Cloning and production of Xylanase B from Paenibacillus barcinonensis in Bacillus subtilis hosts

Xylanase B from Paenibacillus barcinonensis was cloned in shuttle vectors for Escherichia coli and Bacillus subtilis, and expressed in Bacillus hosts. Several recombinant strains were constructed, among which B. subtilis MW15/pRBSPOX20 showed the highest production. This recombinant strain consists of a protease double mutant host containing P. barcinonensis xynB gene under the control of a phage SPO2 strong promoter. Maximum production was found when the strain was cultured in nutrient broth supplemented with xylans. Analysis of xylanase B location in B. subtilis MW15/pRBSPOX20 showed that the enzyme remained cell-associated in young cultures, consistent with its intracellular location in its original host, P. barcinonensis, and the lack of a signal peptide. However, when cultures reached the stationary phase, xylanase B was released to the external medium as a result of cell lysis. The amount of enzyme located in the supernatants of old cultures could account for 50% of total xylanase activity. Analysis by SDS–PAGE showed that xylanase B is an abundant protein found in the culture medium in late stationary phase cultures.     

Expression of a Bacillus subtilis pectate lyase gene in Pichia pastoris

The methylotrophic yeast Pichia pastoris is an attractive heterologous protein expression host, mainly for genes from higher eukaryotes. However, no successful examples for the expression of bacterial gene encoding pectate lyase in P. pastoris have been reported. The present study reports for the first time the cloning and functional expression of the bacterial Bacillus subtilis gene encoding alkaline pectate lyase in P. pastoris. A molecular weight of 43,644 Da was calculated from the deduced amino acid sequence. A pectate lyase activity as high as 100 U/ml was attained in the fermentation broth of P. pastoris GS 115, which was about 10 times higher than when the gene is expressed in Escherichia coli. The recombinant pectate lyase was purified to homogeneity and maximal activity of the enzyme was observed at 65 °C, and pH 9.4. The recombinant enzyme showed a wider pH and thermal stability spectrum than the purified pectate lyase from B. subtilis WSHB04-02. Pectate lyase activity slightly increased in the presence of Mg²⁺ (ion) but decreased in the presence of other metal ions. Analysis of polygalacturonic acid degradation products by electrospray ionization-mass spectrometry revealed that the degradation products were unsaturated trigalacturonic acid and unsaturated bigalacturonic acid, which confirms that the enzyme catalyzes a trans-elimination reaction.     

Purification, characterization and synergistic activity of β-1,3-glucanase and antibiotic extract from an antagonistic Bacillus subtilis NSRS 89-24 against rice blast and sheath blight

Antifungal compounds in the culture filtrate from Bacillus subtilis NSRS 89-24 that inhibited the growth of Pyricularia grisea and Rhizoctonia solani were mainly heat stable as the filter sterilized culture filtrate showed higher activity than an autoclaved one. The heat stable and labile components were due to an antibiotic and a β-1,3-glucanase, respectively. This β-1,3-glucanase was purified and characterized. Glucanase activity in the culture medium of B. subtilis NSRS 89-24 was inducible in the presence of 0.3% chitin, reaching a maximum on day 5. After purification, activity was associated with a protein of molecular mass of approximately 95.5 kDa by both gel filtration and native PAGE. Two major bands of M_r 64.6 and 32.4 kDa were revealed by SDS–PAGE. The enzyme had a K_m of 0.9 mg/ml, and V_max of 0.11 U, the optimal pH was 6.5–9.5 and was stable up to 50 °C. Both the pure enzyme and the antibiotic extract from the culture filtrate of the B. subtilis separately inhibited R. solani and P. grisea with MIC values of 12.5 and 6.25 mU/ml and 3.13 and 1.56 μg/ml, respectively. The glucanase enzyme in combination with the antibiotic showed a strong synergistic inhibitory effect on the hyphal growth of both fungi.     

