Search

Bacterial quorum sensing is a chemical language allowing bacteria to interact through the excretion of molecules called autoinducers, like N-acyl-homoserine lactones (AHLs) produced by Gram-negative Burkholderia and Paraburkholderia bacteria known as opportunistic pathogens. The AHLs differ in their acyl-chain length and may be modified by a 3-oxo or 3-hydroxy substituent, or C = C double bonds at different positions. As the bacterial signal specificity depends on all of these chemical features, their structural characterization is essential to have a better understanding of the population regulation and virulence phenomenon. This study aimed at enabling the localization of the C = C double bond on such specialized metabolites while using significantly lower amounts of biological material. The approach is based on LC-MS/MS analyses of bacterial extracts after in-solution derivatization by a photochemical Paternò-Büchi reaction, leading to the formation of an oxetane ring and subsequently to specific fragmentations when performing MS/MS experiments. The in-solution derivatization of AHLs was optimized on several standards, and then the matrix effect of bacterial extracts on the derivatization was assessed. As a proof of concept, the optimized conditions were applied to a bacterial extract enabling the localization of C = C bonds on unsaturated AHLs., (© 2024. The Author(s), under exclusive licence to Springer-Verlag GmbH, DE part of Springer Nature.)

The development and application of an electrochemical sensor is reported for detection of poly(3-hydroxybutyrate) (P3HB) - a bioplastic derived from agro-industrial residues. To overcome the challenges of molecular imprinting of macromolecules such as P3HB, this study employed methanolysis reaction to break down the P3HB biopolymer chains into methyl 3-hydroxybutyrate (M3HB) monomers. Thereafter, M3HB were employed as the target molecules in the construction of molecularly imprinted sensors. The electrochemical device was then prepared by electropolymerizing a molecularly imprinted poly (indole-3-acetic acid) thin film on a glassy carbon electrode surface modified with reduced graphene oxide (GCE/rGO-MIP) in the presence of M3HB. Electrochemical impedance spectroscopy (EIS), cyclic voltammetry (CV), scanning electron microscopy with field emission gun (SEM-FEG), Raman spectroscopy, attenuated total reflection Fourier-transform infrared (ATR-FTIR) and X-ray Photoelectron Spectroscopy (XPS) were employed to characterize the electrode surface. Under ideal conditions, the MIP sensor exhibited a wide linear working range of 0.1 - 10 nM and a detection limit of 0.3 pM (n = 3). The sensor showed good repeatability, selectivity, and stability over time. For the sensor application, the bioproduction of P3HB was carried out in a bioreactor containing the Burkholderia glumae MA13 strain and sugarcane byproducts as a supplementary carbon source. The analyses were validated through recovery assays, yielding recovery values between 102 and 104%. These results indicate that this MIP sensor can present advantages in the monitoring of P3HB during the bioconversion process., (© 2024. The Author(s), under exclusive licence to Springer-Verlag GmbH Austria, part of Springer Nature.)

The rise of multidrug resistant fungal infections highlights the need to identify and develop novel antifungal agents. Occidiofungin is a nonribosomally synthesized glycolipopeptide that has a unique mechanism of action, disrupting actin-mediated functions and inducing cellular apoptosis. Antifungal activity has been observed in vitro against various fungal species, including multidrug resistant Candida auris , and in vivo efficacy has been demonstrated in a murine vulvovaginal candidiasis model. Occidiofungin, a cyclic glycolipopeptide, is composed of eight amino acids and in previous studies, an asparagine residue was assigned at position 7 (ASN7). In this study, new structural variants of occidiofungin have been characterized which have aspartic acid (ASP7), glutamine (GLN7), or glutamic acid (GLU7) at position 7. The side chain of the ASP7 variant contains the only terminal carboxylic acid in the peptide and provides a useful site for selective chemical modifications. Analogues were synthesized at the ASP7 position and tested for antifungal activity. These analogues were shown to be more active as compared to the ASP7 variant against a panel of Candida species. The naturally occurring variants of occidiofungin with a side chain containing a carboxylic acid at the seventh amino acid position can be used to develop semisynthetic analogues with enhanced therapeutic properties.

We present the isolation and structural characterization of a novel nonionic dirhamnolipid methyl ester produced by the bacterium Burkholderia lata. The structure and the absolute configuration of the isolated dirhamnolipid bearing a symmetrical C 14 -C 14 methyl ester chain were thoroughly investigated through chemical degradation and spectroscopic methods including 1D and 2D NMR analysis, HR-ESI-TOF-MS, chiral GC-MS, and polarimetry. Our work represents the first mention in the literature of a rhamnolipid methyl ester from Burkholderia species., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Charles Gauthier reports financial support was provided by Natural Sciences and Engineering Research Council of Canada. Eric Deziel reports financial support was provided by Natural Sciences and Engineering Research Council of Canada. Serge Lavoie reports financial support was provided by Natural Sciences and Engineering Research Council of Canada. Charles Gauthier reports financial support was provided by Quebec Health Research Fund. Stephan Thies reports financial support was provided by Ministry of Culture and Science (NRW, Germany). Sonja Kubicki reports financial support was provided by Ministry of Culture and Science (NRW, Germany). Sonja Kubicki reports financial support was provided by Mitacs Canada. If there are other authors, they declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier Ltd. All rights reserved.)

Melioidosis is caused by Burkholderia pseudomallei (Bp) acquired from the environment. Conventional identification methods for environmental Bp are challenging due to the presence of closely related species. Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) is accurate for bacterial identification, but has been little used to identify Bp from environmental samples. This study aims to evaluate MALDI-TOF MS for the identification of Bp and closely related species isolated from environmental samples in Thailand using whole-genome sequencing (WGS) as the gold standard, including determining the best sample preparation method for this purpose. We identified Bp (n = 22), Burkholderia spp. (n = 28), and other bacterial species (n = 32) using WGS. MALDI-TOF analysis of all Bp isolates yielded results consistent with WGS. A decision-tree algorithm identified 16 important variable peaks, using the protein extraction method (PEM), demonstrating distinct MALDI-TOF profiles for the three categories (Bp, Burkholderia spp. and "other bacterial species"). Three biomarker peaks (4060, 5196, and 6553 Da) could discriminate Bp from other Burkholderia and closely related species with 100% sensitivity and specificity. Hence, the MALDI-TOF technique has shown its potential as a species discriminatory tool, providing results comparable to WGS for classification and surveillance of environmental Bp., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 The Authors. Published by Elsevier B.V. All rights reserved.)

FK228 (romidepsin) is the only natural histone deacetylases (HDACs) inhibitor approved by FDA to treat cutaneous and peripheral T-cell lymphoma. However, the limited supply and severe cardiotoxicity of FK228 underscore the importance to develop an effective synthetic biology platform for the manufacturing and fine-tuning of this drug lead. In this work, we constructed a Burkholderia chassis for the high-yield production of FK228-family (unnatural) natural products. By virtue of the optimized Burkholderia-specific recombineering system, the biosynthetic gene cluster (BGC) encoding the FK228-like skeleton thailandepsins (tdp) in Burkholderia thailandensis E264 was replaced with an attB integration site to afford the basal chassis KOGC1. The tdp BGC directly captured from E264 was hybridized with the FK228-encoding BGC (dep) using the versatile Red/ET technology. The hybrid BGC (tdp-dep) was integrated into the attB site of KOGC1, resulting in the heterologous expression of FK228. Remarkably, the titer reached 581 mg/L, which is 30-fold higher than that of native producer Chromobacterium violaceum No. 968. This success encouraged us to further engineer the NRPS modules 4 or 6 of hybrid tdp-dep BGC by domain units swapping strategy, and eight new FK228 derivatives (1-8) varying in the composition of amino acids were generated. Especially, the titers of 2 and 3 in KOGC1 were up to 985 mg/L and 453 mg/L, respectively. 2 and 3 displayed stronger cytotoxic activity than FK228. All in all, this work established a robust platform to produce FK228 and its new derivatives in sufficient quantities for anticancer drug development., Competing Interests: Declaration of competing interest The authors declare no competing financial interest(s)., (Copyright © 2022 International Metabolic Engineering Society. Published by Elsevier Inc. All rights reserved.)

