Purification and characterization of alanine carrier isolated from H-proteins of Bacillus subtilis.

Alanine transport carrier was isolated and purified from H-proteins of Bacillus subtilis. The purified carrier preparation was homogeneous in migration on polyacrylamide gels containing urea or sodium dodecyl sulfate. Electrophoresis on polyacrylamide gels containing dodecyl sulfate showed a single band of molecular weight of about 7500. 1 mol alanine was bound/mol carrier protein with a dissociation constant of 0.2 micron. The binding was inhibited by p-chloromercuribenzoate and the inhibition was reversed by dithiothreitol.     

Assay and characterization of an osmolarity inducible promoter newly isolated from Bacillus subtilis

An osmolarity-sensitive promoter fragment, P23423, isolated from Bacillus subtilis was characterized. The expression of β-galactosidase (β-Gal) driven by P23423 was regulated by osmolarity both in Escherichia coli and B. subtilis. The classical conserved region of this prokaryotic promoter was found within the sequence of the cloned fragment, and the putative promoter was identified as the control element of RNA not coding for protein (a RNA molecule that is not translated into a protein). The efficiency and benefit of this promoter was further demonstrated via osmolarity-induced expression of three other heterologous proteins in E. coli. Thus, this approach provided a simple and inexpensive inducible promoter element for the expression of cloned genes.     

Isolation, partial purification and characterization of a bacteriocin produced by a newly isolated Bacillus subtilis strain

A wild type micro-organism producing antibacterial substances has been isolated from a Chinese fermented soybean seasoning and identified as Bacillus subtilis. A crude antibacterial preparation (CABP) was obtained by ammonium sulphate precipitation. Isoelectric focusing assay revealed at least four antimicrobial components in the CABP. However, in SDS-PAGE analysis, only one peptide band displayed antimicrobial activity against pathogenic Bacillus cereus and Listeria monocytogenes. This inhibitory peptide had a molecular weight of approximately 3.4 kDa and a pI value of approximately 4.7. Results of this study suggest that at least one antimicrobial substance produced by this wild type strain of B. subtilis may be a new bacteriocin. Its sensitivity to gastric peptidases and activity against the food-borne pathogens make this bacteriocin potentially useful as an antimicrobial agent in foods.     

Production of elastase in staphylococci isolated from goats

A study was made of elastase production in 273 staphylococcal strains isolated from healthy goats by two different methods. In a soluble elastin medium, 20.2% of the strains tested showed elastolytic activity but no strains showed such activity in an insoluble elastic medium.     

Production and characterization of xylanases of a Bacillus strain isolated from soil

Xylanase production was performed by growing a Bacillus isolate on agricultural by-products, wheat straw, wheat bran, corn cobs and cotton bagasse. A maximum xylanase activity of 180 U/ml was obtained together with a cellulase activity of 0.03 U/ml on 4 (w/v) corn cobs. Electrophoretic analysis showed the presence of three endo--1, 4-xylanases having molecular weights of about 22, 23 and 40 kDa. Xylanolytic activity was stable up to 50 °C in the pH range of 4.5–10 and the highest activity was observed at 70 °C and pH 6.5.     

Production and partial characterization of dehairing protease from Bacillus cereus MCM B-326

Bacillus cereus MCM B-326, isolated from buffalo hide, produced an extracellular protease. Maximum protease production occurred (126.87+/-1.32 U ml(-1)) in starch soybean meal medium of pH 9.0, at 30 degrees C, under shake culture condition, with 2.8 x 10(8) cells ml(-1) as initial inoculum density, at 36 h. Ammonium sulphate precipitate of the enzyme was stable over a temperature range of 25-65 degrees C and pH 6-12, with maximum activity at 55 degrees C and pH 9.0. The enzyme required Ca(2+) ions for its production but not for activity and/or stability. The partially purified enzyme exhibited multiple proteases of molecular weight 45 kDa and 36 kDa. The enzyme could be effectively used to remove hair from buffalo hide indicating its potential in leather processing industry.     

