaocao Liu, Guosong Zheng, Gang Wang, Weihong JiangAbstract:Cyclic dimeric GMP (c-di-GMP) has emerged as the nucleotide second messenger regulating both development and Antibiotic production in high-GC, Gram-positive streptomycetes. Here, a diguanylate cyclase (DGC), CdgD, encoded by SCO5345 from the model strain Streptomyces coelicolor, was functionally identified and characterized to be involved in c-di-GMP synthesis through genetic and biochemical analysis. cdgD overexpression resulted in significantly reduced production of actinorhodin and undecylprodigiosin, as well as completely blocked sporulation or aerial mycelium formation on two different solid media. In the cdgD-overexpression strain, intracellular c-di-GMP levels were 13-27-fold higher than those in the wild-type strain. In vitro enzymatic assay demonstrated that CdgD acts as a DGC, which could efficiently catalyze the synthesis of c-di-GMP from two GTP molecules. Heterologous overproduction of cdgD in two industrial Streptomyces strains could similarly impair developmental transitions as well as Antibiotic Biosynthesis. Collectively, our results combined with previously reported data clearly demonstrated that c-di-GMP-mediated signalling pathway plays a central and universal role in the life cycle as well as secondary metabolism in streptomycetes.
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A Novel Two-Component System, GluR-GluK, Involved in Glutamate Sensing and Uptake in Streptomyces coelicolor.
Journal of bacteriology, 2017Co-Authors: Weihong JiangAbstract:Two-component systems (TCSs), the predominant signal transduction pathways employed by bacteria, play important roles in physiological metabolism in Streptomyces Here, a novel TCS, GluR-GluK (encoded by SCO5778-SCO5779), which is located divergently from the gluABCD operon encoding a glutamate uptake system, was identified as being involved in glutamate sensing and uptake as well as Antibiotic Biosynthesis in Streptomyces coelicolor Under the condition of minimal medium (MM) supplemented with different concentrations of glutamate, deletion of the gluR-gluK operon (gluR-K) resulted in enhanced actinorhodin (ACT) but reduced undecylprodigiosin (RED) and yellow type I polyketide (yCPK) production, suggesting that GluR-GluK plays a differential role in Antibiotic Biosynthesis. Furthermore, we found that the response regulator GluR directly promotes the expression of gluABCD under the culture condition of MM with a high concentration of glutamate (75 mM). Using the biolayer interferometry assay, we demonstrated that glutamate acts as the direct signal of the histidine kinase GluK. It was therefore suggested that upon sensing high concentrations of glutamate, GluR-GluK would be activated and thereby facilitate glutamate uptake by increasing gluABCD expression. Finally, we demonstrated that the role of GluR-GluK in Antibiotic Biosynthesis is independent of its function in glutamate uptake. Considering the wide distribution of the glutamate-sensing (GluR-GluK) and uptake (GluABCD) module in actinobacteria, it could be concluded that the GluR-GluK signal transduction pathway involved in secondary metabolism and glutamate uptake should be highly conserved in this bacterial phylum.IMPORTANCE In this study, a novel two-component system (TCS), GluR-GluK, was identified to be involved in glutamate sensing and uptake as well as Antibiotic Biosynthesis in Streptomyces coelicolor A possible GluR-GluK working model was proposed. Upon sensing high glutamate concentrations (such as 75 mM), activated GluR-GluK could regulate both glutamate uptake and Antibiotic Biosynthesis. However, under a culture condition of MM supplemented with low concentrations of glutamate (such as 10 mM), although GluR-GluK is activated, its activity is sufficient only for the regulation of Antibiotic Biosynthesis. To the best of our knowledge, this is the first report describing a TCS signal transduction pathway for glutamate sensing and uptake in actinobacteria.
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Direct Involvement of the Master Nitrogen Metabolism Regulator GlnR in Antibiotic Biosynthesis in Streptomyces.