Multidrug-resistant pathogens such as Burkholderia cenocepacia have become a hazard in the context of healthcare-associated infections, especially for patients admitted with cystic fibrosis or immuno-compromising conditions. Like other opportunistic Gram-negative bacteria, this pathogen establishes virulence and biofilms through lectin-mediated adhesion. In particular, the superlectin BC2L-C is believed to cross-link human epithelial cells to B. cenocepacia during pulmonary infections. We aimed to obtain glycomimetic antagonists able to inhibit the interaction between the N -terminal domain of BC2L-C (BC2L-C-Nt) and its target fucosylated human oligosaccharides. In a previous study, we identified by fragment virtual screening and validated a small set of molecular fragments that bind BC2L-C-Nt in the vicinity of the fucose binding site. Here, we report the rational design and synthesis of bifunctional C - or N -fucosides, generated by connecting these fragments to a fucoside core using a panel of rationally selected linkers. A modular route starting from two key fucoside intermediates was implemented for the synthesis, followed by evaluation of the new compounds as BC2L-C-Nt ligands with a range of techniques (surface plasmon resonance, isothermal titration calorimetry, saturation transfer difference NMR, differential scanning calorimetry, and X-ray crystallography). This study resulted in a hit molecule with an order of magnitude gain over the starting methyl fucoside and in two crystal structures of antagonist/lectin complexes.

Species of Burkholderia are highly versatile being found not only abundantly in soil, but also as plants and animals' commensals or pathogens. Their complex multireplicon genomes harbour an impressive number of polyketide synthase (PKS) and nonribosomal peptide-synthetase (NRPS) genes coding for the production of antimicrobial secondary metabolites (SMs), which have been successfully deciphered by genome-guided tools. Moreover, genome metrics supported the split of this genus into Burkholderia sensu stricto (s.s.) and five new other genera. Here, we show that the successful antimicrobial SMs producers belong to Burkholderia s.s. Additionally, we reviewed the occurrence, bioactivities, modes of action, structural, and biosynthetic information of thirty-eight Burkholderia antimicrobial SMs shedding light on their diversity, complexity, and uniqueness as well as the importance of genome-guided strategies to facilitate their discovery. Several Burkholderia NRPS and PKS display unusual features, which are reflected in their structural diversity, important bioactivities, and varied modes of action. Up to now, it is possible to observe a general tendency of Burkholderia SMs being more active against fungi. Although the modes of action and biosynthetic gene clusters of many SMs remain unknown, we highlight the potential of Burkholderia SMs as alternatives to fight against new diseases and antibiotic resistance.

Carbohydrate-binding proteins (lectins) are auspicious targets in drug discovery to combat antimicrobial resistance; however, their non-carbohydrate drug-like inhibitors are still unavailable. Here, we present a druggable pocket in a β-propeller lectin BambL from Burkholderia ambifaria as a potential target for allosteric inhibitors. This site was identified employing 19 F NMR fragment screening and a computational pocket prediction algorithm SiteMap. The structure-activity relationship study revealed the most promising fragment with a dissociation constant of 0.3±0.1 mM and a ligand efficiency of 0.3 kcal mol -1  HA -1 that affected the orthosteric site. This effect was substantiated by site-directed mutagenesis in the orthosteric and secondary pockets. Future drug-discovery campaigns that aim to develop small molecule inhibitors can benefit from allosteric sites in lectins as a new therapeutic approach against antibiotic-resistant pathogens., (© 2021 The Authors. Angewandte Chemie International Edition published by Wiley-VCH GmbH.)

Gram-negative, aerobic, rod-shaped, non-spore-forming, motile bacteria, designated CBAS 719  T , CBAS 732 and CBAS 720 were isolated from leaf litter samples, collected in Espírito Santo State, Brazil, in 2008. Sequences of the 16S rRNA, gyrB, lepA and recA genes showed that these strains grouped with Burkholderia plantarii LMG 9035  T , Burkholderia gladioli LMG 2216  T and Burkholderia glumae LMG 2196  T in a clade of phytopathogenic Burkholderia species. Digital DNA-DNA hybridization experiments and ANI analyses demonstrated that strain CBAS 719  T represents a novel species in this lineage that is very closely related with B. plantarii. The genome sequence of the type strain is 7.57 Mbp and its G + C content is 69.01 mol%. The absence of growth on TSA medium supplemented with 3% (w/v) NaCl, citrate assimilation, β-galactosidase (PNPG) activity, and of lipase C14 activity differentiated strain CBAS 719  T from B. plantarii LMG 9035  T , its nearest phylogenetic neighbor. Its predominant fatty acid components were C 16:0 , C 18:1 ω7c, cyclo-C 17:0 and summed feature 3 (C 16:1 ω7c and/or C 15:0 iso 2-OH). Based on these genotypic and phenotypic characteristics, the strains CBAS 719  T , CBAS 732 and CBAS 720 are classified in a novel Burkholderia species, for which the name Burkholderia perseverans sp. nov. is proposed. The type strain is CBAS 719  T (= LMG 31557  T  = INN12 T )., (© 2021. Sociedade Brasileira de Microbiologia.)

Burkholderia territorii , a Gram-negative bacterium, encodes for the ι-class carbonic anhydrase (CA, EC 4.2.1.1) BteCAι, which was recently characterised. It acts as a good catalyst for the hydration of CO 2 to bicarbonate and protons, with a k cat value of 3.0 × 10 5  s -1 and k cat /K M value of 3.9 × 10 7  M -1 s -1 . No inhibition data on this new class of enzymes are available to date. We report here an anion and small molecules inhibition study of BteCAι, which we prove to be a zinc(II)- and not manganese(II)-containing enzyme, as reported for diatom ι-CAs. The best inhibitors were sulphamic acid, stannate, phenylarsonic acid, phenylboronic acid and sulfamide (K I values of 6.2-94 µM), whereas diethyldithiocarbamate, tellurate, selenate, bicarbonate and cyanate were submillimolar inhibitors (K I values of 0.71-0.94 mM). The halides (except iodide), thiocyanate, nitrite, nitrate, carbonate, bisulphite, sulphate, hydrogensulfide, peroxydisulfate, selenocyanate, fluorosulfonate and trithiocarbonate showed K I values in the range of 3.1-9.3 mM.

Inflammasomes are cytosolic multiprotein signaling complexes that are activated upon pattern recognition receptor-mediated recognition of pathogen-derived ligands or endogenous danger signals. Their assembly activates the downstream inflammatory caspase-1 and caspase-4/5 (human) or caspase-11 (mouse), which induces cytokine release and pyroptotic cell death through the cleavage of the pore-forming effector gasdermin D. Pathogen detection by host cells also results in the production and release of interferons (IFNs), which fine-tune inflammasome-mediated responses. IFN-induced guanylate-binding proteins (GBPs) have been shown to control the activation of the noncanonical inflammasome by recruiting caspase-4 on the surface of cytosolic Gram-negative bacteria and promoting its interaction with lipopolysaccharide (LPS). The Gram-negative opportunistic bacterial pathogen Burkholderia thailandensis infects epithelial cells and macrophages and hijacks the host actin polymerization machinery to spread into neighboring cells. This process causes host cell fusion and the formation of so-called multinucleated giant cells (MNGCs). Caspase-1- and IFN-regulated caspase-11-mediated inflammasome pathways play an important protective role against B. thailandensis in mice, but little is known about the role of IFNs and inflammasomes during B. thailandensis infection of human cells, particularly epithelial cells. Here, we report that IFN-γ priming of human epithelial cells restricts B. thailandensis-induced MNGC formation in a GBP1-dependent manner. Mechanistically, GBP1 does not promote bacteriolysis or impair actin-based bacterial motility but acts by inducing caspase-4-dependent pyroptosis of the infected cell. In addition, we show that IFN-γ priming of human primary macrophages confers a more efficient antimicrobial effect through inflammasome activation, further confirming the important role that interferon signaling plays in restricting Burkholderia replication and spread. IMPORTANCE The Gram-negative bacteria of the Burkholderia species are associated with human diseases ranging from pneumonia to life-threatening melioidosis. Upon infection through inhalation, ingestion, or the percutaneous route, these bacteria can spread and establish granuloma-like lesions resulting from the fusion of host cells to form multinucleated giant cells (MNGCs). Burkholderia resistance to several antibiotics highlights the importance to better understand how the innate immune system controls infections. Here, we report that interferons protect human epithelial cells against Burkholderia- induced MNGC formation, specifically through the action of the interferon-induced GBP1 protein. Mechanistically, GBP1 acts by inducing caspase-4-dependent cell death through pyroptosis, allowing the infected cells to be quickly eliminated before bacterial spread and the formation of MNGCs. This study provides evidence that interferon-induced innate immune activation, through GBP1 and caspase-4, confers protection against Burkholderia infection, potentially opening new perspectives for therapeutic approaches.