Isolation and partial characterization of Bacillus subtilis mutants impaired in DNA entry

Transformation-deficient mutants of Bacillus subtilis have been identified either by screening for a nuclease-deficient phenotype on methyl green-DNA agar or for nontransformability on transforming DNA-containing agar. After purification of the mutations causing a reduction in the entry of DNA, a set of isogenic entry-deficient strains was obtained. In addition to being entry deficient to various extents, the strains usually were less capable of association with DNA than the entry-proficient parent. Likewise, the specific transforming activity in the purified mutant strains continued to be less than that in the wild type. With the possible exception of one strain, no evidence was obtained that the mutant strains were impaired in recombination. Since the breakdown of transforming DNA to acid-soluble products correlated fairly well with the residual capacity of the strains to take up DNA, nucleolytic activity is likely to be involved in the entry of DNA in B. subtilis.     

Reconstitution and partial characterization of phospholipid flippase activity from detergent extracts of the Bacillus subtilis cell membrane

In bacteria, phospholipids are synthesized on the inner leaflet of the cytoplasmic membrane and must translocate to the outer leaflet to propagate a bilayer. Transbilayer movement of phospholipids has been shown to be fast and independent of metabolic energy, and it is predicted to be facilitated by membrane proteins (flippases) since transport across protein-free membranes is negligible. However, it remains unclear as to whether proteins are required at all and, if so, whether specific proteins are needed. To determine whether bacteria contain specific proteins capable of translocating phospholipids across the cytoplasmic membrane, we reconstituted a detergent extract of Bacillus subtilis into proteoliposomes and measured import of a water-soluble phospholipid analog. We found that the proteoliposomes were capable of transporting the analog and that transport was inhibited by protease treatment. Active proteoliposome populations were also able to translocate a long-chain phospholipid, as judged by a phospholipase A(2)-based assay. Protein-free liposomes were inactive. We show that manipulation of the reconstitution mixture by prior chromatographic fractionation of the detergent extract, or by varying the protein/phospholipid ratio, results in populations of vesicles with different specific activities. Glycerol gradient analysis showed that the majority of the transport activity sedimented at approximately 4S, correlating with the presence of specific proteins. Recovery of activity in other gradient fractions was low despite the presence of a complex mixture of proteins. We conclude that bacteria contain specific proteins capable of facilitating transbilayer translocation of phospholipids. The reconstitution methodology that we describe provides the basis for purifying a facilitator of transbilayer phospholipid translocation in bacteria.     

Purification and partial characterization of A D-like fragment from human fibrinogen,produced by human leukocyte elastase

Digestion of human fibrinogen with human leukocyte elastase in the presence of Ca²⁺ yields a D-like fragment of M_r 93 000. This fragment was purified by gel filtration on Sephacryl S-200 followed by chromatofocusing. The purified fragment was partially characterized and compared with a fragment termed D-cate, which is produced by plasmin digestion of fibrinogen in the presence of Ca²⁺. The molecular weights of the constituent chains of the D-like fragment and D-cate were similar. The D-like fragment precipitated with antisera directed against D-cate, but not with antisera against fragment E. The name D-elastase for the fragment is suggested. Differences between the D-elastase and D-cate fragments were found in amino-terminal amino acids, in isoelectric point and in the expression of D antigenic determinants. Two major functional differences were demonstrated: fragment D-elastase had a much stronger anticlotting potency than D-cate and the binding of Ca²⁺ by D-elastase and D-cate differed qualitatively and quantitatively. Since it has been suggested that the calcium-binding and anticlotting properties of D-cate are related to a carboxyl-terminal 13 000 stretch of the γ-chain, the present findings for D-elastase indicate that the differences in these properties between D-cate and D-elastase are due to differences in this area of the molecule.     

