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At least 217 records · Page 12Linked to original sources

PubMed2005

A thermodynamic study of mesophilic, thermophilic, and hyperthermophilic L-arabinose isomerases: the effects of divalent metal ions on protein stability at elevated temperatures.

To gain insight into the structural stability of homologous homo-tetrameric l-arabinose isomerases (AI), we have examined the isothermal guanidine hydrochloride (GdnHCl)-induced unfolding of AIs from mesophilic Bacillus halodurans (BHAI), thermophilic Geobacillus stearothermophilus (GSAI), and hyperthermophilic Thermotoga maritima (TMAI) using circular dichroism spectroscopy. The GdnHCl-induced unfolding of the AIs can be well described by a two-state reaction between native tetramers and unfolded monomers, which directly confirms the validity of the linear extrapolation method to obtain the intrinsic stabilities of these proteins. The resulting unfolding free energy (DeltaGU) values of the AIs as a function of temperature were fit to the Gibbs-Helmholtz equation to determine their thermodynamic parameters based on a two-state mechanism. Compared with the stability curves of BHAI in the presence and absence of Mn2+, those of holo GSAI and TMAI were more broadened than those of the apo enzymes at all temperatures, indicating increased melting temperatures (Tm) due to decreased heat capacity (DeltaGp). Moreover, the extent of difference in DeltaCp between the apo and holo thermophilic AIs is larger than that of BHAI. From these studies, we suggest that the metal dependence of the thermophilic AIs, resulting in the reduced DeltaCp, may play a significant role in structural stability compared to their mesophilic analogues, and that the extent of metal dependence of AI stability seems to be highly correlated to oligomerization.

Aldose-Ketose Isomerases↗

PubMed2005

Zinc in lipase L1 from Geobacillus stearothermophilus L1 and structural implications on thermal stability.

Lipase L1 from Geobacillus stearothermophilus L1 contains an unusual extra domain, making a tight intramolecular interaction with the main catalytic domain through a Zn2+-binding coordination. To elucidate the role of the Zn2+, we disrupted the Zn2+-binding site by mutating the zinc-ligand residues (H87A, D61A/H87A, and D61A/H81A/H87A/D238A). The activity vs. temperature profiles of the mutant enzymes showed that the disruption of the Zn2+-binding site resulted in a notable decrease in the optimal temperature for maximal activity from 60 to 45-50 degrees C. The mutations also abolished the Zn2+-induced thermal stabilization. The wild-type enzyme revealed a 34.6-fold increase in stabilization with the addition of Zn2+ at 60 degrees C, whereas the mutant enzymes exhibited no response to Zn2+. Additional circular dichroism spectroscopy studies also confirmed the structural stabilizing role of Zn2+ on lipase L1 at elevated temperatures.

Amino Acid Sequence↗

PubMed2005

Characterization of potential stress responses in ancient Siberian permafrost psychroactive bacteria.

Past studies of cold-acclimated bacteria have focused primarily on organisms not capable of sub-zero growth. Siberian permafrost isolates Exiguobacterium sp. 255-15 and Psychrobacter sp. 273-4, which grow at subzero temperatures, were used to study cold-acclimated physiology. Changes in membrane composition and exopolysaccharides were defined as a function of growth at 24, 4 and -2.5 degrees C in the presence and absence of 5% NaCl. As expected, there was a decrease in fatty acid saturation and chain length at the colder temperatures and a further decrease in the degree of saturation at higher osmolarity. A shift in carbon source utilization and antibiotic resistance occurred at 4 versus 24 degrees C growth, perhaps due to changes in the membrane transport. Some carbon substrates were used uniquely at 4 degrees C and, in general, increased antibiotic sensitivity was observed at 4 degrees C. All the permafrost strains tested were resistant to long-term freezing (1 year) and were not particularly unique in their UVC tolerance. Most of the tested isolates had moderate ice nucleation activity, and particularly interesting was the fact that the Gram-positive Exiguobacterium showed some soluble ice nucleation activity. In general the features measured suggest that the Siberian organisms have adapted to the conditions of long-term freezing at least for the temperatures of the Kolyma region which are -10 to -12 degrees C where intracellular water is likely not frozen.

Adaptation, Physiological↗

PubMed2005

Expression and secretion of the protective antigen of Bacillus anthracis in Bacillus brevis.

