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Plant roots constantly secrete compounds into the soil to interact with neighboring organisms presumably to gain certain functional advantages at different stages of development. Accordingly, it has been hypothesized that the phytochemical composition present in the root exudates changes over the course of the lifespan of a plant. Here, root exudates of in vitro grown Arabidopsis plants were collected at different developmental stages and analyzed using GC-MS. Principle component analysis revealed that the composition of root exudates varied at each developmental stage. Cumulative secretion levels of sugars and sugar alcohols were higher in early time points and decreased through development. In contrast, the cumulative secretion levels of amino acids and phenolics increased over time. The expression in roots of genes involved in biosynthesis and transportation of compounds represented in the root exudates were consistent with patterns of root exudation. Correlation analyses were performed of the in vitro root exudation patterns with the functional capacity of the rhizosphere microbiome to metabolize these compounds at different developmental stages of Arabidopsis grown in natural soils. Pyrosequencing of rhizosphere mRNA revealed strong correlations (p<0.05) between microbial functional genes involved in the metabolism of carbohydrates, amino acids and secondary metabolites with the corresponding compounds released by the roots at particular stages of plant development. In summary, our results suggest that the root exudation process of phytochemicals follows a developmental pattern that is genetically programmed.

Background Bioconversion of d -galacturonic acid to galactaric (mucic) acid has previously been carried out in small scale (50–1000 mL) cultures, which produce tens of grams of galactaric acid. To obtain larger amounts of biologically produced galactaric acid, the process needed to be scaled up using a readily available technical substrate. Food grade pectin was selected as a readily available source of d -galacturonic acid for conversion to galactaric acid. Results We demonstrated that the process using Trichoderma reesei QM6a Δ gar1 udh can be scaled up from 1 L to 10 and 250 L, replacing pure d -galacturonic acid with commercially available pectin. T. reesei produced 18 g L −1 galactaric acid from food-grade pectin (yield 1.00 g [g  d -galacturonate consumed] −1 ) when grown at 1 L scale, 21 g L −1 galactaric acid (yield 1.11 g [g  d -galacturonate consumed] −1 ) when grown at 10 L scale and 14 g L −1 galactaric acid (yield 0.77 g [g  d -galacturonate consumed] −1 ) when grown at 250 L scale. Initial production rates were similar to those observed in 500 mL cultures with pure d -galacturonate as substrate. Approximately 2.8 kg galactaric acid was precipitated from the 250 L culture, representing a recovery of 77% of the galactaric acid in the supernatant. In addition to scaling up, we also demonstrated that the process could be scaled down to 4 mL for screening of production strains in 24-well plate format. Production of galactaric acid from pectin was assessed for three strains expressing uronate dehydrogenase under alternative promoters and up to 11 g L −1 galactaric acid were produced in the batch process. Conclusions The process of producing galactaric acid by bioconversion with T. reesei was demonstrated to be equally efficient using pectin as it was with d -galacturonic acid. The 24-well plate batch process will be useful screening new constructs, but cannot replace process optimisation in bioreactors. Scaling up to 250 L demonstrated good reproducibility with the smaller scale but there was a loss in yield at 250 L which indicated that total biomass extraction and more efficient DSP would both be needed for a large scale process.

Recent studies have shown a relationship between the level of the sialic acid (Sia), N -glycolylneuraminic acid (Neu5Gc) in red meat and its risk in cancer, cardiovascular and inflammatory diseases. Unresolved is the Sia concentration in different organs of piglets during development. Our aim was to determine the level of free and conjugated forms of Neu5Gc, N- acetylneuraminic acid (Neu5Ac) and ketodeoxynonulsonic acid (Kdn) in fresh and cooked spleen, kidney, lung, heart, liver, and skeletal muscle from 3-days-old ( n  = 4–8), 38-days-old ( n  = 10) and adult piglets (n = 4) by LC-MS/MS. Our findings show: (1) Lung tissue from 3 days-old piglets contained the highest level of total Sia (14.6 μmol/g protein) compared with other organs or age groups; (2) Unexpectedly, Neu5Gc was the major Sia in spleen (67–79 %) and adult lung (36–49 %) while free Kdn was the major Sia in skeletal muscle. Conjugated Neu5Ac was the highest Sia in other organs (61–84 %); (3) Skeletal muscle contained the lowest concentration of Neu5Gc in fresh and cooked meat; (4) Kdn accounted for <5 % of the total Sia in most organs; (5) During development, the total Sia concentration showed a 44–79 % decrease in all organs; (6) In adult piglets, the high to low rank order of total Sia was lung, heart, spleen, kidney, liver and skeletal muscle. In conclusion, the high level of Neu5Gc in all organs compared to skeletal muscle is a potential risk factor suggesting that dietary consumption of organ meats should be discouraged in favor of muscle to protect against cancer, cardiovascular and other inflammatory diseases.

