Bacillus Thermoglucosidasius Research Articles

Application of antagonistic rhizobacteria for control of Fusarium seedling blight and basal rot of lily

Three antagonistic bacteria, Streptomyces misionensis strain PMS101, Bacillus thermoglucosidasius strain PMB207, and S. sioyaensis strain PMS502, were tested for sensitivity to the foliar fungicide Sporgon (a.i. 50% prochloraz-Mn complex) and for efficacy in controlling Fusarium diseases of lily. Results showed that the growth of all three antagonistic strains of bacteria was completely suppressed by Sporgon at a concentration of 500 μg/mL, but B. thermoglucosidasius strain PMB207 and S. misionensis strain PMS101 were unaffected at concentrations of 100 μg/mL or lower. A large-scale trial in an automated and environment-controlled commercial greenhouse showed that treatment of scale bulblets of lily with Sporgon (100 μg /mL) and B. thermoglucosidasius strain PMB207 (1–1.2 × 107 cfu/mL) or 100 μg/mL Sporgon and S. misionensis strain PMS101 (1–1.4 × 107 cfu/mL) resulted in a significant reduction (P 0.05). Results of the greenhouse and field experiments showed that treatment of scale bulblets or one-year-old bulbs of lily with B. thermoglucosidasius strain PMB207 (1–1.2 × 108 cfu/mL) or S. misionensis strain PMS101 (1–1.4 × 108 cfu/mL) without Sporgon was also effective in the control of basal rot caused by F. oxysporum f. sp. lilii. These studies reveal that B. thermoglucosidasius strain PMB207 and S. misionensis strain PMS101 are biocontrol agents which have potential for use in the commercial production of lily bulbs, as they can be used alone or in combination with the fungicide Sporgon at low concentration (<100 μg/mL).

Construction of Microbial Fuel Cells Using Thermophilic Microorganisms, Bacillus licheniformis and Bacillus thermoglucosidasius

A systematic study of microbial fuel cells comprised of thermophilic Bacillus licheniformis and Bacillus thermoglucosidasius has been carried out under various operating conditions. Substantial amount of electricity was generated when a redox mediator was used. Being affected by operation temperature, the maximum efficiency was obtained at 50 °C with an open circuit voltage of ca. 0.7 V. While a small change around the optimum temperature did not make much effect on the cell performance, the rapid decrease in performance was observed above 70 °C. It was noticeable that fuel cell efficiency and discharge pattern strongly depended on the kind of carbon sources used in the initial culture medium. In the case of B. thermoglucosidasius, glucose alone was utilized constitutively as a substrate in the microbial fuel cell irrespective of used carbons sources. When B. licheniformis was cultivated with lactose as a carbon source, best charging characteristics were recorded. Trehalose, in particular, showed 41.2% coulombic efficiency when B. thermoglucosidasius was cultured in a starch-containing medium. Relatively good repetitive operation was possible with B. thermoglucosidasius cells up to 12 cycles using glucose as a carbon source, when they were cultured with lactose as an initial carbon source. This study demonstrates that highly efficient thermophilic microbial fuel cells can be constructed by a pertinent modulation of the operating conditions and by carefully selecting carbon sources used in the initial culture medium.

Structural Studies on Flavin Reductase PheA2 Reveal Binding of NAD in an Unusual Folded Conformation and Support Novel Mechanism of Action

The catabolism of toxic phenols in the thermophilic organism Bacillus thermoglucosidasius A7 is initiated by a two-component enzyme system. The smaller flavin reductase PheA2 component catalyzes the NADH-dependent reduction of free FAD according to a ping-pong bisubstrate-biproduct mechanism. The reduced FAD is then used by the larger oxygenase component PheA1 to hydroxylate phenols to the corresponding catechols. We have determined the x-ray structure of PheA2 containing a bound FAD cofactor (2.2 A), which is the first structure of a member of this flavin reductase family. We have also determined the x-ray structure of reduced holo-PheA2 in complex with oxidized NAD (2.1 A). PheA2 is a single domain homodimeric protein with each FAD-containing subunit being organized around a six-stranded beta-sheet and a capping alpha-helix. The tightly bound FAD prosthetic group (K(d) = 10 nm) binds near the dimer interface, and the re face of the FAD isoalloxazine ring is fully exposed to solvent. The addition of NADH to crystalline PheA2 reduced the flavin cofactor, and the NAD product was bound in a wide solvent-accessible groove adopting an unusual folded conformation with ring stacking. This is the first observation of an enzyme that is very likely to react with a folded compact pyridine nucleotide. The PheA2 crystallographic models strongly suggest that reactive exogenous FAD substrate binds in the NADH cleft after release of NAD product. Nanoflow electrospray mass spectrometry data indeed showed that PheA2 is able to bind one FAD cofactor and one FAD substrate. In conclusion, the structural data provide evidence that PheA2 contains a dual binding cleft for NADH and FAD substrate, which alternate during catalysis.

