ABSTRACT
To explore the effect of beef processing on Escherichia coli populations in relation to lactic acid resistance, this study investigated the links among acid response, phylogenetic structure, genome diversity, and genotypes associated with acid resistance of meat plant E. coli. Generic E. coli isolates (n = 700) were from carcasses, fabrication equipment, and beef products. Acid treatment was carried out in Luria-Bertani broth containing 5.5% lactic acid (pH 2.9). Log reductions of E. coli ranged from <0.5 to >5 log CFU/mL (median: 1.37 log). No difference in lactic acid resistance was observed between E. coli populations recovered before and after a processing step or antimicrobial interventions. E. coli from the preintervention carcasses were slightly more resistant than E. coli isolated from equipment, differing by <0.5 log unit. Acid-resistant E. coli (log reduction <1, n = 45) had a higher prevalence of genes related to energy metabolism (ydj, xap, ato) and oxidative stress (fec, ymjC) than the less resistant E. coli (log reduction >1, n = 133). The ydj and ato operons were abundant in E. coli from preintervention carcasses. In contrast, fec genes were abundant in E. coli from equipment surfaces. The preintervention E. coli contained phylogroups A and B1 in relatively equal proportions. Phylogroup B1 predominated (95%) in the population from equipment. Of note, E. coli collected after sanitation shared either the antigens of O8 or H21. Additionally, genome diversity decreased after chilling and equipment sanitation. Overall, beef processing did not select for E. coli resistant to lactic acid but shaped the population structure. IMPORTANCE Antimicrobial interventions have significantly reduced the microbial loads on carcasses/meat products; however, the wide use of chemical and physical biocides has raised concerns over their potential for selecting resistant populations in the beef processing environment. Phenotyping of acid resistance and whole-genome analysis described in this study demonstrated beef processing practices led to differences in acid resistance, genotype, and population structure between carcass- and equipment-associated E. coli but did not select for the acid-resistant population. Results indicate that genes coding for the metabolism of long-chain sugar acids (ydj) and short-chain fatty acids (ato) were more prevalent in carcass-associated than equipment-associated E. coli. These results suggest E. coli from carcasses and equipment surfaces have been exposed to different selective pressures. The findings improve our understanding of the microbial ecology of E. coli in food processing environments and in general.
Subject(s)
Anti-Infective Agents , Disinfectants , Cattle , Animals , Escherichia coli , Lactic Acid , Phylogeny , Meat , Anti-Bacterial Agents/pharmacology , Food Handling , Anti-Infective Agents/pharmacology , Disinfectants/pharmacology , Sugar Acids/analysis , Sugar Acids/pharmacology , Colony Count, Microbial , Food Microbiology , Food Contamination/analysisABSTRACT
As a key precursor of vitamin C, 2-keto-L-gulonic acid (2-KLG) was mainly produced from L-sorbose by mixed fermentation of Ketogulonicigenium vulgare and a helper strain (Bacillus spp.) with a low conversion rate for decades. The aim of this study was to enhance the 2-KLG production by co-culturing K. vulgare and Bacillus megaterium using three-stage temperature control (TSTC) strategy. By investigating the temperature effect on the 2-KLG fermentation, the optimum temperatures for the growths of K. vulgare and B. megaterium were 32 °C and 29 °C, respectively, while the optimum temperature for 2-KLG production was 35 °C. We developed a TSTC process: the temperature was kept at 32 °C during the first 16 h of fermentation, then decreased to 29 °C for the following 14 h, and maintained at 35 °C to the end of fermentation. By using this new process, the productivity and yield of 2-KLG from L-sorbose were obtained at 2.19 ± 0.19 g/L/h and 92.91 ± 1.02 g/L in 20-L fermentors for 5 batches, respectively, which were 22.35% and 6.02% higher than that of the control treatment (the single temperature of 29 °C). The increased cell density of K. vulgare during the exponential phase and the enhanced SDH activity (increased by 25.18% at 36 h, 17.14% at 44 h) in the production stage might be the reasons for enhanced 2-KLG conversion rate and yield. Our results demonstrated the feasibility of the TSTC strategy for 2-KLG production.
