Highly Sensitive Detection of Chemically Modified Thio-Organophosphates by an Enzymatic Biosensing Device: An Automated Robotic Approach.

Pesticides represent some of the most common man-made chemicals in the world. Despite their unquestionable utility in the agricultural field and in the prevention of pest infestation in public areas of cities, pesticides and their biotransformation products are toxic to the environment and hazardous to human health. Esterase-based biosensors represent a viable alternative to the expensive and time-consuming systems currently used for their detection. In this work, we used the esterase-2 from Alicyclobacillus acidocaldarius as bioreceptor for a biosensing device based on an automated robotic approach. Coupling the robotic system with a fluorescence inhibition assay, in only 30 s of enzymatic assay, we accomplished the detection limit of 10 pmol for 11 chemically oxidized thio-organophosphates in solution. In addition, we observed differences in the shape of the inhibition curves determined measuring the decrease of esterase-2 residual activity over time. These differences could be used for the characterization and identification of thio-organophosphate pesticides, leading to a pseudo fingerprinting for each of these compounds. This research represents a starting point to develop technologies for automated screening of toxic compounds in samples from industrial sectors, such as the food industry, and for environmental monitoring.

Cryo-electron Microscopy Structure and Transport Mechanism of a Wall Teichoic Acid ABC Transporter.

The wall teichoic acid (WTA) is a major cell wall component of Gram-positive bacteria, such as methicillin-resistant Staphylococcus aureus (MRSA), a common cause of fatal clinical infections in humans. Thus, the indispensable ABC transporter TarGH, which flips WTA from cytoplasm to extracellular space, becomes a promising target of anti-MRSA drugs. Here, we report the 3.9-Å cryo-electron microscopy (cryo-EM) structure of a 50% sequence-identical homolog of TarGH from Alicyclobacillus herbarius at an ATP-free and inward-facing conformation. Structural analysis combined with activity assays enables us to clearly decode the binding site and inhibitory mechanism of the anti-MRSA inhibitor Targocil, which targets TarGH. Moreover, we propose a "crankshaft conrod" mechanism utilized by TarGH, which can be applied to similar ABC transporters that translocate a rather big substrate through relatively subtle conformational changes. These findings provide a structural basis for the rational design and optimization of antibiotics against MRSA.IMPORTANCE The wall teichoic acid (WTA) is a major component of cell wall and a pathogenic factor in methicillin-resistant Staphylococcus aureus (MRSA). The ABC transporter TarGH is indispensable for flipping WTA precursor from cytoplasm to the extracellular space, thus making it a promising drug target for anti-MRSA agents. The 3.9-Å cryo-EM structure of a TarGH homolog helps us to decode the binding site and inhibitory mechanism of a recently reported inhibitor, Targocil, and provides a structural platform for rational design and optimization of potential antibiotics. Moreover, we propose a "crankshaft conrod" mechanism to explain how a big substrate is translocated through subtle conformational changes of type II exporters. These findings advance our understanding of anti-MRSA drug design and ABC transporters.

Physico-chemical approach to adhesion of Alicyclobacillus cells and spores to model solid materials.

Acidothermophilic bacteria of the genus Alicyclobacillus are frequent contaminants of fruit-based products. This study is the first attempt to characterize the physico-chemical surface properties of two Alicyclobacillus sp. and quantify their adhesion disposition to model materials [diethylaminoethyl (DEAE), carboxyl- and octyl-modified magnetic beads] representing materials with different surface properties used in the food industry. An insight into the mechanism of adhesion was gained through comparison of experimental adhesion intensities with predictions of a colloidal interaction model (XDLVO). Experimental data (contact angles, zeta potentials, size) on interacting surfaces (cells and materials) were used as inputs into the XDLVO model. The results revealed that the most significant adhesion occurred at pH 3. Adhesion of both vegetative cells and spores of two Alicyclobacillus sp. to all materials studied was the most pronounced under acidic conditions, and adhesion was influenced mostly by electrostatic attractions. The most intensive adhesion of vegetative cells and spores at pH 3 was observed for DEAE followed by hydrophobic octyl and hydrophilic carboxyl surfaces. Overall, the lowest rate of adhesion between cells and model materials was observed at an alkaline pH. Consequently, prevention of adhesion should be based on the use of alkaline sanitizers and/or alkaline rinse water.