The dinitrotoluene isomers 2,4 and 2,6-dinitrotoluene (DNT) represent highly toxic, mutagenic, and carcinogenic compounds used in explosive manufacturing and in commercial production of polyurethane foam. Bioremediation, the use of microbes to degrade residual DNT in industry wastewaters, represents a promising, low cost and environmentally friendly alternative technology to landfilling. In the present study, the effect of different bioremediation strategies on the degradation of DNT in a microcosm-based study was evaluated. Biostimulation of the indigenous microbial community with sulphur phosphate (2.3 g/kg sludge) enhanced DNT transformation (82% transformation, from 300 g/L at Day 0 to 55 g/L in week 6) compared to natural attenuation over the same period at 25 °C. The indigenous microbial activity was found to be capable of transforming the contaminant, with around 70% transformation of DNT occurring over the microcosm study. 16S rDNA sequence analysis revealed that while the original bacterial community was dominated by Gammaproteobacteria (30%), the addition of sulphur phosphate significantly increased the abundance of Betaproteobacteria by the end of the biostimulation treatment, with the bacterial community dominated by Burkholderia (46%) followed by Rhodanobacter , Acidovorax and Pseudomonas . In summary, the results suggest biostimulation as a treatment choice for the remediation of dinitrotoluenes and explosives waste.

We report a promoter-assisted glycosidation approach for the stereoselective synthesis of the 6-deoxy-β-d- manno -heptopyranose oligosaccharides. SphosAuNTf 2 -promoted glycosidation of 6-deoxy-d- manno -heptopyranosyl o -hexynylbenzoate with common alcohols afforded a range of 6-deoxy-d- manno -heptosides with good to excellent β-selectivities. The counterion and the ligand of SPhosAuNTf 2 were found to have a dramatic effect on the formation of the 1,2- cis -β-linked 6-deoxy-d- manno -heptosides. This approach was effectively applied to the stereocontrolled synthesis of the 6-deoxy-β-d- manno -heptopyranose oligosaccharides relevant to Burkholderia pseudomallei and Burkholderia mallei .

Objective: Lipases are often used in immobilized form, but commercial immobilized lipases are costly. An alternative is to produce lipases in solid-state fermentation, dry the solids and then use the "dry fermented solids" (DFS) directly. We produced DFS by growing Burkholderia contaminans on a mixture of sugarcane bagasse and sunflower seed meal and used the DFS to esterify oleic acid with ethanol in subcritical and supercritical CO 2 at 40 °C., Results: Compared to a control without CO 2 at atmospheric pressure, subcritical CO 2 at 30 bar improved esterification activity 1.2-fold. Higher pressures, including supercritical pressures up to 150 bar, reduced activity to less than 80% of the control. At 30 bar, the esterification activity was improved a further 1.8-fold with the addition of 9% water (i.e. 9 g water per 100 g oleic acid) to the reaction medium., Conclusion: A subcritical CO 2 atmosphere, with the addition of a small amount of water, improved the esterification activity of DFS containing lipases of Burkholderia contaminans.

Genome mining of one of the protective symbionts (Burkholderia gladioli) of the invasive beetle Lagria villosa revealed a cryptic gene cluster that codes for the biosynthesis of a novel antifungal polyketide with a glutarimide pharmacophore. Targeted gene inactivation, metabolic profiling, and bioassays led to the discovery of the gladiofungins as previously-overlooked components of the antimicrobial armory of the beetle symbiont, which are highly active against the entomopathogenic fungus Purpureocillium lilacinum. By mutational analyses, isotope labeling, and computational analyses of the modular polyketide synthase, we found that the rare butenolide moiety of gladiofungins derives from an unprecedented polyketide chain termination reaction involving a glycerol-derived C3 building block. The key role of an A-factor synthase (AfsA)-like offloading domain was corroborated by CRISPR-Cas-mediated gene editing, which facilitated precise excision within a PKS domain., (© 2020 The Authors. Published by Wiley-VCH GmbH.)

The products of most secondary metabolite biosynthetic gene clusters (BGCs) have yet to be discovered, in part due to low expression levels in laboratory cultures. Reporter-guided mutant selection (RGMS) has recently been developed for this purpose: a mutant library is generated and screened, using genetic reporters to a chosen BGC, to select transcriptionally active mutants that then enable the characterization of the "cryptic" metabolite. The requirement for genetic reporters limits the approach to a single pathway within genetically tractable microorganisms. Herein, we utilize untargeted metabolomics in conjunction with transposon mutagenesis to provide a global read-out of secondary metabolism across large numbers of mutants. We employ self-organizing map analytics and imaging mass spectrometry to identify and characterize seven cryptic metabolites from mutant libraries of two different Burkholderia species. Applications of the methodologies reported can expand our understanding of the products and regulation of cryptic BGCs across phylogenetically diverse bacteria.

Mass spectrometry has become an indispensable tool for the characterization of glycosylation across biological systems. Our ability to generate rich fragmentation of glycopeptides has dramatically improved over the last decade yet our informatic approaches still lag behind. Although glycoproteomic informatics approaches using glycan databases have attracted considerable attention, database independent approaches have not. This has significantly limited high throughput studies of unusual or atypical glycosylation events such as those observed in bacteria. As such, computational approaches to examine bacterial glycosylation and identify chemically diverse glycans are desperately needed. Here we describe the use of wide-tolerance (up to 2000 Da) open searching as a means to rapidly examine bacterial glycoproteomes. We benchmarked this approach using N -linked glycopeptides of Campylobacter fetus subsp. fetus as well as O -linked glycopeptides of Acinetobacter baumannii and Burkholderia cenocepacia revealing glycopeptides modified with a range of glycans can be readily identified without defining the glycan masses before database searching. Using this approach, we demonstrate how wide tolerance searching can be used to compare glycan use across bacterial species by examining the glycoproteomes of eight Burkholderia species ( B. pseudomallei; B. multivorans; B. dolosa; B. humptydooensis; B. ubonensis, B. anthina; B. diffusa; B. pseudomultivorans ). Finally, we demonstrate how open searching enables the identification of low frequency glycoforms based on shared modified peptides sequences. Combined, these results show that open searching is a robust computational approach for the determination of glycan diversity within bacterial proteomes., Competing Interests: Conflict of interest—Authors declare no competing interests., (© 2020 Ahmad Izaham and Scott.)

