Optimization of a sample preparation method for the metabolomic analysis of clinically relevant bacteria

Metabolomics, or metabolite profiling, is an approach that is increasingly used to study the metabolism of diverse organisms, elucidate biological processes and/or find characteristic biomarkers of physiological states. Here, we describe the optimization of a method for global metabolomic analysis of bacterial cultures, with the following steps. Cells are grown to log-phase, starting from an overnight culture and bacterial concentrations are monitored by measuring the optical density of the cultures at 600 nm. At an appropriate density they are harvested by centrifugation, washed three times with NaCl solution and metabolites are extracted using methanol and a bead-mill. Dried extracts are methoxymated and derivatized with methyltrimethylsilyltrifluoroacetamide (MSTFA) then analyzed using gas chromatography coupled to time-of-flight mass spectrometry (GC-MS/TOF). Finally, patterns in the acquired data are examined by multivariate data modeling. This method enabled us to obtain reproducible metabolite profiles of Yersinia pseudotuberculosis, with about 25% compound identification, based on comparison with entries in available GC-MS libraries. To assess the potential utility of the method for comparative analysis of other bacterial species we analyzed cultures of Pseudomonas aeruginosa, Salmonella typhimurium, Escherichia coli and methicillin-sensitive Staphylococcus aureus (MSSA). Multivariate analysis of the acquired data showed that it was possible to differentiate the species according to their metabolic profiles. Our results show that the presented procedure can be used for metabolomic analysis of a wide range of bacterial species of clinical interest.     

Analysis of the glucagon receptor first extracellular loop by the substituted cysteine accessibility method

Glucagon is an important hormone for the prevention of hypoglycemia, and contributes to the hyperglycemia observed in diabetic patients, yet very little is known about its receptor structure and the receptor-glucagon interaction. In related receptors, the first extracellular loop, ECL1, is highly variable in length and sequence, suggesting that it might participate in ligand recognition. We applied a variant of the SCAM (Substituted Cysteine Accessibility Method) to the glucagon receptor ECL1 and sequentially mutated positions 197 to 223 to cysteine. Most of the mutations (15/27) affected the glucagon potency, due either to a modification of the glucagon binding site, or to the destabilization of the active receptor conformation. We reasoned that side chains accessible to glucagon must also be accessible to large, hydrophilic cysteine reagents. We therefore evaluated the accessibility of the introduced cysteines to maleimide-PEO₂-biotin ((+)-biotinyl-3-maleimido-propionamidyl-3,6-dioxa-octanediamine), and tested the effect of pretreatment of intact cells with a large cationic cysteine reagent, MTSET ([2-(trimethylammonium)ethyl]methanethiosulfonate bromide), on glucagon potency. Our results suggest that the second and third transmembrane helices (TM2 and TM3) are extended to position 202 and from position 215, respectively, and separated by a short β stretch (positions 203-209). Glucagon binding induced a conformational change close to TM2: L198C was accessible to the biotin reagent only in the presence of glucagon. Most other mutations affected the receptor activation rather than glucagon recognition, but S217 and D218 (at the top of TM3) were good candidates for glucagon recognition and V221 was very close to the binding site.     

A rapid DNA extraction method for PCR amplification from wetland soils

Aims: We tested a method of rapid DNA extraction from wetland soil samples for use in the polymerase chain reaction. Methods and Results: The glass bead/calcium chloride/SDS method obtained in the present study was compared with the calcium chloride/SDS/enzymatic extraction method and the UltraClean? Soil DNA Isolation Kit. Rapid DNA extraction could be completed within about two hours without purification steps. Conclusions: This study succeeded in establishing a fast soil DNA extraction protocol that can be applied to various environmental sources that are rich in humic acid content. Significance and Impact of the Study: The method provides a technology with high‐quality DNA extraction from soils for testing the diversity of AOB and AOA.     

Enumeration of micro-organisms in processed soy products with an automated most probable number method compared with standard plate method

Aim: The automated TEMPO system (bioMerieux) is based on the most probable number (MPN) method for the enumeration of micro‐organisms in foods. In this study, we evaluated the performance of the TEMPO system as a diagnostic tool in comparison with the standard method in processed soy products. Methods and Results: A verification study was conducted using artificially contaminated soy product samples such as soy protein isolate, water‐soluble soy polysaccharides, soy milk and processed soy food. Five types of micro‐organisms were analysed using the automated MPN method (total aerobic bacteria, total coliforms, Enterobacteriaceae, yeast and mould and Staphylococcus aureus) vs the standard plate method. The results from each of the methods were highly correlated (r > 0·95). Naturally contaminated processed soy products on the market were also studied. There were no discrepancies observed between the respective methods. Conclusions: TEMPO methods were equivalent to the corresponding standard plate methods with very good rates of agreement. Significance and Impact of the Study: The automated MPN method is more practical and reliable for in‐house microbiological testing in processed soy products.     