Subject(s)
Bacillus megaterium/metabolism , Bacteriological Techniques , Rhodobacteraceae/metabolism , Sugar Acids/metabolism , Temperature , Bacillus megaterium/growth & development , Bioreactors , Culture Media/chemistry , Fermentation , Rhodobacteraceae/growth & development , Sorbose/metabolism , Sugar Acids/analysisABSTRACT
2-Keto-L-gulonic acid (2-KLG) is the direct precursor of vitamin C in industrial synthesis. 2-KLG is mainly produced via the classical two-step fermentation route. In the two-step fermentation process, 2-KLG can be synthesized from L-sorbose by Ketogulonicigenium vulgare aided by Bacillus megaterium. There are five sorbose/sorbosone dehydrogenases (SSDHs), SSDA1, SSDA1-P, SSDA2, SSDA3 and SSDB, and two sorbosone dehydrogenases (SNDHs), glucose/sorbosone dehydrogenase (GSNDH) and sorbosone dehydrogenase (SNDH), in K. vulgare, which could play crucial roles in transforming L-sorbose or L-sorbosone to 2-KLG. However, confusion about the catalytic characteristics of the individual SSDHs and SNDHs makes construction of a recombinational strain for the purpose of enhancing 2-KLG production difficult. In this study, the five SSDHs and two SNDHs from K. vulgare WSH-001 were purified, and their optimal pH values and reaction temperatures, kinetic properties, thermostabilities, substrate spectra and effects of electron acceptors on their performances were systematically determined. Among these dehydrogenases, only SSDA1 and SSDA3 have high activity for catalyzing L-sorbose to 2-KLG directly. These data provide more clues for ways to achieve enhanced conversion of L-sorbose in K. vulgare, which could facilitate both the construction of a more efficient one-step fermentation 2-KLG producer and the reconstruction of a one-step fermentation process.
Subject(s)
Bacterial Proteins , Carbohydrate Dehydrogenases , Rhodobacteraceae , Sorbose/analogs & derivatives , Sorbose/metabolism , Ascorbic Acid/analysis , Ascorbic Acid/metabolism , Bacterial Proteins/chemistry , Bacterial Proteins/genetics , Bacterial Proteins/metabolism , Carbohydrate Dehydrogenases/chemistry , Carbohydrate Dehydrogenases/genetics , Carbohydrate Dehydrogenases/metabolism , Enzyme Stability , Metabolic Engineering , Rhodobacteraceae/enzymology , Rhodobacteraceae/genetics , Sugar Acids/analysis , Sugar Acids/metabolismABSTRACT
Brandy de Jerez is the most produced spirit in Spain. The rules of its Regulatory Council require the spirit to age in American oak casks that have previously contained any kind of sherry wine. This use, called seasoning, releases wine compounds into the spirit. Because of the differences among sherries, the organoleptic features of a brandy will be significantly different from any other depending on the seasoning. In addition, its specific features make it different from any other spirit. The chromatographic profiles of Brandy de Jerez are reported to be different depending on the seasonings through their ageing process. Different types of Brandy de Jerez have been characterised, regarding their seasoning, using chromatographic techniques. Applying statistical analysis, correlations between the chromatographic profiles and the seasonings have risen up. In addition, the profiles have demonstrated to possess a high degree of correlation with the ageing time of the samples.
Subject(s)
Alcoholic Beverages/analysis , Phenols/chemistry , Chromatography, High Pressure Liquid , Cluster Analysis , Phenols/analysis , Principal Component Analysis , Spain , Sugar Acids/analysis , Wine/analysisABSTRACT
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 Δgar1 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.
Subject(s)
Batch Cell Culture Techniques/methods , Pectins/metabolism , Sugar Acids/metabolism , Trichoderma/metabolism , Aldehyde Oxidoreductases/genetics , Aldehyde Oxidoreductases/metabolism , Biomass , Bioreactors , Culture Media/chemistry , Hexuronic Acids/metabolism , Promoter Regions, Genetic , Sugar Acids/analysis , Sugar Acids/isolation & purification , Trichoderma/growth & developmentABSTRACT
The diverse gut microbial communities are crucial for host health. How the interactions between microbial communities and between host and microbes influence the host, however, is not well understood. To facilitate gut microbiota research, selective imaging of specific groups of microbiotas in the gut is of great utility but remains technically challenging. Here we present a chemical approach that enables selective imaging of Gram-negative and Gram-positive microbiotas in the mouse gut by exploiting their distinctive cell wall components. Cell-selective labeling is achieved by the combined use of metabolic labeling of Gram-negative bacterial lipopolysaccharides with a clickable azidosugar and direct labeling of Gram-positive bacteria with a vancomycin-derivatized fluorescent probe. We demonstrated this strategy by two-color fluorescence imaging of Gram-negative and Gram-positive gut microbiotas in the mouse intestines. This chemical method should be broadly applicable to different gut microbiota research fields and other bacterial communities studied in microbiology.
