Integration of repeated sequences (pBR322) in the Bacillus subtilis 168 chromosome without affecting the genome structure

The Escherichia coli plasmid pBR322 sequence (4363 bp) was integrated at the met, pro, or leuB locus of the Bacillus subtilis chromosome without duplication of the flanking chromosomal regions. The integrated pBR322 was stably maintained as part of the chromosome regardless of its orientation or location. It was found that a DNA segment as large as 17 kb cloned in pBR322 can be readily transferred to the B. subtilis chromosome by transformation. It was demonstrated that a second pBR322 sequence could be effectively introduced at different regions of the chromosome by sequential transformation using chromosomal DNA isolated from a strain that had already acquired a pBR322 sequence at a different locus. Similarly, a third pBR322 sequence could be introduced. By this method, two or three pBR322 sequences can be incorporated at unlinked loci without affecting the overall structure of the B. subtilis genome.     

Toward a bacterial genome technology: integration of theEscherichia coli prophage lambda genome into theBacillus subtilis 168 chromosome

A novel approach to the cloning large DNAs in theBacillus subtilis chromosome was examined. AnEscherichia coli prophage lambda DNA (48.5 kb) was assembled in the chromosome ofB. subtilis. The lambda DNA was first subcloned in four segments, having partially overlapping regions. Assembly of the complete prophage was achieved by successive transformation using three discrete DNA integration modes: overlap-elongation, Campbell-type integration, and gap-filling. In theB. subtilis chromosome, DNA was elongated, using contiguous DNA segments, via overlap-elongation. Jumping from one end of a contiguous DNA stretch to another segment was achieved by Campbell-type integration. The remaining gap was sealed by gap-filling. The incorporated lambda DNA thus assembled was stably replicated as part of the 4188 kbB. subtilis chromosome under non-selective conditions. The present method can be used to accommodate larger DNAs in theB. subtilis chromosome and possible applications of this technique are discussed.     

一株枯草芽孢杆菌原噬菌体Bsu-yong1的基因组分析

【目的】枯草芽孢杆菌（Bacillus subtilis）是在自然界中广泛存在的革兰氏阳性菌,其抗逆性极强,能抑制大多数有害菌的繁殖,是常用的产酶菌,用其生产的蛋白酶、淀粉酶占全球工业酶产量的50%。原噬菌体（prophage）整合在宿主基因组中,可为宿主提供基因和生物学功能,非常具有研究价值。以往,有关B. subtilis原噬菌体的报道主要集中于缺陷型原噬菌体（defective prophage）,本研究对一株非缺陷型活性原噬菌体（active prophage）的基因组进行解析,以扩充对非缺陷型原噬菌体的认知。【方法】使用丝裂霉素C从枯草芽孢杆菌中诱导一株噬菌体,命名为Bacillus phage Bsu-yong1（简称Bsu-yong1）。对Bsu-yong1进行负染、透射电镜（transmission electron microscopy,TEM）观察,用Illumina MiSeq测定其基因组序列、综合运用生物信息学工具对其进行基因功能注释和系统进化分析。【结果】Bsu-yong1与PBSX类缺陷型原噬菌体在形态上相似,但Bsu-yong1具有完整的噬菌体基因组,这与缺陷型原噬菌体不同,后者在包装过程中不能正确包裹自身的基因组,而是随机包裹一段宿主染色体。Bsu-yong1基因组全长为43 590 bp,G+C含量为41%,含有62个开放阅读框（open reading frame,ORF）,呈模块化分布。Bsu-yong1拥有基因编码T7SS效应器LXG多态性毒素（T7SS effector LXG polymorphic toxin）、ImmA/IrrE蛋白和SMI1/KNR4蛋白。前二者为细菌毒素（toxin）,后者为抗毒素（antitoxin）,toxin-antitoxin是细菌免疫系统重要成员,参与菌间竞争与环境适应。此前,尚未有编码LXG polymorphic toxin的基因在噬菌体中被发现和报道。在基于全基因组比对构建的蛋白谱进化树（proteomic tree）中,Bsu-yong1与噬菌体sv105、rho14、vB_BteM-A9Y聚集形成一个独立的进化支（clade）,基因组比对显示它们基因组的复制与调控模块具有高度保守性,它们共享29个核心基因（core gene）,均具有PBSX样形态特征。Bsu-yong1与其他噬菌体的进化距离较远。将Bsu-yong1与所有噬菌体进行比对,得到的成对序列比较（pairwise sequence comparison,PASC）最大值为46.72%,小于属边界值（70%）。【结论】vB_Bsu-yong1在有尾纲中代表一个新的未知的属;建议构建一个新的科（family）,该科由Bsu-yong1与噬菌体sv105、rho14、vB_BteM-A9Y组成。vB_Bsu-yong携带免疫相关基因,它可能有利于宿主在菌间竞争中获胜和适应环境。本研究丰富了噬菌体基因数据库,拓展了对芽孢杆菌活性原噬菌体的认知。     

