Bacteriophages of Thermophilic 'Bacillus Group' Bacteria-A Systematic Review, 2023 Update.

Bacteriophages associated with thermophiles are gaining increased attention due to their pivotal roles in various biogeochemical and ecological processes, as well as their applications in biotechnology and bionanotechnology. Although thermophages are not suitable for controlling bacterial infections in humans or animals, their individual components, such as enzymes and capsid proteins, can be employed in molecular biology and significantly contribute to the enhancement of human and animal health. Despite their significance, thermophages still remain underrepresented in the known prokaryotic virosphere, primarily due to limited in-depth investigations. However, due to their unique properties, thermophages are currently attracting increasing interest, as evidenced by several newly discovered phages belonging to this group. This review offers an updated compilation of thermophages characterized to date, focusing on species infecting the thermophilic bacilli. Moreover, it presents experimental findings, including novel proteomic data (39 proteins) concerning the model TP-84 bacteriophage, along with the first announcement of 6 recently discovered thermophages infecting Geobacillus thermodenitrificans: PK5.2, PK2.1, NIIg10.1, NIIg2.1, NIIg2.2, and NIIg2.3. This review serves as an update to our previous publication in 2021.

Recombinant TP-84 Bacteriophage Glycosylase-Depolymerase Confers Activity against Thermostable Geobacillus stearothermophilus via Capsule Degradation.

The TP-84 bacteriophage, which infects Geobacillus stearothermophilus strain 10 (G. stearothermophilus), has a genome size of 47.7 kilobase pairs (kbps) and contains 81 predicted protein-coding ORFs. One of these, TP84_26 encodes a putative tail fiber protein possessing capsule depolymerase activity. In this study, we cloned the TP84_26 gene into a high-expression Escherichia coli (E. coli) system, modified its N-terminus with His-tag, expressed both the wild type gene and His-tagged variant, purified the recombinant depolymerase variants, and further evaluated their properties. We developed a direct enzymatic assay for the depolymerase activity toward G. stearothermophilus capsules. The recombinant TP84_26 protein variants effectively degraded the existing bacterial capsules and inhibited the formation of new ones. Our results provide insights into the novel TP84_26 depolymerase with specific activity against thermostable G. stearothermophilus and its role in the TP-84 life cycle. The identification and characterization of novel depolymerases, such as TP84_26, hold promise for innovative strategies to combat bacterial infections and improve various industrial processes.

Selection of spore-specific aptamers for Geobacillus stearothermophilus, a food spoilage bacterium.

Due to their ability to form extremely heat resistant spores, anaerobic bacteria are responsible for frequent food spoilage. The development of rapid and specific methods for the detection and quantification of spore contamination is therefore of major interest. In this paper, we describe for the first time the selection of aptamers specific to spores of Geobacillus stearothermophilus (Gbs), which induce flat sour spoilage in vegetable cans. Eighteen Spore-SELEX cycles were performed including 4 counter-selections with 12 bacteria commonly found in cannery. To optimise candidate amplification, PCR in emulsion was performed, and high-throughput sequencing analysis was applied to follow candidate evolution. Sequencing of aptamers from cycle 18 revealed 43 overrepresented sequences whose copy number exceeds 0.15% of the total obtained sequences. Within this group, the A01 aptamer presented a much higher enrichment with a relative abundance of 17.71%. Affinity and specificity for Gbs spores of the 10 most abundant candidates at cycle 18 were confirmed by PCR assay based on aptamer-spore complex formation and filtration step. Obtaining these aptamers is the starting point for the future development of biosensors dedicated to the detection of Gbs spores.

A novel thermostable TP-84 capsule depolymerase: a method for rapid polyethyleneimine processing of a bacteriophage-expressed proteins.

