scholarly journals Identification of glucose kinase-dependent and -independent pathways for carbon control of primary metabolism, development and antibiotic production inStreptomyces coelicolorby quantitative proteomics

2012 ◽  
Vol 86 (6) ◽  
pp. 1490-1507 ◽  
Author(s):  
Jacob Gubbens ◽  
Marleen Janus ◽  
Bogdan I. Florea ◽  
Herman S. Overkleeft ◽  
Gilles P. van Wezel
Keyword(s):  
Quantitative Proteomics ◽  
Antibiotic Production ◽  
Primary Metabolism ◽  
Carbon Control ◽  
Glucose Kinase

scholarly journals Induction of Holomycin Production and Complex Metabolic Changes by theargRMutation in Streptomyces clavuligerus NP1

2012 ◽  
Vol 78 (9) ◽  
pp. 3431-3441 ◽  
Author(s):  
Hua Yin ◽  
Sihai Xiang ◽  
Jianting Zheng ◽  
Keqiang Fan ◽  
Tingting Yu ◽  
...  
Keyword(s):  
Metabolic Pathways ◽  
Antibiotic Production ◽  
Streptomyces Clavuligerus ◽  
Metabolic Changes ◽  
Primary Metabolism ◽  
Arginine Biosynthesis ◽  
Complex Interplay ◽  
Altered Expression ◽  
Content Type ◽  
Primary And Secondary Metabolism

ABSTRACTIn bacteria, arginine biosynthesis is tightly regulated by a universally conserved regulator, ArgR, which regulates the expression of arginine biosynthetic genes, as well as other important genes. Disruption ofargRinStreptomyces clavuligerusNP1 resulted in complex phenotypic changes in growth and antibiotic production levels. To understand the metabolic changes underlying the phenotypes, comparative proteomic studies were carried out between NP1 and itsargRdisruption mutant (designated CZR). In CZR, enzymes involved in holomycin biosynthesis were overexpressed; this is consistent with its holomycin overproduction phenotype. The effects on clavulanic acid (CA) biosynthesis are more complex. Several proteins from the CA cluster were moderately overexpressed, whereas several proteins from the 5S clavam biosynthetic cluster and from the paralog cluster of CA and 5S clavam biosynthesis were severely downregulated. Obvious changes were also detected in primary metabolism, which are mainly reflected in the altered expression levels of proteins involved in acetyl-coenzyme A (CoA) and cysteine biosynthesis. Since acetyl-CoA and cysteine are precursors for holomycin synthesis, overexpression of these proteins is consistent with the holomycin overproduction phenotype. The complex interplay between primary and secondary metabolism and between secondary metabolic pathways were revealed by these analyses, and the insights will guide further efforts to improve production levels of CA and holomycin inS. clavuligerus.

Possible involvement of the sco2127 gene product in glucose repression of actinorhodin production in Streptomyces coelicolor

2012 ◽  
Vol 58 (10) ◽  
pp. 1195-1201 ◽  
Author(s):  
Angela Forero ◽  
Mauricio Sánchez ◽  
Adán Chávez ◽  
Beatriz Ruiz ◽  
Romina Rodríguez-Sanoja ◽  
...  
Keyword(s):  
Glucose Repression ◽  
Antibiotic Production ◽  
Streptomyces Coelicolor ◽  
Negative Regulator ◽  
Wild Type ◽  
Wild Type Phenotype ◽  
Glucose Sensitivity ◽  
Mutant Complementation ◽  
Glucose Kinase ◽  
Actinorhodin Production

Streptomyces coelicolor mutants resistant to 2-deoxyglucose are insensitive to carbon catabolite repression (CCR). Total reversion to CCR sensitivity is observed by mutant complementation with a DNA region harboring both glucose kinase glkA gene and the sco2127 gene. The sco2127 is located upstream of glkA and encodes a putative protein of 20.1 kDa. In S. coelicolor, actinorhodin production is subject to glucose repression. To explore the possible involvement of both SCO2127 and glucose kinase (Glk) in the glucose sensitivity of actinorhodin production, this effect was evaluated in a wild-type S. coelicolor A3(2) M145 strain and a sco2127 null mutant (Δsco2127) derived from this wild-type strain. In comparison with strain M145, actinorhodin production by the mutant was insensitive to glucose repression. Under repressive conditions, only minor differences were observed in glucose utilization and Glk production between these strains. SCO2127 was detected mainly during the first 36 h of fermentation, just before the onset of antibiotic production, and its synthesis was not related to a particular carbon source. The glucose sensitivity of antibiotic production was restored to wild-type phenotype by transformation with an integrative plasmid containing sco2127. Our results support the hypothesis that SCO2127 is a negative regulator of actinorhodin production and suggest that the effect is independent of Glk.

