RESUMEN

An antibiotic complex was isolated from culture 8-86 referred to Bacillus. The complex consisted of components 8-86A and 8-86B active against gram-negative organisms. By its physico-chemical properties such as IR and UV spectra, amino acid composition, specific rotation and fatty acid composition component 8-86B was shown to be close to polymyxin F.

Asunto(s)

Asunto(s)

RESUMEN

The cell surface of the mesophilic eubacterium Bacillus alvei CCM 2051 is covered by an oblique arranged surface layer glycoprotein. The subunits revealed by sodium dodecyl sulfate - polyacrylamide gel electrophoresis were distinct bands of molecular masses 140,000, 128,000, and 127,000. Proteolytic degradation of the purified S-layer glycoprotein yielded a single glycopeptide fraction with an apparent molecular mass of ca. 25,000. Methylation analysis in conjunction with two-dimensional nuclear magnetic resonance experiments at 500 MHz established the branched trisaccharide (formula; see text) as the repeating unit for this glycan chain.

Asunto(s)

RESUMEN

Nine strains of Bacillus popilliae and Bacillus lentimorbus KLN2 contain N6-methyladenine in GATC sequences, as determined by using the restriction enzymes MboI and DpnI. Among eight other Bacillus species examined, all, except one strain of Bacillus brevis (ATCC 9999), lacked adenine methylation in GATC. A methylase with Escherichia coli dcm site specificity was not present in any of the Bacillus species studied.

Asunto(s)

RESUMEN

Bacillus sphaericus Ts-1 Mosquito larvicidal toxins 42 k Da and 43 k Da were isolated by Sephadex G-200 chromatography. Three strains of highly toxic B. sphaericus and two non toxic strains were screened for toxic proteins using ELISA. The lowest detectable toxin level was 1.56 X 10(-5) mg/ml. Non toxic strains did not produce antigens reacting to either the 42 kDa or the 43 kDa antibodies. Ts-1 cultures were examined at 12 and 24 h by LC50 bioassay against Culex pipiens. The LC50's at 12 h and 24 h were 0.71 ppm and 0.154 ppm, respectively, i.e., the toxin level at 24 h was 4.6 times the level at 12 h. ELISA tests established total toxin at 0.049 mg/ml and 0.225 mg/ml at 12 h and 24 h, respectively, confirming the LC50 study.

Asunto(s)

RESUMEN

The primary and tertiary structures of DNA-binding protein HU from Bacillus stearothermophilus are already known. The primary structure has been previously determined for HU from the closely related B. globigii and the determinations of the sequences from B. caldolyticus and B. subtilis are described here. These bacteria have optimum growth temperatures of greater than 70 degrees C (B. caldolyticus), 65 degrees C (B. stearothermophilus), 37 degrees C (B. subtilis) and 30 degrees C (B. globigii). In vitro measurements from circular dichroic spectra described here give Tm values reflecting these growth temperatures, of 68, 64, 43 and 41 degrees C respectively. We discuss here the relative thermostability of the four proteins in terms of the amino acid differences between the sequences and the three-dimensional model of the B. stearothermophilus HU. The current model for the interaction of the protein with DNA is only discussed in terms of its relevance with regard to thermostability.

Asunto(s)

RESUMEN

Teichuronopeptide is a structural component of the cell wall of alkalophilic Bacillus strain C-125 and is a complex composed of polyglutamate and polyglucuronate. A structural analysis of the polyglucuronic acid moiety was carried out. Periodate oxidation and Smith degradation of the moiety, and enzymic analysis after reduction of glucuronic acid to glucose, revealed that glucuronic acid bound together with alternately alpha- and beta-1,4-linkages.

Asunto(s)

RESUMEN

Bacillus sphaericus strain Ts-1 is highly insecticidal to larvae of the mosquito. It's insecticidal component is toxic proteins. The toxin was extracted from spore-crystal complexes by disruption in a Sonicator Cell Disruptor Model W-220F followed by treatment with 0.05 mol/L NaOH. Fraction recovered from chromatography of the spore-crystal complexes on column of Sephadex G-200 were assayed against mosquito larvae and the toxic fractions from gel chromatography were subjected to SDS-PAGE. The toxic proteins in B. sphaericus Ts-1 spore-crystal complex migrated in position corresponding to 42kD and 43kD. Bioassay of the two purified proteins prepared by PAGE indicated that they were all toxic to mosquito larvae. Toxic protein was further purified by DEAE-cellulose chromatography. The toxic protein with a molecular weight of 42kD was obtained.