We used the Bacillus brevis-pNU212 system to develop a mass production system for the protective antigen (PA) of Bacillus anthracis. A moderately efficient expression-secretion system for PA was constructed by fusing the PA gene from B. anthracis with the B. brevis cell-wall protein signal-peptide encoding region of pNU212, and by introducing the recombinant plasmid, pNU212-mPA, into B. brevis 47-5Q. The clone producing PA secreted about 300 microg of recombinant PA (rPA) per ml of 5PY-erythromycin medium after 4 days incubation at 30 degrees C. The rPA was fractionated from the culture supernatant of B. brevis 47-5Q carrying pNU212-mPA using ammonium sulfate at 70% saturation followed by anion exchange chromatography on a Hitrap Q, a Hiload 16/60 Superdex 200 gel filtration column and a phenyl sepharose hydrophobic interaction column, yielding 70 mg rPA per liter of culture. The N-terminal sequence of the purified rPA was identical to that of native PA from B. anthracis. The purified rPA exhibited cytotoxicity towards J774A.1 cells when combined with lethal factor. The rPA formulated in either Rehydragel HPA or MPL-TDM-CWS adjuvant (Ribi-Trimix) elicited the expression of a large amount of anti-PA and neutralizing antibodies in guinea pigs and completely protected them against a 100 LD50 challenge with fully virulent B. anthracis spores.

Amino Acid Sequence↗

PubMed2004

Cloning and characterization of the Halobacillus trueperi betH gene, encoding the transport system for the compatible solute glycine betaine.

Halobacillus trueperi accumulates glycine betaine under condition of high osmolarity. A fragment of the glycine betaine transporter betH gene was obtained from the genome of H. trueperi with degenerate primers. Through Southern blot hybridization and inverse PCR, a 5.1 kb EcoRI fragment containing the complete betH gene was identified and subsequently sequenced. The betH gene was predicted to encode a 55.2 kDa protein (504 amino acid residues) with 12 transmembrane regions. BetH showed 56% identity to the OpuD of Bacillus subtilis which belongs to the betaine/carnitine/choline transporter (BCCT) family. Its putative promoter region was highly homologous to sigmaB-dependent promoter of B. subtilis. A 2.6 kb fragment containing the betH gene was cloned into pUC18 and transformed into the Escherichia coli MKH13. The accumulation of glycine betaine in transformed E. coli MKH13 bacteria was confirmed using 13C nuclear magnetic resonance spectroscopy.

Amino Acid Sequence↗

PubMed2005

Multilocus sequence typing reveals that Bacillus cereus strains isolated from clinical infections have distinct phylogenetic origins.

Eight strains of Bacillus cereus isolated from bacteremia and soft tissue infections were assigned to seven sequence types (STs) by multilocus sequence typing (MLST). Two strains from different locations had identical STs. The concatenated sequences of the seven STs were aligned with 65 concatenated sequences from reference STs and a neighbor-joining tree was constructed. Two strains were distantly related to all reference STs. Three strains were recovered in a clade that included Bacillus anthracis, B. cereus and rare Bacillus thuringiensis strains while the other three strains were assigned to two STs that were more closely affiliated to most of the B. thuringiensis STs. We conclude that invasive B. cereus strains do not form a single clone or clonal complex of highly virulent strains.

Aged↗

PubMed2005

Validation of low-temperature steam with formaldehyde sterilization for endoscopes, using validation device.

BACKGROUND: Validation of sterilization is an important step before clinical use of medical equipment. Adequate validation of sterilization of the endoscope has not been reported. One reason for this is the lack of suitable devices for validation. METHODS: The VDES (validation device for endoscope sterilization; Olympus prototype model, Olympus, Tokyo, Japan) was designed in two types (type A and type B) and resembles gastroscopes and duodenoscopes, respectively. Each type consists of inner and outer tubing and a central capsule containing a biological indicator. The device was designed to examine the effectiveness of low-temperature sterilizers, such as ethylene oxide gas, hydrogen peroxide gas plasma, and low-temperature steam with formaldehyde (LTSF) sterilizer. The aim of this study was to validate the sterilization of GI endoscopes by the LTSF sterilizer. Sterilization was assessed using both types of VDES after a 60-min application of LTSF. RESULTS: Culture of the biological indicator confirmed the complete eradication of the bacteria in a total of 10 experiments with each type of VDES after LTSF sterilization. CONCLUSIONS: Our results confirm that the LTSF sterilizer may sterilize endoscopes currently distributed by Olympus. Commercialization of VDES will make it possible to evaluate the reliability of sterilization when it is set in the sterilization device with endoscopes.

Bacillaceae↗

PubMed2005

Characterization of microorganisms in Argentinean honeys from different sources.

