A randomly amplified polymorphic DNA marker specific for the Bacillus cereus group is diagnostic for Bacillus anthracis.
Aiming to develop a DNA marker specific for Bacillus anthracis and able to discriminate this species from Bacillus cereus, Bacillus thuringiensis, and Bacillus mycoides, we applied the randomly amplified polymorphic DNA (RAPD) fingerprinting technique to a collection of 101 strains of the genus Bacillus, including 61 strains of the B. cereus group. An 838-bp RAPD marker (SG-850) specific for B. cereus, B. thuringiensis, B. anthracis, and B. mycoides was identified. This fragment included a putative (366-nucleotide) open reading frame highly homologous to the ypuA gene of Bacillus subtilis. The restriction analysis of the SG-850 fragment with AluI distinguished B. anthracis from the other species of the B. cereus group. (+info)
Purification and properties of a low-molecular-weight, high-alkaline pectate lyase from an alkaliphilic strain of Bacillus.
A low-molecular-weight, high-alkaline pectate lyase (pectate transeliminase, EC 184.108.40.206) was found in an alkaline culture of Bacillus sp. strain KSM-P15, purified to homogeneity, and crystallized. The enzyme had a relative molecular weight of approximately 20,300 as measured by sedimentation equilibrium, with a sedimentation coefficient (s20,w0) of 1.73 S. It was a basic protein with an isoelectric point of pH 10.3, and the alpha-helical content was only 6.6%. In the presence of Ca2+ ions, the enzyme degraded polygalacturonic acid in a random manner to yield 4,5-unsaturated oligo-galacturonides and had its optimal activity around pH 10.5 and 50-55 degrees C. It also had a protopectinase-like activity on cotton fibers. The N-terminal amino acid sequences of the intact protein (28 amino acids) and its two lysyl endopeptidase-cleaved peptide fragments (8 and 12 amino acids) had very low sequence similarity with pectate lyases reported to date. These results strongly suggest that the pectate lyase of Bacillus sp. strain KSM-P15 may be a novel enzyme and belongs in a new family. (+info)
Purification and properties of bacteriolytic enzymes from Bacillus licheniformis YS-1005 against Streptococcus mutans.
To find a novel lytic enzyme against cariogenic Streptococci, strains showing strong lytic activity have been screened from soil using Streptococcus mutans. A strain identified as Bacillus licheniformis secreted two kinds of lytic enzymes, which were purified by methanol precipitation, CM-cellulose chromatography, gel filtration, and hydroxyapatite chromatography. The molecular weights of these two enzymes, L27 and L45, were 27,000 and 45,000, respectively. Optimum pH and temperature of both enzymes for lytic activity were pH 8 and 37 degrees C. L27 and L45 digest the peptide linkage between L-Ala and D-Glu in peptidoglycan of Streptococcus mutans. The lytic activity was highly specific for Streptococcus mutans, suggesting their potential use as a dental care product. (+info)
Improving the binding affinity of an antibody using molecular modeling and site-directed mutagenesis.
Activated Factor X releases F1.2, a 271-amino acid peptide, from the amino terminus of prothrombin during blood coagulation. A nine-amino acid peptide, C9 (DSDRAIEGR), corresponding to the carboxyl terminus of F1.2 was synthesized and used to produce a monoclonal antibody, TA1 (K(D)) 1.22 x 10(-6) M). To model the TA1 antibody, we entered the sequence information of the cloned TA1 Fv into the antibody modeling program, ABM, which combines homology methods, conformational search procedures, and energy screening and has proved to be a reliable and reproducible antibody modeling method. Using a novel protein fusion procedure, we expressed the C9 peptide fused to the carboxyl terminus of the PENI repressor protein from Bacillus licheniformis in Escherichia coli. We constructed fusion proteins containing alanine substitutions for each amino acid in the C9 epitope. Binding studies, using the C9 alanine mutants and TA1, and spatial constraints predicted by the modeled TA1 binding cleft enabled us to establish a plausible conformation for C9 complexed with TA1. Furthermore, based on binding results of conservative amino acid substitutions in C9 and mutations in the antibody, we were able to refine the complex model and identify antibody mutations that would improve binding affinity. (+info)
Active site characterization of the exo-N-acetyl-beta-D- glucosaminidase from thermotolerant Bacillus sp. NCIM 5120: involvement of tryptophan, histidine and carboxylate residues in catalytic activity.
