Characterization of the Pyoluteorin Biosynthetic Gene Cluster of Pseudomonas fluorescens Pf-5

ABSTRACT Ten genes (plt) required for the biosynthesis of pyoluteorin, an antifungal compound composed of a bichlorinated pyrrole linked to a resorcinol moiety, were identified within a 24-kb genomic region of Pseudomonas fluorescens Pf-5. The deduced amino acid sequences of eight plt genes were similar to the amino acid sequences of genes with known biosynthetic functions, including type I polyketide synthases (pltB, pltC), an acyl coenzyme A (acyl-CoA) dehydrogenase (pltE), an acyl-CoA synthetase (pltF), a thioesterase (pltG), and three halogenases (pltA,pltD, and pltM). Insertions of the transposon Tn5 or Tn3-nice or a kanamycin resistance gene in each of these genes abolished pyoluteorin production by Pf-5. The presumed functions of the eight plt products are consistent with biochemical transformations involved in pyoluteorin biosynthesis from proline and acetate precursors. Isotope labeling studies demonstrated that proline is the primary precursor to the dichloropyrrole moiety of pyoluteorin. The deduced amino acid sequence of the product of another plt gene, pltR, is similar to those of members of the LysR family of transcriptional activators. pltR and pltM are transcribed divergently from the pltLABCDEFG gene cluster, and a sequence with the characteristics of a LysR binding site was identified within the 486-bp intergenic region separating pltRM frompltLABCDEFG. Transcription of the pyoluteorin biosynthesis genes pltB, pltE, and pltF, assessed with transcriptional fusions to an ice nucleation reporter gene, was significantly greater in Pf-5 than in a pltRmutant of Pf-5. Therefore, PltR is proposed to be a transcriptional activator of linked pyoluteorin biosynthesis genes.

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Genome Sequence-Guided Finding of Lucensomycin Production by Streptomyces achromogenes Subsp. streptozoticus NBRC14001

Microorganisms ◽

10.3390/microorganisms10010037 ◽

2021 ◽

Vol 10 (1) ◽

pp. 37

Author(s):

Sho Nishimura ◽

Kazune Nakamura ◽

Miyako Yamamoto ◽

Daichi Morita ◽

Teruo Kuroda ◽

...

Keyword(s):

Antifungal Activity ◽

Gene Cluster ◽

Genome Sequence ◽

Polyketide Synthase ◽

Macrolide Antibiotic ◽

Biosynthetic Gene Cluster ◽

Antifungal Compound ◽

Type I ◽

Biosynthetic Gene ◽

Polyketide Synthase Gene

Information on microbial genome sequences is a powerful resource for accessing natural products with significant activities. We herein report the unveiling of lucensomycin production by Streptomyces achromogenes subsp. streptozoticus NBRC14001 based on the genome sequence of the strain. The genome sequence of strain NBRC14001 revealed the presence of a type I polyketide synthase gene cluster with similarities to a biosynthetic gene cluster for natamycin, which is a polyene macrolide antibiotic that exhibits antifungal activity. Therefore, we investigated whether strain NBRC14001 produces antifungal compound(s) and revealed that an extract from the strain inhibited the growth of Candida albicans. A HPLC analysis of a purified compound exhibiting antifungal activity against C. albicans showed that the compound differed from natamycin. Based on HR-ESI-MS spectrometry and a PubChem database search, the compound was predicted to be lucensomycin, which is a tetraene macrolide antibiotic, and this prediction was supported by the results of a MS/MS analysis. Furthermore, the type I polyketide synthase gene cluster in strain NBRC14001 corresponded well to lucesomycin biosynthetic gene cluster (lcm) in S. cyanogenus, which was very recently reported. Therefore, we concluded that the antifungal compound produced by strain NBRC14001 is lucensomycin.

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Genetic Engineering of Chromobacterium Vaccinii DSM 25150 for Improved Production of FR900359

10.26434/chemrxiv.14452797.v1 ◽

2021 ◽

Author(s):

Dominik Pistorius ◽

Kathrin Buntin ◽

Caroline Bouquet ◽

Etienne Richard ◽

Eric Weber ◽

...

Keyword(s):

Amino Acid ◽

Genetic Engineering ◽

Gene Cluster ◽

Query Sequence ◽

Genome Mining ◽

Downstream Processing ◽

Biosynthetic Gene Cluster ◽

Amino Acid Sequences ◽

A Genome ◽

Sequence Databases

<a></a>The depsipeptide FR900359 has been first described in literature in 1988 (Fujioka et al, 1988) to be isolated from a methanol extract of the whole plant of Ardisia crenata. FR900359 can be isolated from the leaves of A. crenata, but the very low quantities and the complex matrix prevent access to sufficient amounts of FR900359 to enable drug development efforts and potential commercial manufacturing. Almost two decades later, it has been discovered that FR900359 is in fact produced by a strictly obligate bacterial endosymbiont, Candidatus Burkholderia crenata, of the plant Ardisia crenata (Carlier et al, 2016). This study identified also the DNA sequence of the biosynthetic gene cluster (BGC) of FR900359. In order to identify alternative and scalable methods for production of FR900359, a genome mining effort on bacterial genomes from both public sequence databases and genome sequences generated from internal efforts at Novartis was initiated. Translated amino acid sequences of the FR900359‑BGC from Candidatus B. crenata were used as query sequence. While the query of public sequence databases did not return highly similar sequences, a gene cluster with very high homology in translated amino acid sequence and identical prediction of protein functions was discovered in the genome of Chromobacterium vaccinii DSM 25150, which had been sequenced internally at Novartis. Here we describe the genetic engineering of Chromobacterium vaccinii DSM 25150 resulting in mutants that exhibit improved production of FR900359 and improved characteristics concerning downstream processing and purification.

