Genotypic and phenotypic differentiation of an antifungal biocontrol strain belonging to Bacillus subtilis

Physiological and molecular fingerprints of biotechnologically relevant rhizobacteria are necessary for registration, patenting, recognition and quality checking of the strains. To characterize the biological control agent, Bacillus subtilis B2g, the strain was compared with other plant-associated B. subtilis isolates. Phenotypic characterization included biochemical and nutritional properties, in vitro activity and analysis of potential antagonistic mechanisms towards several plant pathogenic fungi. According to the phenotypic characteristics, it was not possible to differentiate the biocontrol agent from the other strains, although the enzymatic fingerprint was unique. Genotypic diversity among the isolates was characterized by molecular fingerprinting methods using REP-PCR (repetitive extragenomic palindromic PCR), and macrorestriction of genomic DNA and electrophoretic separation of DNA fragments by pulsed-field gel electrophoresis (PFGE). A protocol for PFGE analysis using restriction enzyme SfiI for B. subtilis was developed. PFGE typing of B. subtilis B2g resulted in a unique fingerprint. Therefore, it was possible to differentiate B. subtilis B2g, the biocontrol agent of Phytovit, from other antifungal B. subtilis isolates.     

Features of Bacillus subtilis IMB B-7023 and its streptomycin-resistant strain

Features of phosphate-mobilizing bacteria Bacillus subtilis IMB B-7023 and its streptomycin-resistant strain were investigated. While cultivated in medium with glucose and glycerophosphate, the growth rate of the antibiotic-marked strain was approximately similar to this parameter for Bacillus subtilis IMB B-7023 but cell sizes were 1.3-fold less. Both strains significantly stimulated the germinating of plant seeds, attached to their roots, and insignificantly differed in antagonistic activity toward phytopathogens and quantitative content of cell fatty acids and phosphatase activity. Streptomycin-resistant strain may be used for monitoring of Bacillus subtilis introduced to agroecosystem.     

Unusual Biosynthesis and Structure of Locillomycins from Bacillus subtilis 916

Three families of Bacillus cyclic lipopeptides—surfactins, iturins, and fengycins—have well-recognized potential uses in biotechnology and biopharmaceutical applications. This study outlines the isolation and characterization of locillomycins, a novel family of cyclic lipopeptides produced by Bacillus subtilis 916. Elucidation of the locillomycin structure revealed several molecular features not observed in other Bacillus lipopeptides, including a unique nonapeptide sequence and macrocyclization. Locillomycins are active against bacteria and viruses. Biochemical analysis and gene deletion studies have supported the assignment of a 38-kb gene cluster as the locillomycin biosynthetic gene cluster. Interestingly, this gene cluster encodes 4 proteins (LocA, LocB, LocC, and LocD) that form a hexamodular nonribosomal peptide synthetase to biosynthesize cyclic nonapeptides. Genome analysis and the chemical structures of the end products indicated that the biosynthetic pathway exhibits two distinct features: (i) a nonlinear hexamodular assembly line, with three modules in the middle utilized twice and the first and last two modules used only once and (ii) several domains that are skipped or optionally selected.     

Identification and characterization of a novel Bacillus subtilis strain with potent antifungal activity of a flagellin-like protein

The filamentous fungus Botrytis cinerea is an important agricultural pathogen affecting a wide range of cultivated plants. Since World War II, chemical fungicides have been the go-to method for agricultural pathogen control. However, the potential adverse environmental and health effects of these chemicals have led to an increasing demand for alternative methods of pathogen control, including biological control agents. In this study, we identified a bacterial isolate with strong antagonistic activity against B. cinerea. An analysis of the 16S rRNA gene sequence for this isolate identified it as a novel strain of Bacillus subtilis. Culture media from this isolate were harvested and fractionated using ion exchange and gel filtration chromatography. The fraction exhibiting the highest level of antifungal activity was identified, and its sequence determined by electrospray tandem mass spectrometry had significant similarity to flagellin. This flagellin-like protein was exogenously expressed in Escherichia coli, and screened for antifungal activity against B. cinerea. This flagellin-like protein demonstrated clear antifungal activity of inhibiting B. cinerea growth.     

Expression, purification and characterization of the recombinant ribonuclease P protein component from Bacillus subtilis.

Ribonuclease P is a ribonucleoprotein complex that catalyzes the essential 5' maturation of all precursor tRNA molecules. The protein component both alters the conformation of the RNA component and enhances the substrate affinity and specificity. To facilitate biochemical and biophysical studies, the protein component of Bacillus subtilis ribonuclease P (RNase P) was overproduced in Escherichia coli using the native amino acid sequence with the initial 20 codons optimized for expression in E.coli . A simple purification procedure using consecutive cation exchange chromatography steps in the presence and absence of urea was developed to purify large quantities of P protein without contaminating nucleic acids. The identity of the recombinant protein as a cofactor of RNase P was established by its ability to stimulate the activity of the RNA component in low ionic strength buffer in a 1:1 stoichiometry. Circular dichroism studies indicate that P protein is a combination of alpha-helix and beta-sheet secondary structures and is quite stable, with a T m of 67 degrees C. The described methods facilitated the large scale purification of homogeneous, RNA-free P protein required for high resolution crystallographic analyses and may be useful for the preparation of other RNA binding proteins.     