基于游离质粒的木糖诱导型枯草芽孢杆菌转化体系的建立及其应用

枯草芽孢杆菌（Bacillus subtilis）作为食品级安全菌株,因其具有理化特征清晰、培养发酵方便等特点,广泛应用于异源蛋白质的高效表达以及高附加值物质的合成。传统的B. subtilis遗传转化方法存在操作流程繁琐、效率低等缺点,因此,开发方便高效的遗传转化系统具有重要意义。转录因子ComK被证实能调控B. subtilis感受态的形成,并在B. subtilis高效转化中有重要作用。构建1个含有木糖诱导启动子P_xyl调控comK表达的穿梭质粒pUBC01?P_xyl?comK的菌株B. subtilis K1,经木糖诱导条件优化后,质粒pHY300?p43?egfp的转化效率达到4.8×10³ CFU·μg^-1。此外,质粒pUBC01?P_xyl?comK可在无胁迫条件下连续培养及消除。木糖诱导感受态体系及质粒消除极大地提高了芽孢杆菌基因编辑和菌株改造的便捷性,同时增强了菌株尤其是生产菌株的性状稳定性。     

枯草芽孢杆菌Mn-SOD基因的克隆及原核表达

为了实现Mn-SOD基因在大肠杆菌(E.coli)中的可溶性表达,根据枯草芽孢杆菌(Bacillus subtilis)168sodA核酸序列设计引物,以枯草芽孢杆菌ATCC 9372基因组为模板,PCR扩增获得了Mn-SOD基因.将此基因重组至原核表达载体pET-28a,构建含Mn-SOD基因的重组表达质粒,并转化至大肠杆菌BL21(DE3).异丙基-β-D-硫代半乳糖苷(IPTG)诱导表达获得Mn-SOD,蛋白分子量约为26kD,占全菌蛋白的5.6%.改良的连苯三酚自氧化法测定SOD活力,菌体可溶性总蛋白SOD比活为51.09U·mg^-1,是对照组的.8倍.枯草芽孢杆菌ATCC 9372 Mn-SOD基因在大肠杆菌BL21(DE3)中首次成功表达,产物具有较高的可溶性和活性,为大量制备Mn-SOD奠定了基础.     

Enhanced stability of the cloned Bacillus subtilis alkaline protease gene in alginate-immobilized B. subtilis cells

Expression and stability of the cloned Bacillus subtilis alkaline protease (aprE)gene was monitored throughout the growth of free and alginate-immobilized B. subtilis cells. The time as well as the level of expression of the aprE gene in alginate-immobilized cells was found to be close to that of free cells. The multicopy plasmid that carries the aprE gene was stably maintained in alginate-immobilized cells. Plasmid stability was greatly enhanced, it reached 83% and 8% after ten growth cycles for alginate-immobilized and free cells in the absence of stress, respectively. Data presented demonstrate that immobilization of B. subtilis recombinant cells would partially solve the problem of plasmid instability in B. subtilis.     

Integrated control of Colletotrichum gloeosporioides on mango fruit in Taiwan by the combination of Bacillus subtilis and fruit bagging

Anthracnose disease caused by Colletotrichum gloeosporioides in Jingkwang mango grown in Taiwan was significantly reduced by the integration of fruit bagging with either B. subtilis strain LB5 or fungicides. The combined treatments were most effective in reducing early infection during the 2004 season, leading to 56.4 and 58.3% reduction, respectively, while in 2003 reduction accounted for 51 and 52.3%, respectively. Post-harvest application of B. subtilis strain LB5 cell suspensions on fruits already treated by bagging, bagging+LB5 and baggingfungicides in the field reduced anthracnose incidence significantly at all tested concentrations. These results indicate that biocontrol efficacy of B. subtilis LB5 may be due to the prevention of early fruit infection, thereby reducing significantly anthracnose incidence in ripening fruits to much lower levels than those obtained by using a conventional single post-harvest treatment.     