BACKGROUND: In spite of the fact that recombinant enzymes are preferably biotechnologically obtained using recombinant clones, the purification of proteins from native microorganisms, including those encoded by bacteriophages, continues. The native bacteriophage protein isolation is often troubled by large volumes of the infected bacterial cell lysates needed to be processed, which is highly undesired in scaled-up industrial processing. A well-known ammonium sulphate fractionation is often a method of choice during purification of the native bacteriophage protein. However, this method is time-consuming and cumbersome, and requires large amounts of the relatively expensive reagent. Thus, other effective and inexpensive methods of reversible protein precipitation are highly desirable. We have previously characterized thermophilic TP-84 bacteriophage, defined a new genus TP84virus within Siphoviridae family, conducted the TP-84 genome annotation and proteomic analysis. The longest Open Reading Frame (ORF) identified in the genome is TP84_26. We have previously annotated this ORF as a hydrolytic enzyme depolymerizing the thick polysaccharides host's capsule. RESULTS: The TP84_26 'capsule depolymerase' (depolymerase) is a large, 112 kDa protein, biosynthesized by the infected Geobacillus stearothermophilus 10 (G. stearothermophilus 10) cells. The TP84_26 protein biosynthesis was confirmed by three approaches: (i) purification of the protein of the expected size; (ii) mass spectrometry (LC-MS) analysis and (iii) detection of the enzymatic activity toward G. stearothermophilus polysaccharide capsules. Streptomycin-resistant mutant of the host was generated and microbiological aspects of both the TP-84 and G. stearothermophilus 10 were determined. A new variant of polyethyleneimine (PEI)-mediated purification method was developed, using the novel TP-84 depolymerase as a model. The enzyme was characterized. Three depolymerase forms were detected: soluble, unbound proteins in the bacteriophage/cells lysate and another integrated into the TP-84 virion. CONCLUSIONS: The novel TP-84 depolymerase was purified and characterized. The enzyme exists in three forms. The soluble, unbound forms are probably responsible for the weakening of the capsules of the uninfected bacterial cells. The form integrated into virion particles may generate a local passage for the invading TP-84. The developed PEI purification method appears well suited for the scaled-up or industrial production of bacteriophage proteins.

Novel hyperthermoacidic archaeal enzymes for removal of thermophilic biofilms from stainless steel.

AIMS: To test the efficacy of novel hot/acid hyperthermoacidic enzyme treatments on the removal of thermophilic spore-forming biofilms from stainless steel surfaces. METHODS AND RESULTS: The present study measured the efficacy of hyperthermoacidic enzymes (protease, amylase, and endoglucanase) that are optimally active at low pH (≈3.0) and high temperatures (≈80°C) at removing thermophilic bacilli biofilms from stainless steel (SS) surfaces. Plate counts, spore counts, impedance microbiology, as well as epifluorescence microscopy, and scanning electron microscopy (SEM) were used to evaluate the cleaning and sanitation of biofilms grown in a continuous flow biofilm reactor. Previously unavailable hyperthermoacidic amylase, protease, and the combination of amylase and protease were tested on Anoxybacillus flavithermus and Bacillus licheniformis, and endoglucanase was tested on Geobacillus stearothermophilus. In all cases, the heated acidic enzymatic treatments significantly reduced biofilm cells and their sheltering extracellular polymeric substances (EPS). CONCLUSIONS: Hyperthermoacidic enzymes and the associated heated acid conditions are effective at removing biofilms of thermophilic bacteria from SS surfaces that contaminate dairy plants.

Integrated lactic acid production from lignocellulosic agricultural wastes under thermal conditions.

The production of lactic acid (LA) from agricultural wastes attracts great attention because of the sustainability and abundance of lignocellulosic feedstocks, as well as the increasing demand for biodegradable polylactic acid. In this study, we isolated a thermophilic strain Geobacillus stearothermophilus 2H-3 for use in robust production of L-(+)LA under the optimal conditions of 60 °C, pH 6.5, which were consistent with the whole-cell-based consolidated bio-saccharification (CBS) process. Sugar-rich CBS hydrolysates derived from various agricultural wastes, including corn stover, corncob residue, and wheat straw, were used as the carbon sources for 2H-3 fermentation by directly inoculating 2H-3 cells into the CBS system, without intermediate sterilization, nutrient supplementation, or adjustment of fermentation conditions. Thus, we successfully combined two whole-cell-based steps into a one-pot successive fermentation process to efficiently produce LA with high optical purity (99.5%), titer (51.36 g/L), and yield (0.74 g/gbiomass). This study provides a promising strategy for LA production from lignocellulose through CBS and 2H-3 fermentation integration.

Adenylate kinases of thermophiles Aquifex aeolicus and Geobacillus stearothermophilus: biochemical and kinetic studies.

