scholarly journals Discovery and biosynthesis of gladiochelins: unusual lipodepsipeptide siderophores from Burkholderia gladioli

2020 ◽  
Author(s):  
Yousef Dashti ◽  
Ioanna T. Nakou ◽  
Alex J. Mullins ◽  
Gordon Webster ◽  
Xinyun Jian ◽  
...  
Keyword(s):  
Gene Cluster ◽  
Insertional Mutagenesis ◽  
Carbon Sources ◽  
Biosynthetic Gene Cluster ◽  
Amino Acid Residues ◽  
Opportunistic Pathogens ◽  
Gene Encoding ◽  
Burkholderia Gladioli ◽  
Metabolic Products ◽  
Cas Assay

AbstractBurkholderia is a genus of diverse Gram-negative bacteria that includes several opportunistic pathogens. Siderophores, which transport iron from the environment into microbial cells, are important virulence factors in most pathogenic Burkholderia species. However, it is widely believed that Burkholderia gladioli, which can infect the lungs of cystic fibrosis (CF) sufferers, does not produce siderophores. B. gladioli BCC0238, isolated from the lung of a CF patient, produces two novel metabolites in a minimal medium containing glycerol and ribose as carbon sources. HPLC purification, followed by detailed spectroscopic analyses, identified these metabolites as unusual lipodepsipeptides containing a unique citrate-derived fatty acid and a rare dehydro-β-alanine residue. The absolute configurations of the amino acid residues in the two metabolites was elucidated using Marfey’s method and the gene cluster responsible for their biosynthesis was identified by bioinformatics and insertional mutagenesis. In-frame deletions and enzyme activity assays were used to investigate the functions of several proteins encoded by the biosynthetic gene cluster, which was found in the genomes of most B. gladioli isolates, suggesting that its metabolic products play an important role in the growth and/or survival of the species. The Chrome Azurol S (CAS) assay showed the metabolites bind ferric iron and that this supresses their production when added to the growth medium. Moreover, a gene encoding a TonB-dependent ferric-siderophore receptor is adjacent to the biosynthetic genes. Together, these observations suggest that these metabolites likely function as siderophores in B. gladioli.

scholarly journals Transcriptional Regulator PhlH Modulates 2,4-Diacetylphloroglucinol Biosynthesis in Response to the Biosynthetic Intermediate and End Product

2017 ◽  
Vol 83 (21) ◽  
Author(s):  
Xu Yan ◽  
Rui Yang ◽  
Rui-Xue Zhao ◽  
Jian-Ting Han ◽  
Wen-Juan Jia ◽  
...  
Keyword(s):  
Pseudomonas Fluorescens ◽  
Gene Cluster ◽  
Plant Pathogens ◽  
Feedback Regulation ◽  
Biosynthetic Gene Cluster ◽  
Sequence Motif ◽  
Target Sequence ◽  
Gene Encoding ◽  
Negative Feedback Regulation ◽  
Content Type

