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1 H NMR spectrum recorded at 50 • C of Epol H111-INS in 0.3 M deuterated sodium hydroxide (NaOD) extracted from the biofilm of Burkholderia cenocepacia H111. Anomeric protons have been labelled according to the corresponding residue (A to D), as in Table 1. In the inset, the expansion of the Heteronuclear Single Quantum Coherence Adiabatic (HSQCAD) anomeric region is reported.
Source publication
Biofilms are a multicellular way of life, where bacterial cells are close together and embedded in a hydrated macromolecular matrix which offers a number of advantages to the cells. Extracellular polysaccharides play an important role in matrix setup and maintenance. A water-insoluble polysaccharide was isolated and purified from the biofilm produc...
Contexts in source publication
Context 1
... B. cenocepacia ∆bcsB/pBerA strain is lacking the bcsB gene, and therefore this strain does not produce cellulose, which makes it easier to isolate the Bep polysaccharide. Biofilm was grown on nutrient-yeast extract-glycerol (NYG) agar plates and appeared as a compact wrinkled film which was peeled from each plate in one piece ( Figure S1). The Epol was extracted from the matrix using 0.3 M NaOH, followed by centrifugation to remove insoluble materials, and subjected to dialysis against water in a dialysis bag, where, after reaching the equilibrium, it precipitated as a white powder. ...
Context 2
... repeating unit (RU) structure of Epol H111-INS was investigated at 500 MHz. The 1 H NMR spectrum contains four anomeric signals designated A to D (Figure 1), at 5.29, 5.25, 5.08 and 4.98 ppm, and their peak area integration gave values very close to 1.0. The 1 J H1-H2 values of the first three signals were in agreement with α-anomeric residues, as indicated also by their chemical shifts, while 1 J H1-H2 of the resonance at 4.98 ppm was too small to be measured and attributed to H1 of Man. 1 J C1-H1 were detected in a coupled Gradient Heteronuclear Single Quantum Coherence Adiabatic (gHSQCAD) experiment and the constant values measured of about 170 Hz (Table 1) Table 1. ...
Context 3
... low-density region centered at approximately (ф, ψ) 90 • , −75 • was again disfavoured in water ( Figure S7). Table 3 lists all of the interatomic distances relevant to the NMR experiments as calculated from the MD simulations of each of the linkages in the Epol H111-INS repeating unit in aqueous (TIP4P) solution at room (300 K) temperature (Figures S8-S11). Among the calculated interatomic distances, particularly relevant are H1-'H3 and H1-'H4 (the apostrophe indicates the reducing end residue) for the two disaccharides Man(1→3)Gal and Glc(1→3)Gal, because, contrary to what is usually found, both NOE experimental data and MD simulation indicated smaller values for H1-'H4 than for the two protons across the glycosidic linkages. ...
Citations
... C-di-GMP is a bacterial universal intracellular secondary signaling molecule [36][37][38]. In bacterial biofilm formation, c-di-GMP is known to regulate genes responsible for synthesizing EPS components; extracellular polymeric exoenzymes, polysaccharides, and adhesins [39,40]. In addition, c-di-GMP enhances bacterial adhesion and represses bacterial motility, further promoting biofilm production [32,33,41,42]. ...
... Exopolysaccharides are a major component of most bacterial biofilm matrices [40,88,89]. The exopolysaccharides have been categorized into various forms, such as capsular polysaccharides, free polysaccharides, and lipopolysaccharides (O-antigen) that have a key role in preventing the diffusion of antimicrobial agents within the biofilm community [89][90][91]. ...
Xanthomonas oryzae pv. oryzae (Xoo) causes a devastating bacterial leaf blight in rice. Here, the antimicrobial effects of D-limonene, L-limonene, and its oxidative derivative carveol against Xoo were investigated. We revealed that carveol treatment at ≥ 0.1 mM in liquid culture resulted in significant decrease in Xoo growth rate (> 40%) in a concentration-dependent manner, and over 1 mM, no growth was observed. The treatment with D-limonene and L-limonene also inhibited the Xoo growth but to a lesser extent compared to carveol. These results were further elaborated with the assays of motility, biofilm formation and xanthomonadin production. The carveol treatment over 1 mM caused no motilities, basal level of biofilm formation (< 10%), and significantly reduced xanthomonadin production. The biofilm formation after the treatment with two limonene isomers was decreased in a concentration-dependent manner, but the degree of the effect was not comparable to carveol. In addition, there was negligible effect on the xanthomonadin production mediated by the treatment of two limonene isomers. Field emission-scanning electron microscope (FE-SEM) unveiled that all three compounds used in this study cause severe ultrastructural morphological changes in Xoo cells, showing shrinking, shriveling, and holes on their surface. Moreover, quantitative real-time PCR revealed that carveol and D-limonene treatment significantly down-regulated the expression levels of genes involved in virulence and biofilm formation of Xoo, but not with L-limonene. Together, we suggest that limonenes and carveol will be the candidates of interest in the development of biological pesticides.
