Investigating the biosynthesis and roles of the auxin phenylacetic acid during - pathogenesis.

Several plant-associated microbes synthesize the auxinic plant growth regulator phenylacetic acid (PAA) in culture; however, the role of PAA in plant-pathogen interactions is not well understood. In this study, we investigated the role of PAA during interactions between the phytopathogenic bacterium strain DC3000 (DC3000) and the model plant host, . Previous work demonstrated that indole-3-acetaldehyde dehydrogenase A (AldA) of DC3000 converts indole-3-acetaldehyde (IAAld) to the auxin indole-3-acetic acid (IAA).

Critical analysis of polycyclic tetramate macrolactam biosynthetic gene cluster phylogeny and functional diversity.

Polycyclic tetramate macrolactams (PTMs) are bioactive natural products commonly associated with certain actinobacterial and proteobacterial lineages. These molecules have been the subject of numerous structure-activity investigations since the 1970s. New members continue to be pursued in wild and engineered bacterial strains, and advances in PTM biosynthesis suggest their outwardly simplistic biosynthetic gene clusters (BGCs) belie unexpected product complexity.

Piperazate-Guided Isolation of Caveamides A and B, Cyclohexenylalanine-Containing Nonribosomal Peptides from a Cave Actinomycete.

Hybrid genome-mining/N-NMR was used to target compounds containing piperazate (Piz) residues, leading to the discovery of caveamides A () and B () from sp. strain BE230, isolated from New Rankin Cave (Missouri). Caveamides are highly dynamic molecules containing an unprecedented β-ketoamide polyketide fragment, two Piz residues, and a new -methyl-cyclohexenylalanine residue.

Chemical Stability of Petrichorins.

The structure of petrichorin C1 () converted from petrichorin C () was determined using NMR spectroscopy and X-ray crystallography. The chemical stability of petrichorins A and C ( and ) was investigated by NMR spectroscopy, X-ray crystallography, and calculations.

Discovery of unusual dimeric piperazyl cyclopeptides encoded by a DSM 44664 biosynthetic supercluster.

Rare actinomycetes represent an underexploited source of new bioactive compounds. Here, we report the use of a targeted metabologenomic approach to identify piperazyl compounds in the rare actinomycete Lentzea flaviverrucosa DSM 44664. These efforts to identify molecules that incorporate piperazate building blocks resulted in the discovery and structural elucidation of two dimeric biaryl-cyclohexapeptides, petrichorins A and B.

Multifunctional P450 Monooxygenase CftA Diversifies the Clifednamide Pool through Tandem C-H Bond Activations.

Polycyclic tetramate macrolactams (PTMs) are a class of structurally complex hybrid polyketide-nonribosomal peptide (PK-NRP) natural products produced by diverse bacteria. Several PTMs display pharmaceutically interesting bioactivities, and the early stages of PTM biosynthesis involving polyketide synthase (PKS) and nonribosomal peptide synthetase (NRPS) enzymology are well studied. However, the timing and mechanisms of post PKS-NRPS oxidations by P450 monooxygenases encoded in PTM biosynthetic gene clusters (BGCs) remain poorly characterized.

A comparative metabologenomic approach reveals mechanistic insights into antibiotic crypticity.

genomes harbor numerous, biosynthetic gene clusters (BGCs) encoding for drug-like compounds. While some of these BGCs readily yield expected products, many do not. Biosynthetic crypticity represents a significant hurdle to drug discovery, and the biological mechanisms that underpin it remain poorly understood.

Draft Genome Sequences of Two Polycyclic Tetramate Macrolactam Producers, sp. Strains JV180 and SP18CM02.

Here, we report the draft genome sequences of two related sp. strains, JV180 and SP18CM02. Despite their isolation from soils in Connecticut and Missouri (USA), respectively, they are strikingly similar in gene content.

Genomic discovery of an evolutionarily programmed modality for small-molecule targeting of an intractable protein surface.