ABSTRACT Certain strains of biocontrol bacterium Pseudomonas fluorescens produce the secondary metabolite 2,4-diacetylphloroglucinol (2,4-DAPG) to antagonize soilborne phytopathogens in the rhizosphere. The gene cluster responsible for the biosynthesis of 2,4-DAPG is named phlACBDEFGH and it is still unclear how the pathway-specific regulator phlH within this gene cluster regulates the metabolism of 2,4-DAPG. Here, we found that PhlH in Pseudomonas fluorescens strain 2P24 represses the expression of the phlG gene encoding the 2,4-DAPG hydrolase by binding to a sequence motif overlapping with the −35 site recognized by σ70 factors. Through biochemical screening of PhlH ligands we identified the end product 2,4-DAPG and its biosynthetic intermediate monoacetylphloroglucinol (MAPG), which can act as signaling molecules to modulate the binding of PhlH to the target sequence and activate the expression of phlG. Comparison of 2,4-DAPG production between the ΔphlH, ΔphlG, and ΔphlHG mutants confirmed that phlH and phlG impose negative feedback regulation over 2,4-DAPG biosynthesis. It was further demonstrated that the 2,4-DAPG degradation catalyzed by PhlG plays an insignificant role in 2,4-DAPG tolerance but contributes to bacterial growth advantages under carbon/nitrogen starvation conditions. Taken together, our data suggest that by monitoring and down-tuning in situ levels of 2,4-DAPG, the phlHG genes could dynamically modulate the metabolic loads attributed to 2,4-DAPG production and potentially contribute to rhizosphere adaptation. IMPORTANCE 2,4-DAPG, which is synthesized by biocontrol pseudomonad bacteria, is a broad-spectrum antibiotic against bacteria, fungi, oomycetes, and nematodes and plays an important role in suppressing soilborne plant pathogens. Although most of the genes in the 2,4-DAPG biosynthetic gene cluster (phl) have been characterized, it is still not clear how the pathway-specific regulator phlH is involved in 2,4-DAPG metabolism. This work revealed the role of PhlH in modulating 2,4-DAPG levels by controlling the expression of 2,4-DAPG hydrolase PhlG in response to 2,4-DAPG and MAPG. Since 2,4-DAPG biosynthesis imposes a metabolic burden on biocontrol pseudomonads, it is expected that the fine regulation of phlG by PhlH offers a way to dynamically modulate the metabolic loads attributed to 2,4-DAPG production.

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.

scholarly journals Characterization of FtsH Essentiality in Streptococcus mutans via Genetic Suppression

2021 ◽  
Vol 12 ◽  
Author(s):  
Yaqi Wang ◽  
Wei Cao ◽  
Justin Merritt ◽  
Zhoujie Xie ◽  
Hao Liu
Keyword(s):  
Gene Cluster ◽  
Streptococcus Mutans ◽  
Response Regulator ◽  
Critical Role ◽  
Acid Stress ◽  
Biosynthetic Gene Cluster ◽  
Oral Microbiome ◽  
Regulatory Function ◽  
Biosynthetic Gene ◽  
Gene Encoding

FtsH belongs to the AAA+ ATP-dependent family of proteases, which participate in diverse cellular processes and are ubiquitous among bacteria, chloroplasts, and mitochondria. FtsH is poorly characterized in most organisms, especially compared to other major housekeeping proteases. In the current study, we examined the source of FtsH essentiality in the human oral microbiome species Streptococcus mutans, one of the primary etiological agents of dental caries. By creating a conditionally lethal ftsH mutant, we were able to identify a secondary suppressor missense mutation in the vicR gene, encoding the response regulator of the essential VicRK two-component system (TCS). Transcriptomic analysis of the vicR (G195R) mutant revealed significantly reduced expression of 46 genes, many of which were located within the genomic island Tnsmu2, which harbors the mutanobactin biosynthetic gene cluster. In agreement with the transcriptomic data, deletion of the mutanobactin biosynthetic gene cluster suppressed ftsH essentiality in S. mutans. We also explored the role of FtsH in S. mutans physiology and demonstrated its critical role in stress tolerance, especially acid stress. The presented results reveal the first insights within S. mutans for the pleiotropic regulatory function of this poorly understood global regulator.

A comparison of the clavam biosynthetic gene clusters in Streptomyces antibioticus Tü1718 and Streptomyces clavuligerus

2012 ◽  
Vol 58 (4) ◽  
pp. 413-425 ◽  
Author(s):  
Sarah Goomeshi Nobary ◽  
Susan E. Jensen
Keyword(s):  
Clavulanic Acid ◽  
Gene Cluster ◽  
Gene Clusters ◽  
Streptomyces Clavuligerus ◽  
Biosynthetic Gene Cluster ◽  
Cosmid Library ◽  
Biosynthetic Gene ◽  
Gene Encoding ◽  
Knock Out ◽  
Streptomyces Antibioticus

