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Burkholderia species have emerged as a new source of diverse natural products. This mini-review covers all of the natural products discovered in recent years from Burkholderia sp. by genome-guided approaches-these refer to the use of bacterial genome sequence as an entry point for in silico structural prediction, wet lab experimental design, and execution. While reliable structural prediction based on cryptic biosynthetic gene cluster sequence was not always possible due to noncanonical domains and/or module organization of a deduced biosynthetic pathway, a molecular genetic method was often employed to detect or alter the expression level of the gene cluster to achieve an observable phenotype, which facilitated downstream natural product purification and identification. Those examples of natural product discovery from Burkholderia sp. provide practical guidance for future exploration of Gram-negative bacteria as a new source of natural products. [ABSTRACT FROM AUTHOR]

A 135,638 bp DNA region that encompasses the leinamycin (LNM) biosynthetic gene cluster was sequenced from Streptomyces atroolivaceus S-140. The boundaries of the lnm cluster were defined by systematic inactivation of open reading frames within the sequenced region. The lnm cluster spans 61.3 kb of DNA and consists of 27 genes encoding nonribosomal peptide synthetase (NRPS), polyketide synthase (PKS), hybrid NRPS-PKS, resistance, regulatory, and tailoring enzymes, as well as proteins of unknown function. A model for LNM biosynthesis is proposed, central to which is the LNM hybrid NRPS-PKS megasynthetase consisting of discrete (LnmQ and LnmP) and modular (LnmI) NRPS, acyltransferase-less PKS (LnmG, LnmI, and LnmJ), and PKS modules with unusual domain organization. These studies unveil an unprecedented architectural complexity for the LNM hybrid NRPS-PKS megasynthetase and set the stage to investigate the molecular basis for LNM biosynthesis. [Copyright &y& Elsevier]

Type I polyketide synthases (PKSs) are multifunctional enzymes that are organized into modules, each of which minimally contains a β-ketoacyl synthase, an acyltransferase (AT), and an acyl carrier protein. Here we report that the leinamycin (LNM) biosynthetic gene cluster from Streptomyces atroolivaceus S-140 consists of two PKS genes, Inml and InmJ, that encode six PKS modules, none of which contain the cognate AT domain. The only AT activity identified within the Inm gene cluster is a discrete AT protein encoded by InmG. Inactivation of InmG, Inml, or InmJ in vivo abolished LNM biosynthesis. Biochemical characterization of LnmG in vitro showed that it efficiently and specifically loaded malonyl CoA to all six PKS modules. These findings unveiled a previously unknown PKS architecture that is characterized by a discrete, iteratively acting AT protein that loads the extender units in trans to "AT-less" multifunctional type I PKS proteins for polyketide biosynthesis. This PKS structure provides opportunities for PKS engineering as exemplified by overexpressing InmG to improve LNM production. [ABSTRACT FROM AUTHOR]

In genetic crosses, HC-toxin production in the filamentous fungus Cochliobolus carbonum appears to be controlled by a single locus, TOX2. At the molecular level, TOX2 is composed of at least seven duplicated and coregulated genes involved in HC-toxin biosynthesis, export, and regulation. All copies of four of the TOX2 genes were previously mapped within a 540-kb stretch of DNA in strain SB111. Subsequently, an additional three TOX2 genes, TOXE, TOXF, and TOXG, have been discovered. In this paper we have mapped all copies of the new genes, a total of seven, and show that except for one of the two copies of TOXE, which was previously shown to be on a chromosome of 0.7 Mb in strain SB111, they are all linked to the previously known TOX2 genes within ∼600 kb of each other on a chromosome of 3.5 Mb. We show here that this chromosome also contains at least one non-TOX2 gene, EXG2, which encodes an exo-β1,3-glucanase. EXG2 is still present in strains that have undergone spontaneous deletion of up to ∼1.4 Mb of the 3.5-Mb chromosome. The results contribute to our understanding of the complex organization of the genes involved in HC-toxin biosynthesis and are consistent with the hypothesis that a reciprocal chromosomal translocation accounts for the pattern of distribution of the TOX2 genes in different C. carbonum isolates. [Copyright &y& Elsevier]