Screening and identification of extracellular lipase-producing thermophilic bacteria from a Malaysian hot spring

Seven lipase-producing thermophilic bacteria (ST 1, ST 4, ST 6, ST 7, ST 8, ST 9 and ST 10) were isolated from the Setapak hot spring in Malaysia. The crude extracellular lipases recovered by ultrafiltration of cell-free culture supernatant were reacted in an olive oil mixture and their lipolytic activities were compared. Identification of the bacteria was carried out using the Biolog system and biochemical tests. Strain ST 7 that exhibited the highest lipolytic activity of 4.58 U/ml was identified as belonging to the Bacillus genus. Strain ST 6 with an activity of 3.51 U/ml, was identified as Ralstonia paucula. The lipolytic activities of strains ST 1, ST 4, ST 8, ST 9 and ST 10 were 2.39, 1.84, 2.38, 1.80 and 2.62 U/ml respectively. Strains ST 1, ST 4, and ST 10 were identified as Ralstonia paucula while strains ST 8 and ST 9 were Bacillus spp. Strains ST 7 and ST 9 were tentatively identified as Bacillus thermoglucosidasius, Bacillus stearothermophilus or Bacillus coagulans, whereas strain ST 8 was tentatively identified as Bacillus subtilis.

Isolation and identification of thiocyanate utilizing chemolithotrophs from gold mine soils.

A mixed bacterial culture capable of growing in potassium-thiocyanate containing medium (200 mg KSCN) has been isolated from bacterial suspensions of soil samples collected near gold mines in Kumjung (Korea). The isolates were initially characterized by metabolic profile analysis and were identified as Bacillus thermoglucosidasius, Bacillus cereus, Bacillus licheniformis, Bacillus mycoides, Brevibacterium epidermidis, Brevibacterium otitidis, and Corynebacterium nitrilophilus. One of the seven isolates was initially characterized as Brevibacterium epidermidis, which is not known to degrade thiocyanate. However, using 16S rDNA sequencing, this strain was identified as a member of Klebsiella. The strain shows high similarity values (95.8 to 96.4%) with Klebsiella species, and the closest known relative was found to be K. ornithinolytica ATCC 31898. The result indicates that species of the genus Klebsiella were the closest phylogenetic relatives of the investigated strain. This is the first known report of a member of Klebsiella that is capable of utilizing thiocyanate as sole source of carbon and nitrogen.

Identification of a helix-turn-helix motif of Bacillus thermoglucosidasius HrcA essential for binding to the CIRCE element and thermostability of the HrcA-CIRCE complex, indicating a role as a thermosensor.

In the heat shock response of bacillary cells, HrcA repressor proteins negatively control the expression of the major heat shock genes, the groE and dnaK operons, by binding the CIRCE (controlling inverted repeat of chaperone expression) element. Studies on two critical but yet unresolved issues related to the structure and function of HrcA were performed using mainly the HrcA from the obligate thermophile Bacillus thermoglucosidasius KP1006. These two critical issues are (i) identifying the region at which HrcA binds to the CIRCE element and (ii) determining whether HrcA can play the role of a thermosensor. We identified the position of a helix-turn-helix (HTH) motif in B. thermoglucosidasius HrcA, which is typical of DNA-binding proteins, and indicated that two residues in the HTH motif are crucial for the binding of HrcA to the CIRCE element. Furthermore, we compared the thermostabilities of the HrcA-CIRCE complexes derived from Bacillus subtilis and B. thermoglucosidasius, which grow at vastly different ranges of temperature. The thermostability profiles of their HrcA-CIRCE complexes were quite consistent with the difference in the growth temperatures of B. thermoglucosidasius and B. subtilis and, thus, suggested that HrcA can function as a thermosensor to detect temperature changes in cells.