The Journal of biological chemistry, 2016Co-Authors: Hong Zhu, Guosong Zheng, Pan-pan Liu, Jin Wang, Guoping Zhao, Guo-qiang Zhu, Weihong JiangAbstract:Abstract GlnR, an OmpR-like orphan two-component system response regulator, is a master regulator of nitrogen metabolism in the genus Streptomyces. In this work, evidence that GlnR is also directly involved in the regulation of Antibiotic Biosynthesis is provided. In the model strain Streptomyces coelicolor M145, an in-frame deletion of glnR resulted in markedly increased actinorhodin (ACT) production but reduced undecylprodigiosin (RED) Biosynthesis when exposed to R2YE culture medium. Transcriptional analysis coupled with DNA-binding studies revealed that GlnR represses ACT but activates RED production directly via the pathway-specific activator genes actII-ORF4 and redZ, respectively. The precise GlnR-binding sites upstream of these two target genes were defined. In addition, the direct involvement of GlnR in Antibiotic Biosynthesis was further identified in Streptomyces avermitilis, which produces the important anthelmintic agent avermectin. We found that S. avermitilis GlnR (GlnRsav) could stimulate avermectin but repress oligomycin production directly through the respective pathway-specific activator genes, aveR and olmRI/RII. To the best of our knowledge, this report describes the first experimental evidence demonstrating that GlnR regulates Antibiotic Biosynthesis directly through pathway-specific regulators in Streptomyces. Our results suggest that GlnR-mediated regulation of Antibiotic Biosynthesis is likely to be universal in streptomycetes. These findings also indicate that GlnR is not only a master nitrogen regulator but also an important controller of secondary metabolism, which may help to balance nitrogen metabolism and Antibiotic Biosynthesis in streptomycetes.
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differential regulation of Antibiotic Biosynthesis by drar k a novel two component system in streptomyces coelicolor
Molecular Microbiology, 2012Co-Authors: Hong Zhu, Huarong Tan, Fujun Dang, Weiwen Zhang, Zhongjun Qin, Sheng Yang, Weihong JiangAbstract:A novel two-component system (TCS) designated as DraR-K (sco3063/sco3062) was identified to be involved in differential regulation of Antibiotic Biosynthesis in Streptomyces coelicolor. The S. coelicolor mutants with deletion of either or both of draR and draK exhibited significantly reduced actinorhodin (ACT) but increased undecylprodigiosin (RED) production on minimal medium (MM) supplemented separately with high concentration of different nitrogen sources. These mutants also overproduced a yellow-pigmented type I polyketide (yCPK) on MM with glutamate (Glu). It was confirmed that DraR-K activates ACT but represses yCPK production directly through the pathway-specific activator genes actII-ORF4 and kasO, respectively, while its role on RED Biosynthesis was independent of pathway-specific activator genes redD/redZ. DNase I footprinting assays revealed that the DNA binding sites for DraR were at -124 to -98 nt and -24 to -1 nt relative to the respective transcription start point of actII-ORF4 and kasO. Comparison of the binding sites allowed the identification of a consensus DraR-binding sequence, 5'-AMAAWYMAKCA-3' (M: A or C; W: A or T; Y: C or T; K: G or T). By genome screening and gel-retardation assay, 11 new targets of DraR were further identified in the genome of S. coelicolor. Functional analysis of these tentative targets revealed the involvement of DraR-K in primary metabolism. DraR-K homologues are widely spread in different streptomycetes. Interestingly, deletion of draR-Ksav (sav_3481/sav_3480, homologue of draR-K) in the industrial model strain S. avermitilis NRRL-8165 led to similar abnormal Antibiotic Biosynthesis, showing higher avermectin while slightly decreased oligomycin A production, suggesting that DraR-K-mediated regulation system might be conserved in streptomycetes. This study further reveals the complexity of TCS in regulation of Antibiotic Biosynthesis in Streptomyces.
Charles J. Thompson - One of the best experts on this subject based on the ideXlab platform.