Identification of spoilage by Alicyclobacillus bacteria in tomato-based products by UHPLC-MS/MS.

In recent years, there have been some cases of alteration of tomato derivatives by Alicyclobacilli spp. The spoilage was reported by consumers who felt odors and abnormal tastes ("bad") of products. In this work, an untarget approach with Q-Exactive™ Quadrupole-Orbitrap Mass Spectrometer has been used to identify metabolites associated with the alteration of tomato puree by Alicyclobacillus bacteria. The study was carried out on tomato puree found positive for the presence of Alicyclobacillus spores. The germination and growth of native spores were induced through incubation at 65°C under static conditions for 5 days. For microbial concentration below 100 ufc/mL, negative growth was considered. The samples arising from incubation were submitted to UHPL-HRMS/MS analysis. To find compounds related to microbial growth, data obtained by UHPLC-MS/MS analysis were processed with Thermo Fisher Compound Discoverer™ 2.0 software. We compared samples grouped according to microbial growth: positive growth vs negative growth. Five compounds were identified and confirmed by analysis performed on authentic standards. To other compounds, we have assigned a putative identity. The HRMS approach using Q-Exactive™ Quadrupole-Orbitrap Mass Spectrometer has proven to be an important and powerful tool for identification of compounds which could be used as marker molecules in cases of suspected spoilage by Alicyclobacillus bacteria in tomato derivatives.

Comparison of Alicyclobacillus acidocaldarius o-Succinylbenzoate Synthase to Its Promiscuous N-Succinylamino Acid Racemase/ o-Succinylbenzoate Synthase Relatives.

Studying the evolution of catalytically promiscuous enzymes like those from the N-succinylamino acid racemase/ o-succinylbenzoate synthase (NSAR/OSBS) subfamily can reveal mechanisms by which new functions evolve. Some enzymes in this subfamily have only OSBS activity, while others catalyze OSBS and NSAR reactions. We characterized several NSAR/OSBS subfamily enzymes as a step toward determining the structural basis for evolving NSAR activity. Three enzymes were promiscuous, like most other characterized NSAR/OSBS subfamily enzymes. However, Alicyclobacillus acidocaldarius OSBS (AaOSBS) efficiently catalyzes OSBS activity but lacks detectable NSAR activity. Competitive inhibition and molecular modeling show that AaOSBS binds N-succinylphenylglycine with moderate affinity in a site that overlaps its normal substrate. On the basis of possible steric conflicts identified by molecular modeling and sequence conservation within the NSAR/OSBS subfamily, we identified one mutation, Y299I, that increased NSAR activity from undetectable to 1.2 × 102 M-1 s-1 without affecting OSBS activity. This mutation does not appear to affect binding affinity but instead affects kcat, by reorienting the substrate or modifying conformational changes to allow both catalytic lysines to access the proton that is moved during the reaction. This is the first site known to affect reaction specificity in the NSAR/OSBS subfamily. However, this gain of activity was obliterated by a second mutation, M18F. Epistatic interference by M18F was unexpected because a phenylalanine at this position is important in another NSAR/OSBS enzyme. Together, modest NSAR activity of Y299I AaOSBS and epistasis between sites 18 and 299 indicate that additional sites influenced the evolution of NSAR reaction specificity in the NSAR/OSBS subfamily.

Development of a Polyclonal Antibody-Based Sandwich Enzyme-Linked Immunosorbent Assay for the Detection of Spores of Alicyclobacillus acidoterrestris in Various Fruit Juices.

