Construction and Application of Epitope- and Green Fluorescent Protein-Tagging Integration Vectors for Bacillus subtilis

Here we describe the construction and application of six new tagging vectors allowing the fusion of two different types of tagging sequences, epitope and localization tags, to any Bacillus subtilis protein. These vectors are based on the backbone of pMUTIN2 and replace the lacZ gene with tagging sequences. Fusion of the tagging sequences occurs by PCR amplification of the 3′ terminal part of the gene of interest (about 300 bp), insertion into the tagging vector in such a way that a fusion protein will be synthesized upon integration of the whole vector via homologous recombination with the chromosomal gene. Three of these tagging sequences (FLAG, hemagglutinin, and c-Myc) allow the covalent addition of a short epitope tag and thereby detection of the fusion proteins in immunoblots, while three other tags (green fluorescent protein⁺, yellow fluorescent protein, and cyan fluorescent protein) are helpful in assigning proteins within one of the compartments of the cell. The versatility of these vectors was demonstrated by fusing these tags to the cytoplasmically located HtpG and the inner membrane protein FtsH.     

Versatile Expression and Secretion Vectors for Bacillus subtilis

Most expression systems are based on Escherichia coli as the host strain because of the large availability of all kinds of vector plasmids. However, aside from the obvious advantages of E. coli systems, serious problems can occur during the process of heterologous gene expression and purification. Therefore, low expression rates, formation of inclusion bodies, improper protein-folding, and/or toxicity problems might enforce changing the expression host. Here we describe the construction of two new vectors, pBSMuL1 and pBSMuL2, for overexpression and secretion of heterologous proteins in Bacillus subtilis as an alternative expression host. The new plasmids combine several advantages in comparison to available Bacillus expression systems: an appropriate multiple cloning site consisting of 13 unique restriction sites, one (pBSMuL1) or two (pBSMuL2) strong constitutive promoters, a high efficient signal sequence for protein secretion, and the possibility to express proteins as His-tagged fusions for easy detection and purification. We have demonstrated the applicability of the novel vector plasmids for the production and purification of the heterologous cutinase from Fusarium solani pisi.     

Integration of Deoxyribonuclease-Treated DNA in Bacillus subtilis Transformation

Normal preparations of B. subtilis DNA have weight average native molecular weights of 10 to 30 x 10⁶. For any given preparation the upper and lower 95% size limits may differ by a factor of ten or more. Single-stranded molecular weights indicate an average of 1 to 4 breaks per single strand of the native DNA. The reduction in transforming activity and viscosity following DNAase I digestion can be accounted for by a direct relationship between the transforming activity of a DNA and its single-stranded molecular weight. Uptake studies with DNAase I treated heavy (²H¹⁵N ³H) DNA show that single strand breaks inhibit integration less than transformation. A provisional estimate of the size of the integrated region based on correlating the single strand size of the donor-recipient complex with the donor-recipient density differences following alkali denaturation came to 1530 nucleotides. Using a competent, nonleaky thymine-requiring strain of B. subtilis grown in 5-BU medium before and after transformation, it was shown that (a) No detectable amount of DNA synthesis is necessary for the initial stages of integration, (b) Cells which have recently been replicating DNA are not competent. (c) Cells containing donor DNA show a lag in DNA replication following transformation, (d) When donor DNA is replicated it initially appears in a density region between light and hybrid. This indicates that it includes the transition point formed at the time of reinitiation of DNA synthesis in the presence of 5-BU following transformation. A model is proposed in which donor DNA is integrated at the stationary growing point of the competent cell, which is in a state of suspended DNA synthesis.     

