Use of Tn5-gusA5to investigate environmental and nutritional effects on gene expression in the coronatine biosynthetic gene cluster ofPseudomonas syringaepv.glycinea

1997 ◽  
Vol 43 (6) ◽  
pp. 517-525 ◽  
Author(s):  
David A. Palmer ◽  
Carol L. Bender ◽  
Shashi B. Sharma
Keyword(s):  
Gene Expression ◽  
Amino Acid ◽  
Gene Cluster ◽  
Pseudomonas Syringae ◽  
Nitrogen Sources ◽  
Biosynthetic Gene Cluster ◽  
Amide Bond ◽  
Transcriptional Level ◽  
Coronafacic Acid ◽  
Genes Encoding

Pseudomonas syringae pv. glycinea PG4180 produces coronatine (COR), a chlorosis-inducing phytotoxin that consists of the polyketide coronafacic acid (CFA) coupled via an amide bond to the ethylcyclopropyl amino acid coronamic acid (CMA). Both CFA and CMA function as intermediates in the pathway to coronatine, and genes encoding their synthesis have been localized; however, the precise factors that regulate the production of COR and its precursors remain unclear. In the present study, a λ delivery system for Tn5-gusA5 was developed and used to obtain transcriptional fusions in the COR gene cluster. Selected carbon (fructose and xylose) and amino acid (isoleucine and valine) sources significantly decreased COR biosynthesis at the transcriptional level. Transcriptional activity in the COR gene cluster was temperature dependent with maximal expression at 18–24 °C and significantly less expression at 14 and 30 °C. Interestingly, changes in osmolarity and the addition of complex carbon and nitrogen sources to the growth medium did not significantly affect COR gene expression, although both factors significantly impacted the quantity of COR produced. These results indicate that multiple factors impact COR production and only some of these directly affect transcription in the COR gene cluster.Key words: transcriptional fusion, glucuronidase, gene expression, reporter gene.

scholarly journals Characterization of the Coronatine-Like Phytotoxins Produced by the Common Scab Pathogen Streptomyces scabies

2015 ◽  
Vol 28 (4) ◽  
pp. 443-454 ◽  
Author(s):  
Joanna K. Fyans ◽  
Mead S. Altowairish ◽  
Yuting Li ◽  
Dawn R. D. Bignell
Keyword(s):  
Amino Acid ◽  
Gene Cluster ◽  
Pseudomonas Syringae ◽  
Common Scab ◽  
Biosynthetic Gene Cluster ◽  
Major Product ◽  
Streptomyces Scabies ◽  
Coronafacic Acid ◽  
Thaxtomin A ◽  
Metabolites Production

Streptomyces scabies is an important causative agent of common scab disease of potato tubers and other root crops. The primary virulence factor produced by this pathogen is a phytotoxic secondary metabolite called thaxtomin A, which is essential for disease development. In addition, the genome of S. scabies harbors a virulence-associated biosynthetic gene cluster called the coronafacic acid (CFA)-like gene cluster, which was previously predicted to produce metabolites that resemble the Pseudomonas syringae coronatine (COR) phytotoxin. COR consists of CFA linked to an ethylcyclopropyl amino acid called coronamic acid, which is derived from L-allo-isoleucine. Using a combination of genetic and chemical analyses, we show that the S. scabies CFA-like gene cluster is responsible for producing CFA-L-isoleucine as the major product as well as other minor COR-like metabolites. Production of the metabolites was shown to require the cfl gene, which is located within the CFA-like gene cluster and encodes an enzyme involved in ligating CFA to its amino acid partner. CFA-L-isoleucine purified from S. scabies cultures was shown to exhibit bioactivity similar to that of COR, though it was found to be less toxic than COR. This is the first report demonstrating the production of coronafacoyl phytotoxins by S. scabies, which is the most prevalent scab-causing pathogen in North America.

scholarly journals Identification of the Biosynthetic Gene Cluster for 3-Methylarginine, a Toxin Produced by Pseudomonas syringae pv. syringae 22d/93

2010 ◽  
Vol 76 (8) ◽  
pp. 2500-2508 ◽  
Author(s):  
S. D. Braun ◽  
J. Hofmann ◽  
A. Wensing ◽  
M. S. Ullrich ◽  
H. Weingart ◽  
...  
Keyword(s):  
Amino Acid ◽  
Gene Cluster ◽  
Pseudomonas Syringae ◽  
Biosynthetic Gene Cluster ◽  
Pentanoic Acid ◽  
Promising Candidate ◽  
Biosynthesis Gene Cluster ◽  
E Coli ◽  
Highly Active ◽  
Putative Aminotransferase