Identification and characterization of the dif Site from Bacillus subtilis

Bacteria with circular chromosomes have evolved systems that ensure multimeric chromosomes, formed by homologous recombination between sister chromosomes during DNA replication, are resolved to monomers prior to cell division. The chromosome dimer resolution process in Escherichia coli is mediated by two tyrosine family site-specific recombinases, XerC and XerD, and requires septal localization of the division protein FtsK. The Xer recombinases act near the terminus of chromosome replication at a site known as dif (Ecdif). In Bacillus subtilis the RipX and CodV site-specific recombinases have been implicated in an analogous reaction. We present here genetic and biochemical evidence that a 28-bp sequence of DNA (Bsdif), lying 6 degrees counterclockwise from the B. subtilis terminus of replication (172 degrees ), is the site at which RipX and CodV catalyze site-specific recombination reactions required for normal chromosome partitioning. Bsdif in vivo recombination did not require the B. subtilis FtsK homologues, SpoIIIE and YtpT. We also show that the presence or absence of the B. subtilis SPbeta-bacteriophage, and in particular its yopP gene product, appears to strongly modulate the extent of the partitioning defects seen in codV strains and, to a lesser extent, those seen in ripX and dif strains.     

Purification,characterization and partial amino acid sequences of a novel cephalosporin-C deacetylase from Bacillus subtilis

Cephalosporin-C deacetylase [EC 3.1.1.41] was purified electrophoretically to homogeneity from the newly isolated Bacillus subtilis SHS 0133 (FERM BP-2755). The enzyme was purified about 27-fold with a yield of 9 % and a specific activity of 187.4 U/mg protein. The native enzyme (molecular weight, 280,000) was composed of eight identical subunits with apparent molecular weights of 35,000. The cephalosporin-C deacetylase was stable up to 60°C for 30 min at pH 7.0. The enzyme exhibited Michaelis-Menten kinetics with the substrates cephalosporin C, 7-aminocephalosporanic acid (7-ACA) and p-nitrophenyl acetate; the K_m values were 24.0, 7.9 and 1.0 mM, respectively. One of the reaction products from 7-ACA, deacetyl-7-ACA, was a weak non-competitive inhibitor and other product, acetate, was a weak competitive inhibitor; the K_i values were 171 and 290 mM, respectively. However, these weak product inhibitors did not prevent the completion of the deacylation of 7-ACA. The pI value of the enzyme was determined to be 5.3 using isoelectric focusing. The observed data indicate that the enzyme is different from known cephalosporin-C deacetylases. In addition, amino acid sequencing of the N-terminus and Achromobacter proteinase I-digested peptides yielded no sequences with similarities to other known proteins by a computer search.     

Purification and characterization of catalase-1 from Bacillus subtilis

The catalase activity produced in vegetative Bacillus subtilis, catalase-1, has been purified to homogeneity. The apparent native molecular weight was determined to be 395,000. Only one subunit type with a molecular weight of 65,000 was present, suggesting a hexamer structure for the enzyme. In other respects, catalase-1 was a typical catalase. Protoheme IX was identified as the heme component on the basis of the spectra of the enzyme and of the isolated hemochromogen. The ratio of protoheme/subunit was 1. The enzyme remained active over a broad pH range of 5-11 and was only slowly inactivated at 65 degrees C. It was inhibited by cyanide, azide, and various sulfhydryl compounds. The apparent Km for hydrogen peroxide was 40.1 mM. The amino acid composition was typical of other catalases in having relatively low amounts of tryptophan and cysteine.     