Pathogenic bacteria of the Burkholderia pseudomallei group cause severe infectious diseases such as glanders and melioidosis. Malleicyprols were identified as important bacterial virulence factors, yet the biosynthetic origin of their cyclopropanol warhead has remained enigmatic. By a combination of mutational analysis and metabolomics we found that sulfonium acids, dimethylsulfoniumpropionate (DMSP) and gonyol, known as osmolytes and as crucial components in the global organosulfur cycle, are key intermediates en route to the cyclopropanol unit. Functional genetics and in vitro analyses uncover a specialized pathway to DMSP involving a rare prokaryotic SET-domain methyltransferase for a cryptic methylation, and show that DMSP is loaded onto the NRPS-PKS hybrid assembly line by an adenylation domain dedicated to zwitterionic starter units. Then, the megasynthase transforms DMSP into gonyol, as demonstrated by heterologous pathway reconstitution in E. coli., (© 2020 The Authors. Published by Wiley-VCH Verlag GmbH & Co. KGaA.)

The Burkholderia genus offers a promising potential in medicine because of the diversity of biologically active natural products encoded in its genome. Some pathogenic Burkholderia spp. biosynthesize a specific class of antimicrobial 2-alkyl-4(1 H )-quinolones, i.e., 4-hydroxy-3-methyl-2-alkenylquinolines (HMAQs) and their N -oxide derivatives (HMAQNOs). Herein, we report the synthesis of a series of six HMAQs and HMAQNOs featuring a trans -Δ 2 double bond at the C2-alkyl chain. The quinolone scaffold was obtained via the Conrad-Limpach approach, while the ( E )-2-alkenyl chain was inserted through Suzuki-Miyaura cross-coupling under microwave radiation without noticeable isomerization according to the optimized conditions. Subsequent oxidation of enolate-protected HMAQs cleanly led to the formation of HMAQNOs following cleavage of the ethyl carbonate group. Synthetic HMAQs and HMAQNOs were evaluated in vitro for their antimicrobial activity against different Gram-negative and Gram-positive bacteria as well as against molds and yeasts. The biological results support and extend the potential of HMAQs and HMAQNOs as antimicrobials, especially against Gram-positive bacteria. We also confirm the involvement of HMAQs in the autoregulation of the Hmq system in Burkholderia ambifaria .

The BthA protein from the microorganism Burkholderia thailandensis contains two hemes with axial His/OH 2 and His/Tyr coordinations separated by the closest interheme distance of 14 Å. BthA has a similar structure and belongs to the same family of multiheme cytochrome c peroxidases as MauG, which performs long-range oxidation of the partner protein methylamine dehydrogenase. Magnetic Mössbauer spectroscopy of the diferric state of BthA corroborates previous structural work identifying a high-spin (His/OH 2 ) peroxidatic heme and a low-spin (His/Tyr) electron transfer heme. Unlike MauG, addition of H 2 O 2 fully converts the diferric form of BthA to a stable 2e - oxidized state, allowing a new assessment of this state. The peroxidatic heme is found to be oxidized to a canonical compound II, S = 1 oxoiron(IV) heme. In contrast, the electronic properties of the oxidized His/Tyr heme are puzzling. The isomer shift of the His/Tyr heme (0.17 mm/s) is close to that of the precursor S = 1/2 Fe 3+ heme (0.21 mm/s) which suggests oxidation of the Tyr. However, the spin-dipolar hyperfine coupling constants are found here to be the same as those for the ferryl peroxidatic heme, indicating that the His/Tyr heme is also a compound II, S = 1 Fe 4+ heme and ruling out oxidation of the Tyr. DFT calculations indicate that the unusually high isomer shift is not attributable to the rare axial His/Tyr heme coordination. The calculations are only compatible with spectroscopy for an unusually long Fe 4+ -O Tyr distance, which is presumably under the influence of the protein environment of the His/Tyr heme moiety in the H 2 O 2 oxidized state of the protein. The results offer new insights into how high valence intermediates can be tuned by the protein environment for performing long-range oxidation.

Burkholderia thailandensis has emerged as a model organism for investigating the production and regulation of diverse secondary metabolites. Most of the biosynthetic gene clusters encoded in B. thailandensis are silent, motivating the development of new methods for accessing their products. In the current work, we add to the canon of available approaches using phenotype-guided transposon mutagenesis to characterize a silent biosynthetic gene cluster. Because secondary metabolite biosynthesis is often associated with phenotypic changes, we carried out random transposon mutagenesis followed by phenotypic inspection of the resulting colonies. Several mutants exhibited intense pigmentation and enhanced expression of an iterative type I polyketide synthase cluster that we term org . Disruptions of orgA , orgB , and orgC abolished the biosynthesis of the diffusible pigment, thus linking it to the org operon. Isolation and structural elucidation by HR-MS and 1D/2D NMR spectroscopy revealed three novel, cryptic metabolites, thailandene A-C. Thailandenes are linear formylated or acidic polyenes containing a combination of cis and trans double bonds. Variants A and B exhibited potent antibiotic activity against Staphylococcus aureus and Saccharomyces cerevisiae but not against Escherichia coli . One of the transposon mutants that exhibited an enhanced expression of org contained an insertion upstream of a σ54-dependent transcription factor. Closer inspection of the org operon uncovered a σ54 promoter consensus sequence upstream of orgA , providing clues regarding its regulation. Our results showcase the utility of phenotype-guided transposon mutagenesis in uncovering cryptic metabolites encoded in bacterial genomes.

Plant cell suspension culture systems are valuable for the study of complex biological systems such as inducible defence responses and aspects of plant innate immunity. Perturbations to the cellular metabolome can be investigated using metabolomic approaches in order to reveal the underlying metabolic mechanism of cellular responses. Lipopolysaccharides from the sorghum pathogen, Burkholderia andropogonis (LPS B.a. ), were purified, chemically characterised and structurally elucidated. The lipid A moiety consists of tetra- and penta-acylated 1,4'-bis-phosphorylated disaccharide backbone decorated by aminoarabinose residues, while the O-polysaccharide chain consists of linear trisaccharide repeating units of [→2)-α-Rha3CMe-(1 → 3)-α-Rha-(1 → 3)-α-Rha-(1 → ]. The effect of LPS B.a. in triggering metabolic reprogramming in Sorghum bicolor cells were investigated using untargeted metabolomics with liquid chromatography coupled to mass spectrometry detection. Cells were treated with LPS B.a. and the metabolic changes monitored over a 30 h time period. Alterations in the levels of phytohormones (jasmonates, zeatins, traumatic-, azelaic- and abscisic acid), which marked the onset of defence responses and accumulation of defence-related metabolites, were observed. Phenylpropanoids and indole alkaloids as well as oxylipins that included di- and trihydroxyoctadecedienoic acids were identified as signatory biomarkers, with marked secretion into the extracellular milieu. The study demonstrated that sorghum cells recognise LPS B.a. as a 'microbe-associated molecular pattern', perturbing normal cellular homeostasis. The molecular features of the altered metabolome were associated with phytohormone-responsive metabolomic reconfiguration of primary and secondary metabolites originating from various metabolic pathways, in support of defence and immunity.

The culture broth of Burkholderia rinojensis strain A396 is herbicidal to a number of weed species with greater observed efficacy against broadleaf than grass weeds. A portion of this activity is attributed to romidepsin, a 16-membered cyclic depsipeptide bridged by a 15-membered macrocyclic disulfide. Romidepsin, which is present in small amounts in the broth (18 to 25 μg mL -1 ), was isolated and purified using standard chromatographic techniques. It was established that romidepsin is a natural proherbicide that targets the activity of plant histone deacetylases (HDAC). Assays to measure plant HDAC activity were optimized by testing a number of HDAC substrates. The activity of romidepsin was greater when its macrocyclic-forming disulfide bridge was reduced to liberate a highly reactive free butenyl thiol side chain. Reduction was achieved using 200 mM tris(2-carboxyethyl)phosphine hydrochloride. A similar bioactivation of the proherbicide via reduction of the disulfide bridge of romidepsin was observed in plant-cell-free extracts. Molecular dynamic simulation of the binding of romidepsin to Arabidopsis thaliana HDAC19 indicated the reduced form of the compound could reach deep inside the catalytic domain and interact with an associated zinc atom required for enzyme activity.