... Such analytical assays include traditional biochemical analyses that rely on efforts to solubilize the EPS (e.g., solution nuclear magnetic resonance (NMR) and mass spectrometry). 28,30,31,[42][43][44][45][46][47][48][49][50][51][52][53] Each of these methods yields different information regarding EPS monosaccharide composition, linkage, anomeric configuration, and relative amount in the total biofilm matrix. Typically, approaches using solution NMR and mass spectrometry require processing such as hydrolysis to analyze the EPS sample, which can lead to misidentification of EPS structure. ...
Biofilm formation protects bacteria from antibiotic treatment and host immune responses, making biofilm infections difficult to treat. Within biofilms, bacterial cells are entangled in a self‐produced extracellular matrix that typically includes exopolysaccharides. Molecular‐level descriptions of biofilm matrix components, especially exopolysaccharides, have been challenging to attain due to their complex nature and lack of solubility and crystallinity. Solid‐state nuclear magnetic resonance (NMR) has emerged as a key tool to determine the structure of biofilm matrix exopolysaccharides without degradative sample preparation. In this review, we discuss challenges of studying biofilm matrix exopolysaccharides and opportunities to develop solid‐state NMR approaches to study these generally intractable materials. We specifically highlight investigations of the exopolysaccharide called Pel made by the opportunistic pathogen, Pseudomonas aeruginosa . We provide a roadmap for determining exopolysaccharide structure and discuss future opportunities to study such systems using solid‐state NMR. The strategies discussed for elucidating biofilm exopolysaccharide structure should be broadly applicable to studying the structures of other glycans.
... The latter is the case for cellulose, whose gene clusters is well-known (Carlier et al., 2014;Römling & Galperin, 2015), while cepacian (Cescutti et al., 2000) was produced when the strains used in the present study were grown on yeast extract mannitol (YEM) solid medium which is known to stimulate its production (Sage et al., 1990). Recently two other Epols were isolated from B. cenocepacia H111 biofilms and fully characterized: H111-SOL, a water-soluble polysaccharide rich in Rhamnose (Rha) residues and containing an L-Mannose (Man) residue (Bellich et al., 2021), and H111-INS, a waterinsoluble polymer made of Glucose (Glc), Galactose (Gal) and Mannose (Man) (Bellich et al., 2020) (Fig. 1). In independent investigations, a novel BerA/c-di-GMP regulated exopolysaccharide gene cluster, named bepA-L, was discovered, and its regulation was thoroughly investigated (Fazli et al., 2011;Fazli et al., 2013;Fazli et al., 2017). ...
... In order to establish without doubt which is the product of the bepA-L gene cluster, polysaccharides extracted from pellicles formed by i) a strain overexpressing the transcriptional regulator BerA and ii) two mutant strains carrying a transposon insertion in the bepA-L gene cluster were subjected to chemical and 1D 1 H NMR spectroscopy analyses. The product of the bepA-L gene cluster was then identified after comparison with data of the pure polysaccharides (Bellich et al., 2020;Bellich et al., 2021). Moreover, the type of Epols produced by the B. cenocepacia H111 wild type in pellicles was also elucidated. ...
... One pellicle of each day of growth was hydrolysed with 2 M trifluoroacetic acid (TFA) for 1 h at 125 • C, derivatized to alditol acetates, and subjected to gas liquid chromatography (GLC) analysis for the identification of neutral sugars (Albersheim et al., 1967). The other pellicles were used for recording High-Resolution Magic Angle Spinning (Bellich et al., 2020), H111-SOL (Bellich et al., 2021), cepacian (Cescutti et al., 2000) and cellulose. In circles: sugars and linkages exclusive of each exopolysaccharide which may be used as markers for each polysaccharide. ...