Overexpression of serine alkaline protease encoding gene in Bacillus species: performance analyses

Bacillus species carrying subC gene encoding serine alkaline protease (SAP) enzyme were developed in order to increase the yield and selectivity in the bioprocess for SAP production. For this aim, subC gene was cloned into pHV1431 Escherichia coli–Bacillus shuttle vector, and transferred into nine host Bacillus species, i.e. B. alvei, B. amyloliquefaciens, B. badius, B. cereus, B. coagulans, B. firmus, B. licheniformis, B. sphaericus and B. subtilis. The influence of the host Bacillus species on SAP production on a defined medium with glucose was investigated in bioreactor systems. For each of the recombinant (r-) Bacillus species, effects of initial glucose concentration on cell growth and SAP production were investigated; and, physiological differences and similarities between the wild-type and r-Bacillus species are discussed. The highest biomass concentration was obtained with r-B. coagulans as 3.8 kg m⁻³ at the initial glucose concentration of C_Go=20 kg m⁻³ and the highest volumetric SAP activity was obtained with r-B. amyloliquefaciens as 1650 U cm⁻³ at C_Go=20 kg m⁻³. Overall SAP activity per amount of substrate consumed was the highest for r-B. sphaericus (137 U g⁻¹ cm⁻³) and r-B. licheniformis (130 U g⁻¹ cm⁻³). Among the r-Bacillus species the highest activity increase compared to the wild types was obtained with r-B. sphaericus while the lowest increase was obtained with r-B. amyloliquefaciens and r-B. licheniformis due to high SAP production potential of the wild-type strains. During storage of the host microorganisms, r-B. alvei and r-B. amyloliquefaciens were not able to bear the recombinant plasmid, probably, due to the restriction enzymes synthesized. Due to the highest stable volumetric activities r-B. licheniformis (950 U cm⁻³) and r-B. sphaericus (820 U cm⁻³) appear to be the favorable hosts for the production of SAP. All the r-Bacillus species excreted organic acids oxaloacetic and succinic acids, but, none excreted the amino acid valine. The variations in by-product distributions with each recombinant organism were also discussed.     

Precise molecular weight determination of PCR products of the rRNA intergenic spacer region using electrospray quadrupole mass spectrometry for differentiation of B. subtilis and B. atrophaeus, closely related species of bacilli

Assessment of 16S–23S rRNA intergenic spacer region (ISR) sequence variability is an important supplement to 16S rRNA sequencing for differentiating closely related bacterial species. Species differentiation can also be achieved by determination of approximate size of PCR (polymerase chain reaction) products of ISRs, based on their relative electrophoretic mobility on agarose gels. Closely-related species can have ISR PCR products that are similar in size. More precise molecular weight (M.W.) determination of these products might allow improved discrimination of such species. Electrospray quadrupole mass spectrometry (ESI-Q-MS) has the potential to provide such precision. For ESI-Q-MS analysis, size limitation of PCR products is currently limited to around 130 base pairs (bp). Bacillus subtilis and Bacillus atrophaeus are two closely related species with few distinguishing phenotypic characteristics. B. subtilis has recently been sub-divided into two subgroups, W23 (type strain, W23) and 168 (type strain, 168). PCR products amplified from the ISR including the 5′ terminal end of the 23S rRNA and a conserved portion of the ISR were analyzed by ESI-Q-MS. A 119 or 120 bp PCR product was produced for B. atrophaeus strains. However, strains of B. subtilis subgroups W23 and 168 each produced 114 bp products. In summary, a mass spectrometry method was developed for differentiation of B. subtilis and B. atrophaeus. Also, the genetic similarity of B. subtilis subgroups W23 and 168 was confirmed. Accurate determination of the molecular weight of PCR products from the 16S–23S rRNA intergenic spacer region using electrospray quadrupole mass spectrometry has great potential as a general technique for characterizing closely related bacterial species.     