Seventy polyfloral honeys including commercial samples obtained from supermarkets, harvested from apiaries and purchased in bulk were initially examined for total antibacterial activity. From each sample, numbers of aerobic mesophilic bacteria, total coliforms, moulds and yeasts were determined and the presence of Salmonella spp., Shigella spp., Clostridium sulfite-reducers, Paenibacillus larvae and Bacillus spp. was investigated. Moisture content, pH and total acidity were also determined for all samples. Any honey diluted to concentrations from 75% to 1% (w/v) of full-strength honey showed total antibacterial activity. The numbers of aerobic mesophilic bacteria, moulds and yeasts were less than 10(3) cfu/g for all 70 samples. Faecal coliforms, Escherichia coli, Salmonella spp., Shigella spp. and Clostridium sulfite-reducers were not detected but P. larvae subspp. larvae, Bacillus cereus, Bacillus pumilus and Bacillus laterosporus were found among samples. For commercial, apiary and bulk honey the mean values for moisture content, pH and acidity, respectively, were 17.50%, 17.40% and 17.50%; 4.60, 4.10 and 4.20; and 18.30, 20.60 and 21 meq NaOH/kg. P. larvae was recovered from 35% of apiaries including hives in which the bees did not display symptoms of American foulbrood disease.

Animals↗

PubMed2005

Purification and characterization of an L-arabinose isomerase from an isolated strain of Geobacillus thermodenitrificans producing D-tagatose.

The araA gene, encoding l-arabinose isomerase (AI), from the thermophilic bacterium Geobacillus thermodenitrificans was cloned and expressed in Escherichia coli. Recombinant AI was isolated with a final purity of about 97% and a final specific activity of 2.10 U/mg. The molecular mass of the purified AI was estimated to be about 230 kDa to be a tetramer composed of identical subunits. The AI exhibited maximum activity at 70 degrees C and pH 8.5 in the presence of Mn2+. The enzyme was stable at temperatures below 60 degrees C and within the pH range 7.5-8.0. d-Galactose and l-arabinose as substrate were isomerized with high activities. Ribitol was the strongest competitive inhibitor of AI with a Ki of 5.5mM. The apparent Km and Vmax for L-arabinose were 142 mM and 86 U/mg, respectively, whereas those for d-galactose were 408 mM and 6.9 U/mg, respectively. The catalytic efficiency (kcat/Km) was 48 mM(-1)min(-1) for L-arabinose and 0.5mM(-1)min(-1) for D-galactose. Mn2+ was a competitive activator and increased the thermal stability of the AI. The D-tagatose yield produced by AI from d-galactose was 46% without the addition of Mn2+ and 48% with Mn2+ after 300 min at 65 degrees C.

Aldose-Ketose Isomerases↗

PubMed2006

Purification, characterization, and synergistic action of phytate-resistant alpha-amylase and alpha-glucosidase from Geobacillus thermodenitrificans HRO10.

The alpha-amylase (1, 4-alpha-d-glucanohydrolase; EC 3.2.1.1) and alpha-glucosidase (alpha-d-glucoside glucohydrolase; EC 3.2.1.20) secreted by Geobacillus thermodenitrificans HRO10 were purified to homogeneity (13.6-fold; 11.5% yield and 25.4-fold; 32.0% yield, respectively) through a series of steps. The molecular weight of alpha-amylase was 58kDa, as estimated by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE). The alpha-amylase activity on potato starch was optimal at pH 5.5 and 80 degrees Celsius. In the presence of Ca(2+), the alpha-amylase had residual activity of more than 92% after 1h of incubation at 70 degrees Celsius. The alpha-amylase did not lose any activity in the presence of phytate (a selective alpha-amylase inhibitor) at concentrations as high as 10mM, rather it retained 90% maximal activity after 1h of incubation at 70 degrees Celsius. EGTA and EDTA were strong inhibitory substances of the enzyme. The alpha-amylase hydrolyzed soluble starch at 80 degrees Celsius, with a K(m) of 3.05mgml(-1) and a V(max) of 7.35Uml(-1). The molecular weight of alpha-glucosidase was approximately 45kDa, as determined by SDS-PAGE. The enzyme activity was optimal at pH 6.5-7.5 and 55 degrees Celsius. Phytate did not inhibit G. thermodenitrificans HRO10 alpha-glucosidase activity, whereas pCMB was a potent inhibitor of the enzyme. The alpha-glucosidase exhibited Michaelis-Menten kinetics with maltose at 55 degrees Celsius (K(m): 17mM; V(max): 23micromolmin(-1)mg(-1)). Thin-layer chromatography studies with G. thermodenitrificans HRO10 alpha-amylase and alpha-glucosidase showed an excellent synergistic action and did not reveal any transglycosylation catalyzed reaction by the alpha-glucosidase.