Subject(s)
Diagnostic Techniques, Digestive System , Dysbiosis/diagnostic imaging , Gastrointestinal Microbiome , Gastrointestinal Tract/diagnostic imaging , Gram-Negative Bacteria/isolation & purification , Gram-Positive Bacteria/isolation & purification , Animals , Azides/analysis , Azides/chemistry , Azides/metabolism , Azides/pharmacology , Carbocyanines/analysis , Cell Wall/chemistry , Click Chemistry , Dysbiosis/microbiology , Fluorescent Dyes/analysis , Fluorescent Dyes/chemistry , Fluorescent Dyes/metabolism , Fluorescent Dyes/pharmacology , Gastrointestinal Tract/microbiology , Gram-Negative Bacteria/cytology , Gram-Negative Bacteria/growth & development , Gram-Negative Bacteria/metabolism , Gram-Positive Bacteria/cytology , Gram-Positive Bacteria/growth & development , Gram-Positive Bacteria/metabolism , Lipopolysaccharides/analysis , Lipopolysaccharides/biosynthesis , Lipopolysaccharides/chemistry , Mice, Inbred C57BL , Microbial Viability/drug effects , Optical Imaging , Pilot Projects , Porphobilinogen/analogs & derivatives , Porphobilinogen/analysis , Porphobilinogen/chemistry , Rhodamines/analysis , Rhodamines/chemistry , Specific Pathogen-Free Organisms , Sugar Acids/analysis , Sugar Acids/chemistry , Sugar Acids/metabolism , Sugar Acids/pharmacology , Vancomycin/analogs & derivatives , Vancomycin/analysisABSTRACT
Capsular polysaccharide (CPS) assigned to the K93 type was isolated from the bacterium Acinetobacter baumannii B11911 and studied by sugar analysis along with one- and two-dimensional 1H and 13C 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-Galp-(1â6)-ß-d-Galp-(1â3)-d-GalpNAc trisaccharide fragment in common with the K14 CPS of Acinetobacter nosocomialis LUH 5541 and A. baumannii D46. It also shares the ß-d-Galp-(1â3)-d-GalpNAc disaccharide fragment and the corresponding predicted Gal transferase Gtr5, as well as the initiating GalNAc-1-P transferase ItrA2, with a number of A. baumannii strains.
Subject(s)
Acinetobacter baumannii/metabolism , Bacterial Capsules/metabolism , Multigene Family , Polysaccharides/chemistry , Polysaccharides/genetics , Sugar Acids/analysis , Acinetobacter baumannii/genetics , Carbohydrate Conformation , Carbon-13 Magnetic Resonance Spectroscopy , Genes, Bacterial , Proton Magnetic Resonance SpectroscopyABSTRACT
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.
Subject(s)
Kidney/metabolism , Muscle, Skeletal/metabolism , Myocardium/metabolism , Neuraminic Acids/metabolism , Sialic Acids/metabolism , Sugar Acids/metabolism , Animals , Heart/growth & development , Kidney/growth & development , Lung/growth & development , Lung/metabolism , Mass Spectrometry , Muscle, Skeletal/growth & development , Neuraminic Acids/analysis , Red Meat/standards , Sialic Acids/analysis , Spleen/growth & development , Spleen/metabolism , Sugar Acids/analysis , SwineABSTRACT
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.
Subject(s)
Click Chemistry/methods , Gram-Negative Bacteria/isolation & purification , Immunomagnetic Separation/methods , Azides/analysis , Cell Culture Techniques , Sugar Acids/analysisABSTRACT
Some Bacillus thuringiensis strains secrete type I ß-exotoxin, which is a non-specific insecticidal and thermostable adenine nucleoside oligosaccharide. Toxicity bioassays and HPLC are traditional methods for detecting ß-exotoxin. With the aim of establish a first rapid approach for prediction of type I ß-exotoxin production, two PCR-based methods were successfully evaluated in B. thuringiensis strains and native isolates. In order to validate a reliable technology, results obtained by this method were correlated with that obtained from Musca domestica bioassays.
Subject(s)
Adenosine/analogs & derivatives , Bacillus thuringiensis/metabolism , Polymerase Chain Reaction/methods , Sugar Acids/analysis , Adenosine/analysis , Adenosine/biosynthesisABSTRACT
The sugar 3-deoxy-d-manno-oct-2-ulosonic acid (Kdo) is a characteristic component of bacterial lipopolysaccharide (LPS, endotoxin). It connects the carbohydrate part of LPS with C6 of glucosamine or 2,3-diaminoglucose of lipid A by acid-labile α-ketosidic linkage. The number of Kdo units present in LPS, the way they are connected, and the occurrence of other substituents (P, PEtn, PPEtn, Gal, or ß-l-Ara4N) account for structural diversity of the inner core region of endotoxin. In a majority of cases, Kdo is crucial to the viability and growth of bacterial cells. In this paper, the biosynthesis of Kdo and the mechanism of its incorporation into the LPS structure, as well as the location of this unique component in the endotoxin core structures, have been described.