Purification and characterization of NADH dehydrogenase from Bacillus subtilis

NADH dehydrogenase from Bacillus subtilis W23 has been isolated from membrane vesicles solubilized with 0.1% Triton X-100 by hydrophobic interaction chromatography on an octyl-Sepharose CL-4B column. A 70-fold purification is achieved. No other components could be detected with sodium dodecyl sulphate polyacrylamide gel electrophoresis. Ferguson plots of the purified protein indicated no anomalous binding of sodium dodecyl sulphate and an accurate molecular weight of 63 000 could be determined. From the amino acid composition a polarity of 43.8% was calculated indicating that the protein is not very hydrophobic. Optical absorption spectra and acid extraction of the enzyme chromophore followed by thin-layer chromatography showed that the enzyme contains 1 molecule FAD/molecule. The enzyme was found to be specific for NADH. NADPH is oxidized at a rate which is less than 6% of the rate of NADH oxidation. The activity of the enzyme as determined by NADH:3-(4'-5'-dimethyl-thiazol-2-yl)2,4-diphenyltetrazolium bromide oxidoreduction is optimal at 37 C and pH 7.5-8.0. The purified enzyme has a Kapp for NADH of 60 microM and a V of 23.5 mumol NADH/min X mg protein. These parameters are not influenced by phospholipids. The enzyme activity is hardly or not at all affected by NADH-related compounds such as ATP, ADP, AMP, adenosine, deoxyadenosine, adenine and nicotinic amide indicating the high binding specificity of the enzyme for NADH.     

Isolation and characterization of ribosomes from Bacillus subtilis spores

Bishop, Helen L. (Syracuse University, Syracuse, N.Y.), and Roy H. Doi. Isolation and characterization of ribosomes from Bacillus subtilis spores. J. Bacteriol. 91:695-701. 1966.-The isolation of ribosomes from Bacillus subtilis spores was accomplished by freezing the spores in liquid nitrogen and grinding the spore pellet with an equal weight of levigated alumina. The ribosomes, which were adsorbed to the alumina, were freed by the addition of vegetative-cell ribosomes or bulk ribonucleic acid (RNA) to the crude alumina-ground extract. The spore ribosomes had sedimentation properties and RNA and protein compositions similar to those of vegetative-cell ribosomes. The difficulty encountered in obtaining spore ribosomes by ordinary extraction methods may be the result of nuclease and protease activities which were demonstrated in spore extracts.     

Overexpression and characterization of a lipase from Bacillus subtilis

A novel plasmid, pBSR2, was constructed by incorporating a strong lipase promoter and a terminator into the original pBD64. A mature lipase gene from Bacillus subtilis strain IFFI10210, an existing strain for lipase expression, was cloned into the plasmid pBSR2 and transformed into B. subtilis A.S.1.1655. Thus, an overexpression strain, BSL2, was obtained. The yield of lipase is about 8.6 mg protein/g of wet weight of cell mass and 100-fold higher than that in B. subtilis strain IFFI10210. The recombinant lipase was purified in a three-step procedure involving ammonium sulfate fractionation, ion exchange, and gel filtration chromatography. Characterizations of the purified enzyme revealed a molecular mass of 24 kDa in sodium dodecyl sulfate-polyacrylamide gel electrophoresis, maximum activity at 43 degrees C and pH 8.5 for hydrolysis of p-nitrophenyl caprylate. The values of Km and Vm were found to be 0.37 mM and 303 micromol mg-1 min-1, respectively. The substrate specificity study showed that p-nitrophenyl caprylate is a preference of the enzyme. The metal ions Ca2+, K+, and Mg2+ can activate the lipase, whereas Fe2+, Cu2+, and Co2+ inhibited it. The activity of the lipase can be increased about 48% by sodium taurocholate at the concentration of 7 mM and inhibited at concentrations over 10 mM.     