Bacillaceae↗

PubMed2006

Analysis of reduction of Geobacillus stearothermophilus spores treated with high hydrostatic pressure and mild heat in milk buffer.

Our unpublished experimental results of fractional factorial experiments showed that the significant external factors affecting high pressure processing (HPP) inactivation were pressure, temperature and pressure holding time. Based on these results, response surface methodology (RSM) was employed in the present work and a quadratic equation for HPP inactivation was built. By analyzing the response surface plots and their corresponding contour plots as well as solving the quadratic equation, the experimental values were shown to be significantly in good agreement with predicted values since the adjusted determination coefficient (R(Adj)(2)) was 0.9747. The optimum process parameters for six log-cycles reduction of Geobacillus stearothermophilus spores were obtained as: temperature, 86 degrees C; pressure, 625.0 MPa and pressure holding time, 14.0 min. The adequacy of the model equation for predicting the optimum response values was verified effectively by the validation data.

Bacillaceae↗

PubMed2005

Bacillus cereus infections in Traumatology-Orthopaedics Department: retrospective investigation and improvement of healthcare practices.

OBJECTIVE: To investigate 41 open fractures infected with Bacillus cereus in a Traumatology-Orthopaedy ward and propose a care protocol at admission. METHODS: All B. cereus strains isolated from patients hospitalized in the Traumatology-Orthopaedy ward between March 1997 and August 2001 were submitted to molecular analysis (RAPD and PFGE) in order to investigate a putative outbreak. Susceptibility to the main antibiotics and antiseptics used in this kind of surgery was also evaluated. RESULTS: The B. cereus clinical isolates were mainly isolated from patients who had initially open fractures and were not clonally related. Furthermore, analysis of the clinical data was in favour of a telluric contamination of the wound (wound contamination with terrestrial environments) before admission. Finally, betalactam antibiotics used for prophylactic chemotherapy were not effective against the strains tested as well as the antiseptics who displayed poor effect. CONCLUSION: B. cereus could be termed an emerging pathogen and people need to be aware of its potential importance in orthopaedic trauma cases. In this purpose, a systematic screening for B. cereus at admission should be necessary in front of patients with open fractures associated with telluric contamination. Furthermore, if this bacterium can be isolated, chemotherapy should be based upon ciprofloxacin that would prevent the development of B. cereus infection responsible for deleterious complications.

Adolescent↗

PubMed2005

Bacillus species sepsis in the neonatal intensive care unit.

OBJECTIVES: In 2002 there was an increase in the incidence of Bacillus species sepsis in our NICU that was almost completely resolved in 2003-2004 after the NICU was relocated. Our aims were to identify the source, the risk factors, and to characterize the clinical features of these infections. METHODS: The epidemiological investigation commenced during the outbreak and thereafter. The patient's data were collected retrospectively and a case control study was used to analyze the risk factors. RESULTS: There were eight cases of Bacillus species sepsis: five during 2002, two in 2003, and one in 2004. All infants recovered and salvaging percutaneous central venous catheter (PCVC) was successful in 4/6 of the cases. A case control study identified necrotizing enterocolitis (NEC) and PCVC as risk factors in univariate analysis but only NEC in multivariate analysis. No focal source of Bacillus bacteria was identified, but a high load of bacteria was found in the NICU's air before it was relocated. CONCLUSION: The risk factors for Bacillus species sepsis in our NICU were NEC and PCVC. The clinical course was milder than previously described, and PCVC was successfully salvaged in most cases. The increase in the incidence could be related to the construction work connected with NICU's relocation.

Air Microbiology↗

PubMedDate not supplied

Characterization of Mexican Bacillus thuringiensis strains toxic for lepidopteran and coleopteran larvae.