Taxonomic study of aerobic thermophilic bacilli: descriptions of Geobacillus subterraneus gen. nov., sp. nov. and Geobacillus uzenensis sp. nov. from petroleum reservoirs and transfer of Bacillus stearothermophilus, Bacillus thermocatenulatus, Bacillus thermoleovorans, Bacillus kaustophilus, Bacillus thermodenitrificans to Geobacillus as the new combinations G. stearothermophilus, G. th.

Five hydrocarbon-oxidizing strains were isolated from formation waters of oilfields in Russia, Kazakhstan and China. These strains were moderately thermophilic, neutrophilic, motile, spore-forming rods, aerobic or facultatively anaerobic. The G+C content of their DNA ranged from 49.7 to 52.3 mol%. The major isoprenoid quinone was menaquinone-7; cellular fatty acid profiles consisted of significant amounts of iso-15:0, iso-16:0 and iso-17:0 fatty acids (61.7-86.8% of the total). Based on data from 16S rDNA analysis and DNA-DNA hybridization, the subsurface isolates could be divided into two groups, one of which consisted of strains UT and X and the other of which consisted of strains K, Sam and 34T. The new strains exhibited a close phylogenetic relationship to thermophilic bacilli of 'Group 5' of Ash et al. [Ash, C., Farrow, J. A. E., Wallbanks, S. & Collins, M. D. (1991). Lett Appl Microbiol 13, 202-206] and a set of corresponding signature positions of 16S rRNA. Comparative analysis of the 16S rDNA sequences and fatty acid compositions of the novel isolates and established species of thermophilic bacilli indicated that the subsurface strains represent two new species within a new genus, for which the names Geobacillus subterraneus gen. nov., sp. nov., and Geobacillus uzenensis sp. nov. are proposed. It is also proposed that Bacillus stearothermophilus, Bacillus thermoleovorans, Bacillus thermocatenulatus, Bacillus kaustophilus, Bacillus thermoglucosidasius and Bacillus thermodenitrificans be transferred to this new genus, with Geobacillus stearothermophilus (formerly Bacillus stearothermophilus) as the type species.

Renaturation of Bacillus thermoglucosidasius HrcA repressor by DNA and thermostability of the HrcA-DNA complex in vitro.

HrcA, a negative control repressor for chaperone expression from the obligate thermophile Bacillus thermoglucosidasius KP1006, was purified in a His-tagged form in the presence of 6 M urea but hardly renatured to an intact state due to extreme insolubility. Renaturation trials revealed that the addition of DNA to purified B. thermoglucosidasius HrcA can result in solubilization of HrcA free from the denaturing agent urea. Results from band shift and light scattering assays provided three new findings: (i) any species of DNA can serve to solubilize B. thermoglucosidasius HrcA, but DNA containing the CIRCE (controlling inverted repeat of chaperone expression) element is far more effective than other nonspecific DNA; (ii) B. thermoglucosidasius HrcA renatured with nonspecific DNA bound the CIRCE element in the molecular ratio of 2.6:1; and (iii) B. thermoglucosidasius HrcA binding to the CIRCE element was stable at below 50 degrees C whereas the complex was rapidly denatured at 70 degrees C, suggesting that the breakdown of HrcA is induced by heat stress and HrcA may act as a thermosensor to affect the expression of heat shock regulatory genes. These results will help to determine the nature of HrcA protein molecules.

