Two new exopolysaccharides from a thermophilic bacterium Geobacillus sp. WSUCF1: Characterization and bioactivities.

The production, characterization and bioactivities of exopolysaccharides (EPSs) from a thermophilic bacterium Geobacillus sp. strain WSUCF1 were investigated. Using glucose as a carbon source 525.7 mg/L of exoproduct were produced in a 40-L bioreactor at 60 °C. Two purified EPSs were obtained: EPS-1 was a glucomannan containing mannose and glucose in a molar ratio of 1:0.21, while EPS-2 was composed of mannan only. The molecular weights of both EPSs were estimated to be approximately 1000 kDa, their FTIR and NMR spectra indicated the presence of α-type glycosidic bonds in a linear structure, and XRD analysis indicated a low degree of crystallinity of 0.11 (EPS-1) and 0.27 (EPS-2). EPS-1 and EPS-2 demonstrated high degradation temperatures of 319 °C and 314 °C, respectively, and non-cytotoxicity to HEK-293 cells at 2 and 3 mg/mL, respectively. In addition, both showed antioxidant activities. EPSs from strain WSUCF1 may expand the applications of microorganisms isolated from extreme environments and provide a valuable resource for exploitation in biomedical fields such as drug delivery carriers.

Common mechanism of thermostability in small α- and ß-proteins studied by molecular dynamics.

Protein thermostability is important to evolution, diseases, and industrial applications. Proteins use diverse molecular strategies to achieve stability at high temperature, yet reducing the entropy of unfolding seems required. We investigated five small α-proteins and five ß-proteins with known, distinct structures and thermostability (Tm ) using multi-seed molecular dynamics simulations at 300, 350, and 400 K. The proteins displayed diverse changes in hydrogen bonding, solvent exposure, and secondary structure with no simple relationship to Tm . Our dynamics were in good agreement with experimental B-factors at 300 K and insensitive to force-field choice. Despite the very distinct structures, the native-state (300 + 350 K) free-energy landscapes (FELs) were significantly broader for the two most thermostable proteins and smallest for the three least stable proteins in both the α- and ß-group and with both force fields studied independently (tailed t-test, 95% confidence level). Our results suggest that entropic ensembles stabilize proteins at high temperature due to reduced entropy of unfolding, viz., ΔG = ΔH - TΔS. Supporting this mechanism, the most thermostable proteins were also the least kinetically stable, consistent with broader FELs, typified by villin headpiece and confirmed by specific comparison to a mesophilic ortholog of Thermus thermophilus apo-pyrophosphate phosphohydrolase. We propose that molecular strategies of protein thermostabilization, although diverse, tend to converge toward highest possible entropy in the native state consistent with the functional requirements. We speculate that this tendency may explain why many proteins are not optimally structured and why molten-globule states resemble native proteins so much.

Structural basis of amino acid surveillance by higher-order tRNA-mRNA interactions.

Amino acid availability in Gram-positive bacteria is monitored by T-box riboswitches. T-boxes directly bind tRNAs, assess their aminoacylation state, and regulate the transcription or translation of downstream genes to maintain nutritional homeostasis. Here, we report cocrystal and cryo-EM structures of Geobacillus kaustophilus and Bacillus subtilis T-box-tRNA complexes, detailing their multivalent, exquisitely selective interactions. The T-box forms a U-shaped molecular vise that clamps the tRNA, captures its 3' end using an elaborate 'discriminator' structure, and interrogates its aminoacylation state using a steric filter fashioned from a wobble base pair. In the absence of aminoacylation, T-boxes clutch tRNAs and form a continuously stacked central spine, permitting transcriptional readthrough or translation initiation. A modeled aminoacyl disrupts tRNA-T-box stacking, severing the central spine and blocking gene expression. Our data establish a universal mechanism of amino acid sensing on tRNAs and gene regulation by T-box riboswitches and exemplify how higher-order RNA-RNA interactions achieve multivalency and specificity.

Geobacillin 26 - high molecular weight bacteriocin from a thermophilic bacterium.