A polyclonal rabbit antibody-based sandwich ELISA for the rapid and specific detection of spores of Alicyclobacillus acidoterrestris was established. The reactivity of the antisera with spores was confirmed by immunofluorescence. For a thorough evaluation of the ELISA, 61 strains and isolates of Alicyclobacillus spp. were characterized regarding their guaiacol production ability and genetic variability. The ELISA was highly sensitive, the detection limits were isolate-dependent and ranged from 2.1 × 10(3) - 3.8 × 10(4) spores/mL, except for one isolate, for which a slightly lower sensitivity (5 × 10(5) spores/mL) was observed. Inclusivity tests revealed that the ELISA reacts with all tested A. acidoterrestris, while no cross-reactions with spores of 30 strains of Bacillus spp. and Clostridium spp. were observed. Further on, the assay applicability was tested with orange, apple (clear and unfiltered), tomato, pink grapefruit, pear, and white grape juices. Juices were inoculated with 1 or 10 spores/mL of A. acidoterrestris. After enrichment for 48 h, the established ELISA enabled the reliable and reproducible detection of contaminated samples. The enriched samples could be applied directly to the assay, underlining the robustness of the developed ELISA method.

Deleting the Ig-Like Domain of Alicyclobacillus acidocaldarius Endoglucanase Cel9A Causes a Simultaneous Increase in the Activity and Stability.

Endoglucanase Cel9A from Alicyclobacillus acidocaldarius (AaCel9A) is a monomeric enzyme with 537 residues. This enzyme has an Ig-like domain in the N-terminus of the catalytic domain. In this study, the role of the Ig-like domain on the activity, stability, and structural rigidity of AaCel9A and the effect of calcium on enzyme activity and stability were examined by comparing a truncated enzyme with deletion of the Ig-like domain (AaCel9AΔN) to the wild-type enzyme. Our results showed that the deletion of the Ig-like domain increased the catalytic efficiency of the truncated enzyme up to threefold without any significant changes in the K m of the enzyme. Furthermore, pH and temperature optimum for activity were shifted from 6.5 to 7.5 and from 65 to 60 °C, respectively, by deletion of the Ig-like domain. The thermal stability and fluorescence quenching results indicated that the stability and rigidity of the truncated enzyme have been more than that of the wild-type enzyme. Calcium similarly increased the catalytic efficiency of the enzymes (up to 40 %) and remarkably raised the stability of the AaCel9A compared to the AaCel9AΔN. This shows that Ig-like domain has a role in the increase of the enzyme stability by calcium in the wild-type enzyme.

[Gene cloning, heterologous expression and enzyme characterizations of halo-tolerant endoglucanase from Alicyclobacillus D-1].

Objective: An endoglucanase gene (gluE1) was cloned from a thermalacidophilus (Alicyclobacillus tengchongensis CGMCC1504) isolated from a hot spring, and the sequence and biochemical characterization of enzyme were analyzed. Methods: The full-length gluE1 was obtained based on genome sequencing, analysis of amino acid sequence of GluE1. gluE1 was ligated into pEASY-E2 vector and expressed in Escherichia coli BL21 (DE3) cells. GluE1 was purified to electrophoretic homogeneity by Ni2+-NTA metal chelating affinity chromatography, and then the enzyme characterizations were determined. Results: The 1020 bp full-length gluE1 (50.5% GC content) encodes a 339 residues polypeptide (GluE1: 40.45 kDa). GluE1 showed the highest identity of 97% with endoglucanase in public databases, and <60% identities with other endoglucanase. GluE1 efficiently hydrolyzed CMC-Na, soluble starch and barley-ß-glucan, which showed apparent optimal at pH 6.5 and 55℃. GluE1 was stable and active (>60%) at pH 5.0-10.0, and had a high stability at 37℃; and it exhibited Km, Vmax and kcat values of 8.58 mg/mL, 416.67 U/mg and 280.90 s-1 respectively. GluE1 was strongly inhibited by Ag+, Hg2+ and SDS, partial promoted by ß-Mercaptoethanol, Pb2+, Mg2+, Ca2+ and Na+, 30% NaCl still retains more than 64% of the activity. The residual enzyme activity kept 93% after pre-incubation of the enzyme in 30% NaCl. Conclusion: Endoglucanase gene gluE1 from Alicyclobacillus was first reported, and GluE1 showed a good pH stability and strong halo-tolerant property. GluE1 might have greater potential applications.