Genetic recombination in Bacillus subtilis 168: effect of recN, recF, recH and addAB mutations on DNA repair and recombination

A recN ^– (recN1) strain of Bacillus subtilis was constructed. The effects of this and recF, recH and addAB mutations on recombination proficiency were tested. Mutations in the recN, recF recH and addAB genes, when present in an otherwise Rec⁺ B. subtilis strain, did not affect genetic exchange. Strains carrying different combinations of mutations in these genes were constructed and examined for their sensitivity to 4-nitroquinoline1-oxide (4NQO) and recombination proficiency. The recH mutation did not affect the 4NQO sensitivity of recN and recF cells and it only marginally affected that of addA addB cells. However, it reduced genetic recombination in these cells 10²- to 10⁴-fold. The addA addB mutations increased the 4NQO sensitivity of recF and recN cells, but completely blocked genetic recombination of recF cells and marginally affected recombination in recN cells. The recN mutation did not affect the recombinational capacity of recF cells. These data indicate that the recN gene product is required for, DNA repair and recombination and that the recF, recH and addAB genes provide overlapping activities that compensate for the effects of single mutants proficiency. We proposed that the recF, recH, recB and addA gene products define four different epistatic groups.     

枯草芽胞杆菌降解毒死蜱特性研究

研究生物量、pH、毒死蜱浓度和温度对枯草芽胞杆菌3374菌株(编号为GU086422)在水溶液中降解毒死蜱特性,考察该菌株对白菜上毒死蜱残留的降解特性。结果表明,在毒死蜱质量浓度为240 mg/L、pH7.0、温度30℃的适宜条件下,枯草芽胞杆菌3374菌株对毒死蜱的降解率达到92.48%。该菌株能够有效提高白菜叶面上毒死蜱残留的降解速度,表明其在白菜上具有有效降解毒死蜱的能力,在无公害农产品生产中具有广阔的应用潜力。     

Translational coupling in a penP-lacZ gene fusion in Bacillus subtilis and Escherichia coli: Use of AUA as a restart codon

Summary An out-of-frame fusion between the penicillinase gene (penP) of Bacillus licheniformis and the -galactosidase gene (lacZ) of Escherichia coli was shown to direct the synthesis of an active -galactosidase with the same electrophoretic mobility as the wild-type protein, both in B. subtilis and E. coli. This synthesis was dependent on translation of the truncated penP gene and appeared to result from translational coupling. The fusion point between penP and lacZ contained the sequence AUAG, in which the UAG and AUA codons were in-frame with the penP and lacZ reading units, respectively. N-terminal amino acid sequence analysis of the -galactosidase protein suggested that, both in B. subtilis and E. coli, reinitiation of translation occurred at the AUA codon present at the gene fusion point.     