Bacillus thuringiensis strains C-4, C-9, GM-7, and GM-10, isolated from northeast Mexico and selected for their high toxicity against lepidopteran and coleopteran pests, were characterized following United States Environmental Protection Agency (EPA)'s guidelines. Flagellar serotyping revealed that GM-7 and GM-10 belonged to serotype aizawai, whereas C-4, C-9 corresponded to the kumamotoensis serotype. GM-10 and C-9 were also shown to be the most effective against lepidoptera and coleoptera larvae, respectively. None of the tested strains produced beta-exotoxin or showed activity against mosquitoes. GM-7 and GM-10 were sensitive to R-41 and CP-51 phages. All strains synthesized crystal proteins of 130-140 kDa. PCR analysis showed that C-4, GM-7, and GM-10 strains expressed cry1 genes, and C-9 expressed cry3 and cry7/8 genes, but not cry1. However, the C-9 strain had no cross-reaction with antisera raised against Cry3A and Cry7A proteins. GM-7 and GM-10 were sensitive to R-41 and CP-51 phages. When the delta-endotoxin (crystal) from the four strains was subcutaneously injected to Balb/c mice, alone or in combination with spores, only C-4 and C-9 provoked tissue necrosis similar to that caused by the beta-exotoxin producer HD-41. Tissue necrosis was prevented with the injection of pentoxifylline, an inhibitor of tumor necrosis factor alpha (TNF-alpha) production, suggesting a role of this cytokine in the observed effect. Our results demonstrated that GM-7 and GM-10 strains are effective and suitable for control of lepidopteran pests and safe for mammals under EPA regulations. The potential of the C-9 strain for the control of several coleopteran pests, and the induction of tissue necrosis in mice by C-4 and C-9 strains, are discussed.

Adenosine↗

PubMed2005

Enzyme-substrate complex structures of a GH39 beta-xylosidase from Geobacillus stearothermophilus.

Beta-D-Xylosidases are glycoside hydrolases that catalyse the release of xylose units from short xylooligosaccharides and are engaged in the final breakdown of plant cell-wall hemicelluloses. beta-D-Xylosidases are found in glycoside hydrolase families 3, 39, 43, 52 and 54. The first crystal structure of a GH39 beta-xylosidase revealed a multi-domain organization with the catalytic domain having the canonical (beta/alpha)8 barrel fold. Here, we report the crystal structure of the GH39 Geobacillus stearothermophilus beta-D-xylosidase, inactivated by a point mutation of the general acid-base residue E160A, in complex with the chromogenic substrate molecule 2,5-dinitrophenyl-beta-D-xyloside. Surprisingly, six of the eight active sites present in the crystallographic asymmetric unit contain the trapped covalent glycosyl-enzyme intermediate, while two of them still contain the uncleaved substrate. The structural characterization of these two critical species along the reaction coordinate of this enzyme identifies the residues forming its xyloside-binding pocket as well as those essential for its aglycone recognition.

Bacillaceae↗

PubMed2006

Microbial contamination of endodontic files received from the manufacturer.

This study was conducted to test the sterility of new unused endodontic files received from manufacturers. Fifteen types of hand and rotary files from five manufacturers were tested. Positive microbial cultures were obtained from 13% of the 150 files tested. Autoclaved files were intentionally contaminated with bacterial species recovered from the positive cultures to evaluate a chairside sterilization method. Immersion of contaminated files in 5.25% sodium hypochlorite for five minutes sterilized the files. The results of this study indicate that endodontic files should be sterilized before clinical use. It is also suggested that manufacturers list the sterility state of their endodontic files on their packaging.

Bacillaceae↗

PubMedDate not supplied

[Bacillus licheniformis: an unusual cause of erysipelosis].

The authors report a case of a cutaneous infection due to Bacillus licheniformis. It occurred after a wound due to a wicker splinter. The bacteriological identification was easy thanks to the very typical aspects of culture. First intention antibiotherapy given for bacterial dermo-hypodermatitis may be ineffective because Bacillus licheniformis secretes a biofilm and is frequently resistant to Beta-lactams.

Abscess↗

PubMed2006

Identification and over-expression of a thermostable lipase from Geobacillus thermoleovorans Toshki in Escherichia coli.

A newly isolated thermophilic strain producing thermostable lipase was identified based on 16S rRNA sequencing, where phylogenetic analysis revealed its closeness to Geobacillus thermoleovorans. Thermostable lipase from this bacterium was cloned using consensus degenerate PCR primers. For over-expression in Escherichia coli, the lipase gene was sub-cloned in pET 15b vector with a strong T7 promotor. Lipase activity was approximately 4.5-fold higher than in the wild-type strain. The lipase enzyme was thermostable at 60 degrees C and pH 8, whereas a 30% residual activity was retained when incubated for 1h at 100 degrees C. Optimum lipase expression was obtained in 2 x YT medium after 70min of induction by IPTG.

Bacillaceae↗