Further evidence for the structure of the teichoic acids from Bacillus stearothermophilus B65 and Bacillus subtilis var. niger WM.

Bacillus stearothermophilus B65 and Bacillus subtilis var. niger WM both contain teichoic acids in their walls composed of glycerol, phosphate and glucose. The 13C nuclear magnetic resonance spectrum of B. stearothermophilus teichoic acid showed 13C-31P coupling on the signals from the C-5 and C-6 carbon atoms of the glucose molecule and an alpha-glucosidic linkage between glucose and the C-1 atom of the glycerol moiety. These data are consistent with a poly[glucosylglycerol phosphate] as the cell-wall teichoic acid in this organism. B. subtilis var. niger WM teichoic acid was oxidized by periodate and incubated in glycine buffer at pH 10.5. This treatment did not significantly increase the phosphomonoester content (by beta-elimination of the phosphate groups) of the teichoic acid molecule (7.1 to 9.5%), which is in accordance with earlier data derived from 13C nuclear magnetic resonance spectroscopy [De Boer et al. (1976) Eur. J. Biochem. 62, 1-6], that in this organism the glucose is not an integral part of the polymer chain. Similar treatment of B. stearothermophilus B65 teichoic acid increased the phosphomonoester content of the preparation from 0.15 to 68.1%.     

Characterization of a bacteriocin produced by a newly isolated Bacillus sp. Strain 8 A

AIMS: The aim of this research was to investigate the production of bacteriocins by Bacillus spp. isolated from native soils of south of Brazil. METHODS AND RESULTS: A bacteriocin produced by the bacterium Bacillus cereus 8 A was identified. The antimicrobial activity was produced starting at the exponential growth phase, although maximum activity was at stationary growth phase. A crude bacteriocin obtained from culture supernatant fluid was inhibitory to a broad range of indicator strains, including Listeria monocytogenes, Clostridium perfringens, and several species of Bacillus. Clinically relevant bacteria such as Streptococcus bovis and Micrococcus luteus were also inhibited. Bacteriocin was stable at 80 degrees C, but the activity was lost when the temperature reached 87 degrees C. It was resistant to the proteolytic action of trypsin and papain, but sensitive to proteinase K and pronase E.Bacteriocin activity was observed in the pH range of 6.0-9.0. CONCLUSIONS: A bacteriocin produced by Bacillus cereus 8 A was characterized, presenting a broad spectrum of activity and potential for use as biopreservative in food. SIGNIFICANCE AND IMPACT OF STUDY: The identification of a bacteriocin with large activity spectrum, including pathogens and spoilage microorganisms, addresses an important aspect of food safety.     

Nitrogenase from Bacillus polymyxa. Purification and properties of the component proteins.

A purification procedure is described for the components of Bacillus polymyxa nitrogenase. The procedure requires the removal of interfering mucopolysaccharides before the two nitrogenase proteins can be purified by the methods used with other nitrogenase components. The highest specific activities obtained were 2750 nmol C2H4 formed . min-1 . mg-1 MoFe protein and 2521 nmol C2H4 formed . min-1 . mg-1 Fe protein. The MoFe protein has a molecular weight of 215 000 and contains 2 molybdenum atoms, 33 iron atoms and 21 atoms of acid-labile sulfur per protein molecule. The Fe protein contains 3.2 iron atoms and 3.6 acid-labile sulfur atoms per molecule of 55 500 molecular weight. Each Fe protein binds two ATP molecules. The EPR spectra are similar to those of other nitrogenase proteins. MgATP changes the EPR of the Fe protein from a rhombic to an axial-type signal.     

Substrate-induced deactivation of penicillinases. Studies of beta-lactamase I by hydrogen exchange.

The conformational motility of beta-lactamase I from Bacillus cereus was studied by hydrogen exhange. The time course of the isotopic replacement of peptide hydrogen atoms was followed by 'exchange-in' or 'exchange-out' experiments. Many of the substrates for this enzyme that have o-substituted aromatic or heterocyclic side chains (e.g. methicillin or cloxacillin) are known to effect a decrease in enzymic activity ('substrate-induced deactivation'). There was a marked discontinuity in the exchange-out curve when methicillin or cloxacillin was diffused into the enzyme solution. About one-half of the hydrogen atoms that were probed were affected by the presence of these substrates, and the change in the reactivity of the hydrogen atoms was also large. Substrates that do not bring about deactivation (benzylpenicillin and cephalosporin C) do not affect the hydrogen exchange, nor do reversible competitive inhibitors such as the penicilloic acid or penilloic acid. On the other hand, Zn2+ ions do affect the hydrogen exchange; their effect is similar to that of methicillin or cloxacillin.     