Bacteriocins are ribosomally synthesized peptides/proteins produced by bacteria. These compounds have antibacterial activity against other bacteria that are usually closely related to the producer strain. Here we describe bacteriocin geobacillin 26 from a thermophilic Gram-positive bacterium Geobacillus stearothermophilus 15. We have purified native bacteriocin, determined its amino acid sequence and heterologously expressed in Gram-negative Escherichia coli. Geobacillin 26 is a heat-labile, high molecular weight antibacterial protein belonging to class III bacteriocins. It has a narrow antibacterial spectrum against other thermophilic bacteria. Our study suggests that this bacteriocin is not a cell wall hydrolyzing enzyme as most of high molecular weight bacteriocins. In addition, geobacillin 26 has no amino acid sequence similarities to other known function proteins. No other class III bacteriocin from a thermophilic bacterium has been reported and well characterized before. Geobacillin 26 as a natural antibacterial agent has a great potential in industry where contamination with other thermophilic bacteria is unwanted. Moreover, this study may prompt to disclose more novel geobacillin 26-like antibacterial proteins, which could find their applications in food industry or medicine.

EPS bound flavins driven mediated electron transfer in thermophilic Geobacillus sp.

Through extracellular electron transfer (EET), bacteria are capable of transforming different insoluble materials of geochemical interest into energy-rich molecules for their growth. For this process, bacteria have been depending directly or indirectly on molecules synthesized within the cells or by various synthetics as mediators. Herein, we studied the in-situ change in electrochemistry and supporting components for EET in the extracellular polysaccharide (EPS) producing biofilm of thermophilic Geobacillus sp. The CV and DPV resultsrevealed that the intact biofilm of bacteria was not able to generate any potential at 25 °C /- ≤50 °C. However, at 55 °C (optimal condition), the potential occurred drastically after the EPS production by bacteria. HPLC and MALDI-TOF results revealed that the presence of Flavins, which can able adsorbed to the electrodes from the cell surface. Moreover, the temperature-dependent EPS production and originally conceived ability of flavins to act as electron shuttles suggest that not much complexity in bacteria with minerals. Additionally, the electrochemical potential was severely affected upon removal of EPS/flavin moiety from the intact biofilm, revealed the necessity of EPS bound flavins in transferring the electrons across its thick cell walls. This paradigm shift to electrogenic nature of Geobacillus sp. biofilm will become evident in the adaptation of other microbes during mineral respiration in extreme environments.

Effect of Target Gene Silencing on Calcite Single Crystal Formation by Thermophilic Bacterium Geobacillus thermoglucosidasius NY05.

Geobacillus thermoglucosidasius NY05 catalyzes calcite single crystal formation at 60 °C by using acetate and calcium. Endospores are embedded at the central part of the calcite single crystal and carbon atoms in the calcite lattice are derived from acetate carbon. Here, we synthesized 21-mer antisense DNA oligonucleotides targeting sporulation transcription factor, acetate-CoA ligase, isocitrate lyase, and malate synthase G mRNAs and evaluated the effect of these oligonucleotides on calcite formation in G. thermoglucosidasius NY05. G. thermoglucosidasius NY05 cells containing antisense DNA oligonucleotides targeting sporulation transcription factor, acetate-CoA ligase, isocitrate lyase, and malate synthase G mRNAs had reduced calcite single crystal formation by 18.7, 50.6, 55.7, and 82.3%, respectively, compared with cells without antisense DNA oligonucleotides. These results support that calcite formation needs endospores as the nucleus to grow, and carbon dioxide generated from acetate, which is metabolized via the glyoxylate pathway and glucogenesis, is supplied to the crystal lattice.

An amylopullulanase (ApuNP1) from Geobacillus thermoleovorans NP1: biochemical characterization and its potential industrial applications.