Asunto(s)

RESUMEN

The primary structures of unknown cyclic peptides produced by a Bacillus strain have been determined by fast atom bombardment (FAB) mass spectrometry, which has established the peptides as a new family of the iturin group antibiotics. FAB mass spectra of the intact peptides gave the immonium ions characteristic of constituent amino acids which made it possible to distinguish Asn from Asp and Glu from Gln. Collision-induced dissociation (CID) spectra of protonated molecules provided complete information on the connectivity of amino acid residues but did not reveal the direction of peptide bonding, while those obtained for fragment ions allowed us to make a discrimination between the correct sequence and its retro sequence. The amino acid sequences derived are c(Thr-Xaa-Asn-Tyr-Asn-Ser-Glu-Ser) (Xaa: C14 or C15 beta-amino acid) which are closely related to that of bacillomycin L. The structure is confirmed by the FAB mass spectra of the partial acid hydrolysate and the peptide mixture obtained from its single-step Edman degradation. Fragmentation processes involved in the CID spectra of the cyclic peptides are discussed based on the established amino acid sequence.

Asunto(s)

RESUMEN

Although pyrolysis-mass spectrometry (Py-MS) has been used for bacterial taxonomy, many of the mass spectral peaks used for discrimination of organisms have not been correlated to known biomolecules. This work presents the discrimination of five bacterial species based on Py-MS patterns containing only peaks that can be correlated to fatty acids and fatty acid derivatives. These correlations were confirmed by pyrolysis-tandem mass spectrometry of authentic standards and the organisms. The pattern recognition procedures used gave better results when only the fatty acid peaks were used in the analysis than when full spectra were used.

Asunto(s)

RESUMEN

Protein and antigenic heterogeneity among isolates of Bacillus piliformis, the etiologic agent of Tyzzer's disease, were investigated. The seven isolates utilized in this study were originally isolated from naturally infected animals of different animal species and diverse geographical locations. Isolates were propagated in mammalian cell lines, and bacterial extracts were prepared. Protein and antigenic profiles were compared among isolates, using Coomassie blue-stained polyacrylamide gels and Western blot (immunoblot) analyses, respectively. Results showed differences in protein and antigen banding patterns, indicating diversity among isolates. Western blots probed with serum preabsorbed with a heterologous bacterial extract revealed that numerous antigens have different electrophoretic mobilities among isolates but apparently share common epitopes. Immunodominant cross-reactive antigens may be candidate proteins useful for development of improved serologic diagnostic tests, allowing identification of animals infected with a wide range of B. piliformis isolates.

Asunto(s)

RESUMEN

Bacterial MerR proteins are dimeric DNA-binding proteins that mediate the Hg(II)-dependent induction of mercury resistance operons. Site-directed mutagenesis of the Bacillus sp. RC607 MerR protein reveals that three of four Cys residues per monomer are required for Hg(II) binding at the single high-affinity binding site. Inactive mutant homodimers can exchange subunits to form heterodimers active for Hg(II) binding. Studies of a heterodimer retaining only three of eight cysteine residues per dimer reveal that Cys79 in one subunit and Cys114 and Cys123 in the second subunit are necessary and sufficient for high-affinity Hg(II) binding in an asymmetric, subunit bridging coordination complex.

Asunto(s)

RESUMEN

A procedure for obtaining gas chromatographic (GC) profiles of bacterial cellular monosaccharides was described. Some of the unknown component peaks in these profiles were identified. And, based on the complete linkage cluster analysis with the Euclidean distance coefficient, the interpretation of the resulting cellular monosaccharides of bacteria were performed by mini-computer. By means of this method, the discrimination of 5 species (24 strains) of aerobic endospore-forming bacteria. The results showed that there were defined differences between the profiles of cellular monosaccharides of B. anthracis and B. cereus. This procedure has provided a useful method for the classification and identification of microorganisms, for their physiological and biochemical studies, and for studies on their subcellular components.

Asunto(s)

RESUMEN

Many alkalophilic bacteria were found to produce inhibitors of protein kinases. We isolated a novel inhibitor of protein kinase from an alkalophilic strain of Bacillus species. This substance was A heat-stable peptide with a molecular weight of 13,000 daltons. It was found to be a selective inhibitor of cyclic AMP-dependent protein kinase (A kinase). The inhibition of a kinase by this substance was non-competitive with histone or ATP. It behaved distinctly; other known inhibitors such as H-7, H-8, Staurosporine, K-252 and Erbstatine inhibit protein kinase less selectively and their functions are competitive with either substrate or ATP. This inhibitor was found to bind to the regulatory subunits of A kinase and markedly inhibited the separation of the catalytic subunits from A kinase induced by the binding of cAMP despite of no effect on the binding of cAMP. Thus, the activation step of A kinase was influenced by this inhibitor. This molecule had no effect on the inhibition by cAMP of CHO cell proliferation. This may have been due to the inability of this molecule to reach the target in the cell. Modification of the molecule itself or the administration method is needed for cellular or animal application.