枯草芽孢杆菌抗菌蛋白X98Ⅲ的纯化与性质

枯草芽孢杆菌（Bacillussubtilis）BS－98是一株能强烈抑制苹果轮纹病菌（Physalosporapiricola）等植物病原真菌的拮抗菌株。BS－98菌株培养液经硫酸铵分级盐析、SephadexG－100柱层析和DEAE－纤维素（DE32）柱层析后分离纯化出一种抗菌蛋白,命名为X98Ⅲ。蛋白电泳分析结果表明,此蛋白分子量为59000,等电点为4．50．醋酸纤维膜电泳后经特异染色证明X98Ⅲ含糖及胀。用DNS法测其含糖量为6％。此蛋白对热稳定,对蛋白酶部分敏感。氨基酸组分分析表明,该蛋白含11种不同氨基酸,尤富含谷氨酸和半胱氨酸等,而缺少天冬氨酸等。纯化后的X98Ⅲ对苹果轮纹病菌、芦笋茎枯病菌等有很强的抑制作用。X98Ⅲ的抑菌机理主要是溶解细胞壁,造成菌丝畸形、孢子不发芽或发芽异常。     

Absence of penicillin-binding protein 4 from an apparently normal strain of Bacillus subtilis.

The phenotype of a Bacillus subtilis 168 strain with no detectable penicillin-binding protein 4 was examined. Despite the fact that penicillin-binding protein 4 is one of the most penicillin-sensitive proteins in the species, its apparent loss had no obvious effect on the organism or its susceptibility to various beta-lactam antibiotics.     

水稻内生枯草芽孢杆菌G87抗菌蛋白的分离纯化及理化特性

【目的】为得到枯草芽孢杆菌(Bacillus subtilis)G87的抗菌蛋白,明确其蛋白理化特性。【方法】采用硫酸铵沉淀和柱层析法进行分离纯化。【结果】获得单一抗菌活性蛋白(峰6-2-1),此抗菌蛋白分子量为50.8 kDa,等电点为5.90。经初步分析,抗菌蛋白不含脂,而含有少量(0.62%)糖;其蛋白部分具有脯氨酸或羟脯氨酸,但不含芳香族氨基酸。抗菌蛋白在高温(≥60℃)和较碱(pH8)环境下活性明显下降,但较抗紫外线、氯仿和胰蛋白酶、蛋白酶K、胃蛋白酶。【结论】枯草芽孢杆菌G87的抗菌蛋白为不含芳烃的糖蛋白,对高温和碱性条件敏感,而对蛋白酶类和紫外线等不敏感。     

生防菌Bs-916 合成脂肽类化合物Bac操纵子突变株构建及功能

摘要：生防枯草芽孢杆菌Bs-916对水稻纹枯病有很好的防治效果。【目的】明确其合成脂肽类化合物操纵子Bac功能,为遗传改良提高Bs-916防效奠定基础。【方法】采用同源重组方法成功更换Bac内源启动子为组成性表达强启动子和插入失活Bac启动子,分别得到突变株BGG104和BGG105。突变株生物学活性测定结果表明：BGG104上清液相对Bs-916上清液对几种病原真菌毒力,血红细胞的溶血能力都显著提高;而BGG105则显著降低。采用6 mol/L盐酸沉淀及甲醇抽提方法从Bs-916和突变株培养液中制备脂肽     

Transfer of transposon Tn916 from Bacillus subtilis to Thermus aquaticus

Broad host range conjugating transposon Tn916 has been introduced into the extreme thermophile Thermus by transposon transformation and transposition into the Bacillus subtilis chromosome followed by broth mating with Thermus aquaticus ATCC27634. Tetracycline resistant Thermus transconjugants were obtained at a frequency of 1.4 X 10(-7) per donor and 1.2 X 10(-7) per recipient. Transposon transfer from Thermus to Bacillus subtilis was also demonstrated in similar broth matings. Transfer characteristics were consistent with the conjugation mechanism described for Tn916 in mesophiles.     

Dynamics of backbone conformational heterogeneity in Bacillus subtilis ribonuclease P protein

Interconversion of protein conformations is imperative to function, as evidenced by conformational changes associated with enzyme catalytic cycles, ligand binding and post-translational modifications. In this study, we used 15N NMR relaxation experiments to probe the fast (i.e., ps-ns) and slow (i.e., micros-ms) conformational dynamics of Bacillus subtilis ribonuclease P protein (P protein) in its folded state, bound to two sulfate anions. Using the Lipari-Szabo mapping method [Andrec, M., Montelione, G. T., and Levy, R. M. (2000) J. Biomol. NMR 18, 83-100] to interpret the data, we find evidence for P protein dynamics on the mus-ms time scale in the ensemble. The residues that exhibit these slow internal motions are found in regions that have been previously identified as part of the P protein-P RNA interface. These results suggest that structural flexibility within the P protein ensemble may be important for proper RNase P holoenzyme assembly and/or catalysis.     