枯草芽孢杆菌B2菌株产生的表面活性素变异体的纯化和鉴定

利用6mol/L HCI沉淀枯草芽孢杆菌B2菌株的去细胞培养液,甲醇抽提获得脂肽类抗生素粗提物,过Sephadex LH-20层析柱获得粗纯化物,经MALDI-TOF-MS检测表明B2菌株仅含有表面活性素一种脂肽类抗生素。利用HPLC SMART SYSTEM,将粗纯化物过μPRC C2／C18层析柱对表面活性素变异体进行分离后获得纯化物。经MALDI-TOF-PSD—MS对纯化物的结构分析表明,B2菌株的表面活性素变异体由13、14和15个碳原子的脂肪酸链以及L-Glu-L-Leu—D—Leu—L-Val-L-Asp-D—Leu-L-Leu七环肽组成。     

Microbial metabolism of alkylbenzene sulphonates. Bacterial metabolism of undecylbenzene-p-sulphonate and dodecylbenzene-p-sulphonate

1. A study was made of the biodegradation of alkylbenzene sulphonate homologues, one of the major components of commercially marketed detergents. A Bacillus species was elected for growth on alkylbenzene sulphonate homologues as the sole source of carbon and sulphur. 2. The results from both whole-cell and cell-free systems indicated that the alkyl, aryl and sulphonate moieties of alkylbenzene sulphonate homologues were all further metabolized by the Bacillus species. 3. The alkyl side chain, after a presumed initial oxidation of the terminal methyl group, was subsequently oxidized by a beta-oxidation pathway. Three enzymes of the beta-oxidation pathway, i.e. acyl-CoA synthetase, acyl-CoA dehydrogenase and beta-hydroxyacyl-CoA dehydrogenase, were identified in cell-free extracts of the detergent-grown Bacillus species. The substrate specificity of acyl-CoA synthetase indicated activity towards several alkylbenzene sulphonate homologues. 4. The sulphonate moiety was released as sulphite by a desulphonating enzyme. Some kinetic properties of this enzyme were determined. The sulphite was subsequently metabolized to either sulphate or adenosine 5'-sulphatophosphate. Two enzymes involved in sulphite metabolism, i.e. sulphite-cytochrome c reductase and adenosine 5'-sulphatophosphate-cytochrome c reductase were detected in cell-free extracts of undecylbenzene-p-sulphonate-grown Bacillus species. 5. The combined results of continuous sampling programmes monitored by both t.l.c. and sulphite appearance in the growth medium indicated that desulphonation of the aromatic moiety was the likely first step in the overall biodegradation of several alkylbenzene sulphonate homologues. 6. The presence of p-hydroxyphenylpropionate, p-hydroxybenzoate and 3,4-dihydroxybenzoate in cells after growth on several alkylbenzene sulphonate homologues containing an odd number of carbon atoms in the side chain was confirmed by g.l.c. and t.l.c. analysis. Cells grown on several homologues containing an even number of carbon atoms in the side chain were shown to contain p-hydroxyphenylacetate and 3,4-dihydroxyphenylacetate. 7. The aromatic nucleus obtained from undecylbenzene-p-sulphonate was further metabolized by an oxidation sequence involving an ;ortho-cleavage' route. 8. An overall metabolic pathway for the biodegradation of various alkylbenzene sulphonate homologues by this Bacillus species is proposed.     