The endophyte Burkholderia sp. WYAT7 isolated from the medicinal plant Artemisia nilagirica (Clarke) Pamp. was analyzed for its ability to produce biosurfactant. The evaluation of biosurfactant production was conducted using different screening methods which confirmed the presence of biosurfactant in the culture supernatant. CTAB- methylene blue agar plate method was used for the screening of glycolipid biosurfactant production. The biosurfactant produced by the bacteria effectively metabolized hydrocarbons present in the bacterial culture media. Fourier transform infrared spectroscopic (FTIR) analysis of biosurfactant provided the details regarding OH stretching, stretching vibrations of acyl chain, CO stretching, stretching vibrations of ether and vibrations of glycosidic linkages in the biosurfactant. The stretching vibrations of glycosidic linkage in the fingerprint regions of FTIR spectrum (1200 cm -1 to 800 cm -1 regions) confirms that the biosurfactant produced was a glycolipid. The GC-MS analysis confirmed the methyl and ethyl esters of fatty acids. The biosurfactant from the bacteria exhibited antibacterial activity against bacterial pathogens such as Pseudomonas aeruginosa (MTCC 2453), Escherichia coli (MTCC 1610), Salmonella paratyphi and Bacillus subtilis. The glycolipid biosurfactant had antibiofilm activity as evidenced in Staphylococcus aureus (MTCC 1430). All these results indicated the beneficial effect of the biosurfactant in plant-endophyte interactions. The properties exhibited by the biosurfactant suggest that it can be exploited commercially for the production of novel antibiotics., Competing Interests: Declaration of Competing Interest The authors of the manuscript entitled “Characterization of biosurfactant produced by the endophyte Burkholderia sp. WYAT7 and evaluation of its antibacterial and antibiofilm potentials” declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier B.V. All rights reserved.)

The present study was performed to synthesize, for the first time, the magnesium oxide nanoparticles (MgO NPs) using the cell filtrate of the endobacterium Burkholderia rinojensis. The MgO NPs were characterized by Ultraviolet-visible (UV-Vis), Fourier-transform infrared (FTIR), X-ray diffraction (XRD), Energy dispersive X-ray (EDX), scanning electron microscopy (SEM), transmission electron microscopy (TEM), and zeta potential (ZP). The UV spectrum of the MgO NPs showed a sharp absorption peak at 330 nm. The FTIR results confirm that the bioactive compounds act as reducing and capping agents of synthesized MgO NPs. The XRD pattern showed three major peaks of the crystalline metallic MgO NPs. Presence of magnesium and oxygen were confirmed by EDX profile. Both SEM and TEM revealed the MgO NPs as roughly spherical granular structures, and the size was 26.70 nm. The zeta potential was -32.1 mV, which indicated the stability of the MgO NPs in suspension. The MgO NPs showed considerable antifungal and antibiofilm activities against Fusarium oxysporum f. sp. lycopersici. At the concentration of 15.36 μg/ml, the MgO NPs completely inhibited the mycelial growth of the fungus. The biofilm formation of the pathogen was completely suppressed by MgO NPs at 1.92 μg/ml. The MgO NPs caused severe morphological changes on the hyphal morphology and biofilm formation of the fungus with significant damage on the fungal membrane integrity., Competing Interests: Declaration of competing interest The authors declare no conflict of interest., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Bactobolin is a hybrid natural product with potent cytotoxic activity. Its production from Burkholderia thailandensis was reported as part of a collaboration between the Greenberg and Clardy laboratories in 2010. The collaboration sparked a series of studies leading to the discovery of new analogues and associated structure-activity relationships, the identification of the bactobolin biosynthetic gene cluster and assembly of its unusual amino acid building block, the molecular target of and resistance to the antibiotic, and finally an X-ray crystal structure of the ribosome-bactobolin complex. Herein, we review the collaborations that led to our current understanding of the chemistry and biology of bactobolin.

Ramachandran conformational energy maps have been prepared for all of the glycosidic linkages found in the C1576 exopolysaccharide that constitutes the biofilms of the bacterial species Burkholderia multivorans, a member of the Burkholderia cepacian complex that was isolated from a cystic fibrosis patient. This polysaccharide is a rhamnomannan with a tetrasaccharide repeat unit containing two mannose residues and two rhamnose residues, -[3-α-d-Man-(1→2)-α-d-Man-(1→2)-α-d-Rha-(1→3)-α-d-Rha-(1→] n -, where approximately 50% of the rhamnoses are randomly methylated on their O3 hydroxyl groups, further increasing the overall hydrophobicity of the chains. Because of the methylation, the tetrasaccharide repeat unit actually contains six possible linkages. The conformational energy maps are fully adiabatic relaxed maps in which the energy for each (ϕ,ψ) grid point on the map represents the lowest possible energy for the molecule in that conformation, considering all the combinations of the other degrees of freedom, such as hydroxyl orientations. Molecular dynamics simulations were used to verify that these maps indeed describe the conformational dynamics of these linkages. All six linkages were found to be quite restricted in possible ϕ angles, but to exhibit several possible low-energy ψ angles, suggesting that these chains could be quite flexible., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Pyrrolnitrin (PRN) is a microbial pyrrole halometabolite of immense antimicrobial significance for agricultural, pharmaceutical and industrial implications. The compound and its derivatives have been isolated from rhizospheric fluorescent or non-fluorescent pseudomonads, Serratia and Burkholderia . They are known to confer biological control against a wide range of phytopathogenic fungi, and thus offer strong plant protection prospects against soil and seed-borne phytopathogenic diseases. Although chemical synthesis of PRN has been obtained using different steps, microbial production is still the most useful option for producing this metabolite. In many of the plant-associated isolates of Serratia and Burkholderia , production of PRN is dependent on the quorum-sensing regulation that usually involves N-acylhomoserine lactone (AHL) autoinducer signals. When applied on the organisms as antimicrobial agent, the molecule impedes synthesis of key biomolecules (DNA, RNA and protein), uncouples with oxidative phosphorylation, inhibits mitotic division and hampers several biological mechanisms. With its potential broad-spectrum activities, low phototoxicity, non-toxic nature and specificity for impacts on non-target organisms, the metabolite has emerged as a lead molecule of industrial importance, which has led to developing cost-effective methods for the biosynthesis of PRN using microbial fermentation. Quantum of work narrating focused research efforts in the emergence of this potential microbial metabolite is summarized here to present a consolidated, sequential and updated insight into the chemistry, biology and applicability of this natural molecule.

Microorganisms often grow in communities called biofilms where cells are imbedded in a complex self-produced biopolymeric matrix composed mainly of polysaccharides, proteins, and DNA. This matrix, together with cell proximity, confers many advantages to these microbial communities, but also constitutes a serious concern when biofilms develop in human tissues or on implanted prostheses. Although polysaccharides are considered the main constituents of the matrices, their specific role needs to be clarified. We have investigated the chemical and morphological properties of the polysaccharide extracted from biofilms produced by the C1576 reference strain of the opportunistic pathogen Burkholderia multivorans, which causes lung infections in cystic fibrosis patients. The aim of the present study is the definition of possible interactions of the polysaccharide and the three-dimensional conformation of its chain within the biofilm matrix. Surface plasmon resonance experiments confirmed the ability of the polysaccharide to bind hydrophobic molecules, due to the presence of rhamnose dimers in its primary structure. In addition, atomic force microscopy studies evidenced an extremely compact three-dimensional structure of the polysaccharide which may form aggregates, suggesting a novel view of its structural role into the biofilm matrix., (Copyright © 2019 Elsevier B.V. All rights reserved.)