Comparison and functional characterisation of three homologous intracellular carboxylesterases of Bacillus subtilis

Enzymatic hydrolysis of racemic mixtures may provide an attractive method for the enantiopure production of chiral pharmaceuticals. For example, the carboxylesterase NP of Bacillus subtilis Thai I-8 is an excellent biocatalyst in the kinetic resolution of NSAID esters, such as naproxen and ibuprofen methyl esters. Two homologues of this enzyme were identified when the genome sequence of B. subtilis 168 was revealed in 1997. We characterised one of the homologous, YbfK, as a very enantioselective 1,2-O-isopropylidene-sn-glycerol caprylate esterase, while only modest enantioselectivity towards the naproxen ester was observed. The other homologue, the carboxylesterase NA has not been characterised yet. The purpose of the present study was to fully characterise these three highly homologous esterases with respect to their applicability towards the enantiospecific hydrolysis of a wide range of compounds. The esterase genes were cloned and expressed in B. subtilis using a combination of two strong promotors in a multi-copy vector. After purification of the enzymes from the cytoplasm of B. subtilis, the biochemical and enantioselective properties of the enzymes were determined. Although all carboxylesterases have similar physico-chemical properties, comparison of their specific activities and enantioselectivities towards several compounds revealed rather different substrate specificities. We conclude that carboxylesterase NP and carboxylesterase NA are particularly suited for the enzymatic conversion of naproxen esters, while YbfK offers enantiopure (+)-IPG from its caprylate ester. Given the carboxylesterase activities of the esterases it has been proposed to rename the nap gene of B. subtilis 168 into cesA and the ybfK gene into cesB.     

Development and morphological features of biofilms formed by transgenic and wild type strains of Bacillus subtilis

The study addressed the ability of the transgenic strain (TM) B. subtilis 2335/pBMB105 (Km^rInf⁺) to form biofilms on the surface of liquid media of various compositions, inoculated with vegetative cells and spores. The morphological features of these biofilms do not differ from those of the films formed by the recipient strain (WT) B. subtilis 2335 (Km^s). However, the TM and the natural one differ in the dynamics of biofilm formation and the cellular composition of the films. Biofilms of the TM are formed earlier, develop at a higher rate, but decompose later than the films of the WT. When the medium is inoculated with vegetative cells, sporulation in the biofilms of both strains undergoes glucose repression; no such effect is observed when the medium is inoculated with spores. The TM does not form films when the medium is inoculated with spores and supplemented with glycerin and kanamycin.     

PVA-biocatalyst with entrapped viable Bacillus subtilis cells

Bacillus subtilis cells were entrapped in polyvinyl alcohol (PVA)-cryogel beads without decay in their viability and capability of secretion of proteolytic enzymes (metalloproteinase and subtilisin). Conditions for preparation of the PVA-biocatalyst with suitable stability and viability of B. subtilis cells were optimized. Diffusion of various compounds into the cryogel (sliced beads) has been monitored on-line using image analysis system. Optimal working conditions and kinetic constants for hydrolysis of proteins catalyzed by the PVA-biocatalyst containing whole B. subtilis cells were estimated. The PVA-biocatalyst was applied in the hydrolysis of casein. The productivity of the biocatalyst (expressed as an amount of liberated aromatic amino acids) reached a maximal level of 12 mg g⁻¹ h⁻¹. Composition of mixture of peptides was dependent on pH, concentrations of Na⁺ and glucose, and in the reaction milieu. Protein hydrolysates of desired composition can be obtained using B. subtilis viable cells immobilized in PVA-gel. Incubation of the immobilized cells in a nutrient medium with casein successfully regenerated proteolytic activity of the biocatalyst.     