scholarly journals Secondary Metabolism and Interspecific Competition Affect Accumulation of Spontaneous Mutants in the GacS-GacA Regulatory System in Pseudomonas protegens

mBio ◽  
2018 ◽  
Vol 9 (1) ◽  
Author(s):  
Qing Yan ◽  
Lucas D. Lopes ◽  
Brenda T. Shaffer ◽  
Teresa A. Kidarsa ◽  
Oliver Vining ◽  
...  
Keyword(s):  
Interspecific Competition ◽  
Secondary Metabolism ◽  
Antibiotic Production ◽  
Regulatory System ◽  
Antibiotic Biosynthesis ◽  
Primary Metabolism ◽  
Natural Environments ◽  
Trade Off ◽  
Pure Cultures ◽  
Spontaneous Mutants

ABSTRACTSecondary metabolites are synthesized by many microorganisms and provide a fitness benefit in the presence of competitors and predators. Secondary metabolism also can be costly, as it shunts energy and intermediates from primary metabolism. InPseudomonasspp., secondary metabolism is controlled by the GacS-GacA global regulatory system. Intriguingly, spontaneous mutations ingacSorgacA(Gac−mutants) are commonly observed in laboratory cultures. Here we investigated the role of secondary metabolism in the accumulation of Gac−mutants inPseudomonas protegensstrain Pf-5. Our results showed that secondary metabolism, specifically biosynthesis of the antimicrobial compound pyoluteorin, contributes significantly to the accumulation of Gac−mutants. Pyoluteorin biosynthesis, which poses a metabolic burden on the producer cells, but not pyoluteorin itself, leads to the accumulation of the spontaneous mutants. Interspecific competition also influenced the accumulation of the Gac−mutants: a reduced proportion of Gac−mutants accumulated whenP. protegensPf-5 was cocultured withBacillus subtilisthan in pure cultures of strain Pf-5. Overall, our study associated a fitness trade-off with secondary metabolism, with metabolic costs versus competitive benefits of production influencing the evolution ofP. protegens, assessed by the accumulation of Gac−mutants.IMPORTANCEMany microorganisms produce antibiotics, which contribute to ecologic fitness in natural environments where microbes constantly compete for resources with other organisms. However, biosynthesis of antibiotics is costly due to the metabolic burdens of the antibiotic-producing microorganism. Our results provide an example of the fitness trade-off associated with antibiotic production. Under noncompetitive conditions, antibiotic biosynthesis led to accumulation of spontaneous mutants lacking a master regulator of antibiotic production. However, relatively few of these spontaneous mutants accumulated when a competitor was present. Results from this work provide information on the evolution of antibiotic biosynthesis and provide a framework for their discovery and regulation.

scholarly journals Biosynthesis of Tropolones inStreptomycesspp.: Interweaving Biosynthesis and Degradation of Phenylacetic Acid and Hydroxylations on the Tropone Ring

2018 ◽  
Vol 84 (12) ◽  
pp. e00349-18 ◽  
Author(s):  
Xuefei Chen ◽  
Min Xu ◽  
Jin Lü ◽  
Jianguo Xu ◽  
Yemin Wang ◽  
...  
Keyword(s):  
Carboxylic Acid ◽  
Biosynthetic Pathway ◽  
Phenylacetic Acid ◽  
De Novo ◽  
Biological Activities ◽  
Antibiotic Production ◽  
Degradation Pathway ◽  
Chorismate Mutase ◽  
Primary Metabolism ◽  
Content Type