The phytopathogenic Burkholderia species B. glumae and B. plantarii are the causal agents of bacterial wilt, grain rot, and seedling blight, which threaten the rice industry globally. Toxoflavin and tropolone are produced by these phytopathogens and are considered the most hostile biohazards with a broad spectrum of target organisms. However, despite their nonspecific toxicity, the effects of toxoflavin and tropolone on bacteria remain unknown. RNA-seq based transcriptome analysis was employed to determine the genome-wide expression patterns under phytotoxin treatment. Expression of 2327 and 830 genes was differentially changed by toxoflavin and tropolone, respectively. Enriched biological pathways reflected the down-regulation of oxidative phosphorylation and ribosome function, beginning with the inhibition of membrane biosynthesis and nitrogen metabolism under oxidative stress or iron starvation. Conversely, several systems such as bacterial chemotaxis, flagellar assembly, biofilm formation, and sulfur/taurine transporters were highly expressed as countermeasures against the phytotoxins. In addition, our findings revealed that three hub genes commonly induced by both phytotoxins function as the siderophore enterobactin, an iron-chelator. Our study provides new insights into the effects of phytotoxins on bacteria for better understanding of the interactions between phytopathogens and other microorganisms. These data will also be applied as a valuable source in subsequent applications against phytotoxins, the major virulence factor.

MALDI-TOF (matrix assisted laser desorption ionization-time of flight) mass spectrometry (MS) proved to be a robust tool for the identification of numerous taxonomic groups. However, it has limitations. A key advantage of this technique is the flexibility for the incorporation of protein profiles of microorganisms not included in the commercial database. Due to the prevalence of Burkholderia contaminans in fibrocystic patients in Argentina and the fact that rapid and reliable microbiological diagnosis is crucial in them, MALDI-TOF MS emerges as a strategic tool. The aim of this work was to develop an additional database with peptide spectra of reference isolates of B. contaminans. This database demonstrated to be successful for the identification of 97% of the isolates analyzed. Therefore, MALDI-TOF MS with the extended database was a useful tool for the identification and differentiation of other related species to B. contaminans., (Copyright © 2018 Asociación Argentina de Microbiología. Publicado por Elsevier España, S.L.U. All rights reserved.)

A Rhizopus sp. culture containing an endosymbiont partner ( Burkholderia sp.) was obtained through a citizen-science-based soil-collection program. An extract prepared from the pair of organisms exhibited strong inhibition of Ewing sarcoma cells and was selected for bioassay-guided fractionation. This led to the purification of rhizoxin (1), a potent antimitotic agent that inhibited microtubule polymerization, along with several new (2-5) and known (6) analogues of 1. The structures of 2-6 were established using a combination of NMR data analysis, while the configurations of the new stereocenters were determined using ROESY spectroscopy and comparison of GIAO-derived and experimental data for NMR chemical shift and 3 J HH coupling values. Whereas compound 1 showed modest selectivity for Ewing sarcoma cell lines carrying the EWSR1/ FLI1 fusion gene, the other compounds were determined to be inactive. Chemically, compound 2 stands out from other rhizoxin analogues because it is the first member of this class that is reported to contain a one-carbon-smaller 15-membered macrolactone system. Through a combination of experimental and computational tests, we determined that 2 is likely formed via an acid-catalyzed Meinwald rearrangement from 1 because of the mild acidic culture environment created by the Rhizopus sp. isolate and its symbiont.

Pityriacitrin is a marine alkaloid with typical β-carboline scaffold, and which has been proven to exhibit diverse biological functions. During the course of our research for highly active compounds from natural products, the pityriacitrin have also been isolated and identified from a Chinese Burkholderia sp. NBF227. So, in order to explore the potential functional molecules, a series of β-carboline analogues derived from pityriacitrin were designed and synthesized, and their in vitro cytotoxic activities against SGC-7901, A875, HepG2, and MARC145 cell lines were evaluated. The results demonstrated that some of these β-carboline derivatives exhibited moderate to good cytotoxic activities, especially, compound 9o with a special sulfonyl group presented the highest inhibitory activities against all tested cell lines with the IC 50 values of 6.82 ± 0.98, 8.43 ± 1.93, 7.69 ± 2.17, 7.19 ± 1.43 μM, respectively, which might be used as lead compound for discovery of novel cytotoxic agents., (Copyright © 2019 Elsevier Masson SAS. All rights reserved.)

Since Burkholderia thailandensis is included in the reference spectra of the VITEK MS libraries rather than Burkholderia pseudomallei, B. pseudomallei cannot be correctly identified in the current version of VITEK MS. This study was undertaken to evaluate the utility of matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) with the VITEK MS plus system in the detection of B. pseudomallei and B. thailandensis isolates. For each species, we increased the reference spectra, and then, a SuperSpectrum was created based on the selection of 39 specific masses. In a second step, we validated the SuperSpectra with 106 isolates identified by 16S rRNA gene sequencing. The results showed that there was 100% agreement between the validation strains analyzed by MALDI-TOF MS and those evaluated using 16S rRNA gene sequencing analysis methods. Therefore, MALDI-TOF MS is a promising, rapid, and economical method to monitor the outbreaks and spread of B. pseudomallei and B. thailandensis isolates.

Covering: up to 2018 Burkholderia species are a vast group of human pathogenic, phytopathogenic, and plant- or environment-associated bacteria. B. pseudomallei, B. mallei, and B. cepacia complex are the causative agents of melioidosis, glanders, and cystic fibrosis-related infections, respectively, which are fatal diseases in humans and animals. Due to their high resistance to antibiotics, high mortality rates, and increased infectivity via the respiratory tract, B. pseudomallei and B. mallei have been listed as potential bioterrorism agents by the Centers for Disease Control and Prevention. Burkholderia species are able to produce a large network of surface-exposed polysaccharides, i.e., lipopolysaccharides, capsular polysaccharides, and exopolysaccharides, which are virulence factors, immunomodulators, major biofilm components, and protective antigens, and have crucial implications in the pathogenicity of Burkholderia-associated diseases. This review provides a comprehensive and up-to-date account regarding the structural elucidation and biological activities of surface polysaccharides produced by Burkholderia species. The chemical synthesis of oligosaccharides mimicking Burkholderia polysaccharides is described in detail. Emphasis is placed on the recent research efforts toward the development of glycoconjugate vaccines against melioidosis and glanders based on synthetic or native Burkholderia oligo/polysaccharides.

Burkholderia pseudomallei and B. mallei are the causative agents of melioidosis and glanders, respectively, and are often fatal to humans and animals. Owing to the high fatality rate, potential for spread by aerosolization, and the lack of efficacious therapeutics, B. pseudomallei and B. mallei are considered biothreat agents of concern. In this study, we investigate the proteome of Burkholderia thailandensis , a closely related surrogate for the two more virulent Burkholderia species, during infection of host cells, and compare to that of B. thailandensis in culture. Studying the proteome of Burkholderia spp. during infection is expected to reveal molecular mechanisms of intracellular survival and host immune evasion; but proteomic profiling of Burkholderia during host infection is challenging. Proteomic analyses of host-associated bacteria are typically hindered by the overwhelming host protein content recovered from infected cultures. To address this problem, we have applied bio-orthogonal noncanonical amino acid tagging (BONCAT) to B. thailandensis , enabling the enrichment of newly expressed bacterial proteins from virtually any growth condition, including host cell infection. In this study, we show that B. thailandensis proteins were selectively labeled and efficiently enriched from infected host cells using BONCAT. We also demonstrate that this method can be used to label bacteria in situ by fluorescent tagging. Finally, we present a global proteomic profile of B. thailandensis as it infects host cells and a list of proteins that are differentially regulated in infection conditions as compared to bacterial monoculture. Among the identified proteins are quorum sensing regulated genes as well as homologs to previously identified virulence factors. This method provides a powerful tool to study the molecular processes during Burkholderia infection, a much-needed addition to the Burkholderia molecular toolbox.