ABSTRACT The epiphyte Pseudomonas syringae pv. syringae 22d/93 (Pss22d) produces the rare amino acid 3-methylarginine (MeArg), which is highly active against the closely related soybean pathogen Pseudomonas syringae pv. glycinea. Since these pathogens compete for the same habitat, Pss22d is a promising candidate for biocontrol of P. syringae pv. glycinea. The MeArg biosynthesis gene cluster codes for the S-adenosylmethionine (SAM)-dependent methyltransferase MrsA, the putative aminotransferase MrsB, and the amino acid exporter MrsC. Transfer of the whole gene cluster into Escherichia coli resulted in heterologous production of MeArg. The methyltransferase MrsA was overexpressed in E. coli as a His-tagged protein and functionally characterized (Km , 7 mM; k cat, 85 min−1). The highly selective methyltransferase MrsA transfers the methyl group from SAM into 5-guanidino-2-oxo-pentanoic acid to yield 5-guanidino-3-methyl-2-oxo-pentanoic acid, which then only needs to be transaminated to result in the antibiotic MeArg.

Investigations of the biosynthesis of the phytotoxin coronatine

1994 ◽  
Vol 72 (1) ◽  
pp. 86-99 ◽  
Author(s):  
Ronald J. Parry ◽  
Sunil V. Mhaskar ◽  
Ming-Teh Lin ◽  
Alan E. Walker ◽  
Robson Mafoti
Keyword(s):  
Amino Acid ◽  
Pseudomonas Syringae ◽  
Cyclopropane Ring ◽  
Carbonyl Oxygen ◽  
Oxidative Cyclization ◽  
Amide Bond ◽  
Hydrogen Atoms ◽  
Coronafacic Acid ◽  
Polyketide Chain ◽  
Oxygen Atoms

The biosynthesis of the phytotoxin coronatine has been investigated by administration of isotopically labeled precursors to Pseudomonas syringae pv. glycinea. The structure of coronatine contains two moieties of distinct biosynthetic origin, a bicyclic, hydrindanone carboxylic acid (coronafacic acid) and a cyclopropyl α-amino acid (coronamic acid). Investigations of coronafacic acid biosynthesis have shown that this compound is a polyketide derived from three acetate units, one butyrate unit, and one pyruvate unit. The two carbonyl oxygen atoms of coronafacic acid were found to be derived from the oxygen atoms of acetate. Additional experiments are described that rule out some possible modes for assembly of the polyketide chain. Coronamic acid is shown to be derived from L-isoleucine via the intermediacy of L-alloisoleucine. Examination of the mechanism of the cyclization of L-alloisoleucine to coronamic acid revealed that the formation of the cyclopropane ring takes place with the removal of only two hydrogen atoms from the amino acid, one at C-2 and the other at C-6. The nitrogen atom at C-2 of L-alloisoleucine is shown to be retained. On the basis of these observations, a mechanism is postulated for the cyclization reaction that involves the diversion of an enzymatic hydroxylation reaction into an oxidative cyclization. Finally, a precursor incorporation experiment with deuterium-labeled coronamic acid demonstrated that free coronamic acid can be efficiently incorporated into coronatine. This observation indicates that the cyclization of L-alloisoleucine to coronamic acid can occur before formation of the amide bond between coronafacic acid and coronamic acid.

scholarly journals Characterization of CorR, a Transcriptional Activator Which Is Required for Biosynthesis of the Phytotoxin Coronatine

1998 ◽  
Vol 180 (23) ◽  
pp. 6252-6259 ◽  
Author(s):  
Alejandro Peñaloza-Vázquez ◽  
Carol L. Bender
Keyword(s):  
Gene Expression ◽  
Gene Cluster ◽  
Pseudomonas Syringae ◽  
Transcriptional Start Site ◽  
Regulatory Region ◽  
Dnase I ◽  
Upstream Region ◽  
Start Site ◽  
Large Area ◽  
Coronafacic Acid