ABSTRACTTropolonoids are important natural products that contain a unique seven-membered aromatic tropolone core and exhibit remarkable biological activities. 3,7-Dihydroxytropolone (DHT) isolated fromStreptomycesspecies is a multiply hydroxylated tropolone exhibiting antimicrobial, anticancer, and antiviral activities. In this study, we determined the DHT biosynthetic pathway by heterologous expression, gene deletion, and biotransformation. Ninetrlgenes and some of the aerobic phenylacetic acid degradation pathway genes (paa) located outside thetrlbiosynthetic gene cluster are required for the heterologous production of DHT. ThetrlAgene encodes a single-domain protein homologous to the C-terminal enoyl coenzyme A (enoyl-CoA) hydratase domain of PaaZ. TrlA truncates the phenylacetic acid catabolic pathway and redirects it toward the formation of heptacyclic intermediates. TrlB is a 3-deoxy-d-arabino-heptulosonic acid-7-phosphate (DAHP) synthase homolog. TrlH is an unusual bifunctional protein bearing an N-terminal prephenate dehydratase domain and a C-terminal chorismate mutase domain. TrlB and TrlH enhancedde novobiosynthesis of phenylpyruvate, thereby providing abundant precursor for the prolific production of DHT inStreptomycesspp. Six seven-membered carbocyclic compounds were identified from thetrlC,trlD,trlE, andtrlFdeletion mutants. Four of these chemicals, including 1,4,6-cycloheptatriene-1-carboxylic acid, tropone, tropolone, and 7-hydroxytropolone, were verified as key biosynthetic intermediates. TrlF is required for the conversion of 1,4,6-cycloheptatriene-1-carboxylic acid into tropone. The monooxygenases TrlE and TrlCD catalyze the regioselective hydroxylations of tropone to produce DHT. This study reveals a natural association of anabolism of chorismate and phenylpyruvate, catabolism of phenylacetic acid, and biosynthesis of tropolones inStreptomycesspp.IMPORTANCETropolonoids are promising drug lead compounds because of the versatile bioactivities attributed to their highly oxidized seven-membered aromatic ring scaffolds. Our present study provides clear insight into the biosynthesis of 3,7-dihydroxytropolone (DHT) through the identification of key genes responsible for the formation and modification of the seven-membered aromatic core. We also reveal the intrinsic mechanism of elevated production of DHT and related tropolonoids inStreptomycesspp. The study on DHT biosynthesis inStreptomycesexhibits a good example of antibiotic production in which both anabolic and catabolic pathways of primary metabolism are interwoven into the biosynthesis of secondary metabolites. Furthermore, our study sets the stage for metabolic engineering of the biosynthetic pathway for natural tropolonoid products and provides alternative synthetic biology tools for engineering novel tropolonoids.

Overexpression and inhibition of 3-hydroxy-3-methylglutaryl-CoA synthase affect central metabolic pathways in tobacco

2020 ◽  
Author(s):  
Pan Liao ◽  
Shiu-Cheung Lung ◽  
Wai Lung Chan ◽  
Menglong Hu ◽  
Geoffrey Kwai-Wai Kong ◽  
...  
Keyword(s):  
Metabolic Pathways ◽  
Quantitative Proteomics ◽  
Tricarboxylic Acid Cycle ◽  
Specific Inhibitor ◽  
Primary Metabolism ◽  
Proteomics Analysis ◽  
Acetyl Coa ◽  
Cell Wall Modification ◽  
Mva Pathway ◽  
The Relationship

Abstract Little has been established on the relationship between the mevalonate (MVA) pathway and other metabolic pathways except for the sterol and glucosinolate biosynthesis pathways. In the MVA pathway, 3-hydroxy-3-methylglutaryl-CoA synthase (HMGS) catalyses the condensation of acetoacetyl-CoA and acetyl-CoA to form HMG-CoA. Our previous studies had shown that while the recombinant Brassica juncea HMGS1 (BjHMGS1) mutant S359A displayed 10-fold higher enzyme activity than wild-type (wt) BjHMGS1, transgenic tobacco overexpressing S359A (OE-S359A) exhibited higher sterol content, growth rate and seed yield than OE-wtBjHMGS1. Herein, untargeted proteomics and targeted metabolomics were employed to understand the phenotypic effects of HMGS overexpression in tobacco by examining which other metabolic pathways were affected. SWATH-MS quantitative proteomics analysis on OE-wtBjHMGS1 and OE-S359A identified the misregulation of proteins in primary metabolism and cell wall modification, while some proteins related to photosynthesis and the tricarboxylic acid cycle were upregulated in OE-S359A. Metabolomic analysis indicated corresponding changes in carbohydrate, amino acid and fatty acid contents in HMGS-OEs, and F-244, a specific inhibitor of HMGS, was applied successfully on tobacco to confirm these observations. Finally, the crystal structure of acetyl-CoA-liganded S359A revealed that improved activity of S359A likely resulted from a loss in hydrogen bonding between Ser359 and acyl-CoA which is evident in wtBjHMGS1. This work suggests that regulation of plant growth by HMGS can influence the central metabolic pathways. Furthermore, this study demonstrates that the application of the HMGS-specific inhibitor (F-244) in tobacco represents an effective approach for studying the HMGS/MVA pathway.