Burkholderia thailandensis produces an impressive array of secondary metabolites, most with yet unknown targets. One of these metabolites is thailandamide, a linear polyene natural product that is constitutively synthesized by the corresponding tha gene cluster. Using broad bioactivity screens, we observed strong yet selective antibacterial activity by thailandamide against Gram-positive and cell wall-weakened Gram-negative bacteria. Bacterial cytological profiling and comparison with 10 antibiotics with known modes of action revealed a unique profile for thailandamide, suggesting a distinct mechanism of inhibition. To address the target of the drug, we obtained resistant mutants of Bacillus subtilis and mapped the resistant phenotype to accA, the product of which catalyzes the first committed step in fatty acid biosynthesis. Interestingly, the tha gene cluster encodes an accA homologue with a similar amino acid substitution. Heterologous expression showed that it confers resistance to otherwise susceptible Escherichia coli cultures, indicating that it provides immunity to thailandamide-producing B. thailandensis cells. Aside from moiramide B and andrimid, thailandamide represents only the second class of natural products that inhibits bacterial growth by targeting AccA/AccD.

Background: Despite its ability to grow and produce high-value molecules using renewable carbon sources, two main factors must be improved to use Burkholderia sacchari as a chassis for bioproduction at an industrial scale: first, the lack of molecular tools to engineer this organism and second, the inherently slow growth rate and poly-3-hydroxybutyrate [P(3HB)] production using xylose. In this work, we have addressed both factors., Results: First, we adapted a set of BglBrick plasmids and showed tunable expression in B. sacchari. Finally, we assessed growth rate and P(3HB) production through overexpression of xylose transporters, catabolic or regulatory genes. Overexpression of xylR significantly improved growth rate (55.5% improvement), polymer yield (77.27% improvement), and resulted in 71% of cell dry weight as P(3HB)., Conclusions: These values are unprecedented for P(3HB) accumulation using xylose as a sole carbon source and highlight the importance of precise expression control for improving utilization of hemicellulosic sugars in B. sacchari.

Burkholderia pseudomallei is the causative agent of melioidosis and regarded as a bioterrorism threat. It can adapt to the nutrient-limited environment as the bacteria can survive in triple distilled water for 16 years. Moreover, B. pseudomallei exhibits intrinsic resistance to diverse groups of antibiotics in particular while growing in biofilms. Recently, nutrient-limited condition influenced both biofilm formation and ceftazidime (CAZ) tolerance of B. pseudomallei were found. However, there is no information about how nutrient-limitation together with antibiotics used in melioidosis treatment affects the structure of the biofilm produced by B. pseudomallei. Moreover, no comparative study to investigate the biofilm architectures of B. pseudomallei and the related B. thailandensis under different nutrient concentrations has been reported. Therefore, this study aims to provide new information on the effects of four antibiotics used in melioidosis treatment, viz. ceftazidime (CAZ), imipenem (IMI), meropenem (MEM) and doxycycline (DOX) on biofilm architecture of B. pseudomallei and B. thailandensis with different nutrient concentrations under static and flow conditions using confocal laser scanning microscopy. Impact of nutritional stress on drug susceptibility of B. pseudomallei and B. thailandensis grown planktonically or as biofilm was also evaluated. The findings of this study indicate that nutrient-limited environment enhanced survival of B. pseudomallei in biofilm after exposure to the tested antibiotics. The shedding planktonic B. pseudomallei and B. thailandensis were also found to have increased CAZ tolerance in nutrient-limited environment. However, killing activities of MEM and IMI were stronger than CAZ and DOX on B. pseudomallei and B. thailandensis both in planktonic cells and in 2-day old biofilm. In addition, MEM and IMI were able to inhibit B. pseudomallei and B. thailandensis biofilm formation to a larger extend compared to CAZ and DOX. Differences in biofilm architecture were observed for biofilms grown under static and flow conditions. Under static conditions, biofilms grown in full strength modified Vogel and Bonner's medium (MVBM) showed honeycomb-like architecture while a knitted-like structure was observed under limited nutrient condition (0.1×MVBM). Under flow conditions, biofilms grown in MVBM showed a multilayer structure while merely dispersed bacteria were found when grown in 0.1×MVBM. Altogether, this study provides more insight on the effect of four antibiotics against B. pseudomallei and B. thailandensis in biofilm under different nutrient and flow conditions. Since biofilm formation is believed to be involved in disease relapse, MEM and IMI may be better therapeutic options than CAZ for melioidosis treatment.

Rhamnolipids are biosurfactants consisting of rhamnose (Rha) molecules linked through a β-glycosidic bond to 3-hydroxyfatty acids with various chain lengths, and they have an enormous potential for various industrial applications. The best known native rhamnolipid producer is the human pathogen Pseudomonas aeruginosa, which produces short-chain rhamnolipids mainly consisting of a Rha-Rha-C 10 -C 10 congener. Bacteria from the genus Burkholderia are also able to produce rhamnolipids, which are characterized by their long-chain 3-hydroxyfatty acids with a predominant Rha-Rha-C 14 -C 14 congener. These long-chain rhamnolipids offer different physicochemical properties compared to their counterparts from P. aeruginosa making them very interesting to establish novel potential applications. However, widespread applications of rhamnolipids are still hampered by the pathogenicity of producer strains and-even more important-by the complexity of regulatory networks controlling rhamnolipid production, e.g., the so-called quorum sensing system. To overcome encountered challenges of the wild type, the responsible genes for rhamnolipid biosynthesis in Burkholderia glumae were heterologously expressed in the non-pathogenic Pseudomonas putida KT2440. Our results show that long-chain rhamnolipids from Burkholderia spec. can be produced in P. putida. Surprisingly, the heterologous expression of the genes rhlA and rhlB encoding an acyl- and a rhamnosyltransferase, respectively, resulted in the synthesis of two different mono-rhamnolipid species containing one or two 3-hydroxyfatty acid chains in equal amounts. Furthermore, mixed biosynthetic rhlAB operons with combined genes from different organisms were created to determine whether RhlA or RhlB is responsible to define the fatty acid chain lengths in rhamnolipids.

Burkholderia pseudomallei and its close relative B. mallei are human pathogens that are classified as Tier 1 bio-threat agents. Both organisms have previously been shown to constitutively produce a capsular polysaccharide (CPS) that is both a virulence determinant and protective antigen. Extraction and purification of CPS for use as a potential vaccine candidate requires containment level 3 laboratories which is expensive and time-consuming. B. thailandensis strain E555 is closely related to B. pseudomallei and B. mallei, but is non-pathogenic to humans and based on immunological cross-reactivity has previously been shown to express a B. pseudomallei-like CPS. In this study, capsular polysaccharide isolated from an O-antigen deficient strain of B. thailandensis E555 was identified by 1 H and 13 C NMR spectroscopy as -3-)-2-O-acetyl-6-deoxy-β-d-manno-heptopyranose-(-1, and identical to that produced by B. pseudomallei. This was further substantiated by anti-CPS monoclonal antibody binding. In connection with the production of CPS fragments for use in glycoconjugate vaccines, we set out to assess the importance or otherwise of the CPS 2-OAc groups in immune protection. To this end conjugates of the native and de-O-acetylated CPS with the Hc fragment of tetanus toxin (TetH c ) were used as vaccines in a mouse model of melioidosis. The level of protection provided by deacetylated CPS was significantly lower than that from native, acetylated CPS. In addition, sera from mice vaccinated with the deacetylated CPS conjugate did not recognise native CPS. This suggests that CPS extracted from B. thailandensis can be used as antigen and that the acetyl group is essential for protection., (Crown Copyright © 2017. Published by Elsevier Ltd. All rights reserved.)

The presence of androgen receptor variant 7 (AR-V7) variants becomes a significant hallmark of castration-resistant prostate cancer (CRPC) relapsed from hormonal therapy and is associated with poor survival of CRPC patients because of lacking a ligand-binding domain. Currently, it still lacks an effective agent to target AR-V7 or AR-Vs in general. Here, we showed that a novel class of agents (thailanstatins, TSTs and spliceostatin A analogs) can significantly suppress the expression of AR-V7 mRNA and protein but in a less extent on the full-length AR expression. Mechanistically, TST-D is able to inhibit AR-V7 gene splicing by interfering the interaction between U2AF65 and SAP155 and preventing them from binding to polypyrimidine tract located between the branch point and the 3' splice site. In vivo, TST-D exhibits a potent tumor inhibitory effect on human CRPC xenografts leading to cell apoptosis. The machinery associated with AR gene splicing in CRPC is a potential target for drugs. Based on their potency in the suppression of AR-V7 responsible for the growth/survival of CRPC, TSTs representing a new class of anti-AR-V agents warrant further development into clinical application., (© 2017 UICC.)