Calmodulin-like protein from Bacillus subtilis

The first example of a calmodulin-like activity in a Gram-positive bacterium, Bacillus subtilis, is reported. A calcium ion-dependent, 3', 5' cyclic-AMP phosphodiesterase-stimulating activity was found in the soluble fraction of cell-free extracts of cells sporulating in a chemically-defined medium; activation was reversed by trifluoperazine. The activity was heat stable, bound to phenothiazine-agarose in a calcium ion-dependent manner and was eluted therefrom with buffer containing EGTA, and displaced authentic beef brain calmodulin from its antibody in a radioimmunoassay.     

Homology modeling of an antifungal metabolite plipastatin synthase from the Bacillus subtilis 168

Lipopeptides have a widespread role in different pathways of Bacillus subtilis; they can act as antagonists, spreader and immunostimulators. Plipastatin, an antifungal antibiotic, is one of the most important lipopeptide nonribosomly produced by Bacillus subtilis. Plipastatin has strong fungitoxic activity and involve in inhibition of phospholipase A2 and biofilm formation. For better understanding of the molecule and pathway by which lipopeptide plipastatin is synthesized, we present a computationally predicted structure of plipastatin using homology modeling. Primary and secondary structure analysis suggested that ppsD is a hydrophilic protein containing a significant proportion of alpha helices, and subcellular localization predictions suggested it is a cytoplasmic protein. The tertiary structure of protein (plipastatin synthase subunit D) was predicted by homology modeling. The results suggest a flexible structure which is also an important characteristic of active enzymes enabling them to bind various cofactors and substrates for proper functioning. Validation of 3D structure was done using Ramachandran plot ProsA-web and QMEAN score.This predicted information will help in better understanding of mechanisms underlying plipastatin synthase subunit D synthesis. Plipastatin can be used as an inhibitor of various fungal diseases in plants.     

Purification and characterization of the spoOA protein of Bacillus subtilis from an overproducing strain of Escherichia coli

The spoOA gene of Bacillus subtilis is essential for the earliest stage of sporulation. To purify and characterize the product of the spoOA gene, we constructed a fusion plasmid in which the spoOA coding region was placed under the control of the Ptac promoter. When expression of the spoOA gene was induced in Escherichia coli cells by derepression of Ptac, the SpoOA protein constituted 15% of total cellular protein. The SpoOA protein was purified to homogeneity from these cells. We found that the NH2-terminal amino acid sequence of the purified protein was essentially the same as that of the SpoOA protein (spoOA-cat protein) from B. subtilis, and that the NH2-terminal methionine of the SpoOA protein from E. coli was formylated presumably because of insufficient amounts of the deformylating enzyme. The T signal [Ganoza, M. C., Marliere, P., Kofoid, E. C. and Louis, B. G. (1985) Proc. Natl Acad. Sci. USA 82, 4587-4591], in addition to the Shine-Dalgarno signal to determine the initiation codon of the spoOA gene, is considered to function in E. coli as well as in B. subtilis. We also found that the purified SpoOA protein had a DNA-binding activity. It was preferentially bound to the 175-bp BclI fragment of phi 105 DNA, and was released in the presence of 0.3 M KCl.     

Expression of secreted guanyl-specific ribonuclease genes from Bacillus intermedius and Bacillus pumilus in Bacillus subtilis cells

Plasmids with whole genes for ribonucleases from B. intermedius (binase) and B. pumilis (RNase Bp) assembled with the whole gene of barstar, a specific intracellular inhibitor, are constructed. The resultant plasmids pMZ55 and pMZ56 effectively express binase and RNase Bp genes in B. subtilis cells. A medium for maximum expression of RNase genes by recombinant strains is developed. The expression of binase and RNase Bp genes in B. subtilis cells is negatively regulated by exogenic inorganic phosphate.     

Serine-threonine protein phosphatases from Bacillus subtilis

Gram-positive soil bacterium Bacillus subtilis possesses six eukaryotic-like serine-threonine protein phosphatases. These enzymes play an important role in the cell. The response to environmental or nutrional stress conditions are controlled by three Rsb phosphatases: RsbX, RsbU and RsbP. Phosphatases are also involved in endospore formation process (SpoIIE) and sugar transport (kinase/phosphatase Hpr). Moreover in the cell there are phosphatases with still unknown function (PrpC and PrpE). Cellular processes, presented here are regulated by serine/threonine protein phosphatases and very important for bacterial survival in natural environment. Protein phosphatases must act in cooperation with protein kinases and deserve the same attention as kinases.