The vast majority of intracellular protein targets are refractory toward small-molecule therapeutic engagement, and additional therapeutic modalities are needed to overcome this deficiency. Here, the identification and characterization of a natural product, WDB002, reveals a therapeutic modality that dramatically expands the currently accepted limits of druggability. WDB002, in complex with the FK506-binding protein (FKBP12), potently and selectively binds the human centrosomal protein 250 (CEP250), resulting in disruption of CEP250 function in cells.

Construction of a new integrating vector from actinophage ϕOZJ and its use in multiplex Streptomyces transformation.

Streptomyces and other closely-related actinobacteria are important sources of bioactive molecules. Streptomyces synthetic biology and genetics empower therapeutic and agrichemical development through strain improvement and biosynthetic understanding. Such efforts rely on the availability of developed molecular toolsets.

Bioinformatic and Functional Evaluation of Actinobacterial Piperazate Metabolism.

Piperazate (Piz) is a nonproteinogenic amino acid noted for its unusual N-N bond motif. Piz is a proline mimic that imparts conformational rigidity to peptides. Consequently, piperazyl molecules are often bioactive and desirable for therapeutic exploration.

Draft Genome Sequence of sp. Strain JV178, a Producer of Clifednamide-Type Polycyclic Tetramate Macrolactams.

Here, we report the draft genome sequence of sp. JV178, a strain originating from Connecticut (USA) garden soil. This strain produces the polycyclic tetramate macrolactam compounds clifednamides A and B.

Native and Engineered Clifednamide Biosynthesis in Multiple Streptomyces spp.

Polycyclic tetramate macrolactam (PTM) natural products are produced by actinomycetes and other bacteria. PTMs are often bioactive, and the simplicity of their biosynthetic clusters make them attractive for bioengineering. Clifednamide-type PTMs from Streptomyces sp.

Chemical and Biological Aspects of Nutritional Immunity-Perspectives for New Anti-Infectives that Target Iron Uptake Systems.

Upon bacterial infection, one of the defense mechanisms of the host is the withdrawal of essential metal ions, in particular iron, which leads to "nutritional immunity". However, bacteria have evolved strategies to overcome iron starvation, for example, by stealing iron from the host or other bacteria through specific iron chelators with high binding affinity. Fortunately, these complex interactions between the host and pathogen that lead to metal homeostasis provide several opportunities for interception and, thus, allow the development of novel antibacterial compounds.

Conserved biosynthetic pathways for phosalacine, bialaphos and newly discovered phosphonic acid natural products.

Natural products containing phosphonic or phosphinic acid functionalities often display potent biological activities with applications in medicine and agriculture. The herbicide phosphinothricin-tripeptide (PTT) was the first phosphinate natural product discovered, yet despite numerous studies, questions remain surrounding key transformations required for its biosynthesis. In particular, the enzymology required to convert phosphonoformate to carboxyphosphonoenolpyruvate and the mechanisms underlying phosphorus methylation remain poorly understood.

Cellulolytic Streptomyces strains associated with herbivorous insects share a phylogenetically linked capacity to degrade lignocellulose.

Actinobacteria in the genus Streptomyces are critical players in microbial communities that decompose complex carbohydrates in the soil, and these bacteria have recently been implicated in the deconstruction of plant polysaccharides for some herbivorous insects. Despite the importance of Streptomyces to carbon cycling, the extent of their plant biomass-degrading ability remains largely unknown. In this study, we compared four strains of Streptomyces isolated from insect herbivores that attack pine trees: DpondAA-B6 (SDPB6) from the mountain pine beetle, SPB74 from the southern pine beetle, and SirexAA-E (SACTE) and SirexAA-G from the woodwasp, Sirex noctilio.

Targeted discovery of polycyclic tetramate macrolactams from an environmental Streptomyces strain.