The exo-N-acetyl-beta-d-glucosaminidase (EC 220.127.116.11) from thermotolerant Bacillus sp. NCIM 5120 is a homotetramer with a molecular mass of 240000 kDa. Chemical modification studies on the purified exo-N-acetyl-beta-d-glucosaminidase revealed the involvement of a single tryptophan, histidine and carboxylate, per monomer, in the catalytic activity of the enzyme. Spectral analysis and maintenance of total enzyme activities indicated that N-acetylglucosamine (competitive inhibitor) and p-nitrophenyl-N-acetyl-beta-d-glucosaminide (substrate) prevented the modification of a single essential tryptophan, histidine and carboxylate residue. Kinetic parameters of partially inactivated enzyme (by NBS/HNBB) showed the involvement of tryptophan in substrate binding while that of histidine (by photooxidation/DEPC) and carboxylate (by EDAC/WRK) in catalysis. The Bacillus sp. NCIM 5120 exo-N-acetyl-beta-d-glucosaminidase deviates from the reported N-acetyl-beta-d-glucosaminidases and beta-hexosaminidases that utilize anchimeric assistance in their hydrolytic mechanism. (+info)
Can vector control play a useful supplementary role against bancroftian filariasis?
A single campaign of mass treatment for bancroftian filariasis with diethylcarbamazine (DEC) in Makunduchi, a town in Zanzibar, United Republic of Tanzania, combined with elimination of mosquito breeding in pit latrines with polystyrene beads was followed by a progressive decline over a 5-year period in the microfilarial rate from 49% to 3%. Evidence that vector control had contributed to this long-term decline was obtained by comparison with another town, Moga, where a DEC campaign was used without vector control and where resurgence of microfilariae could be observed 3-6 years after the campaign. In Zanzibar town, treatment of 3844 wet pit latrines and cesspits with polystyrene beads reduced the adult mosquito population in houses by about 65%. Supplementary treatment of open drains and marshes with Bacillus sphaericus produced little or no additional reduction compared to a sector of the town where only pit treatment with polystyrene was carried out. The cost and effort of achieving the 65% reduction in mosquito population could hardly be justified for its impact on filariasis alone, but its noticeable impact on biting nuisance might help to gain community support for an integrated programme. (+info)
Key role of barstar Cys-40 residue in the mechanism of heat denaturation of bacterial ribonuclease complexes with barstar.
The mechanism by which barnase and binase are stabilized in their complexes with barstar and the role of the Cys-40 residue of barstar in that stabilization have been investigated by scanning microcalorimetry. Melting of ribonuclease complexes with barstar and its Cys-82-Ala mutant is described by two 2-state transitions. The lower-temperature one corresponds to barstar denaturation and the higher-temperature transition to ribonuclease melting. The barstar mutation Cys-40-Ala, which is within the principal barnase-binding region of barstar, simplifies the melting to a single 2-state transition. The presence of residue Cys-40 in barstar results in additional stabilization of ribonuclease in the complex. (+info)
Cloning and nucleotide sequence analysis of gyrB of Bacillus cereus, B. thuringiensis, B. mycoides, and B. anthracis and their application to the detection of B. cereus in rice.
As 16S rRNA sequence analysis has proven inadequate for the differentiation of Bacillus cereus from closely related species, we employed the gyrase B gene (gyrB) as a molecular diagnostic marker. The gyrB genes of B. cereus JCM 2152(T), Bacillus thuringiensis IAM 12077(T), Bacillus mycoides ATCC 6462(T), and Bacillus anthracis Pasteur #2H were cloned and sequenced. Oligonucleotide PCR primer sets were designed from within gyrB sequences of the respective bacteria for the specific amplification and differentiation of B. cereus, B. thuringiensis, and B. anthracis. The results from the amplification of gyrB sequences correlated well with results obtained with the 16S rDNA-based hybridization study but not with the results of their phenotypic characterization. Some of the reference strains of both B. cereus (three serovars) and B. thuringiensis (two serovars) were not positive in PCR amplification assays with gyrB primers. However, complete sequencing of 1.2-kb gyrB fragments of these reference strains showed that these serovars had, in fact, lower homology than their originally designated species. We developed and tested a procedure for the specific detection of the target organism in boiled rice that entailed 15 h of preenrichment followed by PCR amplification of the B. cereus-specific fragment. This method enabled us to detect an initial inoculum of 0.24 CFU of B. cereus cells per g of boiled rice food homogenate without extracting DNA. However, a simple two-step filtration step is required to remove PCR inhibitory substances. (+info)