Structural characterization of eight cyclic lipopeptides produced by Bacillus subtilis HSO121

Lipopeptides are amphiphilic compounds which contain both hydrophobic fatty acid moieties and amphiphilic peptide moieties. From the cell-free broth of Bacillus subtilis HSO121, eight cyclic lipopeptides were isolated by reversed-phase high performance liquid chromatography (RP-HPLC). The peptide part of each lipopeptide was elucidated according to electrospray ionization quadruple-time-of-flight mass spectrometry (ESI Q-TOF MS) and the fatty acid part was analyzed by electroionization gas chromatography/mass spectrometry (EI GC/MS). It showed that fractions 1-8 had molecular masses of 1007, 1021, 1021, 1035, 1035, 1035, 1063, and 1049, respectively. Analysis of hydrolyzed lipopeptides revealed that they had invariant amino acid compositions. The differences in molecular weights represent changes in the number of methylene groups and different types of branched chains in fatty acids. Peptide sequences of two of the eight lipopeptides appeared to be N-Asp-Leu-Leu-Val-Glu-Leu-Leu-C, which was different from previously reported lipopeptides. The remaining six had an identical peptide sequence of N-Glu-Leu-Leu-Val-Asp-Leu-Leu-C. The fatty acid parts were found to be mixtures of iso C(12), iso C(13), anteiso C(13), iso C(14), n C(14), iso C(15), anteiso C(15), n C(15), anteiso C(16) and anteiso C(17) beta-hydroxy fatty acids. The structure of each lipopeptide was determined to be the beta-hydroxy fatty acid bonded to the peptide chain.     

Surfactin isoforms from Bacillus coagulans

Bacillus coagulans has been found to produce several surfactins that are powerful lipopeptide surfactants. Four main components with molecular weights 1007, 1021 and 1035 Da were separated. Their structures have been confirmed by spectrometric and spectroscopic studies and by acid hydrolysis. The compounds were found to represent two pairs of surfactin isoforms in which beta-hydroxy-iso-C14 or anteiso-C15 fatty acids are linked to the [Leu7] or [Val7] heptapeptide moiety by both an amide group and a lactone bond.     

Isolation and characterization of a C12-lipopeptide produced by Bacillus subtilis HSO 121.

A new lipopeptide with C12 fatty acid has been isolated from the cell broth of Bacillus subtilis HSO121 by chromatographic methods, which is believed to be the homologue of lipopeptides. The fatty acid portion was methylated and analyzed by GC/MS, ESI Q-TOF MS and 1H-NMR. The peptide portion, of which the amino acid composition was obtained by HPLC combined with a phenyl isothiocyanate (PITC) derivatization methods, was analyzed by ESI Q-TOF MS. Comparing the obtained results with surfactin C13 showed that the new lipopeptide has a peptide moiety similar to that of surfactin and the difference exists in the fatty acid portion, which is an iso-C12 beta-hydroxy fatty acid. The critical micelle concentration (CMC) of this new homologue is estimated to be 6.27 x 10(-5) mol/l in 10 mmol/l phosphate buffer solution (PBS, pH 8.0) at 30 degrees C, and the surface tension at CMC (gamma CMC) achieved is as little as 27.71 mN/m. The hemolytic activities of the C12-lipopeptide on 2% human erythrocytes showed a HC50 of 26.5 micromol/l.     

Antitumoral and Antimicrobial Activity of Surfactin Extracted from Bacillus subtilis KLP2015

International Journal of Peptide Research and Therapeutics - Surfactin lipopeptide (SLP) from Bacillus subtilis KLP2015 (Accession number KT459335) was extracted and purified by ammonium sulphate...     

Surfactin and iturin A effects on Bacillus subtilis surface hydrophobicity

The synthesis of extracellular molecules such as biosurfactants should have major consequences on bacterial adhesion. These molecules may be adsorbed on surfaces and modify their hydrophobicities. Certain strains of Bacillus subtilis synthesize the lipopeptides, which exhibit antibiotic and surface active properties. In this study the high-performance liquid chromatography (HPLC) analysis of the culture supernatants of the seven B. subtilis strains, showed that the lipopeptide profile varied greatly according to the strain. Among the three lipopeptide types, only iturin A was produced by all B. subtilis strains. Bacterial hydrophobicity, evaluated by the water contact angle measurements and the hydrophobic interaction chromatography, varied according to the strain. Two strains (ATCC 15476 and ATCC 15811) showing extreme behaviors in term of hydrophobicity were selected to study surfactin and iturin A effects on bacterial hydrophobicity. The two lipopeptides modified the B. subtilis surface hydrophobicity. Their effects varied according to the bacterial surface hydrophobic character, the lipopeptide type and the concentration. Lipopeptide adsorption increased the hydrophobicity of the hydrophilic strain but decreased that of the hydrophobic. Comparison of lipopeptide effects on B. subtilis surface hydrophobicity showed that surfactin was more effective than iturin A for the two strains tested.     