ABSTRACT Coronatine (COR) is a plasmid-encoded phytotoxin synthesized by several pathovars of phytopathogenic Pseudomonas syringae. The COR biosynthetic gene cluster in P. syringae pv. glycinea PG4180 is encoded by a 32-kb region which contains both the structural and regulatory genes needed for COR synthesis. The regulatory region contains three genes: corP,corS, and corR. corS is thought to function as a histidine protein kinase, whereas corP andcorR show relatedness to response regulators of the two-component regulatory paradigm. In the present study, we investigated whether CorR is a positive activator of COR gene expression. We also studied whether CorR specifically binds the DNA region located upstream of cfl, a gene located at the 5′ end of the gene cluster encoding coronafacic acid, the polyketide portion of COR. Complementation analysis with a corRmutant, PG4180.P2, and transcriptional fusions to a promoterless glucuronidase gene (uidA) indicated that CorR functions as a positive regulator of COR gene expression. Deletion analysis of the 5′ end of the cfl upstream region was used to define the minimal region required for COR gene expression. A 360-bp DNA fragment located over 500 bp upstream from the cfl transcriptional start site was used in DNase I protection assays to define the specific bases bound by CorR. An area extending from −704 to −650 with respect to the cfl transcriptional start site was protected by DNase I footprinting, indicating a rather large area of protection. This area was also conserved in the promoter region forcmaA, which encodes a transcript containing genes for coronamic acid synthesis, another intermediate in the COR biosynthetic pathway. The results obtained in the current study suggest that both the coronafacic acid and the coronamic acid structural genes are controlled by CorR, a positive activator of COR gene expression.

scholarly journals Analysis of Genes Involved in Biosynthesis of Coronafacic Acid, the Polyketide Component of the Phytotoxin Coronatine

1998 ◽  
Vol 180 (13) ◽  
pp. 3330-3338 ◽  
Author(s):  
Vidhya Rangaswamy ◽  
Robin Mitchell ◽  
Matthias Ullrich ◽  
Carol Bender
Keyword(s):  
Gene Cluster ◽  
Pseudomonas Syringae ◽  
Biosynthetic Gene Cluster ◽  
Open Reading Frames ◽  
Plant Pathogenic Bacterium ◽  
Biosynthetic Gene ◽  
Future Studies ◽  
Coronafacic Acid ◽  
Single Transcript ◽  
Reading Frames

ABSTRACT Coronafacic acid (CFA) is the polyketide component of coronatine (COR), a phytotoxin produced by the plant-pathogenic bacteriumPseudomonas syringae. The genes involved in CFA biosynthesis are encoded by a single transcript which encompasses 19 kb of the COR gene cluster. In the present study, the nucleotide sequence was determined for a 4-kb region located at the 3′ end of the CFA biosynthetic gene cluster. Three open reading frames were identified and designated cfa8, cfa9, andtnp1; the predicted translation products of these genes showed relatedness to oxidoreductases, thioesterases, and transposases, respectively. The translational products of cfa8 andcfa9 were overproduced in Escherichia coliBL21; however, tnp1 was not translated in these experiments. Mutagenesis and complementation analysis indicated thatcfa8 is required for the production of CFA and COR. Analysis of a cfa9 mutant indicated that this gene is dispensable for CFA and COR production but may increase the release of enzyme-bound products from the COR pathway; tnp1, however, had no obvious function in CFA or COR biosynthesis. A genetic strategy was used to produce CFA in a P. syringae strain which lacks the COR gene cluster; this approach will be useful in future studies designed to investigate biosynthetic products of the CFA gene cluster.

scholarly journals Characterization of CmaA, an Adenylation-Thiolation Didomain Enzyme Involved in the Biosynthesis of Coronatine

2004 ◽  
Vol 186 (1) ◽  
pp. 35-42 ◽  
Author(s):  
Robin Couch ◽  
Sarah E. O'Connor ◽  
Heather Seidle ◽  
Christopher T. Walsh ◽  
Ronald Parry
Keyword(s):  
Amino Acid ◽  
Carboxylic Acid ◽  
Pseudomonas Syringae ◽  
Cyclopropane Ring ◽  
Polyacrylamide Gel Electrophoresis ◽  
Active Form ◽  
Sodium Dodecyl ◽  
Biosynthetic Gene Cluster ◽  
Amide Bond ◽  
T Domain