Phenol/cresol degradation by the thermophilic Bacillus thermoglucosidasius A7: cloning and sequence analysis of five genes involved in the pathway

Bacillus thermoglucosidasius A7 degraded phenol at 65°C via the meta cleavage pathway. Five enzymes used in the metabolism of phenol were cloned from B. thermoglucosidasius A7 into pUC18. Nine open reading frames were present on the 8.1 kb insert, six of which could be assigned a function in phenol degradation using database homologies and enzyme activities. The phenol hydroxylase is a two-component enzyme encoded by pheA1 and pheA2. The larger component (50 kDa) has 49% amino acid identity with the 4-hydroxyphenylacetate hydroxylase of Escherichia coli, while the smaller component (19 kDa) is most related (30% amino acid identity) to the styrene monoxygenase component B from Pseudomonas fluorescens. Both components were neccessary for activity. The catechol 2,3-dioxygenase encoded by pheB has 45% amino acid identity with dmpB of Pseudomonas sp. CF600 and could be assigned to superfamily I, family 2 and a new subfamily of the Eltis and Bolin grouping. The 2-hydroxymuconic acid semialdehyde hydrolase (2HMSH), encoded by pheC, revealed the highest amino acid identity (36%) to the equivalent enzyme from Pseudomonas sp. strain CF600, encoded by dmpD. Based on sequence identity, pheD and pheE were deduced to encode the 2-hydroxypenta-2,4-dienoate hydratase (2HDH), demonstrating 45% amino acid identity to the gene product of cumE from Pseudomonas fluorescens and the acetaldehyde dehydrogenase (acylating) demonstrating 57% amino acid identity to the gene product of bphJ from Pseudomonas LB400.

Features of dnaK operon genes of the obligate thermophile Bacillus thermoglucosidasius KP1006.

The dnaK gene was cloned from the obligate thermophile Bacillus thermoglucosidasius KP1006, together with the grpE and dnaJ genes in the same operon. The dnaK, grpE and dnaJ genes showed high identity with those of other bacterial strains, particularly with those of Bacillus stearothermophilus NUB36, despite an extremely low homology for the corresponding total genomic DNA. There were significant differences in the proline content of the DnaK operon proteins which is closely correlated with the thermostability of enzyme proteins. The proline content was higher in the GrpE, DnaK and DnaJ proteins of the thermophilic as opposed to the mesophilic strains. The overexpression of the B. thermoglucosidasius DnaK protein in Escherichia coli MV1184 results in extreme filamentation without inhibition on cell growth. The B. thermoglucosidasius DnaK protein seemed to exclusively disturb septation in E. coli cells which suggests that it interacts with key protein(s) involved in cell septation.

Progesterone 6-hydroxylation is catalysed by cytochrome P-450 in the moderate thermophile Bacillus thermoglucosidasius strain 12060

The moderate thermophile, Bacillus thermoglucosidasius, transforms progesterone into four metabolites. These are 6 α- and 6 β-hydroxyprogesterone, androstenedione and testosterone. This is the first report of bacterial 6 α-hydroxylation of steroids. The identity of the progesterone metabolites shows that there are three major types of transforming activity in this organism; C-17–C-20 lyase that cleaves the pregnane side chain of the substrate, C-17 oxidoreductase that interconverts the metabolites androstenedione and testosterone, and 6-hydroxylation. 6-hydroxylation activity was purified virtually to homogeneity and was shown to be catalysed by a cytochrome P-450 monooxygenase enzyme. This is the first report of a thermostable cytochrome P-450.

Clustered proline residues around the active-site cleft in thermostable oligo-1,6-glucosidase of Bacillus flavocaldarius KP1228.

The gene that coded for a cellular oligo-1,6-glucosidase (dextrin 6-alpha-D-glucanohydrolase, EC 3.2.1.10) in Bacillus flavocaldarius KP1228 (FERM-P9542) cells growing at 51-82 degrees C was expressed in Escherichia coli JM109. The enzyme had a half-life of 10 min at 89.2 degrees C. Purification of the enzyme and its characterization showed that the enzyme was identical with the native one. Its primary structure of 529 residues with a molecular weight of 61,469 deduced from the gene was 40-42% identical to the sequences of less thermostable oligo-1,6-glucosidases from Bacillus cereus ATCC 7064, Bacillus coagulans ATCC 7050, and Bacillus thermoglucosidasius KP1006. Sequence analysis showed that the B. flavocaldarius enzyme shared 14 proline residues at the same positions as in the three other enzymes, and that the B. flavocaldarius enzyme had 22 of 33 additional proline residues (cf. 1/5, 5/10, and 9/18 in the respective counterparts) in three long polypeptides constituting the active-site cleft, which connected the third, fourth, and eighth beta-strands to the corresponding third, fourth, and eighth alpha-helices in the (beta/alpha)8-barrel.