Specific In Situ Visualization of Plasma Cells Producing Antibodies against Porphyromonas gingivalis in Gingival Radicular Cyst: Application of the Enzyme-Labeled Antigen Method

The enzyme-labeled antigen method was applied to visualize plasma cells producing antibodies to Porphyromonas gingivalis, flora of the human oral cavity. Antibodies to P. gingivalis have reportedly been detected in sera of patients with periodontitis. Biotinylated bacterial antigens, Ag53, and four gingipain domains (Arg-pro, Arg-hgp, Lys-pro, and Lys-hgp) were prepared by the cell-free protein synthesis system using the wheat germ extract. In paraformaldehyde-fixed frozen sections of rat lymph nodes experimentally immunized with Ag53-positive and Ag53-negative P. gingivalis, plasma cells were labeled with biotinylated Arg-hgp and Lys-hgp. Antibodies to Ag53 were detected only in the nodes immunized with Ag53-positive bacteria. In two of eight lesions of gingival radicular cyst with inflammatory infiltration, CD138-positive plasma cells in frozen sections were signalized for Arg-hgp and Lys-hgp. An absorption study using unlabeled antigens confirmed the specificity of staining. The AlphaScreen method identified the same-type antibodies in tissue extracts but not in sera. Antibodies to Ag53, Arg-pro, and Lys-pro were undetectable. In two cases, serum antibodies to Arg-hgp and Lys-hgp were AlphaScreen positive, whereas plasma cells were scarcely observed within the lesions. These findings indicate the validity of the enzyme-labeled antigen method. This is the very first application of this novel histochemical technique to human clinical samples.     

AFP-Pred: A random forest approach for predicting antifreeze proteins from sequence-derived properties

Some creatures living in extremely low temperatures can produce some special materials called “antifreeze proteins” (AFPs), which can prevent the cell and body fluids from freezing. AFPs are present in vertebrates, invertebrates, plants, bacteria, fungi, etc. Although AFPs have a common function, they show a high degree of diversity in sequences and structures. Therefore, sequence similarity based search methods often fails to predict AFPs from sequence databases. In this work, we report a random forest approach “AFP-Pred” for the prediction of antifreeze proteins from protein sequence. AFP-Pred was trained on the dataset containing 300 AFPs and 300 non-AFPs and tested on the dataset containing 181 AFPs and 9193 non-AFPs. AFP-Pred achieved 81.33% accuracy from training and 83.38% from testing. The performance of AFP-Pred was compared with BLAST and HMM. High prediction accuracy and successful of prediction of hypothetical proteins suggests that AFP-Pred can be a useful approach to identify antifreeze proteins from sequence information, irrespective of their sequence similarity.     

灰翅麦茎蜂蛹的发育分级研究

室内饲养了灰翅麦茎蜂Cephus fumipennis Eyersmann,对该种蛹的发育进行了研究.结果表明,灰翅麦茎蜂蛹期(30±2.08)d,根据蛹体色的显著变化分为5级(Ⅰ,Ⅱ,Ⅲ,Ⅳ,Ⅴ),其历期依次为:(6.5±0.55)d、(3.9±0.48)d、(4.4±0.5)d、(4.6±0.49)d和(10.5±...     

Improvement on the yield of polyhydroxyalkanotes production from cheese whey by a recombinant Escherichia coli strain using the proton suicide methodology

In this work Escherichia coli strain CML3-1 was engineered through the insertion of Cupriavidus necator P(3HB)-synthesis genes, fused to a lactose-inducible promoter, into the chromosome, via transposition-mediated mechanism. It was shown that polyhydroxyalkanotes (PHAs) production by this strain, using cheese whey, was low due to a significant organic acids (OA) synthesis. The proton suicide method was used as a strategy to obtain an E. coli mutant strain with a reduced OA-producing capacity, aiming at driving bacterial metabolism toward PHAs synthesis.Thirteen E. coli mutant strains were obtained and tested in shake flask assays, using either rich or defined media supplemented with lactose. P8-X8 was selected as the best candidate strain for bioreactor fed-batch tests using cheese whey as the sole carbon source. Although cell growth was considerably slower for this mutant strain, a lower yield of OA on substrate (0.04 Cmol_OA/Cmol_lac) and a higher P(3HB) production (18.88 g_P(3HB)/L) were achieved, comparing to the original recombinant strain (0.11 Cmol_OA/Cmol_lac and 7.8 g_P(3HB)/L, respectively). This methodology showed to be effective on the reduction of OA yield by consequently improving the P(3HB) yield on lactose (0.28 Cmol_P(3HB)/Cmol_lac vs 0.10 Cmol_P(3HB)/Cmol_lac of the original strain).     

Exploring the multiscale signaling behavior of phototropin1 from Chlamydomonas reinhardtii using a full‐residue space kinetic Monte Carlo molecular dynamics technique

Devising analysis tools for elucidating the regulatory mechanism of complex enzymes has been a challenging task for many decades. It generally requires the determination of the structural‐dynamical information of protein solvent systems far from equilibrium over multiple length and time scales, which is still difficult both theoretically and experimentally. To cope with the problem, we introduce a full‐residue space multiscale simulation method based on a combination of the kinetic Monte Carlo and molecular dynamics techniques, in which the rates of the rate‐determining processes are evaluated from a biomolecular forcefield on the fly during the simulation run by taking into account the full space of residues. To demonstrate its reliability and efficiency, we explore the light‐induced functional behavior of the full‐length phototropin1 from Chlamydomonas reinhardtii (Cr‐phot1) and its various subdomains. Our results demonstrate that in the dark state the light oxygen voltage‐2‐Jα (LOV2‐Jα) photoswitch inhibits the enzymatic activity of the kinase, whereas the LOV1‐Jα photoswitch controls the dimerization with the LOV2 domain. This leads to the repulsion of the LOV1‐LOV2 linker out of the interface region between both LOV domains, which results in a positively charged surface suitable for cell–membrane interaction. By contrast, in the light state, we observe that the distance between both LOV domains is increased and the LOV1‐LOV2 linker forms a helix–turn–helix (HTH) motif, which enables gene control through nucleotide binding. Finally, we find that the kinase is activated through the disruption of the Jα‐helix from the LOV2 domain, which is followed by a stretching of the activation loop (A‐loop) and broadening of the catalytic cleft of the kinase. Proteins 2014; 82:2018–2040. © 2014 Wiley Periodicals, Inc.