Asunto(s)

RESUMEN

The relationship between the effective dielectric constant that models the electrostatic effect from a charged side chain in a protein was evaluated both experimentally and theoretically. Experimental values were obtained from the shifts in pKa that resulted from point mutations of side chains in subtilisin. Theoretical values were obtained from an iterative solution to Poisson's equation that considers the dielectric response of the protein and the solvent together with charge positions. There is no simple relationship between the effective dielectric constant and the distance from the charge responsible for the interactions. For some charge positions a linear but not a direct proportional relationship of the effective dielectric with distance of separation was observed. Thus, simple models such as a linear distance-dependence for the dielectric response are not suitable to evaluate electrostatic effects in proteins.

Asunto(s)

RESUMEN

Obligate moderately thermophilic bacilli and obligate moderately thermoacidophilic bacilli contained spermine as the major polyamine in addition to putrescine and spermidine. The identity of spermine was confirmed by thin-layer chromatography and high-performance liquid chromatography before and after treatment with putrescine oxidase. Using these methods, thermospermine and spermine can be separated; thermospermine was not present in these organisms. On the other hand, various facultative thermophiles and mesophilic strains of the genus Bacillus, including alkalophiles and halophiles, lack spermine and other tetraamines. No spermine was detected in several strains of mesophilic or facultative slightly thermophilic lactic acid bacteria, Lactobacillus and Streptococcus.

Asunto(s)

RESUMEN

Tyzzer's disease was produced in rats by peroral inoculation with spores of Bacillus piliformis (Tyzzer's organism) of rat origin. After ingestion of 10(6) spores necrotized lesions with intracellular bacterial propagation were seen in the intestines, liver and heart on days 2 to 14 postinoculation (p.i.). A number of B. piliformis were present within enterocytes of the cecum and colon. Infected cells were also seen in the liver, myocardium and intestinal muscle layers on days 3 to 7 p.i. Infective spores were found to be shed in feces during 3 to 10 days p.i.

Asunto(s)

Asunto(s)

RESUMEN

Wild-type subtilisin and several site-specific variants were resolved on a strong cation-exchange column by isocratic elution and a series of sodium chloride concentrations. Changes in primary sequence at the protein surface have an observable effect on the chromatographic behavior of subtilisin. This supports the concept that three-dimensional structure determines which biopolymer surface residues are in position to interact with the stationary phase surface. The retention data fit the stoichiometric displacement model (SDM) of retention. Plots of ln k' vs. ln 1/[NaCl] yield values for the average number of ionic groups (Z) on the protein that interact with the support matrix. Application of the SDM to the chromatographic retention of the variants has uncovered an unusual phenomenon at the protein surface at low ionic strength. A SDM plot normally provides a linear relationship between ln k' and ln 1/[NaCl] with the slope corresponding to the Z number. This study revealed two lines differing in slope and intercept, indicating that the Z number of subtilisin changes at some intermediate ionic strength of the eluent. These results are attributed to some salt-induced protein surface event that triggers a change in structure. Chromatographic detection of this occurrence reflects the connection between the surface-mediated event and mobile phase ionic strength.

Asunto(s)

RESUMEN

Three acidic polymer fractions with molecular masses of about 16 kDa, 35 kDa and 70 kDa were isolated from lysozyme digests of N-acetylated cell walls of Bacillus polymyxa AHU 1385 by ion-exchange chromatography and gel chromatography. These fractions, containing mannosamine, glucosamine and pyruvic acid in a molar ratio of about 1:1:1 together with glycopeptide components, were characterized as polysaccharide-linked glycopeptides with one, two and more polysaccharide chains. On the other hand, treatment of the cell walls with glycine/HC1 buffer, pH 2.5, at 100 degrees C for 10 min followed by separation of water-soluble products on ion-exchange chromatography gave three polysaccharide fractions, PS-I-III, which contained different amounts of pyruvic acid (0,0.6 and 0.9 residue/mannosamine residue) along with equimolar amounts of mannosamine and glucosamine. Pyruvate-free polysaccharides similar to PS-I were also obtained from PS-II, PS-III and polysaccharide-linked glycopeptides by treatment with 10 mM HC1 at 100 degrees C for 1 h. Results of analyses of these polysaccharide preparations by 1H-NMR and 13C-NMR measurement and methylation, together with data from characterization of fragments obtained by hydrogen fluoride hydrolysis, lead to the most likely structure, ----3)[4,6-O-(1-carboxyethylidene)]ManNAc(beta 1----4)GlcNac(beta 1----, for the acidic polysaccharide of this strain.

Asunto(s)