Adenylate kinases (AK) play a pivotal role in the regulation of cellular energy. The aim of our work was to achieve the overproduction and purification of AKs from two groups of bacteria and to determine, for the first time, the comprehensive biochemical and kinetic properties of adenylate kinase from Gram-negative Aquifex aeolicus (AKaq) and Gram-positive Geobacillus stearothermophilus (AKst). Therefore we determined KM and Vmax values, and the effects of temperature, pH, metal ions, donors of the phosphate groups and inhibitor Ap5A for both thermophilic AKs. The kinetic studies indicate that both AKs exhibit significantly higher affinity for substrates with the pyrophosphate group than for adenosine monophosphate. AK activation by Mg2+ and Mn2+ revealed that both ions are efficient in the synthesis of adenosine diphosphate and adenosine triphosphate; however, Mn2+ ions at 0.2-2.0 mmol/L concentration were more efficient in the activation of the ATP synthesis than Mg2+ ions. Our research demonstrates that zinc ions inhibit the activity of enzymes in both directions, while Ap5A at a concentration of 10 µmol/L and 50 µmol/L inhibited both enzymes with a different efficiency. Sigmoid-like kinetics were detected at high ATP concentrations not balanced by Mg2+, suggesting the allosteric effect of ATP for both bacterial AKs.

High level expression and biochemical characterization of an alkaline serine protease from Geobacillus stearothermophilus to prepare antihypertensive whey protein hydrolysate.

BACKGROUND: Proteases are important for hydrolysis of proteins to generate peptides with many bioactivities. Thus, the development of novel proteases with high activities is meaningful to discover bioactive peptides. Because natural isolation from animal, plant and microbial sources is impractical to produce large quantities of proteases, gene cloning and expression of target protease are preferred. RESULTS: In this study, an alkaline serine protease gene (GsProS8) from Geobacillus stearothermophilus was successfully cloned and expressed in Bacillus subtilis. The recombinant GsProS8 was produced with high protease activity of 3807 U/mL after high cell density fermentation. GsProS8 was then purified through ammonium sulfate precipitation and a two-step chromatographic method to obtain the homogeneous protease. The molecular mass of GsProS8 was estimated to be 27.2 kDa by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and 28.3 kDa by gel filtration. The optimal activity of GsProS8 was found to be pH 8.5 and 50 °C, respectively. The protease exhibited a broad substrate specificity and different kinetic parameters to casein and whey protein. Furthermore, the hydrolysis of whey protein using GsProS8 resulted in a large amount of peptides with high angiotensin-I-converting enzyme (ACE) inhibitory activity (IC50 of 0.129 mg/mL). CONCLUSIONS: GsProS8 could be a potential candidate for industrial applications, especially the preparation of antihypertensive peptides.

Airborne Disinfection by Dry Fogging Efficiently Inactivates Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2), Mycobacteria, and Bacterial Spores and Shows Limitations of Commercial Spore Carriers.

Airborne disinfection of high-containment facilities before maintenance or between animal studies is crucial. Commercial spore carriers (CSC) coated with 106 spores of Geobacillus stearothermophilus are often used to assess the efficacy of disinfection. We used quantitative carrier testing (QCT) procedures to compare the sensitivity of CSC with that of surrogates for nonenveloped and enveloped viruses, including severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), mycobacteria, and spores, to an aerosolized mixture of peroxyacetic acid and hydrogen peroxide (aPAA-HP). We then used the QCT methodology to determine relevant process parameters to develop and validate effective disinfection protocols (≥4-log10 reduction) in various large and complex facilities. Our results demonstrate that aPAA-HP is a highly efficient procedure for airborne room disinfection. Relevant process parameters such as temperature and relative humidity can be wirelessly monitored. Furthermore, we found striking differences in inactivation efficacies against some of the tested microorganisms. Overall, we conclude that dry fogging a mixture of aPAA-HP is highly effective against a broad range of microorganisms as well as material compatible with relevant concentrations. Furthermore, CSC are artificial bioindicators with lower resistance and thus should not be used for validating airborne disinfection when microorganisms other than viruses have to be inactivated.IMPORTANCE Airborne disinfection is not only of crucial importance for the safe operation of laboratories and animal rooms where infectious agents are handled but also can be used in public health emergencies such as the current severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic. We show that dry fogging an aerosolized mixture of peroxyacetic acid and hydrogen peroxide (aPAA-HP) is highly microbicidal, efficient, fast, robust, environmentally neutral, and a suitable airborne disinfection method. In addition, the low concentration of dispersed disinfectant, particularly for enveloped viral pathogens such as SARS-CoV-2, entails high material compatibility. For these reasons and due to the relative simplicity of the procedure, it is an ideal disinfection method for hospital wards, ambulances, public conveyances, and indoor community areas. Thus, we conclude that this method is an excellent choice for control of the current SARS-CoV-2 pandemic.