Modeling the effects of temperature and pH on the resistance of Alicyclobacillus acidoterrestris in conventional heat-treated fruit beverages through a meta-analysis approach.

In this work, all publicly-accessible published findings on Alicyclobacillus acidoterrestris heat resistance in fruit beverages as affected by temperature and pH were compiled. Then, study characteristics (protocols, fruit and variety, °Brix, pH, temperature, heating medium, culture medium, inactivation method, strains, etc.) were extracted from the primary studies, and some of them incorporated to a meta-analysis mixed-effects linear model based on the basic Bigelow equation describing the heat resistance parameters of this bacterium. The model estimated mean D* values (time needed for one log reduction at a temperature of 95 °C and a pH of 3.5) of Alicyclobacillus in beverages of different fruits, two different concentration types, with and without bacteriocins, and with and without clarification. The zT (temperature change needed to cause one log reduction in D-values) estimated by the meta-analysis model were compared to those ('observed' zT values) reported in the primary studies, and in all cases they were within the confidence intervals of the model. The model was capable of predicting the heat resistance parameters of Alicyclobacillus in fruit beverages beyond the types available in the meta-analytical data. It is expected that the compilation of the thermal resistance of Alicyclobacillus in fruit beverages, carried out in this study, will be of utility to food quality managers in the determination or validation of the lethality of their current heat treatment processes.

Effect of sporulation medium on wet-heat resistance and structure of Alicyclobacillus acidoterrestris DSM 3922-type strain spores and modeling of the inactivation kinetics in apple juice.

Alicyclobacillus acidoterrestris is a spoilage bacterium in fruit juices leading to high economic losses. The present study evaluated the effect of sporulation medium on the thermal inactivation kinetics of A. acidoterrestris DSM 3922 spores in apple juice (pH3.82±0.01; 11.3±0.1 °Brix). Bacillus acidocaldarius agar (BAA), Bacillus acidoterrestris agar (BATA), malt extract agar (MEA), potato dextrose agar (PDA) and B. acidoterrestris broth (BATB) were used for sporulation. Inactivation kinetic parameters at 85, 87.5 and 90°C were obtained using the log-linear model. The decimal reduction times at 85°C (D85°C) were 41.7, 57.6, 76.8, 76.8 and 67.2min; D87.5°C-values were 22.4, 26.7, 32.9, 31.5, and 32.9min; and D90°C-values were 11.6, 9.9, 14.7, 11.9 and 14.1min for spores produced on PDA, MEA, BATA, BAA and BATB, respectively. The estimated z-values were 9.05, 6.60, 6.96, 6.15, and 7.46, respectively. The present study suggests that the sporulation medium affects the wet-heat resistance of A. acidoterrestris DSM 3922 spores. Also, the dipicolinic acid content (DPA) was found highest in heat resistant spores formed on mineral containing media. After wet-heat treatment, loss of internal volume due to the release of DPA from spore core was observed by scanning electron microscopy. Since, there is no standardized media for the sporulation of A. acidoterrestris, the results obtained from this study might be useful to determine and compare the thermal resistance characteristics of A. acidoterrestris spores in fruit juices.

Molecular dynamics study of the conformational stability of esterase 2 from Alicyclobacillus acidocaldarius.