一株谦逊迷孔菌Daedalea modesta的分离及其杀线虫的作用

为了研究野生担子菌对线虫的作用效果,从腐木桩上采集担子菌,应用常规组织分离法获得纯培养菌丝,以全齿复活线虫作靶标,初步筛选到1株对线虫有高活性的菌株,应用形态学方法和分子生物学方法进行了种的鉴定,测定了其对全齿复活线虫Panagrellus redivivus、松材线虫Bursaphelenchus xylophilus、爪哇根结线虫Meloidogyne javanica的杀线虫活性,并测定了其在不同条件下的发酵液对全齿复活线虫、松材线虫和爪哇根结线虫二龄幼虫的作用效果。经鉴定该菌株为谦逊迷孔菌Daedalea modesta,其菌丝在平板上能够迅速杀死线虫,平板菌落上接种线虫48h,菌丝对全齿复活线虫、松材线虫和爪哇根结线虫的致死率分别为95.61%、85.75%和100%;其马铃薯葡萄糖液体发酵液25℃处理全齿复活线虫、松材线虫和爪哇根结线虫二龄虫24h线虫死亡率均达100%,发酵液稀释10×时,对爪哇根结线虫仍有致死效果。本研究为该菌株用于线虫防治的进一步研究提供了科学参考。     

Do aphids infected with entomopathogenic fungi continue to produce and respond to alarm pheromone?

The response of pea aphids, Acyrthosiphon pisum, to aphid alarm pheromone was not modified by infection with Beauveria bassiana. Approximately 50% of uninfected and infected aphids responded to synthetic alarm pheromone. The simulated attack of aphids infected with B. bassiana did not elicit a response in uninfected aphids. Preliminary air entrainment experiments of both uninfected aphids and aphids at different stages of B. bassiana (generalist pathogen) or P. neoaphidis (obligate pathogen of aphids) demonstrated that B. bassiana infected aphids produced less alarm pheromone than uninfected aphids and, conversely, P. neoaphidis infected aphids produced more alarm pheromone than uninfected aphids. These results are discussed with particular emphasis on the different life history strategies of these two pathogens. We hypothesise that the obligate, specialist pathogen, P. neoaphidis, is under greater selection pressure to increase pathogen transmission and survival resulting in modified host behaviour, than the generalist pathogen, B. bassiana.     

Characterization of signal-sequence-coding regions selected from the Bacillus subtilis chromosome

Signal-sequence-coding regions for protein export were selected from chromosomal Bacillus subtilis DNA. The number of different signals obtained was higher than expected on the basis of known exported proteins in B. subtilis.

Most of the selected regions showed the characteristics of typical signal sequences, including a basic N-terminal region followed by a hydrophobic core and a potential signal-peptidase cleavage site.

The signal-coding regions were functionally interchangeable between the β. licheniformis -amylase and Escherichia coli TEM β-lactamase genes. In addition to the signal-sequence-coding regions, the nature of the host cells, and the mature parts of the reporter proteins contributed to the amounts of protein secreted.     

Poly-γ-glutamate synthetase of Bacillus subtilis

Poly-γ-glutamate (PGA) is a most promising biodegradable polymer. In extracellular mucilage-producing Bacillus subtilis, the pgsBCA genes encode the membrane-associated PGA synthetase complex. It was recently speculated that PGA synthetase consists of both the intact 44 kDa and the in-phase overlapping 33 kDa-ywsC (corresponding to pgsB) gene products. This review covers current research into B. subtilis PGA synthetase and discusses the structural and functional features of the enzyme.     