A targeted polymerase chain reaction (PCR)-based screening approach was used to identify candidate polycyclic tetramate macrolactam (PTM) biosynthetic gene clusters in environmental Streptomyces isolates. Isolation and characterization of the small molecules produced by one of the strains confirmed the production of two new PTMs (clifednamides A, 4, and B, 5) and, more generally, the utility of using a targeted approach for the discovery of new members of this interesting class.

Common biosynthetic origins for polycyclic tetramate macrolactams from phylogenetically diverse bacteria.

A combination of small molecule chemistry, biosynthetic analysis, and genome mining has revealed the unexpected conservation of polycyclic tetramate macrolactam biosynthetic loci in diverse bacteria. Initially our chemical analysis of a Streptomyces strain associated with the southern pine beetle led to the discovery of frontalamides A and B, two previously undescribed members of this antibiotic family. Genome analyses and genetic manipulation of the producing organism led to the identification of the frontalamide biosynthetic gene cluster and several biosynthetic intermediates.

Quorum-sensing-regulated bactobolin production by Burkholderia thailandensis E264.

Bacterial acyl-homoserine lactones upregulated an uncharacterized gene cluster (bta) in Burkholderia thailandensis E264 to produce an uncharacterized polar antibiotic. The antibiotic is identified as a mixture of four bactobolins. Annotation of the bta cluster allows us to propose a biosynthetic scheme for bactobolin and reveals unusual enzymatic reactions for further study.

An unusual carbon-carbon bond cleavage reaction during phosphinothricin biosynthesis.

Natural products containing phosphorus-carbon bonds have found widespread use in medicine and agriculture. One such compound, phosphinothricin tripeptide, contains the unusual amino acid phosphinothricin attached to two alanine residues. Synthetic phosphinothricin (glufosinate) is a component of two top-selling herbicides (Basta and Liberty), and is widely used with resistant transgenic crops including corn, cotton and canola.

Biosynthesis of 2-hydroxyethylphosphonate, an unexpected intermediate common to multiple phosphonate biosynthetic pathways.

Phosphonic acids encompass a common yet chemically diverse class of natural products that often possess potent biological activities. Here we report that, despite the significant structural differences among many of these compounds, their biosynthetic routes contain an unexpected common intermediate, 2-hydroxyethyl-phosphonate, which is synthesized from phosphonoacetaldehyde by a distinct family of metal-dependent alcohol dehydrogenases (ADHs). Although the sequence identity of the ADH family members is relatively low (34-37%), in vitro biochemical characterization of the homologs involved in biosynthesis of the antibiotics fosfomycin, phosphinothricin tripeptide, and dehydrophos (formerly A53868) unequivocally confirms their enzymatic activities.

Unusual transformations in the biosynthesis of the antibiotic phosphinothricin tripeptide.

Phosphinothricin tripeptide (PTT, phosphinothricylalanylalanine) is a natural-product antibiotic and potent herbicide that is produced by Streptomyces hygroscopicus ATCC 21705 (ref. 1) and Streptomyces viridochromogenes DSM 40736 (ref. 2).

Heterologous production of fosfomycin and identification of the minimal biosynthetic gene cluster.

Fosfomycin is a clinically utilized, highly effective antibiotic, which is active against methicillin- and vancomycin-resistant pathogens. Here we report the cloning and characterization of a complete fosfomycin biosynthetic cluster from Streptomyces fradiae and heterologous production of fosfomycin in S. lividans.

Molecular cloning, sequence analysis, and heterologous expression of the phosphinothricin tripeptide biosynthetic gene cluster from Streptomyces viridochromogenes DSM 40736.

A fosmid library from genomic DNA of Streptomyces viridochromogenes DSM 40736 was constructed and screened for the presence of genes known to be involved in the biosynthesis of phosphinothricin tripeptide (PTT). Eight positives were identified, one of which was able to confer PTT biosynthetic capability upon Streptomyces lividans after integration of the fosmid into the chromosome of this heterologous host. Sequence analysis of the 40,241-bp fosmid insert revealed 29 complete open reading frames (ORFs).