Viability of bacterial spores surviving heat-treatment is lost by further incubation at temperature and pH not suitable for growth.

Spores from 21 strains from different genera were heat-treated and stored in different sets of process conditions (4 temperatures and 3 pH levels) defined to prevent growth. In these conditions, spores surviving the heat treatment progressively lost viability during storage. Different inactivation curve shapes (linear, shoulder and tailing) and different sensitivities to storage were observed. B. coagulans showed the fastest inactivation kinetics, with more than 4-log reduction of spore population within 24 h after heating and G. stearothermophilus displayed slower inactivation kinetics, whereas all the anaerobic strains studied (M. thermoacetica and Thermoanaerobacterium spp.) proved resistant to storage conditions, with no destruction detected during 90 days in most cases. Inactivation rates were relatively unaffected by sub-lethal pH but sharply accelerated by temperature: Inactivation became faster as temperature increased (in the 8 °C-55 °C temperature range), with growth blocked by low pH in sub-lethal temperatures. There were changes in surviving spore numbers after the heat-treatment phase. This has implications and applications in canned food industries, as the probability of a retorted sample testing as non-stable, meaning possible spoilage, may decrease with time. In simple terms, a batch of low-acid canned food that tests as non-shelf-stable after an incubation test i.e. positive growth conditions, may later become negative if stored at room temperature (below the minimal growth temperature for thermophilic spores), which may change the marketability of the batch.

Characterization of the substrate scope of an alcohol dehydrogenase commonly used as methanol dehydrogenase.

Many alcohol dehydrogenases (ADHs) catalyze oxidation of a broad scope of alcohols. When an NAD-dependent ADH oxidizes methanol, albeit at a poor rate, it may be treated as methanol dehydrogenase (MDH). One ADH from Geobacillus stearothermophilus DSM 2334 (GsADH) has been widely used as MDH, but its actual substrate scope remains less characterized. Here we purified recombinant GsADH from Escherichia coli and determined its crystal structure. We collected kinetics data of this enzyme towards a number of short chain alcohols, and found that isopropanol is by far the most favorable substrate. Moreover, molecular docking analysis suggested that substrate preference is mainly attributed to the conformer energy of the protein-substrate complex. Our data clarified the substrate scope of GsADH and provided structural insights, which may facilitate more efficient cofactor regeneration and rational metabolic engineering.

A microbiological inhibition method for the rapid, broad-spectrum, and high-throughput screening of 34 antibiotic residues in milk.

In this study, we developed a microbiological inhibition method for the rapid screening of antibiotics in milk with Geobacillus stearothermophilus ATCC12980 as an indicator bacterium and an easy sample pretreatment. We observed that the limits of detection of the kit for 34 common antibiotic residues in milk, including ß-lactams (13), aminoglycosides (6), tetracyclines (4), sulfonamides (6), macrolides (4), lincosamides (1), were lower than or close to the maximum residue limits formulated by the European Union and China. Moreover, the false-positive rate was 1% and the false-negative rates were less than 5%. The ruggedness of the method (the reproducibility of detection capability of different batches of medium) met requirements at determined levels and residual limits. The shelf life of the kit was more than 6 mo at 4°C. Additionally, we observed good correlations between the kit results and ultra-high-performance liquid chromatography-tandem mass spectrometry results for incurred milk (samples taken from animals treated with antibiotics according to the pre-slaughter medication data), which indicated that the kit was reliable for screening antibiotics in incurred samples. In conclusion, the kit has a broad application potential with high sensitivity, specificity, and reproducibility, stability, and reliability, combined with simple operation, low cost, and high-throughput capacity.