解淀粉芽孢杆菌NK10.BAhjaWT抑真菌作用的研究

芽孢杆菌属以多产抗菌素闻名。通过筛选几十株不同来源的芽孢杆菌, 获得1株具有强抑真菌活性的芽孢杆菌。经过16S rDNA检测与Biolog分析, 确定此株菌为解淀粉芽孢杆菌。本实验对摇瓶发酵的条件进行了优化, 经过对发酵上清液硫酸铵盐析、透析、真空冷冻干燥获得粗提蛋白。并对粗蛋白的热稳定性、pH稳定性、抗蛋白酶水解能力、离子稳定性以及抑真菌谱进行了研究, 最后使用扫描电镜对抑真菌机制进行了探索。     

解淀粉芽孢杆菌NK10.BAhjaWT抑真菌作用的研究

芽孢杆菌属以多产抗菌素闻名.通过筛选几十株不同来源的芽孢杆菌,获得1株具有强抑真菌活性的芽孢杆菌.经过16S rDNA检测与Biolog分析,确定此株菌为解淀粉芽孢杆菌.本实验对摇瓶发酵的条件进行了优化,经过对发酵上清液硫酸铵盐析、透析,真空冷冻干燥获得粗提蛋白.并对粗蛋白的热稳定性、pH稳定性、抗蛋白酶水解能力、离子稳定性以及抑真菌谱进行了研究,最后使用扫描电镜对抑真菌机制进行了探索.     

EXAFS study of zinc coordination in bacitracin A.

Bacitracin is a dodecapeptide antibiotic produced by Bacillus sp. The antibacterial activity depends upon the peptide binding a divalent metal. Hitherto, the exact coordination of the cation has not been established. In particular the role played by the sulphur and nitrogen atoms of the thiazoline ring of bacitracin A has not been clear. Here the coordination of Zn2+ by bacitracin A has been studied using extended X-ray absorption fine structure. The experimental data are consistent with a model in which zinc is coordinated by one oxygen and three nitrogen atoms with the sulphur atom of the thiazoline ring not being directly involved in the zinc coordination.     

Characterization of the superoxide dismutase of the denitrifying bacterium,Bacillus halodenitrificans

Summary Bacillus halodenitrificans produced a dimeric, manganese-containing superoxide dismutase constitutively when grown either aerobically or as a denitrifier. The molecular mass of the enzyme was determined by sedimentation equilibrium to be 41.4±3 kDa with each subunit estimated at 26 kDa. Plasma emission spectroscopy indicated the presence of 1.22 mol manganese atoms/mol holoenzyme. The electronic absorption spectrum displayed a broad band centered at approximately 474 nm (=560 mM^–1 · cm^–1) and a shoulder at 595 nm. In the ultraviolet range, the spectrum exhibited split maxima at 278 nm and 283 nm and a shoulder at 291 nm, thus resembling the spectra of superoxide dismutase fromBacillus subtilis andEscherichia coli. The amino acid composition of theB. halodenitrificans enzyme differed slightly quantitatively but little qualitatively from counterpart enzymes from other sources. Like the superoxide dismutases ofMycobacterium lepraemurium and human mitochondria, theB. halodenitrificans enzyme exhibited several cysteine residues. As expected from the capacity superoxide dismutase exhibits for protecting NO as neutrophil cytotoxicity factor, theB. halodenitrificans superoxide dismutase did not interfere with accumulation of NO produced by the organism's nitrite reductase.     

X-ray absorption spectroscopy of xanthine oxidase. The molybdenum centres of the functional and the desulpho forms.

X-ray absorption spectra have been recorded for the molybdenum K-edge region of xanthine oxidase. Both the absorption edge and the extended fine structure (e.x.a.f.s.) regions were investigated. Spectra were obtained for samples of the desulpho enzyme as well as for mixtures of this with the active enzyme. The spectrum of the pure active form was then obtained by difference. The desulpho enzyme shows a pronounced step in the absorption edge, of a type previously associated terminal oxygen ligands. In the active enzyme this step has decreased markedly. Satisfactory simulations of the e.x.a.f.s. spectrum of the desulpho enzyme could be obtained by assuming the molybdenum to be bonded to two terminal oxygen atoms (Mo = O about .175 nm), two sulphur atoms (presumably from cysteine residues, Mo-S about .0250 nm) and one sulphur atom (presumably from a methionine residue, Mo-S about 0.290 nm). E.x.a.f.s. of the active enzyme differed appreciably from this. In keeping with earlier proposals [Gutteridge, Tanner & Bray (1978) Biochem. J. 175, 887-897], the spectrum of the active enzyme could be simulated if a sulphur atom at about 0.225 nm (i.e. presumably a terminal sulphur atom) replaced one of the terminal oxygen atoms of the desulpho from, with small changes in the other bond distances. Validity of the interpretative procedures, which involved phase shift and amplitude calculations ab initio, was demonstrated by using low molecular weight compounds of known structure.     

