Biological control against bacterial wilt and colonization of mulberry by an endophytic Bacillus subtilis strain

Forty-five bacterial isolates were collected from surface-sterilized leaves of mulberry ( Morus alba L.). By screening their antagonistic activities against Ralstonia solanacearum in vitro , four isolates showed a remarkable inhibitory effect. The evaluation of the antagonistic strains against bacterial wilt of mulberry indicated that the strain Lu144 effectively reduced disease incidence. In the greenhouse, Lu144 displayed effective biological control against bacterial wilt of mulberry when it was applied to sterile or nonsterile soil before the infection by the pathogen. Based on bacteriological properties and 16S rRNA gene sequencing, Lu144 was identified as a strain of Bacillus subtilis . The endophytic population and infection process of Lu144 in mulberry seedlings was explored following recovery of the green fluorescent protein (GFP)-labeled Lu144 and examination of the labeled strain by confocal laser scanning microscopy. Interestingly, the infection of GFP-labeled Lu144 cells into the mulberry seedlings occurred through the cracks formed at the lateral root junctions and the zone of differentiation and elongation, and the cells were able to develop and transfer in mulberry and mainly in the intercellular spaces of different tissues. The population of the GFP-labeled Lu144 inoculant was larger and more stable in leaves than that in roots and stems.     

Characterization of an antimicrobial material from a newly isolated Bacillus amyloliquefaciens from mangrove for biocontrol of Capsicum bacterial wilt

Understanding the mechanisms of the antagonistic endophytic bacteria is helpful in controlling plant diseases. An endophytic bacterium, Bg-C31, from mangrove was found to be antagonistic to some fungal and bacterial pathogens of plants and to be effective in the biocontrol of Capsicum bacterial wilt in pot and field trials. Bg-C31 was identified as Bacillus amyloliquefaciens by biochemical and physiological tests as well as sequences of 16S rDNA and the LCI gene. The antimicrobial substance produced by Bg-C31 was identified as a protein, which is resistant to protease k and heat, by ammonium sulfate precipitation and butanol extraction. The antagonistic gene was located in the chromosome by plasmid curing. A 29 kDa fusion protein of the LCI gene was expressed. Antimicrobial activity of the fusion protein to Ralstonia solanacearum was detected on gels in situ, indicating that the LCI gene could potentially be used to produce transgenic plants that are resistant to bacterial infection.     

H2, a temperate bacteriophage isolated from Bacillus amyloliquefaciens strain H

Bacillus amyloliquefaciens strain H is lysogenic for a large temperate phage we call H2. H2 has a polyhedral head 85 nm in diameter and a tail of about 17 x 434 nm. H2 lysogenizes Bacillus subtilis between the tyrA and metB genes, and gives specialized transduction of metB and, at lower frequencies, of ilvD and ilvA. The phage carries a thymidylate synthase gene and converts thymine auxotrophs of B. subtilis to prototrophy. The H2 genome is a linear DNA molecule about 129 kb in length. DNA extracted from phage particles grown in B. subtilis is not cut by the restriction endonucleases HaeIII, Fnu4HI, Bsp1286I, and BamHI; the latter enzyme is produced by B. amyloliquefaciens strain H. The prophage in lysogenic B. subtilis cells can be cut by these enzymes. We have isolated H2 mutants that carry the transposon Tn917, or a mutation resulting in clear-plaque morphology, or both.     

Characterization of a novel PepF-like oligopeptidase secreted by Bacillus amyloliquefaciens 23-7A

An oligopeptidase from Bacillus amyloliquefaciens 23-7A was characterized along with its biochemical activities and structural gene. The protein's amino acid sequence and enzymatic activities were similar to those of other bacterial PepFs, which belong to metallopeptidase family M3. While most bacterial PepFs are cytoplasmic endopeptidases, the identified PepFBa oligopeptidase is a secreted protein and may facilitate the process of sporulation.     

Production of a thermostable uricase by a novel Bacillus thermocatenulatus strain

A novel uricase-producing bacterium was identified based on its 16S rRNA sequence as Bacillus thermocatenulatus. The kinetic constants for this uricase, determined with uric acid as the substrate, were a V(max) of 0.99U/ml of enzyme and a K(m) of 0.25mM. After heat treatment at 75 degrees C for 45min, the uricase retained about 100% of its initial activity. The uric acid showed to be an inducer for uricase production. The effects of different factors on the enzyme production were studied. Pretreated cane molasses and corn steep liquor were the most promising carbon and nitrogen sources, respectively. When the strain was cultured at 30 degrees C at pH 7.0 for 30-36h, the uricase activity peaked at 1.25U/ml.     