An amylopullulanase was produced by Geobacillus thermoleovorans NP1. The optimum enzyme production occurred at 45°C and pH 7.0 (12 hr). NP1 amylopullulanase (ApuNP1) exhibited the maximal activity at 50°C and pH 6.0 and was stable between 30-50°C, and pH 3.0-12.0 for 24 hr. The enzyme showed two bands with molecular weights of 112 and 107 kDa in sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The amylopullulanase retained 100% of its activity in the presence of 10 mM of Ca2+, Ba2+, Zn2+, Mg2+, Cu2+, EDTA, and PMSF. While the enzyme showed resistance to 5% of TritonX-100, Tween 20, and Tween 80, the activity was inhibited by 5% ß-mercaptoethanol and H2O2. While the hydrolysis products of pullulan were maltose, maltotriose, and maltodextrin, the starch was hydrolyzed to maltose, maltotriose, and maltodextrin units. This shows that NP1 pullulanase is a type II pullulanase (amylopullulanase). After the liquefaction assay, 12% glucose content was measured with a refractometer in the presence of 20% starch. According to the wash performance tests, the mixture of ApuNP1 and 1% detergent removed almost all of the stains. This novel thermo-acidic amylopullulanase has a potency to be used in detergent, starch, food, baking, textile, and cosmetic industries.

N-Terminal Domain Truncation and Domain Insertion-Based Engineering of a Novel Thermostable Type I Pullulanase from Geobacillus thermocatenulatus.

A novel thermostable type I pullulanase gene ( pul GT) from Geobacillus thermocatenulatus DSMZ730 was cloned. It has an open reading frame of 2154 bp encoding 718 amino acids. G. thermocatenulatus pullulanase (PulGT) was found to be optimally active at pH 6.5 and 70 °C. It exhibited stable activity in the pH range of 5.5-7.0. PulGT lacked three domains (CBM41 domain, X25 domain, and X45 domain) compared with the pullulanase from Bacillus acidopullulyticus ( 2WAN ). Different N-terminally domain truncated (730T) or spliced (730T-U1 and 730T-U2) mutants were constructed. Truncating the N-terminal 85 amino acids decreased the Km value and did not change its optimum pH, an advantageous biochemical property in some applications. Compared with 2WAN , PulGT can be used directly for maize starch saccharification without adjusting the pH, which reduces cost and improves efficiency.

Crystallographic study of dioxygen chemistry in a copper-containing nitrite reductase from Geobacillus thermodenitrificans.

Copper-containing nitrite reductases (CuNIRs) are multifunctional enzymes that catalyse the one-electron reduction of nitrite (NO2-) to nitric oxide (NO) and the two-electron reduction of dioxygen (O2) to hydrogen peroxide (H2O2). In contrast to the mechanism of nitrite reduction, that of dioxygen reduction is poorly understood. Here, results from anaerobic synchrotron-radiation crystallography (SRX) and aerobic in-house radiation crystallography (iHRX) with a CuNIR from the thermophile Geobacillus thermodenitrificans (GtNIR) support the hypothesis that the dioxygen present in an aerobically manipulated crystal can bind to the catalytic type 2 copper (T2Cu) site of GtNIR during SRX experiments. The anaerobic SRX structure showed a dual conformation of one water molecule as an axial ligand in the T2Cu site, while previous aerobic SRX GtNIR structures were refined as diatomic molecule-bound states. Moreover, an SRX structure of the C135A mutant of GtNIR with peroxide bound to the T2Cu atom was determined. The peroxide molecule was mainly observed in a side-on binding manner, with a possible minor end-on conformation. The structures provide insights into dioxygen chemistry in CuNIRs and hence help to unmask the other face of CuNIRs.

Cloning and sequencing of the gene encoding the enzyme for the reductive cleavage of diaryl ether bonds of 2,3,7,8-tetrachlorodibenzo-p-dioxin in Geobacillus thermodenitrificans UZO 3.