Physicochemical characterization of the four-iron-four-sulphide ferredoxin from Bacillus stearothermophilus.

1. A stable ferredoxin was prepared from Bacillus stearothermophilus and purified by chromatography on DEAE-cellulose and by electrophoresis. 2. The minimum molecular weight determined from the amino acid composition was about 7900 and this was in reasonable agreement with a value of 8500 determined by polyacrylamide-gel electrophoresis. The ferredoxin contained four iron atoms and four labile sulphide groups per molecule. 3. The optical absorption, optical-rotatory-dispersion and circular-dichroism spectra are typical of ferredoxins containing 4Fe-4S clusters. 4. Oxidation-reduction titrations, combined with electron-paramagnetic-resonance (e.p.r.) spectroscopy, showed that the protein has a mid-point potential, at pH8, of -280 +/- 10mV, and that only one electron-accepting paramagnetic species is present. 5. The e.p.r. spectrum of the reduced ferredoxin is more readily saturated with microwave power at low temperatures than those of the eight-iron ferredoxins, indicating that there is another mechanism of electron-spin relaxation in the latter. 6. Mossbauer spectra of both redox states were observed over a range of temperatures and in magnetic fields. At high temperatures (77 degrees K and above) both redox states appear as quadrupole-split doublets; in the reduced state two resolved doublets are seen, suggesting appreciable localization of the additional reducing electron. 7. The average chemical shift indicates formal valences of two Fe3+ and two Fe2+ in the oxidized state and three Fe2+ and one Fe3+ in the reduced state. However, the spectra indicate that there are differing degrees of electron delocalization over the iron atoms. 8. At low temperatures (4.2 degrees K) the oxidized form shows no hyperfine magnetic interaction, even in an applied magnetic field, evidence that the oxidized ferredoxin is in a non-magnetic state as a result of antiferromagnetic coupling between the iron atoms. 9. At 4.2 degrees K the reduced form shows a broad asymmetric pattern resulting from magnetic hyperfine interaction. This contrasts with the reduced ferredoxin of Clostridium pasteurianum, which shows a doublet, suggesting that in the latter there may be interaction between the two 4Fe-4S centres. 10. In large applied magnetic fields, positive and negative hyperfine fields are seen in the Mossbauer spectra of the reduced ferredoxin, evidence for antiferromagnetic coupling between the iron atoms in the 4Fe-4S centre. The high-field spectra of the reduced ferredoxin of B. stearothermophilus are similar to those of the reduced ferredoxin of C. pasteurianum.     

Partial characterization of polyfermenticin SCD, a newly identified bacteriocin of Bacillus polyfermenticus

AIMS: To characterize polyfermenticin SCD, a newly identified bacteriocin of Bacillus polyfermenticus SCD. METHODS AND RESULTS: Bacillus polyfermenticus SCD was identified as a bacteriocin producer with a bactericidal activity against Bacillus subtilis IFO 12113. Polyfermenticin SCD, named tentatively as the bacteriocin produced by B. polyfermenticus SCD, showed a narrow spectrum of activity against Gram-positive and Gram-negative bacteria, a yeast and moulds. Production of polyfermenticin SCD in a 5 l jar fermenter followed typical kinetics of primary metabolite synthesis. The antibacterial activity of polyfermenticin SCD on sensitive indicator cells disappeared completely by treatment with proteinase K, which indicates its proteinaceous nature. Polyfermenticin SCD seemed to be very stable throughout the pH range of 2.0 to 9.0, and it was relatively heat labile compared with other bacteriocins. Direct detection of polyfermenticin SCD activity on SDS-PAGE suggested that it had an apparent molecular mass of about 14.3 kDa. CONCLUSIONS: Bacillus polyfermenticus SCD produced relatively heat-labile polyfermenticin SCD with a narrow spectrum of activity. SIGNIFICANCE AND IMPACT OF THE STUDY: Bacillus polyfermenticus SCD is a commercial probiotic which has been used for the treatment of long-term intestinal disorders. New findings on polyfermenticin SCD will be valuable in the evaluation of commercial probiotics. Polyfermenticin SCD can be used to control Bacillus spoilage organisms as a biological control agent.     