Subject(s)
Bacteria/chemistry , Endotoxins/biosynthesis , Endotoxins/chemistry , Sugar Acids/analysis , Sugar Acids/metabolism , Bacteria/metabolism , Endotoxins/metabolism , Sugar Acids/chemistryABSTRACT
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 â3)-ß-GalpNAc-(1â5)-ß-Kdop-(2â and the other containing galactose alone with the structure â5)-ß-Galf-(1â. 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 â3)-ß-GalpNAc-(1â5)-ß-Kdop-(2â and a separate exopolysaccharide with the structure â5)-ß-Galf-(1â. The polysaccharide capsule and the exopolysaccharide require distinct genetic loci for surface localization.
Subject(s)
Bacterial Capsules/chemistry , Genes, Bacterial/genetics , Kingella kingae/chemistry , Polysaccharides, Bacterial/chemistry , DNA Primers/genetics , Galactosamine/analysis , Galactose/analysis , Gas Chromatography-Mass Spectrometry , Gene Deletion , Kingella kingae/genetics , Magnetic Resonance Spectroscopy , Sugar Acids/analysisABSTRACT
N-acetylneuraminic acid (NANA) consumes toxic hydrogen peroxide (H2O2) under physiological conditions and is oxidized by an equimolar amount of H2O2 to yield its decarboxylated product 4-(acetylamino)-2,4-dideoxy-d-glycero-d-galacto-octonic acid (ADOA). Highly sensitive analytical methods are required to detect ADOA in the human body. We labeled NANA and ADOA with 4-(N,N-dimethylaminosulfonyl)-7-(2-aminoethylamino)-2,1,3-benzoxadiazole (DBD-ED) to enable their fluorometric detection, and developed a method using HPLC with fluorometric detection (HPLC-FD) for the simultaneous determination of the derivatized NANA and ADOA. The derivatized NANA and ADOA were separated by a hydrophilic interaction liquid chromatography (HILIC) column using an H2O/CH3CN/HCOOH (10/90/0.35) mobile phase. Fluorescence was monitored at excitation and emission wavelengths of 450nm and 560nm, respectively. Both intra- and inter-day (n=6) repeat determinations of the DBD-ED-derivatized NANA and ADOA gave relative standard deviations of less than 5%. The calibration curves for standard solutions of DBD-ED-derivatized NANA and ADOA were linear over the ranges from 576fmol to 2.0nmol and 556fmol to 2.0nmol, respectively. The method developed was highly specific and sensitive for NANA and ADOA. The presence of ADOA in biological samples was revealed for the first time using this method.
Subject(s)
Chromatography, High Pressure Liquid/methods , N-Acetylneuraminic Acid/analysis , Saliva/chemistry , Sugar Acids/analysis , Fluorescent Dyes/analysis , Fluorometry/methods , Humans , Limit of Detection , Oxidation-ReductionABSTRACT
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.
Subject(s)
Arabidopsis/chemistry , Gene Expression Regulation, Developmental/physiology , Gene Expression Regulation, Plant/physiology , Plant Exudates/analysis , Plant Roots/chemistry , Arabidopsis/growth & development , Base Sequence , Carbohydrates/analysis , Gas Chromatography-Mass Spectrometry , Gene Expression Profiling , Gene Expression Regulation, Developmental/genetics , Gene Expression Regulation, Plant/genetics , Metagenome/genetics , Molecular Sequence Data , Principal Component Analysis , Reverse Transcriptase Polymerase Chain Reaction , Rhizosphere , Sequence Analysis, DNA , Sugar Acids/analysisABSTRACT
Ketogulonicigenium vulgare is used widely during the industrial production of 2-keto-l-gulonic acid (2-KLG), the precursor of vitamin C, in a coculture with Bacillus megaterium. We analyzed the sulfate and coenzyme A metabolic module in the genome-scale metabolic model (GSMM) iWZ663 and found that the poor growth of K. vulgare was due to a deficiency in key reductases in the sulfate metabolic pathway. To carefully investigate the metabolism of sulfate, we developed a chemically defined medium (CDM) to produce pure cultures of K. vulgare. The addition of glutathione and l-cysteine to a flask culture of K. vulgare increased the cell growth, 2-KLG titer, and the intracellular coenzyme A level by 38.7%, 45.5%, and 85.3%, respectively, with glutathione, and by 25.6%, 35.8%, and 44.7%, respectively, with l-cysteine. The addition of glutathione to a 7-L fermenter culture of K. vulgare and B. megaterium increased the 2-KLG productivity by 20.9%. This study shows that the analysis of a specific metabolic module in GSMM can provide a potential strategy for optimizing microbial physiological functions.