We have previously reported that a cell-free extract prepared from Geobacillus thermodenitrificans UZO 3 reductively cleaves diaryl ether bonds of 2,3,7,8-tetrachlorodibenzo-p-dioxin (2,3,7,8-TCDD), a dioxin with the highest toxicity, in a sequential fashion producing 3',4',4,5-tetrachloro-2-hydroxydiphenyl ether (TCDE) as the intermediate, and 3,4-dichlorophenol (DCP) as the final reaction product. The detection of TCDE implicated the discovery of an unprecedented dioxin-degrading enzyme that reductively cleaves the diaryl ether bonds. In this study, we report the cloning and sequencing of the dioxin reductive etherase gene dreE which codes for the 2,3,7,8-TCDD-degrading enzyme. We showed that dreE was expressed in Escherichia coli and that the product of the expression could reductively cleave diaryl ether bonds of 2,3,7,8-TCDD to produce TCDE. Furthermore, we established that the amino acid sequence encoded by dreE was homologous to an enzyme with yet unknown function that is encoded by a gene located in the riboflavin (vitamin B2) biosynthesis operon in Bacillus subtilis. We also showed that the amino acid sequence possesses a coenzyme A (CoA) binding site that is conserved in the N-acyltransferase superfamily. For the first time, the degradation of 2,3,7,8-TCDD at the molecular level using a enzyme of bacterial origin has been demonstrated. A novel mechanism model for the reductive cleavage of diaryl ether bond of 2,3,7,8-TCDD was also proposed.

Characterizing a thermostable Cas9 for bacterial genome editing and silencing.

CRISPR-Cas9-based genome engineering tools have revolutionized fundamental research and biotechnological exploitation of both eukaryotes and prokaryotes. However, the mesophilic nature of the established Cas9 systems does not allow for applications that require enhanced stability, including engineering at elevated temperatures. Here we identify and characterize ThermoCas9 from the thermophilic bacterium Geobacillus thermodenitrificans T12. We show that in vitro ThermoCas9 is active between 20 and 70 °C, has stringent PAM-preference at lower temperatures, tolerates fewer spacer-protospacer mismatches than SpCas9 and its activity at elevated temperatures depends on the sgRNA-structure. We develop ThermoCas9-based engineering tools for gene deletion and transcriptional silencing at 55 °C in Bacillus smithii and for gene deletion at 37 °C in Pseudomonas putida. Altogether, our findings provide fundamental insights into a thermophilic CRISPR-Cas family member and establish a Cas9-based bacterial genome editing and silencing tool with a broad temperature range.

A Novel Benzoquinone Compound Isolated from Deep-Sea Hydrothermal Vent Triggers Apoptosis of Tumor Cells.

Microorganisms are important sources for screening bioactive natural products. However, natural products from deep-sea microbes have not been extensively explored. In this study, the metabolites of bacteriophage GVE2 -infected (Geobacillus sp. E263 virus) thermophilic bacterium Geobacillus sp. E263, which was isolated from a deep-sea hydrothermal vent, were characterized. A novel quinoid compound, which had anti-tumor activity, was isolated from the phage-challenged thermophile. The chemical structure analysis showed that this novel quinoid compound was 2-amino-6-hydroxy-[1,4]-benzoquinone. The results indicated that 2-amino-6-hydroxy-[1,4]-benzoquinone and its two derivatives could trigger apoptosis of gastric cancer cells and breast cancer cells by inducing the accumulation of intracellular reactive oxygen species. Therefore, our study highlighted that the metabolites from the phage-challenged deep-sea microbes might be a kind of promising sources for anti-tumor drug discovery, because of the similarity of metabolic disorder between bacteriophage-infected microbes and tumor cells.

Cloning and characterization of thermostable GroEL/GroES homologues from Geobacillus thermopakistaniensis and their applications in protein folding.

The chaperonin genes encoding GroELGt (ESU72018) and GroESGt (ESU72017), homologues of bacterial GroEL and GroES, from Geobacillus thermopakistaniensis were cloned and expressed in Escherichia coli. The purified gene products possessed the ATPase activity similar to other bacterial and eukaryal counterparts. Recombinant GroELGt and GroESGt were able to refold the denatured insoluble aggregates of α-amylase from Bacillus licheniformis into soluble and active form. Furthermore, GroELGt and GroESGt successfully enhanced the thermostability of porcine heart malate dehydrogenase. Expression of GroELGt gene in E. coli cells enhanced the thermotolerance of the host. Furthermore, soluble production of recombinant alcohol dehydrogenase from Bacillus subtilis strain R5 in E. coli, initially produced as insoluble aggregates, was achieved by co-expressing the gene with GroELGt. Our results implied that GroELGt could assist folding of nascent protein in E. coli with the help of host co-chaperonin without requiring additional ATP. This system can be used for soluble production of recombinant proteins which otherwise are produced in insoluble form in E. coli. To the best of our knowledge this is the first report on functional characterization and applications of chaperonins from genus Geobacillus.