Spiruchostatins A and B are members of the FK228-family of natural products with potent histone deacetylase inhibitory activities and antineoplastic activities. However, their production in the wild-type strain of Pseudomonas sp. Q71576 is low. To improve the yield, the spiruchostatin biosynthetic gene cluster ( spi) was first identified by rapid genome sequencing and characterized by genetic mutations. This spi gene cluster encodes a hybrid biosynthetic pathway similar to that encoded by the FK228 biosynthetic gene cluster ( dep) in Chromobacterium violaceum No. 968. Each gene cluster contains a pathway regulatory gene ( spiR vs. depR), but these two genes encode transcriptional activators of different classes. Overexpression of native spiR or heterologous depR in the wild-type strain of Pseudomonas sp. Q71576 resulted in 268 or 1,285 % increase of the combined titer of spiruchostatins A and B, respectively. RT-PCR analysis indicates that overexpression of heterologous depR upregulates the expression of native spiR. [ABSTRACT FROM AUTHOR]

Abstract: The bacterial protease ClpP consists of 14 subunits that assemble into two stacked heptameric rings. The central degradation chamber can be accessed via axial pores. In free ClpP, these pores are obstructed by the N-terminal regions of the seven subunits at either end of the barrel. Acyldepsipeptides (ADEPs) are antibacterial compounds that bind in hydrophobic clefts surrounding the pore region, causing the pores to open up. The ensuing uncontrolled degradation of intracellular proteins is responsible for the antibiotic activity of ADEPs. Recently published X-ray structures yielded conflicting models regarding the conformation adopted by the N-terminal regions in the open state. Here, we use hydrogen/deuterium exchange (HDX) mass spectrometry to obtain complementary insights into the ClpP behavior with and without ADEP1. Ligand binding causes rigidification of the equatorial belt, accompanied by destabilization in the vicinity of the binding clefts. The N-terminal regions undergo rapid deuteration with only minor changes after ADEP1 binding, revealing a lack of stable H-bonding. Our data point to a mechanism where the pore opening mechanism is mediated primarily by changes in the packing of N-terminal nonpolar side chains. We propose that a “hydrophobic plug” causes pore blockage in ligand-free ClpP. ADEP1 binding provides new hydrophobic anchor points that nonpolar N-terminal residues can interact with. In this way, ADEP1 triggers the transition to an open conformation, where nonpolar moieties are clustered around the rim of the pore. This proposed mechanism helps reconcile the conflicting models that had been put forward earlier. [Copyright &y& Elsevier]

Thailandepsin A is natural product of Burkholderia thailandensis E264 with potent histone deacetylase inhibitory activities and promising anticancer activities. The titer of thailandepsin A is very low (less than 10 mg/l) from limited empirical fermentation. To facilitate preclinical evaluations and potentially clinical development of thailandepsin A, systematic optimization and extractive fermentation of thailandepsin A from B. thailandensis E264 culture in flasks were investigated in this pilot study. The main fermentation parameters-28°C, pH 7.0, inoculum ratio 1% (v/v), incubation duration 60 h, medium volume 26%, shaking speed 170 rpm, and chloroform as extracting solvent-were determined by single factor experiments. Polyaromatic adsorbent resin Diaion HP-20, when added at a concentration of 4% (w/v), was most effective to reduce feedback inhibition of thailandepsin A and to significantly increase the titer of target product. Central composite design was used to further optimize the fermentation medium for B. thailandensis E264. The optimized medium contains glucose 17.89 g/l, tryptone 34.98 g/l, potassium phosphate 24.84 g/l, and sodium citrate 0.01 g/l, which resulted in a large increase of the titer of thailandepsin A to 236.7 mg/l. Finally kinetic models based on the modified logistic and Luedeking-Piret equations were developed, delivering a good description of temporal variations of biomass, product, and substrate in the fermentation process, which could be used as references for developing large-scale fermentation. [ABSTRACT FROM AUTHOR]