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Pleiotropic Functions of a Streptomyces pristinaespiralis Autoregulator Receptor in Development, Antibiotic Biosynthesis, and Expression of a Superoxide Dismutase
The Journal of biological chemistry, 2001Co-Authors: Marc Folcher, Hélène Gaillard, Lieu T. Nguyen, Kien T. Nguyen, Patricia Lacroix, Nathalie Bamas-jacques, Monique Rinkel, Charles J. ThompsonAbstract:Abstract In Streptomyces, a family of related butyrolactones and their corresponding receptor proteins serve as quorum-sensing systems that can activate morphological development and Antibiotic Biosynthesis. Streptomyces pristinaespiraliscontains a gene cluster encoding enzymes and regulatory proteins for the Biosynthesis of pristinamycin, a clinically important streptogramin Antibiotic complex. One of these proteins, PapR1, belongs to a well known family of Streptomyces Antibiotic regulatory proteins. Gel shift assays using crude cytoplasmic extracts detected SpbR, a developmentally regulated protein that bound to thepapR1 promoter. SpbR was purified, and its gene was cloned using reverse genetics. spbR encoded a 25-kDa protein similar to Streptomyces autoregulatory proteins of the butyrolactone receptor family, including scbR fromStreptomyces coelicolor. In Escherichia coli, purified SpbR and ScbR produced bound sequences immediately upstream ofpapR1, spbR, and scbR. SpbR DNA-binding activity was inhibited by an extracellular metabolite with chromatographic properties similar to those of the well known γ-butyrolactone signaling compounds. DNase I protection assays mapped the SpbR-binding site in the papR1 promoter to a sequence homologous to other known butyrolactone autoregulatory elements. A nucleotide data base search showed that these binding motifs were primarily located upstream of genes encoding StreptomycesAntibiotic regulatory proteins and butyrolactone receptors in variousStreptomyces species. Disruption of the spbRgene in S. pristinaespiralis resulted in severe defects in growth, morphological differentiation, pristinamycin Biosynthesis, and expression of a secreted superoxide dismutase.
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Role of acid metabolism in Streptomyces coelicolor morphological differentiation and Antibiotic Biosynthesis.
Journal of bacteriology, 2001Co-Authors: Patrick H. Viollier, Wolfgang Minas, Glenn E. Dale, Marc Folcher, Charles J. ThompsonAbstract:Studies of citrate synthase (CitA) were carried out to investigate its role in morphological development and Biosynthesis of Antibiotics in Streptomyces coelicolor. Purification of CitA, the major vegetative enzyme activity, allowed characterization of its kinetic properties. The apparent Km values of CitA for acetyl coenzyme A (acetyl-CoA) (32 μM) and oxaloacetate (17 μM) were similar to those of citrate synthases from other gram-positive bacteria and eukaryotes. CitA was not strongly inhibited by various allosteric feedback inhibitors (NAD+, NADH, ATP, ADP, isocitrate, or α-ketoglutarate). The corresponding gene (citA) was cloned and sequenced, allowing construction of a citA mutant (BZ2). BZ2 was a glutamate auxotroph, indicating that citA encoded the major citrate synthase allowing flow of acetyl-CoA into the tricarboxylic acid (TCA) cycle. Interruption of aerobic TCA cycle-based metabolism resulted in acidification of the medium and defects in morphological differentiation and Antibiotic Biosynthesis. These developmental defects of the citA mutant were in part due to a glucose-dependent medium acidification that was also exhibited by some other bald mutants. Unlike other acidogenic bald strains, citA and bldJ mutants were able to produce aerial mycelia and pigments when the medium was buffered sufficiently to maintain neutrality. Extracellular complementation studies suggested that citA defines a new stage of the Streptomyces developmental cascade.