Circular dichroism and differential scanning calorimetry measurements showed that esterase 2 from the thermophilic microorganism Alicyclobacillus acidocaldarius, EST2, and its variant in which the first 35 residues have been deleted, EST2-36 del, unfold reversibly on increasing temperature, and possess two cooperative and coupled domains [12]. Structural features of the α/ß hydrolase fold of EST2, with nine α-helices packed against the central twisted ß-sheet, do not allow a straightforward identification of these two cooperative and coupled domains. Molecular dynamics simulations, each one 20 ns long, have been performed at 300, 400 and 500 K, on both proteins in explicit water. Suitable analysis of MD trajectories has allowed a reliable identification of the two cooperative domains (i.e., the less stable one corresponds to external α-helices, whereas the more stable one corresponds to the central twisted ß-sheet) and the attribution of the key coupling role to the last and long α-helix of EST2.

[Identification of the catalytic residues of mannanase from Alicyclobacillus acidocaldarius].

OBJECTIVE: To identify the catalytic residues of mannanase AaManA from Alicyclobacillus acidocaldarius. METHODS: Based on the sequence alignment by ClustalX and ESPript and the structure information of GH -53 family, the possible catalytic residues were selected and mutated by overlap extension PCR. The protein of wild type and mutant were expressed in E. coli BL21 (DE3) and ordinal purified by Ni - NTA affinity chromatography, gel - filtrate chromatography and ion - exchange chromatography. The purified protein was analyzed by thin layer chromatography (TLC) and the dinitrosalicylic acid (DNS) methods for enzyme assay. RESULTS: Seven mutants, E151A, E159A, E231A, C150A, E151Q, E231Q and double mutation E151Q&E231Q were successful constructed. Mutant E159A showed similar activities with wild type, and C150A mutation resulted in only a 3 -fold reduction in the activities, but mutations E151A, E231A, E151Q, E231Q and E151Q&E231Q resulted in sharp decreases or loss in the activities, indicating that Glu151 and Glu231 play critical roles in AaManA activity. Furthermore, the presence of Glu151 at the C terminus of beta4 and Glu231 at the C terminus of beta7 was entirely consistent with the positions of the acid/base catalyst and the nucleophile catalyst of a GH - A enzyme, respectively. CONCLUSION: By combining the results of TLC and enzyme assay of those mutants and the structural comparisons, it was confirmed that Glu151 and Glu231 fulfilled the roles of an acid/base catalyst and nucleophile catalyst in AaManA, respectively.

N-acylated bacteriohopanehexol-mannosamides from the thermophilic bacterium Alicyclobacillus acidoterrestris.

Identification of molecular species of various N-acylated bacteriohopanehexol-mannosamides from the thermophilic bacterium Alicyclobacillus acidoterrestris by semipreparative HPLC and by RP-HPLC with ESI is described. We used triple-quadrupole type mass spectrometer, (1)H and (13)C NMR for analyzing this complex lipid. CD spectra of two compounds (model compound--7-deoxy-D: -glycero-D: -allo-heptitol obtained by stereospecific synthesis, and an isolated derivative of hopane) were also measured and the absolute configuration of both compounds was determined. On the basis of all the above methods, we identified the full structure of a new class of bacteriohopanes, represented by various N-acylated bacteriohopanehexol-mannosamides.

Alicyclobacillus acidoterrestris in pasteurized exotic Brazilian fruit juices: isolation, genotypic characterization and heat resistance.