枯草芽孢杆菌表面展示技术用于黏膜疫苗的研究进展

枯草杆菌全名枯草芽孢杆菌(Bacillus subtilis),因其优秀的益生特性及芽孢良好的抗逆性而备受研究者青睐,由于芽孢的特殊结构及独特的生理特性,是酶和免疫原等外源蛋白的理想锚定点。采用枯草杆菌进行芽孢表面展示被认为是表达高活性和高稳定性的外源蛋白的方法之一。本文主要对枯草杆菌芽孢表面展示抗原蛋白以生产黏膜疫苗的策略和应用前景进行综述。     

一株抑制产气荚膜梭菌的枯草芽孢杆菌BS-2特性

【背景】感染产气荚膜梭菌会引起动物坏死性肠炎,通常使用抗生素进行预防和治疗。随着我国饲料禁抗、养殖减抗的实施,寻找绿色微生态制剂及其代谢产物成为当前研究的热点。【目的】旨在研究前期筛选的一株抑制产气荚膜梭菌的枯草芽孢杆菌BS-2特性。【方法】检测了菌株生长曲线、代谢物质的抑菌特性及细菌素基因簇mRNA表达。【结果】枯草芽孢杆菌BS-2代谢物质对革兰氏阴性菌无抑制作用,而对革兰氏阳性菌具有较强的抑菌性能,并且对产气荚膜梭菌的抑菌性能在2-12 h内迅速增长,在12-24 h内抑菌性能较稳定;该抑菌性能不受胃蛋白酶、胰蛋白酶、蛋白酶K的影响,具有良好的热稳定性;进一步分析抑菌物质基因簇mRNA表达,发现枯草芽孢杆菌BS-2抑制产气荚膜梭菌的活性可能与表面活性素(surfactin)和美杀菌素(mersacidin)表达有关。【结论】枯草芽孢杆菌BS-2对产气荚膜梭菌具有较强的抑制作用,可能通过抑菌物质surfactin和mersacidin表达发挥作用。     

Molecular characterization of thirteen gyrA mutations conferring nalidixic acid resistance in Bacillus subtilis

We isolated 607 independent nalidixic acid-resistant mutants from Bacillus subtilis. A 163 by DNA segment from a 5 portion of the gyrA gene was amplified from the DNA of each mutant strain. After heat denaturation, the product was subjected to gel electrophoresis to detect conformational polymorphism of single-strand DNA (PCR-SSCP analysis). Mobility patterns of the two DNA strands from all the mutant strains examined differed from those of the parental wild-type strains. The patterns were classified into 13 types, and the DNA sequence of each type was determined. A unique sequence alteration was found in mutants belonging to each of the 13 types, defining 13 gyrA alleles. Eight were single base pair substitutions, four were substitutions of two consecutive base pairs, and one was a substitution of three consecutive base pairs. Only three amino acid residues (Ser-84, Ala-85, and Glu-88) were altered in the deduced amino acid sequences of the mutated genes. We conclude that molecular typing based on the PCR-SSCP method is a powerful technique for the exhaustive identification of allelic variants among mutants selected for a phenotypic trait.     

枯草芽孢杆菌(Bacillus subtilis)Tpb55灌根与烟草根围细菌多样性变化的相关性北大核心CSCD

【目的】利用454高通量测序技术分析生防菌株枯草芽孢杆菌(Bacillus subtilis)Tpb55对烟草根围土壤细菌群落的影响;同时测定施加Tpb55处理对烟草黑胫病的大田防效。【方法】试验设置Tpb55菌剂108 CFU/m L灌根和空白对照2个处理,分别在处理0、10、22 d采集烟草根围土壤,提取土壤细菌总DNA,扩增细菌16S r DNA V1-V3区,对扩增产物进行454高通量测序,使用Qiime软件分析不同处理的细菌群落结构。【结果】对照和处理样品测序后共获得了41207个优质序列,鉴定属于细菌的25个门。所有样品中优势菌群均为放线菌门(Actinobacteria)、变形菌门(Proteobacteria)和酸杆菌门(Acidobacteria)。在病情发展过程中,放线菌门的细菌丰度逐渐下降,变形菌门含量呈上升趋势。施用Tpb55后,酸杆菌门含量明显上升并高于对照。对照中芽孢杆菌科(Bacillaceae)和多样性指示菌草酸杆菌科(Oxalobacteraceae)的细菌丰度均明显下降,Tpb55处理的样品中芽孢杆菌科含量不断上升,草酸杆菌科含量相对稳定。Chao1、ACE和Shannon指数分析表明,Tpb55处理的样品中细菌多样性和丰富度不断提高且高于对照。Tpb55处理后10 d和22 d,OTU序列数据库中与Tpb55 16S r DNA V1-V3区PCR产物高度同源OTU数目分别为31和45。Tpb55处理的烟草黑胫病病情指数(5.29)显著低于对照(38.52)。【结论】施用Tpb55可以提高根围土壤细菌多样性和群落结构稳定性,这可能是其发挥良好生防作用的重要机制之一。     