Recent evidence indicates that Kingella kingae produces a polysaccharide capsule. In an effort to determine the composition and structure of this polysaccharide capsule, in the current study we purified capsular material from the surface of K. kingae strain 269-492 variant KK01 using acidic conditions to release the capsule and a series of steps to remove DNA, RNA, and protein. Analysis of the resulting material by gas chromatography and mass spectrometry revealed N-acetyl galactosamine (GalNAc), 3-deoxy-D-manno-oct-2-ulosonic acid (Kdo), and galactose (Gal). Further analysis by NMR demonstrated two distinct polysaccharides, one consisting of GalNAc and Kdo with the structure →3)-β-GalpNAc-(1→5)-β-Kdop-(2→ and the other containing galactose alone with the structure →5)-β-Galf-(1→. Disruption of the ctrA gene required for surface localization of the K. kingae polysaccharide capsule resulted in elimination of GalNAc and Kdo but had no effect on the presence of Gal in bacterial surface extracts. In contrast, deletion of the pamABCDE locus involved in production of a reported galactan exopolysaccharide eliminated Gal but had no effect on the presence of GalNAc and Kdo in surface extracts. Disruption of ctrA and deletion of pamABCDE resulted in a loss of all carbohydrates in surface extracts. These results establish that K. kingae strain KK01 produces a polysaccharide capsule with the structure →3)-β-GalpNAc-(1→5)-β-Kdop-(2→ and a separate exopolysaccharide with the structure →5)-β-Galf-(1→. The polysaccharide capsule and the exopolysaccharide require distinct genetic loci for surface localization.

Currently, identification of pathogenic bacteria present at very low concentration requires a preliminary culture-based enrichment step. Many research efforts focus on the possibility to shorten this pre-enrichment step which is needed to reach the minimal number of cells that allows efficient identification. Rapid microbiological controls are a real public health issue and are required in food processing, water quality assessment or clinical pathology. Thus, the development of new methods for faster detection and isolation of pathogenic culturable bacteria is necessary. Here we describe a specific enrichment technique for culturable Gram negative bacteria, based on non-lethal click chemistry and the use of magnetic beads that allows fast detection and isolation. The assimilation and incorporation of an analog of Kdo, an essential component of lipopolysaccharides, possessing a bio-orthogonal azido function (Kdo-N3), allow functionalization of almost all Gram negative bacteria at the membrane level. Detection can be realized through strain-promoted azide-cyclooctyne cycloaddition, an example of click chemistry, which interestingly does not affect bacterial growth. Using E. coli as an example of Gram negative bacterium, we demonstrate the excellent specificity of the technique to detect culturable E. coli among bacterial mixtures also containing either dead E. coli, or live B. subtilis (as a model of microorganism not containing Kdo). Finally, in order to specifically isolate and concentrate culturable E. coli cells, we performed separation using magnetic beads in combination with click chemistry. This work highlights the efficiency of our technique to rapidly enrich and concentrate culturable Gram negative bacteria among other microorganisms that do not possess Kdo within their cell envelope.

Capsular polysaccharide (CPS) assigned to the K93 type was isolated from the bacterium Acinetobacter baumannii B11911 and studied by sugar analysis along with oneand two-dimensional 1 H and 13 C NMR spectroscopy. The CPS was found to contain a derivative of pseudaminic acid, and the structure of the branched tetrasaccharide repeating unit was established. Genes in the KL93 capsule biosynthesis locus were annotated and found to be consistent with the CPS structure established. The K93 CPS has the α-D-Gal p -(1→6)-β-D-Gal p -(1→3)-D-Gal p NAc trisaccharide fragment in common with the K14 CPS of Acinetobacter nosocomialis LUH 5541 and A. baumannii D46. It also shares the β-D-Gal p -(1→3)-DGal p NAc disaccharide fragment and the corresponding predicted Gal transferase Gtr5, as well as the initiating GalNAc-1P transferase ItrA2, with a number of A. baumannii strains.