The production of clavam metabolites has been studied previously in Streptomyces clavuligerus , a species that produces clavulanic acid as well as 4 other clavam compounds, but the late steps of the pathway leading to the specific end products are unclear. The present study compared the clavam biosynthetic gene cluster in Streptomyces antibioticus , chosen because it produces only 2 clavam metabolites and no clavulanic acid, with that of S. clavuligerus. A cosmid library of S. antibioticus genomic DNA was screened with a clavaminate synthase-specific probe based on the corresponding genes from S. clavuligerus, and 1 of the hybridizing cosmids was sequenced in full. A clavam gene cluster was identified that shows similarities to that of S. clavuligerus but also contains a number of novel genes. Knock-out mutation of the clavaminate synthase gene abolished clavam production in S. antibioticus, confirming the identity of the gene cluster. Knock-out mutation of a novel gene encoding an apparent oxidoreductase also abolished clavam production. A potential clavam biosynthetic pathway consistent with the genes in the cluster and the metabolites produced by S. antibioticus, and correspondingly different from that of S. clavuligerus, is proposed.

scholarly journals Mutagenesis of NosM Leader Peptide Reveals Important Elements in Nosiheptide Biosynthesis

2016 ◽  
Vol 83 (4) ◽  
Author(s):  
Liang Jin ◽  
Xuri Wu ◽  
Yanjiu Xue ◽  
Yue Jin ◽  
Shuzhen Wang ◽  
...  
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Gene Cluster ◽  
Genetic Manipulation ◽  
Fold Increase ◽  
Biosynthetic Gene Cluster ◽  
Leader Peptide ◽  
Amino Acid Residues ◽  
Precursor Peptide

ABSTRACT Nosiheptide, a typical member of the ribosomally synthesized and posttranslationally modified peptides (RiPPs), exhibits potent activity against multidrug-resistant Gram-positive bacterial pathogens. The precursor peptide of nosiheptide (NosM) is comprised of a leader peptide with 37 amino acids and a core peptide containing 13 amino acids. To pinpoint elements in the leader peptide that are essential for nosiheptide biosynthesis, a collection of mutants with unique sequence features, including N- and C-terminal motifs, peptide length, and specific sites in the leader peptide, was generated by mutagenesis in vivo. The effects of various mutants on nosiheptide biosynthesis were evaluated. In addition to the necessity of a conserved motif LEIS box, native length and the N-terminal 12 amino acid residues were indispensable, and single-site substitutions of these 12 amino acid residues resulted in changes ranging from a greater-than-5-fold decrease to a 2-fold increase of nosiheptide production, depending on the sites and substituted residues. Moreover, although the C-terminal motif is not conservative, significant effects of this portion on nosiheptide production were also evident. Taken together, the present results further highlight the importance of the leader peptide in nosiheptide biosynthesis, and provide new insights into the diversity and specificity of leader peptides in the biosynthesis of various RiPPs. IMPORTANCE As a representative thiopeptide, nosiheptide exhibits excellent antibacterial activity. Although the biosynthetic gene cluster and several modification steps have been revealed, the presence and roles of the leader peptide within the precursor peptide of the nosiheptide gene cluster remain elusive. Thus, identification of specific elements in the leader peptide can significantly facilitate the genetic manipulation of the gene cluster for increasing nosiheptide production or generating diverse analogues. Given the complexity of the biosynthetic process, the instability of the leader peptide, and the unavailability of intermediates, cocrystallization of intermediates, leader peptide, and modification enzymes is currently not feasible. Therefore, a mutagenesis approach was used to construct a series of leader peptide mutants to uncover a number of crucial and characteristic elements affecting nosiheptide biosynthesis, which moves a considerable distance toward a thorough understanding of the biosynthetic machinery for thiopeptides.

scholarly journals The enduracidin biosynthetic gene cluster from Streptomyces fungicidicus