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Genetic Engineering of Chromobacterium Vaccinii DSM 25150 for Improved Production of FR900359

10.26434/chemrxiv.14452797 ◽

2021 ◽

Author(s):

Dominik Pistorius ◽

Kathrin Buntin ◽

Caroline Bouquet ◽

Etienne Richard ◽

Eric Weber ◽

...

Keyword(s):

Amino Acid ◽

Genetic Engineering ◽

Gene Cluster ◽

Query Sequence ◽

Genome Mining ◽

Downstream Processing ◽

Biosynthetic Gene Cluster ◽

Amino Acid Sequences ◽

A Genome ◽

Sequence Databases

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Characterization of algG encoding C5-epimerase in the alginate biosynthetic gene cluster of Pseudomonas fluorescens

Gene ◽

10.1016/s0378-1119(01)00685-0 ◽

2001 ◽

Vol 278 (1-2) ◽

pp. 107-114 ◽

Cited By ~ 12

Author(s):

Antonella Morea ◽

Kalai Mathee ◽

Michael J. Franklin ◽

Alessio Giacomini ◽

Michael O'Regan ◽

...

Keyword(s):

Pseudomonas Fluorescens ◽

Gene Cluster ◽

Biosynthetic Gene Cluster ◽

Biosynthetic Gene

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

Applied and Environmental Microbiology ◽

10.1128/aem.00666-09 ◽

2010 ◽

Vol 76 (8) ◽

pp. 2500-2508 ◽

Cited By ~ 20

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.

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

Applied and Environmental Microbiology ◽

10.1128/aem.01419-17 ◽

2017 ◽

Vol 83 (21) ◽

Cited By ~ 9

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.

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

10.26434/chemrxiv.13142786 ◽

2020 ◽

Cited By ~ 1

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 Streptomyces cacaoi subsp. cacaoi 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.

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Characterization of the Biosynthesis Gene Cluster for the Pyrrole Polyether Antibiotic Calcimycin (A23187) inStreptomyces chartreusisNRRL 3882

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.01130-10 ◽

2010 ◽

Vol 55 (3) ◽

pp. 974-982 ◽

Cited By ~ 33

Author(s):

Qiulin Wu ◽

Jingdan Liang ◽

Shuangjun Lin ◽

Xiufen Zhou ◽

Linquan Bai ◽

...

Keyword(s):

Gene Cluster ◽

Mutational Analysis ◽

Divalent Cations ◽

Biological Effects ◽

Biosynthetic Gene Cluster ◽

Type I ◽

Biosynthesis Gene Cluster ◽

Biosynthesis Gene ◽

Hydroxyanthranilic Acid

ABSTRACTThe pyrrole polyether antibiotic calcimycin (A23187) is a rare ionophore that is specific for divalent cations. It is widely used as a biochemical and pharmacological tool because of its multiple, unique biological effects. Here we report on the cloning, sequencing, and mutational analysis of the 64-kb biosynthetic gene cluster fromStreptomyces chartreusisNRRL 3882. Gene replacements confirmed the identity of the gene cluster, andin silicoanalysis of the DNA sequence revealed 27 potential genes, including 3 genes for the biosynthesis of the α-ketopyrrole moiety, 5 genes that encode modular type I polyketide synthases for the biosynthesis of the spiroketal ring, 4 genes for the biosynthesis of 3-hydroxyanthranilic acid, anN-methyltransferase tailoring gene, a resistance gene, a type II thioesterase gene, 3 regulatory genes, 4 genes with other functions, and 5 genes of unknown function. We propose a pathway for the biosynthesis of calcimycin and assign the genes to the biosynthesis steps. Our findings set the stage for producing much desired calcimycin derivatives using genetic modification instead of chemical synthesis.

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Insights into the Selective Pressures Restricting Pelargonium Flower Break Virus Genome Variability: Evidence for Host Adaptation

Journal of Virology ◽

10.1128/jvi.00603-06 ◽

2006 ◽

Vol 80 (16) ◽

pp. 8124-8132 ◽

Cited By ~ 43

Author(s):

Patricia Rico ◽

Pilar Ivars ◽

Santiago F. Elena ◽

Carmen Hernández

Keyword(s):

Amino Acid ◽

Leucine Zipper ◽

Critical Role ◽

Chenopodium Quinoa ◽

Virus Genome ◽

Acid Sites ◽

Genomic Region ◽

Amino Acid Sequences ◽

Nucleotide Substitutions ◽

Selective Pressures

ABSTRACT The molecular diversity of Pelargonium flower break virus (PFBV) was assessed using a collection of isolates from different geographical origins, hosts, and collecting times. The genomic region examined was 1,828 nucleotides (nt) long and comprised the coding sequences for the movement (p7 and p12) and the coat (CP) proteins, as well as flanking segments including the entire 3′ untranslated region (3′ UTR). Some constraints limiting viral heterogeneity could be inferred from sequence analyses, such as the conservation of the amino acid sequences of p7 and of the shell domain of the CP, the maintenance of a leucine zipper motif in p12, and the preservation of a particular folding in the 3′ UTR. A remarkable covariation, involving five specific amino acid sites, was found in the CP of isolates largely propagated in the local lesion host Chenopodium quinoa and in the progeny of a PFBV variant subjected to serial passages in this host. Concomitant with this covariation, up to 30 nucleotide substitutions in a 1,428-nt region of the viral RNA could be attributable to C. quinoa-specific adaptation, representing one of the most outstanding cases of host-driven genome variation for a plant virus. Globally, the results indicate that the selective pressures exerted by the host play a critical role in shaping PFBV populations and that these populations are likely being selected for at both protein and RNA levels.

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