Enhanced Production of Surfactin from Bacillus subtilis by Continuous Product Removal and Metal Cation Additions

The lipopeptide, surfactin, is produced by Bacillus subtilis. A study has been made on large-scale production of this surfactant. A good yield was obtained from a glucose substrate fermentation by continuously removing the product by foam fractionation. The surfactin could be easily recovered from the collapsed foam by acid precipitation. The yield was also improved by the addition of either iron or manganese salts. Hydrocarbon addition to the medium, which normally increases biosurfactant production, completely inhibited surfactin production by B. subtilis.     

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。     

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.     

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.     

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.     

Bacillus natto TK-1 产脂肽的结构鉴定及其诱导MCF-7细胞凋亡的研究

利用酸沉、醇提和薄层层析等方法从Bacillus natto TK-1发酵液中分离脂肽,经过HPLC、ESI MS和IR分析可知分离物是分子量为1036Da的脂肪酸链上有15个碳的环状脂肽surfactin。细胞增殖抑制实验显示surfactin在体外能抑制肿瘤细胞MCF-7的增殖,并且呈现出浓度和时间依赖关系,其中细胞被处理48 h时的IC50是38.77mg/L。通过HE染色观察和TUNEL实验发现surfactin可诱导MCF-7细胞凋亡,而且这种抑制作用呈现明显的时间依赖性。     

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.     

Surfactin from Bacillus subtilis displays anti-proliferative effect via apoptosis induction, cell cycle arrest and survival signaling suppression

The effect of surfactin on the proliferation of LoVo cells, a human colon carcinoma cell line, was examined. Surfactin strongly blocked the proliferation of LoVo cells by inducing pro-apoptotic activity and arresting the cell cycle, according to several lines of evidence on DNA fragmentation, Annexin V staining, and altered levels of poly (ADP-ribose) polymerase, caspase-3, p21(WAF1/Cip1), p53, CDK2 and cyclin E. The anti-proliferative activity of surfactin was mediated by inhibiting extracellular-related protein kinase and phosphoinositide 3-kinase/Akt activation, as assessed by phosphorylation levels. Therefore, our data suggest that surfactin may have anti-cancer properties as a result of its ability to downregulate the cell cycle and suppress its survival.     

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.     

Isolation and characterization of four plasmids from Bacillus subtilis.

Nineteen Bacillus subtilis isolates obtained from type culture collections were examined for the presence of covalently closed circular duplex deoxyribonucleic acid molecules by the technique of cesium chloride-ethidium bromide density gradient centrifugation. Four of the 19 strains tested carried covalently closed circular molecules. Two of these strains (IFO3022, IFO3215) harbored a similar plasmid with a molecular weight of 5.4 X 10(6). The other two strains (IAM1232, IAM1261) carried 4.9 C 10(6)-and 5.3 X 10(6)-dalton plasmids, respectively. These plasmid-harboring strains did not show phenotypic traits such as antibiotic resistance orbacteriocin production. The plasmid deoxyribonucleic acids were digested by three restriction endonucleases, EcoRI, HindIII, and BamNI, and were classified into three different types from their electrophoretic patterns in agarose gels.     

Purification and characterization of an arabinofuranosidase from Bacillus polymyxa expressed in Bacillus subtilis

Two polypeptides showing -l-arabinofuranosidase activity have been purified to homogeneity from culture supernatants of a Bacillus subtilis clone harbouring the xynD gene [Gosalbes et al. (1991) J Bacteriol 173: 7705–7710] from Bacillus polymyxa. Both polypeptides, with determined molecular masses of 64 kDa and 53 kDa, share the same sequence at their N termini, which also coincides with the sequence deduced for the mature protein from the previously determined sequence of nucleotides (Gosalbes et al. 1991). The two polypeptides have been biochemically characterized. Arabinose is the unique product released from arabinose-containing xylans which are substrates for both enzyme forms. Other natural arabinose-containing polysaccharides, such as arabinogalactans, are not attacked by them but some artificial arabinose derivatives are good substrates for both polypeptides. Their arabinose-releasing activity on arabinoxylans facilitates the hydrolysis of the xylan backbone by some endoxylanases from Bacillus polymyxa.