The catalytic metal atoms of cobalt substituted liver alcohol dehydrogenase.

The catalytic and non-catalytic Zn atom pairs of horse liver alcohol dehydrogenase (LADH) have been replaced sequentially either by ⁶⁵Zn, Co or ⁶⁵Zn and Co. The Co derivatives exhibit characteristic spectra. When Co replaces the Zn atoms which exchange secondly, enzymatic activity is altered, and both imidazole and 1,10-phenanthroline (OP) significantly modify the spectrum of the catalytic Co atoms. Further, due to the removal of cobalt, the instantaneous and reversible OP inhibition of the native enzyme becomes time-dependent and irreversible. Jointly, these data identify the pair of metal atoms of LADH which exchange secondly under the present conditions as the catalytic one. The approach described provides a basis for the differentiation of catalytic and non-catalytic metal atoms of multichain metalloenzymes.     

On the DNA binding protein II from Bacillus stearothermophilus. I. Purification, studies in solution, and crystallization

DNA binding protein II from Bacillus stearothermophilus has been purified as a single species from the nonribosomal cell fraction by a combination of gel filtration and ion exchange chromatography. The protein occurs in solution as a tetramer and is able to bind to 30 S, 50 S, and 70 S ribosomal particles. Circular dichroism studies show that the protein has approximately 45% alpha-helix. The secondary structure of the Bacillus protein is considerably more resistant to the effects of increasing temperature and urea concentration than the homologous protein (NS1 and NS2) from Escherichia coli. Proton magnetic resonance experiments show that the protein has a well folded, compact tertiary structure. The DNA binding protein has been crystallized from several precipitants as monoclinic needles and triclinic plates. The monoclinic form diffracts to at least 3.5 A and oscillation data from the native crystals have been collected. The protein is able to bind to both single- and double-stranded oligodeoxyribonucleotides. Upon binding, several changes occur in the protein NMR spectrum which may be used for further analysis of the mechanism of interaction.     

The histidine residues of phospholipase C from Bacillus cereus.

The inactivation of phospholipase C from Bacillus cereus at pH6 by diethyl pyrocarbonate parallelled the N-ethoxyformylation of a single histidine residue in the enzyme. The inactivation arose from a decrease in the maximum velocity of the enzymic reaction with no effect on the Km value. The inactivation did not apparently alter the ability of the enzyme to bind to a substrate-based affinity gel. The native enzyme contained only one reactive histidine residue. Removal of the two zinc atoms from the enzyme increased the number of reactive histidine residues to five, whereas in the totally denatured enzyme nearly eight such residues were available for reaction with diethyl pyrocarbonate. The enzyme thus appears to contain one histidine residue that is essential for catalytic activity and four that may be involved in co-ordinating the zinc atoms in the structure.     

Survival of spacecraft-associated microorganisms under simulated martian UV irradiation

Spore-forming microbes recovered from spacecraft surfaces and assembly facilities were exposed to simulated Martian UV irradiation. The effects of UVA (315 to 400 nm), UVA+B (280 to 400 nm), and the full UV spectrum (200 to 400 nm) on the survival of microorganisms were studied at UV intensities expected to strike the surfaces of Mars. Microbial species isolated from the surfaces of several spacecraft, including Mars Odyssey, X-2000 (avionics), and the International Space Station, and their assembly facilities were identified using 16S rRNA gene sequencing. Forty-three Bacillus spore lines were screened, and 19 isolates showed resistance to UVC irradiation (200 to 280 nm) after exposure to 1,000 J m(-2) of UVC irradiation at 254 nm using a low-pressure mercury lamp. Spores of Bacillus species isolated from spacecraft-associated surfaces were more resistant than a standard dosimetric strain, Bacillus subtilis 168. In addition, the exposure time required for UVA+B irradiation to reduce the viable spore numbers by 90% was 35-fold longer than the exposure time required for the full UV spectrum to do this, confirming that UVC is the primary biocidal bandwidth. Among the Bacillus species tested, spores of a Bacillus pumilus strain showed the greatest resistance to all three UV bandwidths, as well as the total spectrum. The resistance to simulated Mars UV irradiation was strain specific; B. pumilus SAFR-032 exhibited greater resistance than all other strains tested. The isolation of organisms like B. pumilus SAFR-032 and the greater survival of this organism (sixfold) than of the standard dosimetric strains should be considered when the sanitation capabilities of UV irradiation are determined.     