scholarly journals SILAC-based quantitative proteomics reveals pleiotropic, phenotypic modulation in primary murine macrophages infected with the protozoan pathogen Leishmania donovani

2019 ◽  
Author(s):  
Despina Smirlis ◽  
Florent Dingli ◽  
Pascale Pescher ◽  
Eric Prina ◽  
Damarys Loew ◽  
...  
Keyword(s):  
Quantitative Proteomics ◽  
Leishmania Donovani ◽  
Ex Vivo ◽  
Mononuclear Phagocytes ◽  
Mammalian Host ◽  
Primary Metabolism ◽  
Strong Anion Exchange ◽  
Significant Enrichment ◽  
Bona Fide ◽  
Underlying Mechanisms

ABSTRACTLeishmaniases are major vector-borne tropical diseases responsible for great human morbidity and mortality, caused by protozoan, trypanosomatid parasites of the genus Leishmania. In the mammalian host parasites survive and multiply within mononuclear phagocytes, especially macrophages. However, the underlying mechanisms by which Leishmania spp affect their host, are not fully understood. Herein, proteomic alterations of primary bone marrow-derived, BALB/c macrophages are documented after 72 h of infection with Leishmania donovani insect-stage promastigotes, with the use of a SILAC-based, quantitative proteomics approach. The protocol was optimised by combining strong anion exchange and gel electrophoresis fractionation that displayed similar depth of analysis (>5500 proteins). Our analyses revealed 86 differentially modulated proteins (35 showing increased and 51 decreased abundance) in response to Leishmania donovani infection. The proteomics results were validated by analysing the abundance of selected proteins. Intracellular Leishmania donovani infection led to changes in various host cell biological processes, including primary metabolism and catabolic process, with a significant enrichment in lysosomal organisation. Overall, our analysis allows new technical insight into the challenges of quantitative proteomics applied on primary cells, and establishes the first proteome of bona fide primary macrophages infected ex vivo with Leishmania donovani, revealing new mechanisms acting at the host/pathogen interface.

scholarly journals The Phosphinomethylmalate Isomerase Genepmi, Encoding an Aconitase-Like Enzyme, Is Involved in the Synthesis of Phosphinothricin Tripeptide inStreptomyces viridochromogenes

2001 ◽  
Vol 67 (8) ◽  
pp. 3603-3609 ◽  
Author(s):  
E. Heinzelmann ◽  
G. Kienzlen ◽  
S. Kaspar ◽  
J. Recktenwald ◽  
W. Wohlleben ◽  
...  
Keyword(s):  
Antibiotic Production ◽  
Tca Cycle ◽  
Biosynthetic Gene Cluster ◽  
Amino Acid Sequences ◽  
Insertion Mutagenesis ◽  
Primary Metabolism ◽  
Enzymatic Reactions ◽  
Gene Encoding ◽  
Iron Regulatory Proteins ◽  
Bacteria And Fungi

ABSTRACT Streptomyces viridochromogenes Tü494 produces the antibiotic phosphinothricin tripeptide (PTT). In the postulated biosynthetic pathway, one reaction, the isomerization of phosphinomethylmalate, resembles the aconitase reaction of the tricarboxylic acid (TCA) cycle. It was speculated that this reaction is carried out by the corresponding enzyme of the primary metabolism (C. J. Thompson and H. Seto, p. 197–222,in L. C. Vining and C. Stuttard, ed.,Genetics and Biochemistry of Antibiotic Production,1995). However, in addition to the TCA cycle aconitase gene, a gene encoding an aconitase-like protein (the phosphinomethylmalate isomerase gene, pmi) was identified in the PTT biosynthetic gene cluster by Southern hybridization experiments, using oligonucleotides which were derived from conserved amino acid sequences of aconitases. The deduced protein revealed high similarity to aconitases from plants, bacteria, and fungi and to iron regulatory proteins from eucaryotes. Pmi and the S. viridochromogenes TCA cycle aconitase, AcnA, have 52% identity. By gene insertion mutagenesis, a pmi mutant (Mapra1) was generated. The mutant failed to produce PTT, indicating the inability of AcnA to carry out the secondary-metabolism reaction. A His-tagged protein (Hispmi*) was heterologously produced inStreptomyces lividans. The purified protein showed no standard aconitase activity with citrate as a substrate, and the corresponding gene was not able to complement an acnAmutant. This indicates that Pmi and AcnA are highly specific for their respective enzymatic reactions.