Microbiology ◽  
2006 ◽  
Vol 152 (10) ◽  
pp. 2969-2983 ◽  
Author(s):  
Xihou Yin ◽  
T. Mark Zabriskie
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Gene Cluster ◽  
Genetic Locus ◽  
Biosynthetic Gene Cluster ◽  
Antibiotic Biosynthesis ◽  
Peptide Antibiotic ◽  
Amino Acid Residues ◽  
Biosynthetic Gene ◽  
Condensation Domain

The biosynthetic gene cluster for the 17 aa peptide antibiotic enduracidin has been cloned and sequenced from Streptomyces fungicidicus ATCC 21013. The 84 kb gene cluster contains 25 ORFs and is located within a 116 kb genetic locus that was fully sequenced. Targeted disruption of non-ribosomal peptide synthetase (NRPS) genes in the cluster abolished enduracidin production and confirmed function. The cluster includes four genes, endA-D, encoding two-, seven-, eight- and one-module NRPSs, respectively, and includes unique modules for the incorporation of citrulline and enduracididine. The NRPS organization generally follows the collinearity principle, and starts with a condensation domain (C domain) similar to those found in other lipopeptide systems for the coupling of an acyl group to the starting amino acid. The sixth module of EndB, corresponding to Thr8, is missing an adenylation domain (A domain) and this module is presumed to be loaded in trans by the single module protein EndD. The most striking feature of the NRPS organization is the lack of epimerization domains (E domains) in light of the fact that the product has seven d-amino acid residues. Sequence analysis reveals that C domains following modules corresponding to d-amino acids belong to a unique subset of C domains able to catalyse both epimerization and condensation reactions. Other genes directing lipid modification and activation, and formation of the non-proteinogenic amino acids 4-hydroxyphenylglycine and enduracididine are readily identified, as are genes possibly involved in regulation of antibiotic biosynthesis and export. These findings provide the basis to further genetically manipulate and improve lipodepsipeptide antibiotics via combinatorial and chemical methods.

scholarly journals Molecular Cloning and Physical Mapping of the Daptomycin Gene Cluster from Streptomyces roseosporus

1998 ◽  
Vol 180 (1) ◽  
pp. 143-151 ◽  
Author(s):  
Margaret A. Mchenney ◽  
Thomas J. Hosted ◽  
Bradley S. Dehoff ◽  
Paul R. Rosteck ◽  
Richard H. Baltz
Keyword(s):  
Molecular Cloning ◽  
Gene Cluster ◽  
Physical Mapping ◽  
Insertional Mutagenesis ◽  
Biosynthetic Gene Cluster ◽  
Biosynthetic Gene ◽  
Linear Chromosome ◽  
Coding Regions ◽  
Streptomyces Roseosporus ◽  
Peptide Synthetase

ABSTRACT The daptomycin biosynthetic gene cluster of Streptomyces roseosporus was analyzed by Tn5099 mutagenesis, molecular cloning, partial DNA sequencing, and insertional mutagenesis with cloned segments of DNA. The daptomycin biosynthetic gene cluster spans at least 50 kb and is located about 400 to 500 kb from one end of the ∼7,100-kb linear chromosome. We identified two peptide synthetase coding regions interrupted by a 10- to 20-kb region that may encode other functions in lipopeptide biosynthesis.

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

2017 ◽  
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 <i>Anabaena</i>. 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 (<b>1</b>), from <i>Sphaerospermopsis</i> sp. LEGE 00249. The anacyclamide biosynthetic gene cluster (<i>acy</i>) encoding the novel macrocyclic prenylated cyanobactin, was sequenced. Heterologous expression of the acy gene cluster in <i>Escherichia</i> <i>coli</i> established the connection between genomic and mass spectrometric data. Unambiguous establishment of the type and site of prenylation required the full structural elucidation of <b>1</b> using Nuclear Magnetic Resonance (NMR), which demonstrated that a forward prenylation occurred on the tyrosine residue. Compound <b>1</b> was tested in pharmacologically or ecologically relevant biological assays and revealed moderate antimicrobial activity towards the fouling bacterium <i>Halomonas aquamarina</i> CECT 5000.<br>

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