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Pleiotropic effects of cAMP on germination, Antibiotic Biosynthesis and morphological development in Streptomyces coelicolor
Molecular microbiology, 1998Co-Authors: Urs Süsstrunk, Josette Pidoux, Stefan Taubert, Agnes Ullmann, Charles J. ThompsonAbstract:In wild-type Streptomyces coelicolor MT1110 cultures, cyclic adenosine 3',5' monophosphate (cAMP) was synthesized throughout the developmental programme with peaks of accumulation both during germination and later when aerial mycelium and actinorhodin were being produced. Construction and characterization of an adenylate cyclase disruption mutant (BZ1) demonstrated that cAMP facilitated these developmental processes. Although pulse-labelling experiments showed that a similar germination process was initiated in BZ1 and MT1110, germ-tube emergence was severely delayed in BZ1 and never occurred in more than 85% of the spores. Studies of growth and development on solid glucose minimal medium (SMMS, buffered or unbuffered) showed that MT1110 and BZ1 produced acid during the first rapid growth phase, which generated substrate mycelium. Thereafter, on unbuffered SMMS, only MT1110 resumed growth and produced aerial mycelium by switching to an alternative metabolism that neutralized its medium, probably by reincorporating and metabolizing extracellular acids. BZ1 was not able to neutralize its medium or produce aerial mycelium on unbuffered SMMS; these defects were suppressed by high concentrations (>1 mM) of cAMP during early growth or on buffered medium. Other developmental mutants (bldA, bldB, bldC, bldD, bldG) also irreversibly acidified this medium. However, these bald mutants were not suppressed by exogenous cAMP or neutralizing buffer. BZ1 also differentiated when it was cultured in close proximity to MT1110, a property observed in cross-feeding experiments between bald mutants and commonly thought to reflect diffusion of a discrete positively acting signalling molecule. In this case, MT1110 generated a more neutral pH environment that allowed BZ1 to reinitiate growth and form aerial mycelium. The fact that actinorhodin synthesis could be induced by concentrations of cAMP (< 20 microM) found in the medium of MT1110 cultures, suggested that it may serve as a diffusible signalling molecule to co-ordinate Antibiotic Biosynthesis.
Max J Cryle - One of the best experts on this subject based on the ideXlab platform.
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Redesign of Substrate Selection in Glycopeptide Antibiotic Biosynthesis Enables Effective Formation of Alternate Peptide Backbones.
ACS chemical biology, 2020Co-Authors: Milda Kaniusaite, Tiia Kittilä, Robert J. A. Goode, Ralf B. Schittenhelm, Max J CryleAbstract:Nonribosomal peptide synthesis is capable of utilizing a wide range of amino acid residues due to the selectivity of adenylation (A)-domains. Changing the selectivity of A-domains could lead to new bioactive nonribosomal peptides, although remodeling efforts of A-domains are often unsuccessful. Here, we explored and successfully reengineered the specificity of the module 3 A-domain from glycopeptide Antibiotic Biosynthesis to change the incorporation of 3,5-dihydroxyphenylglycine into 4-hydroxyphenylglycine. These engineered A-domains remain selective in a functioning peptide assembly line even under substrate competition conditions and indicate a possible application of these for the future redesign of GPA Biosynthesis.
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investigating cytochrome p450 specificity during glycopeptide Antibiotic Biosynthesis through a homologue hybridization approach
Journal of Inorganic Biochemistry, 2018Co-Authors: Clara Brieke, Anja Greule, Michał Tarnawski, Max J CryleAbstract:Accepted manuscript: Investigating Cytochrome P450 specificity during glycopeptide Antibiotic Biosynthesis through a homologue hybridization approach Clara Brieke, Miroslaw Tarnawski, Anja Greule and Max J. CryleJournal of Inorganic BiochemistryCytochrome P450 enzymes perform an impressive range of oxidation reactions against diverse substrate scaffolds whilst generally maintaining a conserved tertiary structure and active site chemistry. Within secondary metabolism, P450 enzymes play widespread and important roles in performing crucial modifications of precursor molecules, with one example of the importance of such reactions being found in the Biosynthesis of the glycopeptide Antibiotics (GPAs). In GPA Biosynthesis P450s, known as Oxy enzymes, are key players in the cyclization of the linear GPA peptide precursor, which is a process that is both essential for their Antibiotic activity and is the source of the synthetic challenge of these important Antibiotics. In this work, we developed chimeric P450 enzymes from GPA Biosynthesis based on two homologues from different GPA Biosynthesis pathways – vancomycin and teicoplanin – as an approach to explore the divergent catalytic behavior of the two parental homologues. We could generate, crystalize and explore the activity of new hybrid P450 enzymes from GPA Biosynthesis and show that the unusual in vitro behavior of the vancomycin OxyB homologue does not stem from the major regions of the P450 active site, and that additional regions in and around the P450 active site must contribute to the unusual properties of this P450 enzyme. Our results further show that it is possible to successfully transplant entire regions of secondary structure between such P450s and retain P450 expression and activity, which opens the door to use such targeted approaches to generate and explore novel biosynthetic P450 enzymes.