scholarly journals ppGpp Controlled by the Gac/Rsm Regulatory Pathway Sustains Biocontrol Activity in Pseudomonas fluorescens CHA0

2012 ◽  
Vol 25 (11) ◽  
pp. 1440-1449 ◽  
Author(s):  
Kasumi Takeuchi ◽  
Kosumi Yamada ◽  
Dieter Haas
Keyword(s):  
Pseudomonas Fluorescens ◽  
Secondary Metabolism ◽  
Plant Protection ◽  
Antibiotic Production ◽  
Krebs Cycle ◽  
Signal Transduction Pathway ◽  
Primary Metabolism ◽  
Metabolomic Profiling ◽  
Biocontrol Activity ◽  
Negative Mutant

In Pseudomonas fluorescens CHA0 and other fluorescent pseudomonads, the Gac/Rsm signal transduction pathway is instrumental for secondary metabolism and biocontrol of root pathogens via the expression of regulatory small RNAs (sRNAs). Furthermore, in strain CHA0, an imbalance in the Krebs cycle can affect the strain's ability to produce extracellular secondary metabolites, including biocontrol factors. Here, we report the metabolome of wild-type CHA0, a gacA-negative mutant, which has lost Gac/Rsm activities, and a retS-negative mutant, which shows strongly enhanced Gac/Rsm-dependent activities. Capillary electrophoresis-based metabolomic profiling revealed that the gacA and retS mutations had opposite effects on the intracellular levels of a number of central metabolites, suggesting that the Gac/Rsm pathway regulates not only secondary metabolism but also primary metabolism in strain CHA0. Among the regulated metabolites identified, the alarmone guanosine tetraphosphate (ppGpp) was characterized in detail by the construction of relA (for ppGpp synthase) and spoT (for ppGpp synthase/hydrolase) deletion mutants. In a relA spoT double mutant, ppGpp synthesis was completely abolished, the expression of Rsm sRNAs was attenuated, and physiological functions such as antibiotic production, root colonization, and plant protection were markedly diminished. Thus, ppGpp appears to be essential for sustaining epiphytic fitness and biocontrol activity of strain CHA0.

scholarly journals Gamma-Butyrolactone Regulatory System of Streptomyces chattanoogensis Links Nutrient Utilization, Metabolism, and Development

2011 ◽  
Vol 77 (23) ◽  
pp. 8415-8426 ◽  
Author(s):  
Yi-Ling Du ◽  
Xue-Ling Shen ◽  
Pin Yu ◽  
Lin-Quan Bai ◽  
Yong-Quan Li
Keyword(s):  
Antibiotic Production ◽  
Regulatory System ◽  
Nutrient Utilization ◽  
Transcriptional Analysis ◽  
Growth Defect ◽  
Primary Metabolism ◽  
Uptake System ◽  
Submerged Cultures ◽  
Content Type ◽  
Streptomyces Chattanoogensis

ABSTRACTGamma-butyrolactones (GBLs) produced by severalStreptomycesspecies have been shown to serve as quorum-sensing signaling molecules for activating antibiotic production. The GBL system ofStreptomyces chattanoogensisL10, a producer of antifungal agent natamycin, consists of three genes:scgA,scgX, andscgR. BothscgAandscgXcontribute to GBL production, whilescgRencodes a GBL receptor.ΔscgAandΔscgXmutants ofS. chattanoogensisbehaved identically: they had a growth defect in submerged cultures and delayed or abolished the morphological differentiation and secondary metabolites production on solid medium. ScgR could bind to the promoter region ofscgAand repress its transcription. Moreover,scgAseems also to be controlled by a GBL-mediated negative-feedback system. Hence, it is apparent that GBL biosynthesis is tightly controlled to ensure the correct timing for metabolic switch. An additional direct ScgR-target genegbdAwas identified by genomic SELEX and transcriptional analysis. Comparative proteomic analysis between L10 and itsΔscgAmutant revealed that the GBL system affects the expression of more than 50 proteins, including enzymes involved in carbon uptake system, primary metabolism, and stress response, we thus conclude thatscgR-scgA-scgXconstitute a novel GBL regulatory system involved in nutrient utilization, triggering adaptive responses, and finally dictating the switch from primary to secondary metabolism.

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