内生枯草芽孢杆菌JL4在葡萄叶上的定殖及其对葡萄霜霉病的防治

为了明确枯草芽孢杆菌JL4在葡萄叶表面和内部的定殖情况,研究定殖与防治效果的关系,采用电击转化的方法将含有GFP基因的质粒pGFP78导入枯草芽孢杆菌JL4中,并得到成功表达GFP 的生防菌JL4-gfp,测试了标记菌株的稳定性及其对葡萄霜霉病菌的抑制作用.采用叶片喷雾法接种,用抗生素平板稀释分离回收,检测生防菌JL4-gfp在葡萄叶片的定殖情况,并将采回的叶片在室内接种葡萄霜霉菌孢子囊悬浮液进行生防测定.结果表明: 标记菌株在经过10次传代培养后,仍具有良好的发光表型,能稳定表达GFP蛋白,并且标记菌株JL4-gfp对葡萄霜霉菌保持了原有的抑菌作用;用抗生素平板稀释分离回收,检测到JL4-gfp菌株在葡萄叶片表面的定殖量在接种后的0、3和7 d分别为3.6×10⁵、2.7×10⁵和3.1×10³ CFU·g^-1;叶片内部的定殖在接种3 d后达到最大(9.6×10⁴ CFU·g^-1),然后下降,14 d后已经检测不到接种菌株;室内生防测定结果显示,喷雾后3 d对葡萄霜霉病的防治效果达88.0%以上,但7 d后则无明显防效.JL4-gfp的定殖量与其防治葡萄霜霉病的效果呈正相关,其有效定殖量临界值为10⁵ CFU·g^-1.     

Preparation and characterization of konjac glucomannan/poly(diallydimethylammonium chloride) antibacterial blend films

A novel antibacterial film was prepared by blending konjac glucomannan (KGM) and poly(diallydimethylammonium chloride) (PDADMAC) in an aqueous system. The antibacterial activity of the films against Staphylococcus aureus, Bacillus subtilis, Escherichia coli, Pseudomonas aeruginosa, and Saccharomyces were measured by the halo zone test and the double plate method. The films exhibited an excellent antibacterial activity against B. subtilis and S. aureus but not against E. coli, P. aeruginosa or Saccharomyces. The miscibility, morphology, thermal stability, water vapour permeability and mechanical properties of the blend films were investigated by density determination, SEM, ATR-IR, XRD, DSC, TGA, WVA and tensile tests. The results of density determination predicted that the blends of KGM and PDADMAC were miscible when the PDADMAC content was less than 70 wt%. Moreover, SEM and XRD confirmed the result. ATR-IR showed that strong intermolecular hydrogen bonds and electrostatic interactions occurred between KGM and PDADMAC in the blends. The tensile strength and the break elongation of the blends were improved largely to 106.5 MPa and 32.04% and the water vapour permeability decreased when the PDADMAC content was 20 wt%. The thermal stability of the blends was higher than pure KGM. The blends should be good antibacterial materials.     

典型黑土区主要树种根系构型特征及其对固土能力的影响

为量化典型黑土区主要树种根系构型特征,探究其对固土能力的影响,以该区分布较广的榆叶梅、小叶锦鸡儿、白桦、糖槭、红皮云杉、樟子松单株个体为研究对象,采用全根挖掘和WinRHIZO Pro LA2004分析系统相结合对其根系空间分布、几何形态、分形等特征进行测定,同时采用原位整株根系拉拔的方法量化根系垂直拉拔力。结果表明: 榆叶梅以倾斜根为主,小叶锦鸡儿、白桦、糖槭和红皮云杉以水平根为主,樟子松根系在水平和垂直分布上较为均衡;除白桦总根表面积和红皮云杉总根长外,灌木树种总根长、总根表面积显著大于乔木,落叶阔叶乔木总根长、总根表面积显著大于针叶常绿乔木,白桦总根体积显著大于小叶锦鸡儿、糖槭、红皮云杉和樟子松;榆叶梅、小叶锦鸡儿和白桦根系分形维数和分形丰度显著大于红皮云杉和樟子松;榆叶梅、小叶锦鸡儿和糖槭整株根系平均最大垂直拉拔力显著大于白桦、樟子松和红皮云杉。主要受根系总根长、总根表面积和倾斜根数量的影响,榆叶梅、小叶锦鸡儿和糖槭根系表现出较强的固土能力,可作为典型黑土区水土保持植被构建中优先选择的树种。