Toward functional genomics in bacteria: analysis of gene expression in Escherichia coli from a bacterial artificial chromosome library of Bacillus cereus.
(17/1270)
As the study of microbes moves into the era of functional genomics, there is an increasing need for molecular tools for analysis of a wide diversity of microorganisms. Currently, biological study of many prokaryotes of agricultural, medical, and fundamental scientific interest is limited by the lack of adequate genetic tools. We report the application of the bacterial artificial chromosome (BAC) vector to prokaryotic biology as a powerful approach to address this need. We constructed a BAC library in Escherichia coli from genomic DNA of the Gram-positive bacterium Bacillus cereus. This library provides 5.75-fold coverage of the B. cereus genome, with an average insert size of 98 kb. To determine the extent of heterologous expression of B. cereus genes in the library, we screened it for expression of several B. cereus activities in the E. coli host. Clones expressing 6 of 10 activities tested were identified in the library, namely, ampicillin resistance, zwittermicin A resistance, esculin hydrolysis, hemolysis, orange pigment production, and lecithinase activity. We analyzed selected BAC clones genetically to identify rapidly specific B. cereus loci. These results suggest that BAC libraries will provide a powerful approach for studying gene expression from diverse prokaryotes. (+info)
Crystal structure of beta-amylase from Bacillus cereus var. mycoides at 2.2 A resolution.
(18/1270)
The crystal structure of beta-amylase from Bacillus cereus var. mycoides was determined by the multiple isomorphous replacement method. The structure was refined to a final R-factor of 0.186 for 102,807 independent reflections with F/sigma(F) > or = 2.0 at 2.2 A resolution with root-mean-square deviations from ideality in bond lengths, and bond angles of 0.014 A and 3.00 degrees, respectively. The asymmetric unit comprises four molecules exhibiting a dimer-of-dimers structure. The enzyme, however, acts as a monomer in solution. The beta-amylase molecule folds into three domains; the first one is the N-terminal catalytic domain with a (beta/alpha)8 barrel, the second one is the excursion part from the first one, and the third one is the C-terminal domain with two almost anti-parallel beta-sheets. The active site cleft, including two putative catalytic residues (Glu172 and Glu367), is located on the carboxyl side of the central beta-sheet in the (beta/alpha)8 barrel, as in most amylases. The active site structure of the enzyme resembles that of soybean beta-amylase with slight differences. One calcium ion is bound per molecule far from the active site. The C-terminal domain has a fold similar to the raw starch binding domains of cyclodextrin glycosyltransferase and glucoamylase. (+info)
Nosocomial pseudoepidemic caused by Bacillus cereus traced to contaminated ethyl alcohol from a liquor factory.
(19/1270)
From September 1990 to October 1990, 15 patients who were admitted to four different departments of the National Taiwan University Hospital, including nine patients in the emergency department, three in the hematology/oncology ward, two in the surgical intensive care unit, and one in a pediatric ward, were found to have positive blood (14 patients) or pleural effusion (1 patient) cultures for Bacillus cereus. After extensive surveillance cultures, 19 additional isolates of B. cereus were recovered from 70% ethyl alcohol that had been used as a skin disinfectant (14 isolates from different locations in the hospital) and from 95% ethyl alcohol (5 isolates from five alcohol tanks in the pharmacy department), and 10 isolates were recovered from 95% ethyl alcohol from the factory which supplied the alcohol to the hospital. In addition to these 44 isolates of B. cereus, 12 epidemiologically unrelated B. cereus isolates, one Bacillus sphaericus isolate from a blood specimen from a patient seen in May 1990, and two B. sphaericus isolates from 95% alcohol in the liquor factory were also studied for their microbiological relatedness. Among these isolates, antibiotypes were determined by using the disk diffusion method and the E test, biotypes were created with the results of the Vitek Bacillus Biochemical Card test, and random amplified polymorphic DNA (RAPD) patterns were generated by arbitrarily primed PCR. Two clones of the 15 B. cereus isolates recovered from patients were identified (clone A from 2 patients and clone B from 13 patients), and all 29 isolates of B. cereus recovered from 70 or 95% ethyl alcohol in the hospital or in the factory belonged to clone B. The antibiotype and RAPD pattern of the B. sphaericus isolate from the patient were different from those of isolates from the factory. Our data show that the pseudoepidemic was caused by a clone (clone B) of B. cereus from contaminated 70% ethyl alcohol used in the hospital, which we successfully traced to preexisting contaminated 95% ethyl alcohol from the supplier, and by another clone (clone A) without an identifiable source. (+info)
Role of hemolysin BL in the pathogenesis of extraintestinal Bacillus cereus infection assessed in an endophthalmitis model.