DepR, a LysR-type transcriptional regulator encoded by the last gene of the putative min operon (orf21-20-19-depR) located at the downstream region of the anticancer agent FK228 biosynthetic gene cluster in Chromobacterium violaceum No. 968, positively regulates the biosynthesis of FK228. In this work, the mechanism underlining this positive regulation was probed by multiple approaches. Electrophoretic mobility shift assay (EMSA) and DNase I footprinting assay (DIFA) identified a conserved 35-nt DNA segment in the orf21-orf22 intergenic region where the purified recombinant DepR binds to. Quantitative reverse transcription PCR (RT-qPCR) and green fluorescent protein (GFP) promoter probe assays established that transcription of phasin gene orf22 increases in the depR deletion mutant of C. violaceum (CvΔdepR) compared to the wild-type strain. FK228 production in the orf22-overexpressed strain C. violaceum was reduced compared with the wild-type strain. DepR has two conserved cysteine residues C199 and C208 presumed to form a disulfide bridge upon sensing oxidative stress. C199X point mutations that locked DepR in a reduced conformation decreased the DNA-binding affinity of DepR; T232A or R278A mutation also had a negative impact on DNA binding of DepR. Complementation of CvΔdepR with any of those versions of depR carrying a single codon mutation was not able to restore FK228 production to the level of wild-type strain. All evidences collectively suggested that DepR positively regulates the biosynthesis of FK228 through indirect metabolic networking. [ABSTRACT FROM AUTHOR]

Histone deacetylase (HDAC) inhibitors are an emerging class of targeted therapy against cancers. Thailandepsin A (TDP-A) is a recently discovered class I HDAC inhibitor with broad anti-proliferative activities. In the present study, we aimed to investigate the potential of TDP-A in the treatment of breast cancer. We demonstrated that TDP-A inhibited cell proliferation and induced apoptosis in breast cancer cells at low nanomolar concentrations. TDP-A activated the intrinsic apoptotic pathway through increase of pro-apoptotic protein Bax, decrease of anti-apoptotic Bcl-2, and cleavage of caspase-3 and poly (ADP-ribose) polymerase (PARP). TDP-A also induced cell cycle arrest at the G2/M phase, and promoted the production of reactive oxygen species (ROS). We have successfully encapsulated TDP-A into our recently developed disulfide cross-linked micelles (DCMs), improving its water solubility and targeted delivery. TDP-A loaded DCMs (TDP-A/DCMs) possess the characteristics of high loading capacity (>20%, w/w), optimal and monodisperse particle size (16 ± 4 nm), outstanding stability with redox stimuli-responsive disintegration, sustained drug release, and preferential uptake in breast tumors. In the MDA-MB-231 breast cancer xenograft model, TDP-A/DCMs were more efficacious than the FDA-approved FK228 at well-tolerated doses. Furthermore, TDP-A/DCMs exhibited synergistic anticancer effects when combined with the proteasome inhibitor bortezomib (BTZ) loaded DCMs (BTZ/DCMs). Our results indicate that TDP-A nanoformulation alone or in combination with BTZ nanoformulation are efficacious against breast cancer. [ABSTRACT FROM AUTHOR]

Abstract: Background: Anaplastic thyroid cancer (ATC) remains refractory to available surgical and medical interventions. Histone deacetylase (HDAC) inhibitors are an emerging targeted therapy with antiproliferative activity in a variety of thyroid cancer cell lines. Thailandepsin A (TDP-A) is a novel class I HDAC inhibitor whose efficacy remains largely unknown in ATC. Therefore, we aimed to characterize the effect of TDP-A on ATC. Methods: Human-derived ATC cells were treated with TDP-A. IC50 was determined by a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) rapid colorimetric assay, and cell proliferation was measured by viable cell count. Molecular mechanisms of cell growth inhibition were investigated by Western blot analysis of canonical apoptosis markers, intrinsic and extrinsic apoptosis regulators, and cell cycle regulatory proteins. Cell cycle staging was determined with propidium iodide flow cytometry. Results: TDP-A dose- and time-dependently reduced cell proliferation. Increased cleavage of the apoptosis markers Caspase-9, Caspase-3, and poly adenosine diphosphate ribose polymerase were observed with TDP-A treatment. Levels of the intrinsic apoptosis pathway proteins BAD, Bcl-XL, and BAX remained unchanged. Importantly, the extrinsic apoptosis activator cleaved Caspase-8 increased dose-dependently, and the antiapoptotic proteins Survivin and Bcl-2 decreased. Among the cell cycle regulatory proteins, levels of CDK inhibitors p21/WAF1 and p27/KIP increased. Flow cytometry showed that ATC cells were arrested in G2/M phase with diminished S phase after TDP-A treatment. Conclusions: TDP-A induces a notable dose- and time-dependent antiproliferative effect on ATC, which is mainly attributed to extrinsic apoptosis with concomitant cell cycle arrest. TDP-A therefore warrants further preclinical and clinical investigations. [Copyright &y& Elsevier]