Rapid identification of heterotrophic, thermophilic, spore-forming bacteria isolated from hot composts.

The restriction enzyme profiles of 16S ribosomal DNAs (rDNAs) amplified by PCR from thermophilic heterotrophic bacterial strains isolated from composts were compared with those of reference strains. This allowed us to assign all but 1 of 16 strains to four different Bacillus species (namely, Bacillus stearothermophilus, Bacillus pallidus, Bacillus thermoglucosidasius, and "Bacillus thermodenitrificans"). This study showed that PCR restriction analysis of 16S rDNA contributes to rapid and reliable identification of newly isolated strains belonging to recognized species.

Analysis of the critical sites for protein thermostabilization by proline substitution in oligo-1,6-glucosidase from Bacillus coagulans ATCC 7050 and the evolutionary consideration of proline residues.

To identify the critical sites for protein thermostabilization by proline substitution, the gene for oligo-1,6- glucosidase from a thermophilic Bacillus coagulans strain, ATCC 7050, was cloned as a 2.4-kb DNA fragment and sequenced. In spite of a big difference in their thermostabilities, B. coagulans oligo-1,6-glucosidase had a large number of points in its primary structure identical to respective points in the same enzymes from a mesophilic Bacillus cereus strain, ATCC 7064 (57%), and an obligately thermophilic Bacillus thermoglucosidasius strain, KP1006 (59%). The number of prolines (19 for B. cereus oligo-1,6-glucosidase, 24 for B. coagulans enzyme, and 32 for B. thermoglucosidasius enzyme) was observed to increase with the rise in thermostabilities of the oligo-1,6-glucosidases. Classification of proline residues in light of the amino acid sequence alignment and the protein structure revealed by X-ray crystallographic analysis also supported this tendency. Judging from proline residues occurring in B. coagulans oligo-1,6-glucosidase and the structural requirement for proline substitution (second site of the beta turn and first turn of the alpha helix) (K. Watanabe, T. Masuda, H. Ohashi, H. Mihara, and Y. Suzuki, Eur. J. Biochem. 226:277-283, 1994), the critical sites for thermostabilization were found to be Lys-121, Glu-290, Lys-457, and Glu-487 in B. cereus oligo-1,6-glucosidase. With regard to protein evolution, the oligo-1,6-glucosidases very likely follow the neutral theory. The adaptive mutations of the oligo-1,6-glucosidases that appear to increase thermostability are consistent with the substitution of proline residues for neutrally occurring residues. It is concluded that proline substitution is an important factor for the selection of thermostability in oligo-1,6-glucosidases.

“Bacillus thermoantarcticus” sp. nov., from Mount Melbourne, Antarctica: a novel thermophilic species

A novel thermophilic Gram-positive bacillus, “Bacillus thermoantarcticus”, isolated from geothermal soil near the crater of Mount Melbourne, is described. The organism grows at an optimal temperature of 63°C at pH 6.0, is oxidase-positive, catalase-negative and produces an exopolysaccharide, an exocellular xylanase, an intracellular alcohol dehydrogenase and exo- and endocellularα-glucosidase(s). The sequence of 16S rDNA is very similar to that of “Bacillus thermoglucosidasius”; however, the guanine-plus-cytosine (G + C) content is 8 mol% higher. The type strain is “Bacillus thermoantarcticus” (DSM 9572).