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understanding the crucial interactions between cytochrome p450s and non ribosomal peptide synthetases during glycopeptide Antibiotic Biosynthesis
Current Opinion in Structural Biology, 2016Co-Authors: Madeleine Peschke, Max J Cryle, Melanie Gonsior, Roderich D. SüssmuthAbstract:The importance of Cytochrome P450-catalyzed modifications of natural products produced by non-ribosomal peptide synthetase machineries is most apparent during glycopeptide Antibiotic Biosynthesis: specifically, the formation of essential amino acid side chains crosslinks in the peptide backbone of these clinically relevant Antibiotics. These cyclization reactions take place whilst the peptide substrate remains bound to the non-ribosomal peptide synthetase in a process mediated by a conserved domain of previously unknown function — the X-domain. This review addresses recent advances in understanding P450 recruitment to non-ribosomal peptide synthetase-bound substrates and highlights the importance of both carrier proteins and the X-domain in different P450-catalyzed reactions.
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f o g ring formation in glycopeptide Antibiotic Biosynthesis is catalysed by oxye
Scientific Reports, 2016Co-Authors: Madeleine Peschke, Clara Brieke, Max J CryleAbstract:The glycopeptide Antibiotics are peptide-based natural products with impressive Antibiotic function that derives from their unique three-dimensional structure. Biosynthesis of the glycopeptide Antibiotics centres of the combination of peptide synthesis, mediated by a non-ribosomal peptide synthetase, and the crosslinking of aromatic side chains of the peptide, mediated by the action of a cascade of Cytochrome P450s. Here, we report the first example of in vitro activity of OxyE, which catalyses the F-O-G ring formation reaction in teicoplanin Biosynthesis. OxyE was found to only act after an initial C-O-D crosslink is installed by OxyB and to require an interaction with the unique NRPS domain from glycopeptide Antibiotic – the X-domain – in order to display catalytic activity. We could demonstrate that OxyE displays limited stereoselectivity for the peptide, which mirrors the results from OxyB-catalysed turnover and is in sharp contrast to OxyA. Furthermore, we show that activity of a three-enzyme cascade (OxyB/OxyA/OxyE) in generating tricyclic glycopeptide Antibiotic peptides depends upon the order of addition of the OxyA and OxyE enzymes to the reaction. This work demonstrates that complex enzymatic cascades from glycopeptide Antibiotic Biosynthesis can be reconstituted in vitro and provides new insights into the Biosynthesis of these important Antibiotics.
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regulation of the p450 oxygenation cascade involved in glycopeptide Antibiotic Biosynthesis
Journal of the American Chemical Society, 2016Co-Authors: Madeleine Peschke, Clara Brieke, Kristina Haslinger, Jochen Reinstein, Max J CryleAbstract:Glycopeptide Antibiotics (GPAs) are nonribosomal peptides rich in modifications introduced by external enzymes. These enzymes act on the free peptide aglycone or intermediates bound to the nonribosomal peptide synthetase (NRPS) assembly line. In this process the terminal module of the NRPS plays a crucial role as it contains a unique recruitment platform (X-domain) interacting with three to four modifying Cytochrome P450 (P450) enzymes that are responsible for cyclizing bound peptides. However, whether these enzymes share the same binding site on the X-domain and how the order of the cyclization steps is orchestrated has remained elusive. In this study we investigate the first two reactions in teicoplanin aglycone maturation catalyzed by the enzymes OxyBtei and OxyAtei. We demonstrate that both enzymes interact with the X-domain via the identical interaction site with similar affinities, irrespective of the peptide modification stage, while their catalytic activity is restricted to the correctly cross-lin...
George P. C. Salmond - One of the best experts on this subject based on the ideXlab platform.
