The Biosynthetic Gene Cluster for the β-Lactam Antibiotic Tabtoxin in Pseudomonas syringae

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.

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Identification of a Biosynthetic Gene Cluster and the Six Associated Lipopeptides Involved in Swarming Motility of Pseudomonas syringae pv. tomato DC3000

Journal of Bacteriology ◽

10.1128/jb.00725-07 ◽

2007 ◽

Vol 189 (17) ◽

pp. 6312-6323 ◽

Cited By ~ 88

Author(s):

Andrew D. Berti ◽

Nathan J. Greve ◽

Quin H. Christensen ◽

Michael G. Thomas

Keyword(s):

Secondary Metabolites ◽

Gene Cluster ◽

Pseudomonas Syringae ◽

Biosynthetic Gene Cluster ◽

Nonribosomal Peptides ◽

Biosynthetic Gene ◽

Tomato Dc3000 ◽

Metabolic Potential ◽

Swarming Motility ◽

Nonribosomal Peptide

ABSTRACT Pseudomonas species are known to be prolific producers of secondary metabolites that are synthesized wholly or in part by nonribosomal peptide synthetases. In an effort to identify additional nonribosomal peptides produced by these bacteria, a bioinformatics approach was used to “mine” the genome of Pseudomonas syringae pv. tomato DC3000 for the metabolic potential to biosynthesize previously unknown nonribosomal peptides. Herein we describe the identification of a nonribosomal peptide biosynthetic gene cluster that codes for proteins involved in the production of six structurally related linear lipopeptides. Structures for each of these lipopeptides were proposed based on amino acid analysis and mass spectrometry analyses. Mutations in this cluster resulted in the loss of swarming motility of P. syringae pv. tomato DC3000 on medium containing a low percentage of agar. This phenotype is consistent with the loss of the ability to produce a lipopeptide that functions as a biosurfactant. This work gives additional evidence that mining the genomes of microorganisms followed by metabolite and phenotypic analyses leads to the identification of previously unknown secondary metabolites.

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Anacyclamide D8P, a prenylated cyanobactin from a Sphaerospermopsis sp. cyanobacterium

10.26434/chemrxiv.5346739.v1 ◽

2017 ◽

Cited By ~ 1

Author(s):

Joana Martins ◽

Niina Leikoski ◽

Matti Wahlsten ◽

Joana Azevedo ◽

Jorge Antunes ◽

...

Keyword(s):

Gene Cluster ◽

Cyclic Peptides ◽

Biosynthetic Gene Cluster ◽

The Novel ◽

Tyrosine Residue ◽

Biosynthetic Gene ◽

Post Translational Modification ◽

Biological Assays ◽

Mass Spectrometric Data ◽

Spectrometric Data

Cyanobactins are a family of linear and cyclic peptides produced through the post-translational modification of short precursor peptides. Anacyclamides are macrocyclic cyanobactins with a highly diverse sequence that are common in the genus Anabaena. A mass spectrometry-based screening of potential cyanobactin producers led to the discovery of a new prenylated member of this family of compounds, anacyclamide D8P (1), from Sphaerospermopsis sp. LEGE 00249. The anacyclamide biosynthetic gene cluster (acy) encoding the novel macrocyclic prenylated cyanobactin, was sequenced. Heterologous expression of the acy gene cluster in Escherichia coli established the connection between genomic and mass spectrometric data. Unambiguous establishment of the type and site of prenylation required the full structural elucidation of 1 using Nuclear Magnetic Resonance (NMR), which demonstrated that a forward prenylation occurred on the tyrosine residue. Compound 1 was tested in pharmacologically or ecologically relevant biological assays and revealed moderate antimicrobial activity towards the fouling bacterium Halomonas aquamarina CECT 5000.

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