Burkholderia cenocepacia is an opportunistic pathogen isolated from cystic fibrosis patients where it causes infections that are extremely difficult to treat with antibiotics, and sometimes have a fatal outcome. Biofilm is a virulence trait of B. cenocepacia, and is associated with infection persistence and increased tolerance to antibiotics. In biofilms exopolysaccharides have an important role, conferring mechanical stability and antibiotic tolerance. Two different exopolysaccharides were isolated from B. cenocepacia H111 biofilms: a water-soluble polysaccharide rich in rhamnose and containing an L-Man residue, and a water-insoluble polymer made of glucose, galactose and mannose. In the present work, the product encoded by B. cenocepacia H111 bepA-L gene cluster was identified as the water-insoluble exopolysaccharide, using mutant strains and NMR spectroscopy of the purified polysaccharides. It was also demonstrated that the B. cenocepacia H111 wild type strain produces the water-insoluble exopolysaccharide in pellicles, thus underlining its potential importance in in vivo infections.
... Overexpressing wspH increases cell surface hydrophobicity. The product of the bep gene cluster in B. cenocepacia H111 was recently identified as a water insoluble exopolysaccharide, named Bep (Burkholderia cepacia exopolysaccharide), and consisting of repeated tetrasaccharide units containing glucose, galactose and mannose ( (24). Pellicle biofilms need to float on the ALI (18) and it is therefore logic to hypothesize that inducing wspH would coincide with increased formation of Bep. ...
... Contents of 5 tubes were pooled, pelleted by centrifugation Exopolysaccharide analysis by NMR spectroscopy. Exopolysaccharide purification was performed as described in Bellich et al. (24). In brief, pellicle material, collected as described for adherence to hydrocarbons measurement, was freeze-dried and polysaccharides extracted by stirring in 0.3 M NaOH for 3 h at 12°C. ...
The chemosensory signal transduction system Wsp regulates biofilm formation and related phenotypes by influencing cyclic-di-GMP (c-di-GMP) levels in bacterial cells. This is typically achieved by activation of the diguanylate cyclase WspR, through phosphorylation of its phosphoreceiver domain. The Wsp system of Burkholderia cenocepacia J2315 is in one operon with the hybrid response regulator/histidine kinase wspH, but lacks the diguanylate cyclase wspR which is located in a different operon. The expression of wspH, the first gene in the B. cenocepacia Wsp operon as well as pellicle biofilm formation are epigenetically regulated in B. cenocepacia J2315. To investigate whether WspH regulates pellicle biofilm formation, several mutants with altered expression of wspH were constructed. Mutants with increased expression of wspH showed accelerated pellicle biofilm formation, reduced swimming motility and increased c-di-GMP levels. This was independent of WspR phosphorylation, showing that WspR is not the cognate response receiver for histidine kinase WspH.
IMPORTANCE Biofilms are surface-attached or suspended aggregates of cells, that are problematic in the context of bacterial infections, as they provide protection from antibiotic treatment. Burkholderia cenocepacia can colonize the lung of immunocompromised patients and forms biofilms that increase its recalcitrance to antibiotic treatment. Pellicles are biofilms which form at an air-liquid interface to take advantage of the higher oxygen concentrations in this environment. How quickly pellicles are formed is crucial for the fitness of obligate aerobic bacteria such as B. cenocepacia. Cyclic-di-GMP (c-di-GMP) levels determine the transition between planktonic and biofilm lifestyle, and WspH controls c-di-GMP production. WspH is therefore important for the fitness of B. cenocepacia in environments with gradients in oxygen concentration, such as the human lung.
... Good correlation between NOE data and calculated interatomic distances by molecular dynamics simulations validated potential energy functions used for calculations https://www.mdpi.com/1422-0067/21/5/1702 (accessed on 2 March 2020) [11]. ...
The study of carbohydrates has a long history: for two centuries, the researches performed the way from “sweet matter” to glycomics [...]
... B. cenocepacia encodes the capacity to produce various polysaccharides. The best known of these is cepacian (composed of rhamnose, mannose, glucose, galactose, and glucuronic acid) (31), but others include Bep (Burkholderia extracellular polysaccharide) and galactan-deoxyd-manno-octulosonic acid (32)(33)(34)(35). We hypothesized that the different binding and aggregation properties of rpfR mutants related to the production of the components in these exopolysaccharides of varied composition. ...