One Intact Transmembrane Substrate Binding Site Is Sufficient for the Function of the Homodimeric Type I ATP-Binding Cassette Importer for Positively Charged Amino Acids Art(MP)2 of Geobacillus stearothermophilus.

ATP-binding cassette (ABC) transport systems comprise two transmembrane domains/subunits that form a translocation path and two nucleotide-binding domains/subunits that bind and hydrolyze ATP. Prokaryotic canonical ABC import systems require an extracellular substrate-binding protein for function. Knowledge of substrate-binding sites within the transmembrane subunits is scarce. Recent crystal structures of the ABC importer Art(QN)2 for positively charged amino acids of Thermoanerobacter tengcongensis revealed the presence of one substrate molecule in a defined binding pocket in each of the transmembrane subunits, ArtQ (J. Yu, J. Ge, J. Heuveling, E. Schneider, and M. Yang, Proc Natl Acad Sci U S A 112:5243-5248, 2015, https://doi.org/10.1073/pnas.1415037112). This finding raised the question of whether both sites must be loaded with substrate prior to initiation of the transport cycle. To address this matter, we first explored the role of key residues that form the binding pocket in the closely related Art(MP)2 transporter of Geobacillus stearothermophilus, by monitoring consequences of mutations in ArtM on ATPase and transport activity at the level of purified proteins embedded in liposomes. Our results emphasize that two negatively charged residues (E153 and D160) are crucial for wild-type function. Furthermore, the variant Art[M(L67D)P]2 exhibited strongly impaired activities, which is why it was considered for construction of a hybrid complex containing one intact and one impaired substrate-binding site. Activity assays clearly revealed that one intact binding site was sufficient for function. To our knowledge, our study provides the first biochemical evidence on transmembrane substrate-binding sites of an ABC importer.IMPORTANCE Canonical prokaryotic ATP-binding cassette importers mediate the uptake of a large variety of chemicals, including nutrients, osmoprotectants, growth factors, and trace elements. Some also play a role in bacterial pathogenesis, which is why full understanding of their mode of action is of the utmost importance. One of the unsolved problems refers to the chemical nature and number of substrate binding sites formed by the transmembrane subunits. Here, we report that a hybrid amino acid transporter of G. stearothermophilus, encompassing one intact and one impaired transmembrane binding site, is fully competent in transport, suggesting that the binding of one substrate molecule is sufficient to trigger the translocation process.

Effect of storage temperature on the lag time of Geobacillus stearothermophilus individual spores.

The lag times (λ) of Geobacillus stearothermophilus single spores were studied at different storage temperatures ranging from 45 to 59 °C using the Bioscreen C method. A significant variability of λ was observed among individual spores at all temperatures tested. The storage temperature affected both the position and the spread of the λ distributions. The minimum mean value of λ (i.e. 10.87 h) was observed at 55 °C, while moving away from this temperature resulted in an increase for both the mean and standard deviation of λ. A Cardinal Model with Inflection (CMI) was fitted to the reverse mean λ, and the estimated values for the cardinal parameters Tmin, Tmax, Topt and the optimum mean λ of G. stearothermophilus were found to be 38.1, 64.2, 53.6 °C and 10.3 h, respectively. To interpret the observations, a probabilistic growth model for G. stearothermophilus individual spores, taking into account λ variability, was developed. The model describes the growth of a population, initially consisting of N0 spores, over time as the sum of cells in each of the N0 imminent subpopulations originating from a single spore. Growth simulations for different initial contamination levels showed that for low N0 the number of cells in the population at any time is highly variable. An increase in N0 to levels exceeding 100 spores results in a significant decrease of the above variability and a shorter λ of the population. Considering that the number of G. stearothermophilus surviving spores in the final product is usually very low, the data provided in this work can be used to evaluate the probability distribution of the time-to-spoilage and enable decision-making based on the "acceptable level of risk".

Partial purification and characterization of lipase from Geobacillus stearothermophilus AH22.