Fractionation and partial characterization of the products of autolysis of cell walls of Bacillus subtilis

Young, Frank E. (Western Reserve University, Cleveland, Ohio). Fractionation and partial characterization of the products of autolysis of cell walls of Bacillus subtilis. J. Bacteriol. 92:839-846. 1966.-Autolysis of the cell wall of Bacillus subtilis by an indigenous autolytic enzyme results in solubilization of 90% of the cell wall. The solubilized cell wall (supernatant fraction) was fractionated by the combination of ion-exchange chromatography on diethylaminoethyl cellulose and gel filtration on Sephadex G-25 into polysaccharides (composed of N-acyl glucosamine and N-acyl muramic acid), mucopeptides, peptides, and teichoic acid. The chemical composition of the products of autolysis confirms the proposed mechanism of autolysis and establishes the autolytic enzyme as an N-acyl muramyl-l-alanine amidase. The heteropolymers in the cell wall are linked by peptide bridges. Two peptides which account for 70% of the peptides of the cell wall have a molar ratio of 1.0:0.9:1.3 for diaminopimelic acid, glutamic acid, and alanine, respectively. Other minor peptides contain diaminopimelic acid, glutamic acid, and alanine in molar ratios of 1.0:0.9:1.5, 1.0:0.5:1.0, and 1.0:1.5:1.7, respectively. The procedures employed in this study should be applicable to the fractionation of heteropolymers in cell walls of other gram-positive organisms and thereby aid in the study of the structure of antigenic determinants and endotoxins.     

Cloning of fibrinolytic enzyme gene from Bacillus subtilis isolated from Cheonggukjang and its expression in protease-deficient Bacillus subtilis strains

Bacillus subtilis CH3-5 was isolated from cheonggukjang prepared according to traditional methods. CH3-5 secreted at least four different fibrinolytic proteases (63, 47, 29, and 20 kDa) into the culture medium. A fibrinolytic enzyme gene, aprE2, encoding a 29 kDa enzyme was cloned from the genomic DNA of CH3-5, and the DNA sequence determined. aprE2 was overexpressed in heterologous B. subtilis strains deficient in extracellular proteases using a E. coli-Bacillus shuttle vector. A 29 kDa AprE2 band was observed and AprE2 seemed to exhibit higher activities towards fibrin rather than casein.     

Production and characterization of a new cyclodextrin glycosyltransferase from Bacillus firmus isolated from Brazilian soil

A new CGTase was obtained from Bacillus firmus, strain 7B, isolated from oat soil culture, using a high alkaline pH medium containing 1% Na₂CO₃. The enzyme was characterized in soluble form, for pH 5–11, temperature from 30 to 85 °C, using a 1% maltodextrin substrate solution and appropriate buffers. It produced mainly β-CD and the cell-free supernatant had a precipitating activity measured by the trichloroethylene method that is a 100-fold greater than that of the enzyme of Bacillus firmus, strain 37, previously studied by our group. The molecular weight of the pure protein was measured as 56,230 Da with SDS-PAGE. The optimum temperature for the enzyme activity was 50 °C and it was most active at pH 6.0. Thermal deactivation was noticeable above 65 °C and the enzyme was highly stable below 60 °C. The influence of substrate or product concentration on the initial rate of CD production was studied and the kinetic parameters were determined. The enzyme showed cyclization activity on different raw and hydrolyzed starches and hydrolyzed cornstarch gave the highest activity.     

Cloning and characterization of two plasmids from Bacillus thuringiensis in Bacillus subtilis

Bacillus thuringiensis subspecies israliensis plasmids pTX14-1 and pTX14-3 were cloned and analyzed by Southern blot hybridization for their replication mechanism in Bacillus subtilis. The cloning of pTX14-1 into the replicon deficient vector pBOE335 showed the usual characteristics of single-stranded DNA plasmids, i.e., it generated circular single-stranded DNA and high molecular weight (HMW) multimers. The other plasmid, pTX14-3, behaved differently; it generated neither single-stranded DNA nor HMW multimers. Treatment with rifampicin did not result in the accumulation of single-stranded DNA. However, deletion of an EcoRI-PstI fragment resulted in the accumulation of both single-stranded DNA and HMW multimers. From various deletion derivatives, we have mapped the minus origin and the locus responsible for suppression of HMW multimer formation. Full activity of the minus origin and of the locus suppressing HMW formation was only observed on the native replicon, indicating a coupling to the plus strand synthesis.