In this study, the population of Alicyclobacillus spp. was estimated in pasteurized exotic Brazilian fruit juices using the most probable number (MPN) technique followed by biochemical tests. Pasteurized passion fruit (n = 57) and pineapple (n = 50) juices were taken directly from Brazilian manufacturers. While Alicyclobacillus spp. was isolated from passion fruit juice, the microorganism was not found in any pineapple juice samples. A higher incidence of Alicyclobacillus was observed in samples taken in June and July (dry months in Brazil) in comparison to the other months (March, April, May and August), and the highest Alicyclobacillus counts were recovered from these samples(>23 MNP/100 mL). Sixteen (n = 16) Alicyclobacillus strains were typed using the randomly amplified polymorphic DNA method (RAPD-PCR). RAPD-PCR revealed great genetic similarity between the passion fruit juice strains and Alicyclobacillus acidoterrestris DSM 2498. The heat resistance of three isolates was determined, and the mean D(95°) (1.7 min) and z (7.6 °C) values in the passion fruit juice were not significantly different (p > 0.05) from those obtained for the DSM 2498 strain (D(95°) = 1.5 min and z = 7.1 °C). This is the first report on the isolation of A. acidoterrestris from exotic fruit juices such as passion fruit juice. It is worth pointing out the importance of applying good agricultural practices in the field and applying controls for the fruit selection and washing steps, as well as controlling the time/temperature conditions for pasteurization so as to reduce the incidence and chances of A. acidoterrestris spoilage in these juices.

A novel family 9 beta-1,3(4)-glucanase from thermoacidophilic Alicyclobacillus sp. A4 with potential applications in the brewing industry.

An endo-beta-1,3(4)-glucanase gene, Agl9A, was cloned from Alicyclobacillus sp. A4 and expressed in Pichia pastoris. Its deduced amino acid sequence shared the highest identity (48%) with an endo-beta-1,4-glucansae from Alicyclobacillus acidocaldarius that belongs to family 9 of the glycoside hydrolases. The purified recombinant Agl9A exhibited relatively wide substrate specificity, including lichenan (109%), barley beta-glucan (100%), CMC-Na (15.02%), and laminarin (6.19%). The optimal conditions for Agl9A activity were pH 5.8 and 55 degrees C. The enzyme was stable over a broad pH range (>60% activity retained after 1-h incubation at pH 3.8-11.2) and at 60 degrees C (>70% activity retained after 1-h incubation). Agl9A was highly resistant to various neutral proteases (e.g., trypsin, alpha-chymotrypsin, and collagenase) and Neutrase 0.8L (Novozymes), a protease widely added to the mash. Under simulated mashing conditions, addition of Agl9A (20 U/ml) or a commercial xylanase (200 U/ml) reduced the filtration rate (26.71% and 20.21%, respectively) and viscosity (6.12% and 4.78%, respectively); furthermore, combined use of Agl9A (10 U/ml) and the xylanase (100 U/ml) even more effectively reduced the filtration rate (31.73%) and viscosity (8.79%). These characteristics indicate that Agl9A is a good candidate to improve glucan degradation in the malting and brewing industry.

Extremely acidic beta-1,4-glucanase, CelA4, from thermoacidophilic Alicyclobacillus sp. A4 with high protease resistance and potential as a pig feed additive.

An acidic endo-beta-1,4-glucanase, denoted CelA4 ( approximately 48 kDa), was purified from thermoacidophilic Alicyclobacillus sp. A4. Two internal peptides of CelA4 showed strong sequence identity to the Alicyclobacillus acidocaldarius cellulase precursor and contained the conserved domain and catalytic region of glycoside hydrolase family 51 beta-1,4-glucanases, and the N-terminal and three other internal peptides had no close glucanase or cellulase relatives, suggesting that the enzyme might be novel. CelA4 had broad substrate specificity, exhibited maximum activity at 65 degrees C and pH 2.6, was stable over pH 1.8-7.6, and showed strong resistance to acidic and neutral proteases, notably pepsin. In comparison to the commercial endo-beta-1,3-1,4-glucanase, CelA4 was more stable, released more reducing sugar from barley beta-glucan, and under simulated gastric conditions, decreased the viscosity of barley-soybean feed to a greater extent. These properties make CelA4 a good candidate as a new commercial glucanase to improve the nutrient bioavailability of pig feed.