Acyldepsipeptides ( ADEPs) antibiotics bind to Escherichia coli ClpP mimicking the interactions that the IGL/ F loops in ClpA or ClpX ATPases establish with the hydrophobic pockets surrounding the axial pore of the tetradecamer that the protease forms. ADEP binding induces opening of the gates blocking the axial channel of ClpP and allowing protein substrates to be translocated and hydrolysed in the degradation chamber. To identify the structural determinants stabilizing the open conformation of the axial channel for efficient substrate translocation, we constructed ClpP variants with amino acid substitutions in the N-terminal region that forms the axial gates. We found that adoption of a β-hairpin loop by this region and the integrity of the hydrophobic cluster at the base of this loop are necessary elements for the axial gate to efficiently translocate protein substrates. Analysis of ClpP variants from Bacillus subtilis suggested that the identified structural requirements of the axial channel for efficient translocation are conserved between Gram-positive and Gram-negative bacteria. These findings provide mechanistic insights into the activation of ClpP by ADEPs as well as the gating mechanism of the protease in the context of the ClpAP and ClpXP complexes. [ABSTRACT FROM AUTHOR]

Summary: ClpP is a cylindrical serine protease whose ability to degrade proteins is regulated by the unfoldase ATP-dependent chaperones. ClpP on its own can only degrade small peptides. Here, we used ClpP as a target in a high-throughput screen for compounds, which activate the protease and allow it to degrade larger proteins, hence, abolishing the specificity arising from the ATP-dependent chaperones. Our screen resulted in five distinct compounds, which we designate as Activators of Self-Compartmentalizing Proteases 1 to 5 (ACP1 to 5). The compounds are found to stabilize the ClpP double-ring structure. The ACP1 chemical structure was considered to have drug-like characteristics and was further optimized to give analogs with bactericidal activity. Hence, the ACPs represent classes of compounds that can activate ClpP and that can be developed as potential novel antibiotics. [ABSTRACT FROM AUTHOR]

Summary: In ClpXP and ClpAP complexes, ClpA and ClpX use the energy of ATP hydrolysis to unfold proteins and translocate them into the self-compartmentalized ClpP protease. ClpP requires the ATPases to degrade folded or unfolded substrates, but binding of acyldepsipeptide antibiotics (ADEPs) to ClpP bypasses this requirement with unfolded proteins. We present the crystal structure of Escherichia coli ClpP bound to ADEP1 and report the structural changes underlying ClpP activation. ADEP1 binds in the hydrophobic groove that serves as the primary docking site for ClpP ATPases. Binding of ADEP1 locks the N-terminal loops of ClpP in a β-hairpin conformation, generating a stable pore through which extended polypeptides can be threaded. This structure serves as a model for ClpP in the holoenzyme ClpAP and ClpXP complexes and provides critical information to further develop this class of antibiotics. [ABSTRACT FROM AUTHOR]

The article presents a comparative analysis of the large conformational change of the pseudomerohedrally twinned monoclinic structure of unfolded free nonactin upon the encapsulation of alkali metal ions. It observes the presence of symmetry-independent half-molecules of nonactin in the asymmetric unit residing on a crystallographic two-fold axis during crystallization. Also observed is the low-temperature high-precision ordered structure of free nonactin possessing an unfolded and more planar geometry but globular when encapsulating a cation.

Summary: Disulfide bonds are rare in bacterial natural products, and the mechanism of disulfide bond formation in those products is unknown. Here we characterize a gene and its product critical for a disulfide bond formation in FK228 anticancer depsipeptide in Chromobacterium violaceum. Deletion of depH drastically reduced FK228 production, whereas complementation of the depH-deletion mutant with a copy of depH on a medium copy-number plasmid not only fully restored the FK228 production but also significantly increased the FK228 yield. Purified 6xHis-tagged DepH fusion protein in native form is a homodimer of 71.0 kDa, with each monomer containing one molecule of FAD. DepH efficiently converts an immediate FK228 precursor to FK228 in the presence of NADP+. We conclude that DepH is an FAD-dependent pyridine nucleotide-disulfide oxidoreductase, specifically and efficiently catalyzing a disulfide bond formation in FK228. [Copyright &y& Elsevier]

Presents a study to address the requirement for any pectin-degrading ability in pathogenesis by Cochliobolus carbonum. Methodology used to conduct study; Indication of findings; Discussion on results.