The lipase was partially purified by ion exchange chromatography and gel filtration column chromatography, and was characterized from Geobacillus stearothermophilus AH22 strain. The lipase was purified 18.3-folds with 19.7% recovery. The lipase activity was determined by using p-nitrophenyl esters (C2-C12) as substrates. The Km values of the enzyme for these substrates were found as 0.16, 0.02, 0.19 and 0.55 mM, respectively, while Vmax values were 0.52, 1.03, 0.72 and 0.15 U mg(-1). The enzyme showed maximum activity at 50 °C and between pH 8.0 and 9.0. The enzyme was found to be quite stable at pH range of 4.0-10.0, and thermal stability between 50 and 60 °C. It was found that the best inhibitory effect of the enzyme activity was of Hg(2+). The inhibitory effect as orlistat, catechin, propyl paraben, p-coumaric acid, 3,4-dihydroxy hydro-cinnamic acid was examined. These results suggest that G. stearothermophilus AH22 lipase presents very suitable properties for industrial applications.

Development and application of Geobacillus stearothermophilus growth model for predicting spoilage of evaporated milk.

The presence of Geobacillus stearothermophilus spores in evaporated milk constitutes an important quality problem for the milk industry. This study was undertaken to provide an approach in modelling the effect of temperature on G. stearothermophilus ATCC 7953 growth and in predicting spoilage of evaporated milk. The growth of G. stearothermophilus was monitored in tryptone soy broth at isothermal conditions (35-67 °C). The data derived were used to model the effect of temperature on G. stearothermophilus growth with a cardinal type model. The cardinal values of the model for the maximum specific growth rate were Tmin = 33.76 °C, Tmax = 68.14 °C, Topt = 61.82 °C and µopt = 2.068/h. The growth of G. stearothermophilus was assessed in evaporated milk at Topt in order to adjust the model to milk. The efficiency of the model in predicting G. stearothermophilus growth at non-isothermal conditions was evaluated by comparing predictions with observed growth under dynamic conditions and the results showed a good performance of the model. The model was further used to predict the time-to-spoilage (tts) of evaporated milk. The spoilage of this product caused by acid coagulation when the pH approached a level around 5.2, eight generations after G. stearothermophilus reached the maximum population density (Nmax). Based on the above, the tts was predicted from the growth model as the sum of the time required for the microorganism to multiply from the initial to the maximum level ( [Formula: see text] ), plus the time required after the [Formula: see text] to complete eight generations. The observed tts was very close to the predicted one indicating that the model is able to describe satisfactorily the growth of G. stearothermophilus and to provide realistic predictions for evaporated milk spoilage.

Die another day: Fate of heat-treated Geobacillus stearothermophilus ATCC 12980 spores during storage under growth-preventing conditions.

Geobacillus stearothermophilus spores are recognized as one of the most wet-heat resistant among aerobic spore-forming bacteria and are responsible for 35% of canned food spoilage after incubation at 55 °C. The purpose of this study was to investigate and model the fate of heat-treated survivor spores of G. stearothermophilus ATCC 12980 in growth-preventing environment. G. stearothermophilus spores were heat-treated at four different conditions to reach one or two decimal reductions. Heat-treated spores were stored in nutrient broth at different temperatures and pH under growth-preventing conditions. Spore survival during storage was evaluated by count plating over a period of months. Results reveal that G. stearothermophilus spores surviving heat treatment lose their viability during storage under growth-preventing conditions. Two different subpopulations were observed during non-thermal inactivation. They differed according to the level of their resistance to storage stress, and the proportion of each subpopulation can be modulated by heat treatment conditions. Finally, tolerance to storage stress under growth-preventing conditions increases at refrigerated temperature and neutral pH regardless of heat treatment conditions. Such results suggest that spore inactivation due to heat treatment could be completed by storage under growth-preventing conditions.

Structure-function relationships in Gan42B, an intracellular GH42 ß-galactosidase from Geobacillus stearothermophilus.