ABSTRACT Several pathovars of Pseudomonas syringae produce the phytotoxin coronatine (COR), which contains an unusual amino acid, the 1-amino-2-ethylcyclopropane carboxylic acid called coronamic acid (CMA), which is covalently linked to a polyketide-derived carboxylic acid, coronafacic acid, by an amide bond. The region of the COR biosynthetic gene cluster proposed to be responsible for CMA biosynthesis was resequenced, and errors in previously deposited cmaA sequences were corrected. These efforts allowed overproduction of P. syringae pv. glycinea PG4180 CmaA in P. syringae pv. syringae FF5 as a FLAG-tagged protein and overproduction of P. syringae pv. tomato CmaA in Escherichia coli as a His-tagged protein; both proteins were in an enzymatically active form. Sequence analysis of CmaA indicated that there were two domains, an adenylation domain (A domain) and a thiolation domain (T domain). ATP-32PPi exchange assays showed that the A domain of CmaA catalyzes the conversion of branched-chain l-amino acids and ATP into the corresponding aminoacyl-AMP derivatives, with a kinetic preference for l-allo-isoleucine. Additional experiments demonstrated that the T domain of CmaA, which is posttranslationally modified with a 4′-phosphopantetheinyl group, reacts with the AMP derivative of l-allo-isoleucine to produce an aminoacyl thiolester intermediate. This covalent species was detected by incubating CmaA with ATP and l-[G-3H]allo-isoleucine, followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis. It is postulated that the l-allo-isoleucine covalently tethered to CmaA serves as the substrate for additional enzymes in the CMA biosynthetic pathway that catalyze cyclopropane ring formation, which is followed by thiolester hydrolysis, yielding free CMA. The availability of catalytically active CmaA should facilitate elucidation of the details of the subsequent steps in the formation of this novel cyclopropyl amino acid.

scholarly journals Pentaminomycins F and G, First Non-Ribosomal Peptides Containing 2-Pyridylalanine

2020 ◽  
Author(s):  
Daniel Carretero Molina ◽  
Francisco Javier Ortiz-Lopez ◽  
Jesús Martín ◽  
Ignacio González ◽  
Marina Sánchez-Hidalgo ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Amino Acid ◽  
Gene Cluster ◽  
Biosynthetic Gene Cluster ◽  
Biosynthetic Gene ◽  
Peptide Synthetase ◽  
Non Ribosomal Peptides

Pentaminomycins F-H, a group of three new hydroxyarginine-containing cyclic pentapeptides, were isolated from cultures of a <i>Streptomyces cacaoi</i> subsp. <i>cacaoi</i> strain along with the known pentaminomycins A-E. The structures of the new peptides were determined by a combination of mass spectrometry and NMR and Marfey's analyses. Among them, pentaminomycins F and G were shown to contain in their structures the rare amino acid 3-(2-pyridyl)-alanine. This finding represents the first reported example of non-ribosomal peptides containing this residue. The LDLLD chiral sequence found for the three compounds was in agreement with that reported for previously isolated pentaminomycins and consistent with the epimerization domains present in the putative non-robosomal peptide synthetase (NRPS) biosynthetic gene cluster.<br>

scholarly journals Unraveling the regulation of sophorolipid biosynthesis in Starmerella bombicola

2020 ◽  
Vol 20 (3) ◽  
Author(s):  
Sofie Lodens ◽  
Sophie L K W Roelants ◽  
Goedele Luyten ◽  
Robin Geys ◽  
Pieter Coussement ◽  
...  
Keyword(s):  
Gene Expression ◽  
Stationary Phase ◽  
Gene Cluster ◽  
Biosynthetic Gene Cluster ◽  
Exponential Phase ◽  
Biosynthetic Gene ◽  
Reporter System ◽  
Gfp Expression ◽  
Qpcr Analysis ◽  
Starmerella Bombicola

ABSTRACT Starmerella bombicola very efficiently produces the secondary metabolites sophorolipids (SLs). Their biosynthesis is not-growth associated and highly upregulated in the stationary phase. Despite high industrial and academic interest, the underlying regulation of SL biosynthesis remains unknown. In this paper, potential regulation of SL biosynthesis through the telomere positioning effect (TPE) was investigated, as the SL gene cluster is located adjacent to a telomere. An additional copy of this gene cluster was introduced elsewhere in the genome to investigate if this results in a decoy of regulation. Indeed, for the new strain, the onset of SL production was shifted to the exponential phase. This result was confirmed by RT-qPCR analysis. The TPE effect was further investigated by developing and applying a suitable reporter system for this non-conventional yeast, enabling non-biased comparison of gene expression between the subtelomeric CYP52M1- and the URA3 locus. This was done with a constitutive endogenous promotor (pGAPD) and one of the endogenous promotors of the SL biosynthetic gene cluster (pCYP52M1). A clear positioning effect was observed for both promotors with significantly higher GFP expression levels at the URA3 locus. No clear GFP upregulation was observed in the stationary phase for any of the new strains.