Calcium-dependent catalytic activity of a novel phytase from Bacillus amyloliquefaciens DS11

The thermostable phytase from Bacillus amyloliquefaciens DS11 hydrolyzes phytate (myo-inositol hexakisphosphate, IP6) to less phosphorylated myo-inositol phosphates in the presence of Ca2+. In this report, we discuss the unique Ca2+-dependent catalytic properties of the phytase and its specific substrate requirement. Initial rate kinetic studies of the phytase indicate that the enzyme activity follows a rapid equilibrium ordered mechanism in which binding of Ca2+ to the active site is necessary for the essential activation of the enzyme. Ca2+ turned out to be also required for the substrate because the phytase is only able to hydrolyze the calcium-phytate complex. In fact, both an excess amount of free Ca2+ and an excess of free phytate, which is not complexed with each other, can act as competitive inhibitors. The Ca2+-dependent catalytic activity of the enzyme was further confirmed, and the critical amino acid residues for the binding of Ca2+ and substrate were identified by site-specific mutagenesis studies. Isothermal titration calorimetry (ITC) was used to understand if the decreased enzymatic activity was related to poor Ca2+ binding. The pH dependence of the Vmax and Vmax/Km consistently supported these observations by demonstrating that the enzyme activity is dependent on the ionization of amino acid residues that are important for the binding of Ca2+ and the substrate. The Ca2+-dependent activation of enzyme and substrate was found to be different from other histidine acid phytases that hydrolyze metal-free phytate.     

解淀粉芽孢杆菌SWB16菌株脂肽类代谢产物对球孢白僵菌的拮抗作用

【目的】筛选对球孢白僵菌(Beauveria bassiana)具有较强拮抗作用的细菌菌株及检测菌株脂肽类代谢产物的拮抗活性。【方法】通过形态学观察、生理生化实验、16S rRNA和gyrA基因序列分析鉴定目标菌株;用滤纸片扩撒法(K-B法)测定抑菌圈的直径;采用甲醇萃取菌株发酵液以提取脂肽类代谢产物,并显微观察提取物对白僵菌分生孢子及菌丝的拮抗作用;高效液相色谱-质谱联用方法及靶基因克隆技术检测菌株脂肽类代谢产物的主要成分和基因。【结果】从植物盾叶薯蓣(Dioscorea zingiberensis C.H.Wright)组织内分离得到了一株对球孢白僵菌具有较强拮抗活性的菌株SWB16,该菌株属于解淀粉芽孢杆菌(Bacillus amyloliquefaciens),其脂肽类提取物对球孢白僵菌分生孢子的发芽和菌丝生长均具有明显的抑制作用,质谱检测表明提取物的主要成分是芬枯草菌素和伊枯草菌素,从菌株基因组中克隆到编码芬枯草菌素和伊枯草菌素的fenB基因和ituA基因。【结论】解淀粉芽孢杆菌(B.amyloliquefaciens)SWB16菌株能产生脂肽类抗生素并对球孢白僵菌具有拮抗作用,拮抗活性显示该菌株对防治家蚕等经济昆虫的白僵病具有潜在的应用价值。     

一株拮抗姜瘟青枯假单胞杆菌的链霉菌的分离及鉴定

从生姜田土中分离到一株对姜瘟青枯假单胞杆菌(PseudomonassolanacarumSmith)有强拮抗作用的链霉菌菌株SR 11,研究拮抗性表明,对革兰氏阳性细菌、革兰氏阴性细菌以及多种病原真菌均有很强的抑制作用。对该菌株进行形态特征、培养特征、生理生化、细胞壁组分分析及16SrDNA序列分析。基内菌丝无横隔、不断裂,气生菌丝多分枝;孢子丝波曲至螺旋形,孢子椭圆形,表面光滑。细胞壁化学组分Ⅰ型,糖型C。在培养成熟后,气丝变为灰色,可闻到浓烈的土味。以16SrDNA序列为基础构建了包括13株相关种属细菌在内的系统发育树,其中,与12个模式链霉菌株的16SrDNA序列的同源性为96 5 %～98 3%。     

Biotransformation of eugenol to vanillin by a novel strain Bacillus safensis SMS1003

Due to the extensive applications of vanillin as flavored compound and increasing consumers concern for its natural and environment friendly mode of production, present work was focused on the selection of bacterial isolate capable of producing vanillin using eugenol biotransformation. Bacterial strain SMS1003 is evidenced as the potential strain for vanillin production and identified as Bacillus safensis (GeneBank accession no. MG561863) using biochemical tests and molecular phylogenic analysis of its 16S rDNA gene sequence. Molar yield of vanillin reached up to 10.7% (0.055?g/L) at 96?h of biotransformation using growing culture of B. safensis SMS1003 in following culture conditions: eugenol concentration 500?mg/L; temperature 37?°C; initial pH 7.0; inoculum volume 4%; volume of culture media 10%; and shaking speed 180?rpm. Vanillin was detected as the single metabolite with a molar yield of 26% (0.12?g/L) at 96?h using resting cells of B. safensis SMS1003. Product confirmation was based on spectral scan using photodiode array detector, Fourier-transform infrared spectroscopy, high-performance liquid chromatography, and mass spectroscopy.     