Subject(s)
Glutathione/pharmacology , Models, Biological , Rhodobacteraceae/drug effects , Rhodobacteraceae/metabolism , Sugar Acids/metabolism , Biotechnology/methods , Cell Proliferation , Fermentation , Industrial Microbiology , Metabolic Networks and Pathways , Pantothenic Acid , Sugar Acids/analysis , Sulfates/metabolismABSTRACT
The thuringiensin abiotic degradation processes in aqueous solution under different conditions, with a pH range of 5.0-9.0 and a temperature range of 10-40°C, were systematically investigated by an exponential decay model and a radius basis function (RBF) neural network model, respectively. The half-lives of thuringiensin calculated by the exponential decay model ranged from 2.72 d to 16.19 d under the different conditions mentioned above. Furthermore, an RBF model with accuracy of 0.1 and SPREAD value 5 was employed to model the degradation processes. The results showed that the model could simulate and predict the degradation processes well. Both the half-lives and the prediction data showed that thuringiensin was an easily degradable antibiotic, which could be an important factor in the evaluation of its safety.
Subject(s)
Adenosine/analogs & derivatives , Models, Chemical , Neural Networks, Computer , Sugar Acids/chemistry , Water Pollutants, Chemical/chemistry , Adenosine/analysis , Adenosine/chemistry , Half-Life , Hydrogen-Ion Concentration , Sugar Acids/analysis , Temperature , Water Pollutants, Chemical/analysisABSTRACT
The co-cultures of Ketogulonicigenium vulgare and Bacillus cereus were subcultured daily for a total of 150 transfers. The yield of 2-keto-gulonic acid (2-KGA) and medium pH in the co-cultures were measured. We found that the serial subcultivation increased the yield of 2-KGA from 77% (original co-culture) to 93% (the 150th transfer of transferred co-culture). The resulted strains are of industrial interests for vitamin C production.
Subject(s)
Bacillus cereus/metabolism , Biotechnology/methods , Cell Culture Techniques/methods , Rhodobacteraceae/metabolism , Sugar Acids/metabolism , Ascorbic Acid/biosynthesis , Bacillus cereus/growth & development , Fermentation , Rhodobacteraceae/growth & development , Sorbose/analysis , Sugar Acids/analysisABSTRACT
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.
Subject(s)
Adenosine/analogs & derivatives , Bacillus thuringiensis/chemistry , Bacterial Toxins/analysis , Crops, Agricultural/chemistry , Sugar Acids/analysis , Tandem Mass Spectrometry/methods , Adenosine/analysis , Chromatography, Liquid/methods , Exotoxins/analysis , Insecticides/chemistry , Limit of Detection , Magnetic Resonance Spectroscopy , Vegetables/chemistryABSTRACT
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.
Subject(s)
Actinomycetales/chemistry , Sugar Acids/analysis , Teichoic Acids/analysis , Uronic Acids/analysis , Carbohydrate Sequence , Cell Wall/chemistry , Nuclear Magnetic Resonance, Biomolecular , Sialic Acids/chemistry , Sugar Acids/chemistry , Teichoic Acids/chemistry , Uronic Acids/chemistryABSTRACT
Structure of the O-specific polysaccharide chain of the lipopolysaccharide (LPS) of Shewanella japonica KMM 3601 was elucidated. The initial and O-deacylated LPS as well as a trisaccharide representing the O-deacetylated repeating unit of the O-specific polysaccharide were studied by sugar analysis along with 1H and 13C NMR spectroscopy. The polysaccharide was found to contain a rare higher sugar, 5,7-diacetamido-3,5,7,9-tetradeoxy-D-glycero-D-talo-non-2-ulosonic acid (a derivative of 4-epilegionaminic acid, 4eLeg). The following structure of the trisaccharide repeating unit was established: â4)-α-4eLegp5Ac7Ac-(2â4)-ß-D-GlcpA3Ac-(1â3)-ß-D-GalpNAc-(1â.