Met349 Mutations Enhance the Activity of 1,4-α-Glucan Branching Enzyme from Geobacillus thermoglucosidans STB02.

1,4-α-Glucan branching enzyme (GBE, EC 2.4.1.18) is used to increase the number of α-1,6 branch points in starch and glycogen. On the basis of a multiple sequence alignment of the GBEs from a variety of bacteria, residue 349 (Geobacillus thermoglucosidans STB02 numbering) in region III is generally methionine in bacteria with higher identity, while it is threonine or serine in bacteria with lower identity. Four mutants (M349T, M349S, M349H, and M349Y) were constructed by site-directed mutagenesis and characterized. M349T and M349S showed 24.5% and 21.1% increases in specific activity compared with that of wild-type GBE, respectively. In addition, M349T and M349S displayed 24.2% and 17.6% enhancements in the α-1,6-glycosidic linkage ratio of potato starch samples, respectively. However, M349Y displayed a significant reduction in activity. Moreover, the mutations at M349 have a negligible effect on substrate specificity. Thus, M349T and M349S are more suitable for industrial applications than wild-type GBE.

Cryogenic optical localization provides 3D protein structure data with Angstrom resolution.

We introduce Cryogenic Optical Localization in 3D (COLD), a method to localize multiple fluorescent sites within a single small protein with Angstrom resolution. We demonstrate COLD by determining the conformational state of the cytosolic Per-ARNT-Sim domain from the histidine kinase CitA of Geobacillus thermodenitrificans and resolving the four biotin sites of streptavidin. COLD provides quantitative 3D information about small- to medium-sized biomolecules on the Angstrom scale and complements other techniques in structural biology.

What Can and Cannot Be Learned from Molecular Dynamics Simulations of Bacterial Proton-Coupled Oligopeptide Transporter GkPOT?

We have performed an extensive set of all-atom molecular dynamics (MD) simulations of a bacterial proton-coupled oligopeptide transporter (POT) in an explicit membrane environment. We have characterized both the local and global conformational dynamics of the transporter upon the proton and/or substrate binding, within a statistical framework. Our results reveal a clearly distinct behavior for local conformational dynamics in the absence and presence of the proton at the putative proton binding residue E310. Particularly, we find that the substrate binding conformation is drastically different in the two conditions, where the substrate binds to the protein in a lateral/vertical manner, in the presence/absence of the proton. We do not observe any statistically significant distinctive behavior in terms of the global conformational changes in different simulation conditions, within the time scales of our simulations. Our extensive simulations and analyses call into question the implicit assumption of many MD studies that local conformational changes observed in short simulations could provide clues to the global conformational changes that occur on much longer time scales. The linear regression analysis of quantities associated with the global conformational fluctuations, however, provides an indication of a mechanism involving the concerted motion of the transmembrane helices, consistent with the rocker-switch mechanism.

Characteristics and applications of recombinant thermostable amylopullulanase of Geobacillus thermoleovorans secreted by Pichia pastoris.

The 3'-deleted amylopullulanase gene from the extreme thermophile Geobacillus thermoleovorans (Gt-apuΔC) was expressed extracellularly in Pichia pastoris under both methanol-inducible AOX1 and constitutive GAP promoters. The expression of the gene (Gt-apuΔC) was higher under GAP promoter (36.2 U ml-1, α-amylase; 33.5 U ml-1, pullulanase) than that under AOX1 promoter (32.5 and 28.6 U ml-1). The heavily glycosylated Gt-apuΔC from the recombinant P. pastoris displays higher substrate specificity, thermal stability and starch saccharification efficiency than that expressed in Escherichia coli. The enzyme hydrolyses maltotriose and maltotetraose unlike that expressed in E. coli. The enzyme action on wheat bran liberates maltose and glucose without detectable amount(s) of maltooligosaccharides. The sugars released from wheat bran (glucose and maltose) could be fractionated by ultrafiltration, as confirmed by TLC and HPLC analysis. This is the first report on the production of recombinant amylopullulanase extracellularly in P. pastoris.