Purification, amino-acid sequence and some properties of the ferredoxin isolated from Bacillus acidocaldarius

Ferredoxin was isolated from the aerobic, thermophilic and acidophilic bacterium Bacillus acidocaldarius and its sequence of 78 amino acids completely determined by automated Edman degradation of the protein and of peptides derived from chemical cleavage between aspartic acid and proline and from enzymatic digestions. The optical spectrum of the oxidized protein has a broad maximum around 400 nm. The ferredoxin is thermostable: its absorbance begins to decrease only at incubation over 71 degrees C. The number of iron and inorganic sulphur atoms per molecule was determined to be 5.3 and 5.0, respectively. The calculated molar extinction coefficient was 23 000 M-1 X cm-1, the molecular mass of the apoferredoxin 8 872 Da. Contrary to all expectations, the sequence of B. acidocaldarius ferredoxin shows very little homology to that of B. stearothermophilus but closely resembles that of Thermus thermophilus.     

Tertiary structure of Bacillus thermoproteolyticus [4Fe-4S] ferredoxin. Evolutionary implications for bacterial ferredoxins

The structure of a low-potential [4Fe-4S] ferredoxin from Bacillus thermoproteolyticus has been solved using anomalous scattering data from iron atoms in the diffraction data of native crystals and refined partially to a crystallographic R-factor of 0.33, with 2.3 A (1 A = 0.1 nm) resolution data. The least-squares refinement based on the Bijvoet differences has determined that the four iron atoms in the cluster are an equal distance, approximately 2.8 A, apart. The NH ... S hydrogen bonds between polypeptide nitrogen atoms, and both cysteine and inorganic sulfur atoms, are present, as in ferrodoxin from Peptococcus aerogenes. The polypeptide chain of the B. thermoproteolyticus ferredoxin has a fold closely similar to that of 2[4Fe-4S] ferredoxin from P. aerogenes. The structural correspondence indicates strongly that both types of ferredoxin evolved from a common ancestor. The second cluster-binding region in P. aerogenes ferredoxin corresponds to the alpha-helix in B. thermoproteolyticus ferredoxin. The secondary-structure predictions strongly suggest that the alpha-helix is generally present in the monocluster-type ferredoxins. The conformational change to alpha-helix, insertions of a loop and a protrusion, as well as the absence of the second cluster in B. thermoproteolyticus ferredoxin, result in the lack of 2-fold symmetry present in P. aerogenes ferredoxin. So, the track of gene duplication is no longer detectable in the tertiary structure alone. The evolutionary events that may have occurred in the ferredoxins with the [4Fe-4S] cluster are discussed.     

Isolation of Bacillus strains from the rhizosphere of cereals and in vitro screening for antagonism against phytopathogenic, food-borne pathogenic and spoilage micro-organisms

Bacillus strains were isolated from the rhizosphere of cereals in order to be used as natural biocontrol agents (BCAs). They were screened for antagonism in vitro against various test micro-organisms. The isolates showing antagonism were identified to species level. A combination of techniques was employed for the isolation of Bacillus species. Using the direct method, only one of the 25 isolates screened showed antagonistic properties. This strain (IFS-01) was identified by means of API test strips and the ATB Plus computer programme. It proved to be Bacillus subtilis and consequently has been designated as Bacillus subtilis IFS-01. This strain produced either a broad spectrum antimicrobial compound or several compounds with different activities. The fungi and Gram-positive bacteria were more sensitive to the antagonistic isolate than the Gram-negative bacteria. A Bacillus strain producing BCAs which can be used as biopesticides or organic preservatives has been isolated and identified.