Bacillus subtilis secretes a foreign protein by the signal sequence of Bacillus amyloliquefaciens neutral protease

Summary We constructed a secretion plasmid in which a truncated penicillinase gene of Bacillus licheniformis was introduced at the end of the signal peptide coding region of a Bacillus amyloliquefaciens neutral protease gene. A Bacillus subtilis recombinant secreted about 140 mg/liter of the penicillinase into the medium. Analysis of the purified product revealed that it was a mixture of two penicillinases containing one or two additional amino acids at the NH₂-terminus of B. licheniformis exo-small penicillinase.     

A process for extracellular thermostable lipase production by a novel Bacillus thermoamylovorans strain

A lipolytic enzyme-producing thermophilic microorganism, recently isolated from a hot spring in Galicia (North Western Spain), has been investigated. First, the strain was genetically identified and tentatively named Bacillus thermoamylovorans CH6B. It produced significant levels (around 450 U/L) of extracellular lipolytic activity in shake flask cultures, and the most suitable conditions for this biological process were found at temperatures between 50 and 55 °C, and an initial pH value around 7.0. Next, a preliminary scaling up of the process was carried out in a 5-L stirred tank bioreactor, and it was concluded that operation at agitation and aeration rates of 300 rpm and 0.33 vvm, respectively, were advisable. In both type of cultures, the results were successfully fitted to logistic equations, and the relationship between lipase production and cell growth was investigated. Furthermore, some relevant properties of the crude lipolytic enzyme extracts were assessed. The crude biocatalyst preferentially hydrolysed p-nitrophenyl esters of medium and long-chain fatty acids. Thermal stability in aqueous solution of the produced enzyme was also promising, and the deactivation profiles were fitted to a series-type deactivation model.     

Studies of plant colonisation by closely related Bacillus amyloliquefaciens biocontrol agents using strain specific quantitative PCR assays

Certain strains of Bacillus amyloliquefaciens can colonize plants and improve growth and stress management. In order to study these effects, bacterial growth dynamics on plants and in the rhizosphere are of interest calling for specific analytical tools. For that purpose, quantitative real-time PCR (qPCR) assays were developed in order to differentiate among three closely related B. amyloliquefaciens subsp. plantarum strains (UCMB5033, UCMB5036, UCMB5113) and to determine their levels with high accuracy. Oligonucleotide primers were designed for strain unique gene sequences and used for SYBR green based qPCR analysis. Standard curves covered a wide linear range (10⁶) of DNA amounts with the lowest detection level at 50 fg. Post-reaction melting curve analysis showed only a single product. Accurate threshold cycles were obtained, even in the presence of high excess of related Bacillus strains and total bacterial DNA from soil. Analysis of Bacillus colonisation after seed treatment of two oilseed rape cultivars (Oase and Ritz) grown on agar support showed a time dependent effect but that the bacteria mostly were found on root tissues and little on green tissues. The colonisation on plants grown in soil varied among the Bacillus strains where Oase seemed to house more bacteria than Ritz. Applied as a mixture, all three Bacillus strains co-existed on the roots of plants grown in soil. The qPCR assay in combination with other techniques will be a powerful tool to study plant interactions of these B. amyloliquefaciens biocontrol agents to further understand the requirements for successful interactions and improvement of plant properties.     

Production, purification and characterization of beta-1,4-endoglucanase from a novel bacterial strain CTP-09 of a Bacillus sp

Bacillus strain CTP-09 yielded maximum productivity (1120 IU/L.h) of extracellular endoglucanase (CMCase) on 0.5% cellobiose after 10 h fermentation at 55 degrees C. The purified enzyme is mono-meric in nature and exhibits stability up to 80 degrees C and over a pH range (6.0-9.0). Activation energy, enthalpy and entropy of catalysis, and inactivation indicated that this CMCase is highly thermos-table. Purified enzyme possessed high power of defibrillation of textile and was minutely inhibited by anionic detergent and oxidizing agent comparable with inhibition by commercial enzyme. This polypeptide could be exploited for mass production and application in local industries.