碱性蛋白酶SubC在枯草芽孢杆菌中的高效异源表达

【背景】碱性蛋白酶是工业用酶中占比最大的酶类,广泛应用于清洁、食品、医疗等行业。近期研究发现碱性蛋白酶在生产生物活性肽方面有巨大潜力,这将进一步拓宽其在保健食品领域中的应用。【目的】利用枯草芽孢杆菌异源表达地衣芽孢杆菌来源的碱性蛋白酶SubC。【方法】通过筛选3种枯草芽孢杆菌宿主菌株(Bacillus subtilis 1A751、MA07、MA08)和6种信号肽(AmyE、AprE、NprE、Pel、YddT、YoqM),同时优化诱导剂浓度、发酵培养基和发酵时长,最终得到最优重组菌株MA08-AmyE-subCopt。【结果】重组菌株MA08-AmyE-subCopt的胞外酶活力为3.33×103 AU/mL,胞外蛋白分泌量为胞内可溶蛋白表达量的4倍,与携带野生型信号肽的对照组菌株WT相比,酶活提高了73.4%。【结论】异源碱性蛋白酶SubC在枯草芽孢杆菌中成功表达,为碱性蛋白酶SubC的表达和在保健食品领域的工业化应用提供了理论基础。     

菌株YN145对稻瘟病菌黑色素合成的影响及全基因组分析

【背景】枯草芽孢杆菌YN145是一株从湖南省桃江县的健康稻株中分离的细菌,前期研究中该菌对稻瘟病菌拮抗效果显著,在生物防治方面有很大的应用潜力。【目的】深入研究该菌株的生防机制并挖掘次级代谢产物基因资源。【方法】在4株稻瘟病菌生防菌中,选择其胞外抗菌物质抑制稻瘟病菌黑色素合成效果最佳的菌株YN145,采用紫外-可见分光光度计在波长400 nm处测定胞外和菌丝体内黑色素液的吸光度值,采用菌丝生长抑制平板法和分生孢子萌发抑制法测定抑菌活性。采用PacBio第三代测序和IlluminaHiSeq第二代测序相结合的技术对菌株YN145进行全基因组测序,并对测序数据进行组装,注释预测基因的功能,分析次级代谢产物合成基因簇。【结果】菌株YN145的胞外抗菌物质能较好地抑制稻瘟病菌黑色素合成、分生孢子萌发和菌丝生长。菌株YN145全基因组大小为4 167 871 bp,GC含量为43.86%,编码序列(coding sequence, CDS)数量为4 294个;共找到85个tRNA、30个rRNA和92个sRNA。同时预测到5个已知的次级代谢产物合成基因簇,分别编码合成bacillaene、bac...     

Nucleotide sequence homology of the tetracycline-resistance determinant naturally maintained in Bacillus subtilis Marburg 168 chromosome and the tetracycline-resistance gene of B. subtilis plasmid pNS1981

The nucleotide sequence (1579 bp) of tetracycline-resistance determinant and flanking regions of the cloned 5.1 kb DNA fragment from Bacillus subtilis GSY908 chromosome (Sakaguchi, R. and Shishido, K. (1988) Biochim. Biophys. Acta 949, 49–57) were determined and compared with those of the B. subtilis tetracycline-resistance plasmid pNS1981. The tetracycline-resistance structural (tet) genes of the B. subtilis GSY908 chromosome (tet BS908) and pNS1981 (tet pNS1981) were found to be highly homologous (80% identical). Both tet genes were composed of 1374 bp and 458 amino-acid residues initiating from a GTG codon preceded by a ribosome-binding site (RBS-2). Upstream from tet BS908 there exists a short open reading frame (20 amino acids) initiating from a ATG codon preceded by its own RBS (RBS-1). This leader sequence was also highly homologous to that of tet pNS1981 except for a deletion of one bp between the RBS-1 and the ATG codon.     