Geobacillus stearothermophilus T-6 is a Gram-positive thermophilic soil bacterium that contains a battery of degrading enzymes for the utilization of plant cell-wall polysaccharides, including xylan, arabinan and galactan. A 9.4 kb gene cluster has recently been characterized in G. stearothermophilus that encodes a number of galactan-utilization elements. A key enzyme of this degradation system is Gan42B, an intracellular GH42 ß-galactosidase capable of hydrolyzing short ß-1,4-galactosaccharides into galactose units, making it of high potential for various biotechnological applications. The Gan42B monomer is made up of 686 amino acids, and based on sequence homology it was suggested that Glu323 is the catalytic nucleophile and Glu159 is the catalytic acid/base. In the current study, the detailed three-dimensional structure of wild-type Gan42B (at 2.45 Šresolution) and its catalytic mutant E323A (at 2.50 Šresolution), as determined by X-ray crystallography, are reported. These structures demonstrate that the three-dimensional structure of the Gan42B monomer generally correlates with the overall fold observed for GH42 proteins, consisting of three main domains: an N-terminal TIM-barrel domain, a smaller mixed α/ß domain, and the smallest all-ß domain at the C-terminus. The two catalytic residues are located in the TIM-barrel domain in a pocket-like active site such that their carboxylic functional groups are about 5.3 Šfrom each other, consistent with a retaining mechanism. The crystal structure demonstrates that Gan42B is a homotrimer, resembling a flowerpot in general shape, in which each monomer interacts with the other two to form a cone-shaped tunnel cavity in the centre. The cavity is ∼35 Šat the wide opening and ∼5 Šat the small opening and ∼40 Šin length. The active sites are situated at the interfaces between the monomers, so that every two neighbouring monomers participate in the formation of each of the three active sites of the trimer. They are located near the small opening of the cone tunnel, all facing the centre of the cavity. The biological relevance of this trimeric structure is supported by independent results obtained from gel-permeation chromatography. These data and their comparison to the structural data of related GH42 enzymes are used for a more general discussion concerning structure-activity aspects in this GH family.

Genotypic and phenotypic characterization of foodborne Geobacillus stearothermophilus.

Geobacillus stearothermophilus is the main thermophilic spore former involved in flat sour spoilage of canned foods. Three typing methods were tested and applied to differentiate strains at intra-species level: panC sequence analysis, REP-PCR and M13-PCR. panC gene was highly conserved within the studied strains, suggesting a low intra-specific diversity. This was supported by REP-PCR primary assays and M13-PCR results. M13-PCR profile analysis succeeded in differentiating six closely related groups (at 79% threshold similarity) among 127 strains from a range of spoiled canned food products and from different canneries. Phenotypic traits were investigated among 20 selected strains representing groups and origins. Ranges of growth under different temperatures (from 40 °C to 70 °C), pH (from 5.0 to 6.5), NaCl concentrations (from 1 to 5%) and sporulation conditions poorly differed between strains, but wet heat resistance of spores showed a 20-fold variation between strains. Furthermore, in this study, strains that belonged to the same M13-PCR genetic group did not share phenotypic characteristics or common origin. The work emphasizes a low diversity within the G. stearothermophilus species but data from this study may contribute to a better control of G. stearothermophilus spoilage in canned food.

Study of the combined effect of electro-activated solutions and heat treatment on the destruction of spores of Clostridium sporogenes and Geobacillus stearothermophilus in model solution and vegetable puree.

The combined effect of heat treatment and electro-activated solution (EAS) on the heat resistance of spores of Clostridium sporogenes and Geobacillus stearothermophilus was assessed under various heating and exposure time combinations. The acid and neutral EAS showed the highest inhibitory activity, indicating that these solutions may be considered as strong sporicidal disinfectants. These EAS were able to cause a reduction of ≥6 log of spores of C. sporogenes at 60 °C in only 1 min of exposition. For G. stearothermophilus spores, a reduction of 4.5 log was observed at 60 °C in 1 min, while in 5 min, ≥7 log CFU/ml reduction was observed. Inoculated puree of pea and corn were used as a food matrix for the determination of the heat resistance of these spores during the treatments in glass capillaries. The inactivation kinetics of the spores was studied in an oil bath. Combined treatment by EAS and temperature demonstrated a significant decrease in the heat resistance of C. sporogenes. The D100°C in pea puree with NaCl solution was 66.86 min while with acid and neutral EAS it was reduced down to 3.97 and 2.19 min, respectively. The spore of G. stearothermophilus displayed higher heat resistance as confirmed by other similar studies. Its D130°C in pea puree showed a decrease from 1.45 min in NaCl solution down to 1.30 and 0.93 min for acid and neutral EAS, respectively. The differences between the spores of these species are attributable to their different sensitivities with respect to pH, Redox potential and oxygen.