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virulence and prodigiosin Antibiotic Biosynthesis in serratia are regulated pleiotropically by the ggdef eal domain protein pigx
Journal of Bacteriology, 2007Co-Authors: Peter C. Fineran, Neil R. Williamson, Kathryn S. Lilley, George P. C. SalmondAbstract:Gram-negative bacteria of the genus Serratia are opportunistic human, plant, and insect pathogens. Serratia sp. strain ATCC 39006 secretes pectinases and cellulases and produces the secondary metabolites carbapenem and prodigiosin. Mutation of a gene (pigX) resulted in an extremely pleiotropic phenotype: prodigiosin Antibiotic Biosynthesis, plant virulence, and pectinase production were all elevated. PigX controlled secondary metabolism by repressing the transcription of the target prodigiosin biosynthetic operon (pigA-pigO). The transcriptional start site of pigX was determined, and pigX expression occurred in parallel with Pig production. Detailed quantitative intracellular proteome analyses enabled the identification of numerous downstream targets of PigX, including OpgG, mutation of which reduced the production of the plant cell wall-degrading enzymes and virulence. The highly pleiotropic PigX regulator contains GGDEF and EAL domains with noncanonical motifs and is predicted to be membrane associated. Genetic evidence suggests that PigX might function as a cyclic dimeric GMP phosphodiesterase. This is the first characterization of a GGDEF and EAL domain protein in Serratia and the first example of the regulation of Antibiotic production by a GGDEF/EAL domain protein.
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Virulence and Prodigiosin Antibiotic Biosynthesis in Serratia Are Regulated Pleiotropically by the GGDEF/EAL Domain Protein, PigX
Journal of Bacteriology, 2007Co-Authors: Peter C. Fineran, Neil R. Williamson, Kathryn S. Lilley, George P. C. SalmondAbstract:Gram-negative bacteria of the genus Serratia are opportunistic human, plant, and insect pathogens. Serratia sp. strain ATCC 39006 secretes pectinases and cellulases and produces the secondary metabolites carbapenem and prodigiosin. Mutation of a gene (pigX) resulted in an extremely pleiotropic phenotype: prodigiosin Antibiotic Biosynthesis, plant virulence, and pectinase production were all elevated. PigX controlled secondary metabolism by repressing the transcription of the target prodigiosin biosynthetic operon (pigA-pigO). The transcriptional start site of pigX was determined, and pigX expression occurred in parallel with Pig production. Detailed quantitative intracellular proteome analyses enabled the identification of numerous downstream targets of PigX, including OpgG, mutation of which reduced the production of the plant cell wall-degrading enzymes and virulence. The highly pleiotropic PigX regulator contains GGDEF and EAL domains with noncanonical motifs and is predicted to be membrane associated. Genetic evidence suggests that PigX might function as a cyclic dimeric GMP phosphodiesterase. This is the first characterization of a GGDEF and EAL domain protein in Serratia and the first example of the regulation of Antibiotic production by a GGDEF/EAL domain protein.
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Molecular genetics of carbapenem Antibiotic Biosynthesis
Antonie van Leeuwenhoek, 1999Co-Authors: Simon J Mcgowan, Matthew T. G. Holden, Barrie W. Bycroft, George P. C. SalmondAbstract:Carbapenems are potent β-lactam Antibiotics with a broad spectrum of activity against both Gram positive and Gram negative bacteria. As naturally produced metabolites, they have been isolated from species of Streptomyces, Erwinia and Serratia. The latter two members of the Enterobacteriaceae have proved to be genetically amenable and a growing body of research on these organisms now exists concerning the genes responsible for carbapenem Biosynthesis and the regulatory mechanisms controlling their expression. A cluster of nine carbapenem (car) genes has been identified on the chromosome of Erwinia carotovora. These genes encode the enzymes required for construction of carbapenem and the proteins responsible for a novel β-lactam resistance mechanism, conferring carbapenem immunity in the producing host. Although sharing no homology with the well known enzymes of penicillin Biosynthesis, two of the encoded proteins are apparently similar to enzymes of the clavulanic acid biosynthetic pathway implying a common mechanism for construction of the β-lactam ring. In addition, a transcriptional activator is encoded as the first gene of the carbapenem cluster and this allows positive expression of the remaining downstream genes in response to a quorum sensing, N-acyl homoserine lactone, signalling molecule.