Significance
Many organisms, including bacteria, live in fluctuating environments that require attachment and dispersal. These lifestyle decisions require processing of multiple external signals by several genetic pathways, but how they are integrated is largely unknown. We conducted multiple evolution experiments totaling >20,000 generations with Burkholderia cenocepacia populations grown in a model of the biofilm life cycle and identified parallel mutations in one gene, rpfR , that is a conserved central regulator. Because RpfR has multiple sensor and catalytic domains, different mutations can produce different ecological strategies that can coexist and even increase net growth. This study demonstrates that a single gene may coordinate complex life histories in biofilm-dwelling bacteria and that selection in defined environments can reshape niche breadth by single mutations.
The development of deuterium‐incorporation method is eagerly desired. A solution of sodium deuteroxide in deuterium oxide (NaOD in D2O) was simply prepared by mixing D2O and t‐BuONa. Subsequently, the t‐BuOD layer, generated under conditions of the salting‐out effect, was separate. The purity (volume of the contaminated t‐BuOD as side‐product) of the homemade NaOD in D2O was evaluated by ¹H NMR and Raman analyses. NaOD in D2O was used for the one‐pot synthesis of mono‐deuterated trans‐stilbene (stilbene‐d1) in the aqueous Horner‐Wadsworth‐Emmons reaction. The present method can be widely applied to synthesize the various mono‐deuterated stilbene derivatives with high deuterium content. Additionally, the obtained mono‐deuterated stilbene derivatives could be successfully converted to various deuterated compounds.
The incidence of melioidosis cases caused by the gram-negative pathogen Burkholderia pseudomallei (BP) is seeing an increasing trend that has spread beyond its previously known endemic regions. Biofilms produced by BP have been associated with antimicrobial therapy limitation and relapse melioidosis, thus making it urgently necessary to understand the mechanisms of biofilm formation and their role in BP biology. Microbial cells aggregate and enclose within a self-produced matrix of extracellular polymeric substances (EPSs) to form biofilm. The transition mechanism of bacterial cells from planktonic state to initiate biofilm formation, which involves the formation of surface attachment microcolonies and the maturation of the biofilm matrix, is a dynamic and complex process. Despite the emerging findings on the biofilm formation process, systemic knowledge on the molecular mechanisms of biofilm formation in BP remains fractured. This review provides insights into the signaling systems, matrix composition, and the biosynthesis regulation of EPSs (exopolysaccharide, eDNA and proteins) that facilitate the formation of biofilms in order to present an overview of our current knowledge and the questions that remain regarding BP biofilms.
The polysaccharide Bep is essential for in vitro biofilm formation of the opportunistic pathogen Burkholderia cenocepacia . We found that the Burkholderia diffusible signaling factor (BDSF) quorum sensing receptor RpfR is a negative regulator of the bep gene cluster in B. cenocepacia . An rpfR mutant formed wrinkled colonies, whereas additional mutations in the bep genes or known bep regulators like berA and berB restored the wild-type smooth colony morphology. We found that there is a good correlation between intracellular c-di-GMP levels and bep expression when the c-di-GMP level is increased or decreased through ectopic expression of a diguanylate cyclase or a c-di-GMP phosphodiesterase, respectively. However, when the intracellular c-di-GMP level is changed by site directed mutagenesis of the EAL or GGDEF domain of RpfR there is no correlation between intracellular c-di-GMP levels and bep expression. Except for rpfR, deletion mutants of all 25 c-di-GMP phosphodiesterase and diguanylate cyclase genes encoded by B. cenocepacia showed no change to berA and bep gene expression. Moreover, bacterial two-hybrid assays provided evidence that RpfR and BerB physically interact and give specificity to the regulation of the bep genes. We suggest a model where RpfR binds BerB at low c-di-GMP levels to sequester this RpoN-dependent activator to an RpfR/RpfF complex. If the c-di-GMP levels rise, possibly by the enzymatic action of RpfR, BerB binds c-di-GMP and is released from the RpfR/RpfF complex and associates with RpoN to activate transcription of berA , and the BerA protein subsequently activates transcription of the bep genes.