Bacillus stearothermophilus ATCC12016 α-glucosidase specific for α-1,4 bonds of maltosaccharides and α-glucans shows high amino acid sequence similarities to seven α-d-glucohydrolases with different substrate specificity

We have sequenced the gene encoding Bacillus stearothermophilus ATCC12016 α-glucosidase (α-d-glucoside glucohydrolase, EC 3.2.1.20) specific for non-reducing terminal α-1,4 bonds of maltosaccharides and α-glucans. The amino acid sequence of the enzyme deduced from the nucleotide sequence of the gene (1665 base pairs) consisted of 555 residues with a molecular mass of 65233. The enzyme showed 40%–57% sequence similarities to α-d-glucohydrolases with very different substrate specificity, such as Bacillus cereus ATCC7064 oligo-1,6-glucosidase, Bacillus thermoglucosidasius KP1006 oligo-1,6-glucosidase, Saccharomyces carlsbergensis CB11 α-glucosidase, Bacillus sp. F5 α-glucosidase, Streptococcus mutans (Ingbritt strain) dextran glucosidase, Bacillus sp. SAM1606 α-glucosidase and Escherichia coli ECL116 trehalose-6-phosphate hydrolase. All these enzymes had sequences equivalent to secondary elements revealed in B. cereus oligo-1,6-glucosidase by X-ray crystallography. We have suggested that the B.stearothermophilus enzyme adopts the same polypeptide folding, i.e. an (α/β)8-barrel in the N-terminal active-site domain, as the B.cereus enzyme and other α-glucohydrolases.

Characterization and identification of a new strain of esterase-producing Bacillus thermoglucosidasius “EAEC” from an aerated thermophilic sewag

Enzymes and especially esterases become increasingly important as tools for the organic chemist. Searching for esterase-producing microorganisms a thermophilic, rodshaped, endospore-forming bacterium, code-named “EAEC”, was isolated from an aerated thermophilic sewage sludge by enrichment on ethyl acetate as the sole carbon source. Phenotypic and genotypic investigations and comparisons led to the conclusion that this isolate belongs to the species Bacillus thermoglucosidasius.

Physiology of growth and carboxyl esterase secretion of a newly-isolated Bacillus thermoglucosidasius strain “EAEC”, enriched on ethyl acetate

The physiological aspects of growth and extracellular esterase activity of a Bacillus species, isolated from an enrichment culture on ethyl acetate and identified recently as B. thermoglucosidasius strain “EAEC”, were investigated by growing the organism in batch, fed-batch and continuous culture regarding the carbon source, bio-availability of iron, phosphate and ammonium concentrations in the medium. Pulse experiments in continuous culture revealed the necessity of adding complexed Fee 2+ to the medium to guarantee sufficient bioavailable iron. Extracellular esterase activity was low in complex media, only weakly induced by ethyl acetate, and very high in cultures grown on glucose and sucrose. The major part of esterase activity was secreted into the medium in the late exponential and in the stationary phase, implying that catabolite-repression is a regulatory mechanism for the expression of the extracellular esterase activity.

Phylogenetic Evidence for the Relationship of Saccharococcus thermophilus to Bacillus stearothermophilus

Summary Comparative analysis of the 16S rDNA of Saccharococcus thermophilus with those of other thermophilic and mesophilic members of the domain Bacteria indicates that this Gram-positive, thermophilic, aerobic, heterotrophic non-sporeforming coccus is closely related to members of the genus Bacillus. Its nearest phylogenetic neighbors are the three recognised species of Group 5 bacilli, namely the thermophilic Bacillus thermoglucosidasius, Bacillus kaustophilus and Bacillus stearothermophilus. This relationship is supported by the presence of common 16S rDNA signature nucleotides as well as by phenotypic similarities.

Cloning and expression in Escherichia coli of an extremely thermostable oligo-1,6-glucosidase gene from Bacillus thermoglucosidasius.

The gene for an extremely thermostable oligo-1,6-glucosidase (dextrin-6-alpha-D-glucanohydrolase; EC 3.2.1.10) of obligately thermophilic Bacillus thermoglucosidasius KP1006 was cloned within a 4.2-kilobase HindIII-PvuII fragment of DNA by using the plasmid pUC19 as a vector and Escherichia coli C600 as a host. The gene was transcribed, presumably from its own promoter, in E. coli. E. coli with the hybrid plasmid accumulated oligo-1,6-glucosidase mainly in the cytoplasm. The level of enzyme production was comparable to that observed for B. thermoglucosidasius. The enzyme coincided absolutely with the B. thermoglucosidasius enzyme in its molecular weight (60,000), in its electrophoretic behavior on denaturing and nondenaturing polyacrylamide gels, in the temperature dependency of its stability and activity, and in its antigenic determinants.