Direct observation of a deoxyadenosyl radical in an active enzyme environment.

5'-deoxyadenosyl radicals have been proposed as the first common intermediate in the molecular reaction mechanism of the family of radical S-adenosyl-l-methionine (SAM) enzymes. However, this radical species has not yet been directly observed in a catalytically active enzyme environment. In a reduced and SAM-containing C140A mutant of the spore photoproduct lyase from Geobacillus thermodenitrificans, a mutant with altered catalytic activity, we were able to identify an organic radical with pronounced hyperfine structure using electron paramagnetic resonance spectroscopy. Guided by quantum-chemical computations at the density functional theory level of theory, this radical could be tentatively assigned to a deoxyadenosyl radical, which provides first experimental evidence for this intermediate in the reaction mechanism of radical SAM enzymes.

The quorum-quenching lactonase from Geobacillus caldoxylosilyticus: purification, characterization, crystallization and crystallographic analysis.

Lactonases are enzymes that are capable of hydrolyzing various lactones such as aliphatic lactones or acyl-homoserine lactones (AHLs), with the latter being used as chemical signaling molecules by numerous Gram-negative bacteria. Lactonases therefore have the ability to quench the chemical communication, also known as quorum sensing, of numerous bacteria, and in particular to inhibit behaviors that are regulated by this system, such as the expression of virulence factors or the production of biofilms. A novel representative from the metallo-ß-lactamase superfamily, dubbed GcL, was isolated from the thermophilic bacterium Geobacillus caldoxylosilyticus. Because of its thermophilic origin, GcL may constitute an interesting candidate for the development of biocontrol agents. Here, we show that GcL is a thermostable enzyme with a half-life at 75°C of 152.5 ± 10 min. Remarkably, it is also shown that GcL is among the most active lactonases characterized to date, with catalytic efficiencies (kcat/Km) against AHLs of greater than 10(6) M(-1) s(-1). The structure of GcL is expected to shed light on the catalytic mechanism of the enzyme and the molecular determinants for the substrate specificity in this class of lactonases. Here, the expression, purification, characterization, crystallization and X-ray diffraction data collection to 1.6 Šresolution of GcL are reported.

Magnesium-Calcite Crystal Formation Mediated by the Thermophilic Bacterium Geobacillus thermoglucosidasius Requires Calcium and Endospores.

Fresh Geobacillus thermoglucosidasius cells grown on soybean-casein digest nutrient agar were inoculated as a parent colony 1 cm in diameter on the surface of an agar gel containing acetate and calcium ions (calcite-promoting hydrogel) and incubated at 60 °C for 4 days, after which magnesium-calcite single crystals of 50-130 µm in size formed within the parent colony. Addition of EDTA, polyacrylic acid or N,N-dicyclohexylcarbodiimide to the calcite-forming hydrogel inhibited the parent colony from forming magnesium-calcite crystals. Inoculation of G. thermoglucosidasius on calcite-forming hydrogel containing 5 µM cadmium and 20 µM zinc resulted in a decrease in the sporulation rate from 55 to 7-8 %. Magnesium-calcite synthesis decreased relative to the sporulation rate. G. thermoglucosidasius exhibited higher adsorption/absorbance of calcium than other Geobacillus sp. that do not mediate calcite formation and higher levels of magnesium accumulation. Calcium ions contained in the calcite-promoting hydrogel and magnesium ions concentrated in G. thermoglucosidasius cells serve as the elements for magnesium-calcite synthesis. The observed decreases in sporulation rate and magnesium-calcite formation support the hypothesis that endospores act as nuclei for the synthesis of magnesium-calcite single crystals.