The long held but challenged view that plants do not synthesize sialic acids was re-evaluated using two different procedures to isolate putative sialic acid containing material from plant tissues and cells. The extracts were reacted with 1,2-diamino-4,5-methylene dioxybenzene and the fluorescently labelled 2-keto sugar acids analysed by reversed phase and normal phase HPLC and by HPLC-electrospray tandem mass spectrometry. No N-glycolylneuraminic acid was found in the protein fraction from Arabidopsis thaliana MM2d cells. However, we did detect 3-deoxy-d-manno-octulosonic acid and trace amounts (3-18 pmol/g fresh weight) of a compound indistinguishable from N-acetylneuraminic acid by its retention time and its mass spectral fragmentation pattern. Thus, plant cells and tissues contain five orders of magnitude less sialic acid than mammalian tissues such as porcine liver. Similar or lower amounts of N-acetylneuraminic acid were detected in tobacco cells, mung bean sprouts, apple and banana. Yet even yeast and buffer blanks, when subjected to the same isolation procedures, apparently contained the equivalent of 5 pmol of sialic acid per gram of material. Thus, we conclude that it is not possible to demonstrate unequivocally that plants synthesize sialic acids because the amounts of these sugars detected in plant cells and tissues are so small that they may originate from extraneous contaminants.

The subject of the present review is the structural diversity and abundance of cell wall teichuronic and teichulosonic acids of representatives of the order Actinomycetales. Recently found teichulosonic acids are a new class of natural glycopolymers with ald-2-ulosonic acid residues: Kdn (3-deoxy-D- glycero -D- galacto -non-2-ulosonic acid) or di-N-acyl derivatives of Pse (5,7-diamino-3,5,7,9-tetradeoxy-L- glycero -L- manno -non-2-ulosonic or pseudaminic acid) as the obligatory component. The structures of teichuronic and teichulosonic acids are presented. Data are summarized on the occurrence of the glycopolymers of different nature in the cell wall of the studied actinomycetes. The biological role of the glycopolymers and their possible taxonomic implication are discussed. The comprehensive tables given in the Supplement show 13 C NMR spectroscopic data of teichuronic and teichulosonic acids obtained by the authors.

Background: Duarte galactosemia (DG) is frequently detected in newborn-screening programs. DG patients do not manifest the symptoms of classic galactosemia, but whether they require dietary galactose restriction is controversial. We sought to assess the relationships of selected galactose metabolites (plasma galactose, plasma galactitol, erythrocyte (RBC) galactitol, RBC galactonate, and urine galactitol and galactonate) to RBC galactose 1-phosphate (Gal-1-P), dietary galactose intake, and neurodevelopmental/clinical outcomes in DG children. Methods: We studied 30 children 1–6 years of age who had DG galactosemia and were on a regular diet. All participants underwent a physical and ophthalmologic examination and a neurodevelopmental assessment. RBC galactitol, RBC galactonate, RBC Gal-1-P, plasma galactose, plasma galactonate, and urine galactitol and galactonate concentrations were measured. Results: RBC galactitol and galactonate concentrations were about 2 and 6 times higher, respectively, than control values. Plasma galactose and galactitol concentrations were also about twice the control values. The mean values for RBC Gal-1-P and urine galactitol were within the reference interval. We found a relationship between plasma and urine galactitol concentrations but no relationship between RBC galactose metabolites and urine galactitol. There was a significant relationship between galactose intake and RBC galactose metabolites, especially RBC galactitol (P < 0.0005) and RBC galactonate (P < 0.0005). Galactose intake was not related to the urine galactitol, plasma galactose, or plasma galactitol concentration. RBC galactitol, RBC galactonate, plasma galactose, plasma galactitol, and urine galactonate concentrations showed no relationship with clinical or developmental outcomes. Conclusions: DG children on a regular diet have RBC Gal-1-P concentrations within the reference interval but increased concentrations of other galactose metabolites, including RBC galactitol and RBC galactonate. These increased concentrations correlate with galactose intake and neither cause any developmental or clinical pathology during early childhood nor oblige a lactose-restricted diet.

Bacterial products based on Bacillus thuringiensis are registered in many countries as plant protection products (PPPs) and are widely used as insecticides and nematocides. However, certain B. thuringiensis strains produce harmful toxins and are therefore not allowed to be used as PPPs. The serotype B. thuringiensis thuringiensis produces the beta-exotoxin thuringiensin (ßeT) which is considered to be toxic for almost all forms of life including humans (WHO 1999). The use of a non-registered PPP based on B. thuringiensis thuringiensis called bitoxybacillin was established through the determination of ßeT. First, an analytical reference standard of ßeT was characterized by nuclear magnetic resonance, liquid chromatography–high-resolution mass spectrometry and liquid chromatography–tandem mass spectrometry (LC-MS/MS). Then, a confirmatory quantitative method for the determination of ßeT in PPPs and selected greenhouse crops based on LC-MS/MS was developed and validated. A limit of quantitation of 0.028 mg/kg was established, and average recoveries ranged from 85.6 % to 104.8 % with repeatability (RSDr) of 1.5–7.7 % and within-lab reproducibility (RSD WLR ) of 17 %. The method was used for analysis of >100 samples. ßeT was found in leaves of ornamentals, but no evidence was found for use in edible crops. HMBC NMR spectrum of beta exotoxin thuringiensin