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Molecular genetics of carbapenem Antibiotic Biosynthesis.
Antonie van Leeuwenhoek, 1999Co-Authors: Simon J Mcgowan, Matthew T. G. Holden, Barrie W. Bycroft, George P. C. SalmondAbstract:Carbapenems are potent beta-lactam Antibiotics with a broad spectrum of activity against both Gram positive and Gram negative bacteria. As naturally produced metabolites, they have been isolated from species of Streptomyces, Erwinia and Serratia. The latter two members of the Enterobacteriaceae have proved to be genetically amenable and a growing body of research on these organisms now exists concerning the genes responsible for carbapenem Biosynthesis and the regulatory mechanisms controlling their expression. A cluster of nine carbapenem (car) genes has been identified on the chromosome of Erwinia carotovora. These genes encode the enzymes required for construction of carbapenem and the proteins responsible for a novel beta-lactam resistance mechanism, conferring carbapenem immunity in the producing host. Although sharing no homology with the well known enzymes of penicillin Biosynthesis, two of the encoded proteins are apparently similar to enzymes of the clavulanic acid biosynthetic pathway implying a common mechanism for construction of the beta-lactam ring. In addition, a transcriptional activator is encoded as the first gene of the carbapenem cluster and this allows positive expression of the remaining downstream genes in response to a quorum sensing, N-acyl homoserine lactone, signalling molecule.
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Small molecule mediated autoinduction of Antibiotic Biosynthesis in the plant pathogen Erwinia carotovora.
Biochemical Society transactions, 1995Co-Authors: Pan F. Chan, Barrie W. Bycroft, George P. C. Salmond, Nigel J. Bainton, Mavis Daykin, Michael K. Winson, Siri Ram Chhabra, Gordon S. A. B. Stewart, Paul WilliamsAbstract:Bacteria have evolved sophisticated sensing mechanisms which facilitate adaptation to fluctuating environmental conditions. Commonly these sensory systems consist of a histidine protein kinase which senses changes in a specific environmental parameter and transmits the information to the regulator protein by phosphorylating it. However not all types of sensor-regulator circuits relay information via phosphoryl transfer. The lux operon which confers a bioluminescent phenotype on Vibrio fischeri includes lunR, the product of which has previously been characterised as a member of the response-regulator superfanuly [ I ] . LuxR responds to a small diffusible signal molecule, N-(3-oxohexanoyl)-L-homoserine lactone (OHHL) the Biosynthesis of which is directed by the lux1 gene product (Fig. I(i)). Characteristically, the OHHL-LuxR mediated induction of bioluminescence is cell density dependent and provides a mechanism by which a strong co-ordinated response can be achieved by a population of bacterial cells i.e. it represents an intercellular communication device [ 1.21. We have recently demonstrated that the plant pathogen Envinia carotovora also employs OHHL in the regulation of both the synthesis of the b-lactam Antibiotic l-carbapen-2-em-3-carboxylic acid and exoenzyme virulence determinants [3-5] (Fig.l(ii)). In this paper we: (i) demonstrate the temporal relationship between OHHL production, carhapenem Biosynthesis and cell population density, (ii) characterise the Envinia 1rtxI homologue, car1 and (iii) provide evidencc to suggest that the expression of car/ and hence carbapenem is autoinducible. In V.fischeri, bioluminescence is regulated in a cell density dependent manner mediated through the accumulation, to a critical threshold concentration. of the autoinducer OHHL (21. Since Envinia employs the same small molecule to regulate carbapenem Antibiotic Biosynthesis, we wished to determine whether OHIjL production was constitutive throughout growth or exhibited the same cell density dependency seen in V.fischeri. By transforming the carbapenem producer strain E. carofovora GSlOl with the recombinant lux sensor pSB315, we followed the in vivo production of OHHL by monitoring the induction of bioluminescence. Results clearly demonstrate that OHHL synthesis in Envinia is cell density dependent [6].