Purpose: The purpose of the present study was to obtain a better understanding of the relationship between cell surface physiology and outer cellular envelope permeability for hydrophobic substances in mucoid and non-mucoid B. multivorans strains, as well as in two capsule-deficient derivatives of a mucoid parental strain., Methodology: Cell surface hydrophobicity properties were determined using the hydrocarbon adherence method, while outer cell envelope accessibility and permeability for non-polar compounds were measured using hydrophobic antimicrobial agent susceptibility and fluorescent probe assays. Extracellular polysaccharide (EPS) production was assessed by cultivating strains of disparate origin on yeast extract agar (YEA) containing different sugars, while the resultant colonial and cellular morphological parameters were assessed macro- and microscopically, respectively.Results/Key findings. The cell surfaces of all the strains were hydrophilic, impermeable to mechanistically disparate hydrophobic antibacterial agents and inaccessible to the hydrophobic probe N-phenyl-1-napthylamine, regardless of EPS phenotype. Supplementation of basal YEA with eight different sugars enhanced macroscopic EPS expression for all but one non-mucoid strain, with mannose potentiating the greatest effect. Despite acquisition of the mucoid phenotype, non-mucoid strains remained non-capsulated and capsulation of a hyper-mucoid strain and its two non-mucoid derivative strains was unaffected, as judged by microscopic observation., Conclusion: These data support the conclusion that EPS expression and the consistent mucoid phenotype are not necessarily associated with the ability of the outer cell surface to associate with non-polar substances or cellular capsulation.

β-Aminopeptidases are a unique group of enzymes that have the unusual capability to hydrolyze N-terminal β-amino acids from synthetic β-peptides. β-Peptides can form secondary structures mimicking α-peptide-like structures that are resistant to degradation by most known proteases and peptidases. These characteristics of β-peptides give them great potential as peptidomimetics. Here, the X-ray crystal structure of BcA5-BapA, a β-aminopeptidase from a Gram-negative Burkholderia sp. that was isolated from activated sludge from a wastewater-treatment plant in Australia, is reported. The crystal structure of BcA5-BapA was determined to a resolution of 2.0 Å and showed a tetrameric assembly typical of the β-aminopeptidases. Each monomer consists of an α-subunit (residues 1-238) and a β-subunit (residues 239-367). Comparison of the structure of BcA5-BapA with those of other known β-aminopeptidases shows a highly conserved structure and suggests a similar proteolytic mechanism of action.

Biofilms are a collective mode of bacterial life in which a self-produced matrix confines cells in close proximity to each other. Biofilms confer many advantages, including protection from chemicals (including antibiotics), entrapment of useful extracellular enzymes and nutrients, as well as opportunities for efficient recycling of molecules from dead cells. Biofilm matrices are aqueous gel-like structures composed of polysaccharides, proteins, and DNA stabilized by intermolecular interactions that may include non-polar connections. Recently, polysaccharides extracted from biofilms produced by species of the Burkholderia cepacia complex were shown to possess clusters of rhamnose, a 6-deoxy sugar with non-polar characteristics. Molecular dynamics simulations are well suited to characterizing the structure and dynamics of polysaccharides, but only relatively few such studies exist of their interaction with non-polar molecules. Here we report an investigation into the hydrophobic properties of the exopolysaccharide produced by Burkholderia multivorans strain C1576. Fluorescence experiments with two hydrophobic fluorescent probes established that this polysaccharide complexes hydrophobic species, and NMR experiments confirmed these interactions. Molecular simulations to model the hydrodynamics of the polysaccharide and the interaction with guest species revealed a very flexible, amphiphilic carbohydrate chain that has frequent dynamic interactions with apolar molecules; both hexane and a long-chain fatty acid belonging to the quorum-sensing system of B. multivorans were tested. A possible role of the non-polar domains of the exopolysaccharide in facilitating the diffusion of aliphatic species toward specific targets within the biofilm aqueous matrix is proposed., (© 2017 by The American Society for Biochemistry and Molecular Biology, Inc.)

Species of genus Burkholderia display different interaction profiles in the environment, causing either several diseases in plants and animals or being beneficial to some plants, promoting their growth, and suppressing phytopathogens. Burkholderia spp. also produce many types of biomolecules with antimicrobial activity, which may be commercially used to protect crops of economic interest, mainly against fungal diseases. Herein we have applied matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI-MSI) to investigate secondary metabolites produced by B. seminalis TC3.4.2R3 in monoculture and coculture with plant pathogen Fusarium oxysporum. The siderophore pyochelin and the rhamnolipid Rha-Rha-C15-C14 were detected in wild-type B. seminalis strain, and their productions were found to vary in mutant strains carrying disruptions in gene clusters associated with antimicrobial compounds. Two mycotoxins were detected in F. oxysporum. During coculture with B. seminalis, metabolites probably related to defense mechanisms of these microorganisms were observed in the interspecies interaction zone. Our findings demonstrate the effective application of MALDI-MSI in the detection of bioactive molecules involved in the defense mechanism of B. seminalis, and these findings suggest the potential use of this bacterium in the biocontrol of plant diseases caused by F. oxysporum. Graphical Abstract ᅟ.

Biopolymers as polyhydroxyalkanoates (PHA) composed by different co-monomers 3-hydroxybutyrate and 3-hydroxyhexanoate [P(3HB-co-3HHx)] has attracted interest since its properties are similar to low density polyethylene. Burkholderia sacchari produces this copolymer with a very low 3HHx molar fraction, about 2 mol%. B. sacchari mutant (unable to produce polymer) was engineered to host PHA biosynthesis genes (phaPCJ) from Aeromonas sp. In addition, a two-step bioprocess to increase biopolymer production was developed. The combination of these techniques resulted in the production of P(3HB-co-3HHx) with 3HHx content up to 20 mol%. The PHA content was about 78% of dry biomass, resulting in PHA volumetric productivities around 0.45gl -1 h -1 . The P(3HB-co-3HHx) containing 20 mol% of 3HHx presented an elongation at brake of 945%, higher than reported before for this PHA composition. Here we have described an approach to increase 3HHx content into the copolymer, allowing the precise control of the 3HHx molar fractions., (Copyright © 2017 Elsevier B.V. All rights reserved.)

A disaccharide repeating unit of the O-antigen from Burkholderia ambifaria, 6-deoxy-β-d-Alt-(1 → 4)-α-d-Rha-O(CH 2 ) 3 NH 2 (1), and its dimer and trimer, 6-deoxy-β-d-Alt-(1 → 4)-α-d-Rha-(1 → 3)-6-deoxy-β-d-Alt-(1 → 4)-α-d-Rha-O(CH 2 ) 3 NH 2 (2) and 6-deoxy-β-d-Alt-(1 → 4)-α-d-Rha-(1 → 3)-6-deoxy-β-d-Alt-(1 → 4)-α-d-Rha-(1 → 3)-6-deoxy-β-d-Alt-(1 → 4)-α-d-Rha-O(CH 2 ) 3 NH 2 (3), were synthesized via a convergent strategy. The key disaccharyl thioglycoside 4 as a glycosyl donor was stereoselectively assembled by glycosylation of rhammnosyl acceptor 5 with 6-deoxy-altrosyl trichloroacetimidate donor 6b. The glycosidation of 4 with 3-azidopropanol followed by global deprotection afforded the target disaccharide 1. Further elongation of the carbohydrate chain of this glycosidation product with the disaccharyl donor 4 followed by global deprotection generated rapidly the dimeric tetrasaccharide 2 and the trimeric hexasaccharide 3 in a convergent [2 + 2] and [2 + 2 + 2] manner, respectively., (Copyright © 2017 Elsevier Ltd. All rights reserved.)