Industrial scale of optimization for the production of carboxymethylcellulase from rice bran by a marine bacterium, Bacillus subtilis subsp. subtilis A-53

Rice bran and yeast extract were found to be the best combination of carbon and nitrogen sources for the production of carboxymethycellulase (CMCase) by Bacillus subtilis subsp. subtlis A-53. Optimal concentrations of rice bran and yeast extract for the production of CMCase were 5.0% (w/v) and 0.10% (w/v), respectively. Optimal temperature and initial pH of medium for cell growth of B. subtilus subsp. subtilis A-53 were 35 °C and 7.3, whereas those for the production of CMCase by B. subtilus subsp. subtilis A-53 were 30 °C and 6.8. Optimal agitation speed and aeration rate in a 7 L bioreactor were 300 rpm and 1.0 vvm, respectively. The optimal agitation speed and aeration rate for the production of CMCase by B. subtilus subsp. subtilis A-53 were lower than those for cell growth. The highest productions of CMCase by B. subtilus subsp. subtilis A-53 in 7 and 100 L bioreactors were 150.3 and 196.8 U mL⁻¹, respectively.     

芽胞杆菌产生的环脂肽类物质的研究进展

环脂肽类物质具有抗菌、抗肿瘤、抗病毒等多种生物活性,其潜在的应用价值已逐渐引起了人们的注意。主要针对芽胞杆菌产生的环脂肽,综述了环脂肽类物质的结构及分类、生理活性、生物合成机制。由于环脂肽结构的不同,分离纯化及鉴定方法也会有所差异,因此对环脂肽的分离纯化及鉴定方法方面也做了简单的综述。最后展望了我国对于环脂肽研究的不足及未来的发展前景。     

Cloning and expression of the gene for neutral protease of Bacillus amyloliquefaciens in Bacillus subtilis

A Bacillus amyloliquefaciens neutral protease gene was cloned and expressed in Bacillus subtilis.The chromosomal DNA of B. amyloliquefaciens strain F was partially digested with restriction endonuclease Sau3AI, and 2 to 9 kb fragments isolated were ligated into the BamHI site of plasmid pUB110. Then, B. subtilis strain 1A289 was transformed with the hybrid plasmids by the method of protoplast transformation and kanamycin-resistant transformants were screened for the formation of large halo on a casein plate. A transformant that produced a large amount of an extracellular neutral protease harbored a plasmid, designated as pNP150, which contained a 1.7 kb insert.The secreted neutral protease of the transformant was found to be indistinguishable from that of DNA donor strain B. amyloliquefaciens by double immunodiffusion test and SDS-polyacrylamide gel electrophoresis.The amount of the neutral protease activity excreted into culture medium by the B. subtilis transformed with pNP150 was about 50-fold higher than that secreted by B. amyloliquefaciens. The production of the neutral protease in the transformant was partially repressed by addition of glucose to the medium.     

Purification and characterization of carboxymethylcellulase isolated from a marine bacterium, Bacillus subtilis subsp. subtilis A-53

The microorganism hydrolyzing carboxymethylcellulose (CMC) was isolated from seawater, identified as Bacillus subtilis subsp. subtilis by analyses of 16S rDNA and partial sequences of the gyrA gene, and named as B. subtilis subsp. subtilis A-53. The molecular weight of the purified carboxymethylcellulase (CMCase) was estimated to be about 56 kDa with the analysis of SDS-PAGE. The purified CMCase hydrolyzed carboxymethylcellulose (CMC), cellobiose, filter paper, and xylan, but not avicel, cellulose, and p-nitrophenyl-β-d-glucospyranoside (PNPG). Optimal temperature and pH for the CMCase activity were determined to be 50 °C and 6.5, respectively. More than 70% of original CMCase activity was maintained at relative low temperatures ranging from 20 to 40 °C after 24 h incubation at 50 °C. The CMCase activity was enhanced by EDTA and some metal ions in order of EDTA, K⁺, Ni²⁺, Sr²⁺, Pb²⁺, and Mn²⁺, but inhibited by Co²⁺ and Hg²⁺.