scholarly journals Amino Acid Transport and Metabolism in Mycobacteria: Cloning, Interruption, and Characterization of anl-Arginine/γ-Aminobutyric Acid Permease inMycobacterium bovis BCG

2000 ◽  
Vol 182 (4) ◽  
pp. 919-927 ◽  
Author(s):  
Anjali Seth ◽  
Nancy D. Connell
Keyword(s):  
Amino Acid ◽  
Mutant Strain ◽  
Nitrogen Sources ◽  
Single Copy ◽  
Aminobutyric Acid ◽  
Cosmid Library ◽  
Wild Type ◽  
Carbon And Nitrogen ◽  
Genes Encoding ◽  
Type Gene

ABSTRACT Genes encoding l-arginine biosynthetic and transport proteins have been shown in a number of pathogenic organisms to be important for metabolism within the host. In this study we describe the cloning of a gene (Rv0522) encoding an amino acid transporter fromMycobacterium bovis BCG and the effects of its deletion onl-arginine transport and metabolism. The Rv0522 gene of BCG was cloned from a cosmid library by using primers homologous to therocE gene of Bacillus subtilis, a putative arginine transporter. A deletion mutant strain was constructed by homologous recombination with the Rv0522 gene interrupted by a selectable marker. The mutant strain was complemented with the wild-type gene in single copy. Transport analysis of these strains was conducted using 14C-labeled substrates. Greatly reduced uptake of l-arginine and γ-aminobutyric acid (GABA) but not of lysine, ornithine, proline, or alanine was observed in the mutant strain compared to the wild type, grown in Middlebrook 7H9 medium. However, when the strains were starved for 24 h or incubated in a minimal salts medium containing 20 mM arginine (in which even the parent strain does not grow),l-[14C]arginine uptake by the mutant but not the wild-type strain increased strongly. Exogenousl-arginine but not GABA, lysine, ornithine, or alanine was shown to be toxic at concentrations of 20 mM and above to wild-type cells growing in optimal carbon and nitrogen sources such as glycerol and ammonium. l-Arginine supplied in the form of dipeptides showed no toxicity at concentrations as high as 30 mM. Finally, the permease mutant strain showed no defect in survival in unactivated cultured murine macrophages compared with wild-type BCG.

scholarly journals Different Biosynthetic Pathways to Fosfomycin in Pseudomonas syringae and Streptomyces Species

2012 ◽  
Vol 56 (8) ◽  
pp. 4175-4183 ◽  
Author(s):  
Seung Young Kim ◽  
Kou-San Ju ◽  
William W. Metcalf ◽  
Bradley S. Evans ◽  
Tomohisa Kuzuyama ◽  
...  
Keyword(s):  
Gene Cluster ◽  
Pseudomonas Syringae ◽  
Citrate Synthase ◽  
Wide Spectrum ◽  
The United States ◽  
Biosynthetic Gene Cluster ◽  
Gene Encoding ◽  
Acute Cystitis ◽  
Streptomyces Species ◽  
Content Type

ABSTRACTFosfomycin is a wide-spectrum antibiotic that is used clinically to treat acute cystitis in the United States. The compound is produced by several strains of streptomycetes and pseudomonads. We sequenced the biosynthetic gene cluster responsible for fosfomycin production inPseudomonas syringaePB-5123. Surprisingly, the biosynthetic pathway in this organism is very different from that inStreptomyces fradiaeandStreptomyces wedmorensis. The pathways share the first and last steps, involving conversion of phosphoenolpyruvate to phosphonopyruvate (PnPy) and 2-hydroxypropylphosphonate (2-HPP) to fosfomycin, respectively, but the enzymes converting PnPy to 2-HPP are different. The genome ofP. syringaePB-5123 lacks a gene encoding the PnPy decarboxylase found in theStreptomycesstrains. Instead, it contains a gene coding for a citrate synthase-like enzyme, Psf2, homologous to the proteins that add an acetyl group to PnPy in the biosynthesis of FR-900098 and phosphinothricin. Heterologous expression and purification of Psf2 followed by activity assays confirmed the proposed activity of Psf2. Furthermore, heterologous production of fosfomycin inPseudomonas aeruginosafrom a fosmid encoding the fosfomycin biosynthetic cluster fromP. syringaePB-5123 confirmed that the gene cluster is functional. Therefore, two different pathways have evolved to produce this highly potent antimicrobial agent.

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