(20/1270)
Bacillus cereus is a rare cause of serious human infection but, paradoxically, causes one of the most severe posttraumatic or endogenous infections of the eye, endophthalmitis, which frequently results in blindness. The virulence of B. cereus endophthalmitis historically has been attributed to toxin production. We therefore sought to examine the contribution of the dermonecrotic toxin, hemolysin BL, to the pathogenesis of B. cereus infection in an endophthalmitis system that is highly amenable to study. The pathogenesis of infection resulting from intravitreal injection of 10(2) CFU of either a clinical ocular isolate of B. cereus producing hemolysin BL (HBL+) or an isogenic mutant in this trait (HBL-) was assessed bacteriologically and by slit lamp biomicroscopy, electroretinography, histology, and inflammatory cell enumeration. Both HBL+ and HBL- strains evoked severe intraocular inflammatory responses as early as 12 h postinfection, with complete loss of retinal responsiveness by 12 h. The infections caused by both strains spread of the infection to adjacent tissues by 18 h. No significant differences in intraocular bacterial growth (P >/= 0.21) or inflammatory changes (P >/= 0.21) were observed in eyes infected with either HBL+ or HBL- strains during the course of infection. The level of retinal responsiveness was greater in HBL- infected eyes than in HBL+-infected eyes at 6 h only (P = 0.01). These results indicate that hemolysin BL makes no essential contribution to the severe and rapid course of infection in the endophthalmitis model. (+info)
Specific detection of the gene for the extracellular neutral protease of Bacillus cereus by PCR and blot hybridization.
(21/1270)
A pair of primers and a gene probe for the amplification and detection of the Bacillus cereus neutral protease gene (NPRC) were developed. Specificity for the npr genes of the B. cereus group members B. cereus, B. mycoides, and B. thuringiensis was shown. Restriction polymorphism patterns of the PCR products confirmed the presence of the NPRC gene in all three species. (+info)
Roles of asp126 and asp156 in the enzyme function of sphingomyelinase from Bacillus cereus.
(22/1270)
To elucidate the roles of conserved Asp residues of Bacillus cereus sphingomyelinase (SMase) in the kinetic and binding properties of the enzyme toward various substrates and Mg2+, the kinetic data on mutant SMases (D126G and D156G) were compared with those of wild type (WT) enzyme. The stereoselectivity of the enzyme in the hydrolysis of monodispersed short-chain sphingomyelin (SM) analogs and the binding of Mg2+ to the enzyme were not affected by the replacement of Asp126 or Asp156. The pH-dependence curves of kinetic parameters (1/Km and kcat) for D156G-catalyzed hydrolysis of micellar SM mixed with Triton X-100 (1:10) and of micellar 2-hexadecanoylamino-4-nitrophenylphosphocholine (HNP) were similar in shape to those for WT enzyme-catalyzed hydrolysis. On the other hand, the curves for D126G lacked the transition observed for D156G and WT enzymes. Comparison of the values and the shape of pH-dependence curves of kinetic parameters indicated that Asp126 of WT SMase enhances the enzyme's catalytic activity toward both substrates and its binding of HNP but not SM. The deprotonation of Asp126 enhances the substrate binding and slightly suppresses the catalytic activity toward both substrates. Asp156 of WT SMase acts to decrease the binding of both substrates and the catalytic activity to HNP but not SM. From the present study and the predicted three-dimensional structure of B. cereus SMase, Asp126 was thought to be located close to the active site, and its ionization was shown to affect the catalytic activity and substrate binding. (+info)
Relationship of Bacillus subtilis DNA polymerase III to bacteriophage PBS2-induced DNA polymerase and to the replication of uracil-containing DNA.
(23/1270)
In vivo studies of PBS2 phage replication in a temperature-sensitive Bacillus subtilis DNA polymerase III (Pol III) mutant and a temperature-resistant revertant of this mutant have suggested the possible involvement of Pol III in PBS2 DNA synthesis. Previous results with 6-(p-hydroxyphenylazo)-uracil (HPUra), a specific inhibitor of Pol III and DNA replication in uninfected cells, suggest that Pol III is not involved in phage DNA replication, due to its resistance to this drug. Experiments were designed to examine possible explanations for this apparent contradiction. First, assays of the host Pol III and the phage-induced DNA polymerase activities in extracts indicated that a labile Pol III did not result in a labile phage-induced enzyme, suggesting that this new polymerase is not a modified HPUra-resistant form of Pol III. Indeed the purified phage-induced enzyme was resistant to the active, reduced form of HPUra under all assay conditions tested. Since in vitro Pol III was capable of replicating the uracil-containing DNA found in this phage, the sensitivity of the purified enzyme to reduced HPUra was examined using phage DNA as template-primer and dUTP as substrate; these new substrates did not affect the sensitivity of the host enzyme to the drug. (+info)
Ionophoretic properties and mitochondrial effects of cereulide: the emetic toxin of B. cereus.
(24/1270)
The emetic toxin of Bacillus cereus, found to cause immobilization of spermatozoa and swelling of their mitochondria, was purified and its structure found to be identical to the earlier known toxin cereulide. It increased the conductance in black-lipid membranes in KCl solutions in an ionophore-like manner. It formed adducts with K+, Na+, and NH4+ but the conductance was highly selective for K+ in relation to Na+ and H+ (three orders of magnitude). The increase in the kinetics of conductance indicated a stoichiometric ratio between the cereulide and K+. Its ionophoretic properties are thus similar to those of valinomycin. In addition, its effects on rat liver mitochondria were similar: it stimulated swelling and respiration in respiring mitochondria in the presence but not in the absence of K+, it reduced the transmembrane potential under these conditions. In nonrespiring mitochondria, swelling was seen in KNO3- but not in NaNO3-containing media, less in acetate. In NaNO3 media addition of the cereulide caused a transient diffusion potential which was reduced by adding K+. It is concluded that the toxic effects of cereulide are due to it being a K+ ionophore. (+info)