Incompatibility of outer membrane proteins OmpA and OmpF of Escherichia coli with secretion in Bacillus subtilis: Fusions with secretable peptides

The secretion of the outer membrane proteins OmpA and OmpF of Escherichia coli has previously been found to be blocked at an early intracellular step, when these proteins were fused to a bacillar signal sequence and expressed in Bacillus subtilis. We have now fused these proteins to long secretable polypeptides, the amino-terminal portions of alpha-amylase or beta-lactamase. In spite of this, no secretion of the fusion proteins was detected in B. subtilis. With the exception of a small fraction of the beta-lactamase fusion, the proteins were cell-bound with uncleaved signal sequences. Protease accessibility indicated that the fusion proteins were not even partially exposed on the outer surface of the cytoplasmic membrane. Thus there was no change of the location compared to the OmpA or OmpF fused to the signal sequence only. We conclude that, like OmpA and OmpF, the fusion proteins fold into an export-incompatible conformation in B. subtilis before the start of translocation, which we postulate to be a late post-translational event.     

绿色荧光蛋白基因标记野生型生防枯草芽孢杆菌的研究

根据绿色荧光蛋白基因和枯草芽孢杆菌木糖诱导型启动子PxylR 序列,分别设计两对特异引物primers PxyF/R和primers gfpF/R,扩增获得了完整的启动子PxylR和-gfp基因序列。进一步以上述产物混合物为模板,以primer PxyF/primer gfpR做引物进行重迭PCR,获得了PxylR-gfp重组翻译融合表达盒。经SphⅠ和KpnⅠ完全酶切后,将PxylR-gfp表达盒分别插入大肠杆菌_苏云金芽孢杆菌穿梭载体pHT315和大肠杆菌枯草芽孢杆菌穿梭载体pRP22。相应的重组表达质粒pGFP315和 pGFP22转化枯草芽孢杆菌感受态细胞。前者在标准菌株168中得到良好发光表型,后者则在标准菌株168和野生目标菌株B916中均得到良好的发光表型。室内平板抑菌实验结果显示B916生防效果与出发菌株没有明显差异,遗传稳定性研究表明连续稀释培养约175代后,工程菌株稳定性为94%,质粒丢失频率低于3.5×10^-4/代。     

Integration and amplification of plasmid DNA in the Bacillus subtilis chromosome

Some Bacillus subtilis strains demonstrate a high degree of pHV14 and pP1251 plasmid integration into the chromosome (1.10(-1]. According to the data of genetical analysis the plasmids are integrated into several regions of the same chromosome. A significant plasmid amplification in the composition of chromosome is observed when breeding on an increasing chloramphenicol concentration (20-300 micrograms/ml). Both tandem repetitions and single plasmid DNA copies and their fragments are found in the bacterial chromosomal DNA.     

Visualization of Differential Gene Expression by Improved Cyan Fluorescent Protein and Yellow Fluorescent Protein Production in Bacillus subtilis

The distinguishable cyan and yellow fluorescent proteins (CFP and YFP) enable the simultaneous in vivo visualization of different promoter activities. Here, we report new cloning vectors for the construction of cfp and yfp fusions in Bacillus subtilis. By extending the N-terminal portions of previously described CFP and YFP variants, 20- to 70-fold-improved fluorescent-protein production was achieved. Probably, the addition of sequences encoding the first eight amino acids of the N-terminal part of ComGA of B. subtilis overcomes the slow translation initiation that is provoked by the eukaryotic codon bias present in the original cfp and yfp genes. Using these new vectors, we demonstrate that, within an isogenic population of sporulating B. subtilis cells, expression of the abrB and spoIIA genes is distinct in individual cells.     

用pGAP启动子在P.pastoris中组成型表达漆酶

目的:用pGAP启动子在P.pastoris中组成型表达漆酶.方法:用PCR从枯草芽孢杆菌基因组中扩增漆酶基因lac2.用Not和EcoRⅠ双酶切将lac2基因重组于表达载体PGAP9K.通过电转法将其转化于P.pastoris基因组,筛选高G418抗性以及高表达Lac2酶的重组子作为工程菌Gs115( pGAP9k - lac2).用甘油作为碳源在50L生物反应器中表达重组漆酶Lac2.用ABTS法测定发酵液中的漆酶活力.结果:在发酵30h时,其Lac2酶的表达达峰值,其活性为136.67U/L.其峰值的Lac2酶的表达量为50mg/L.表达产物具有分解ABTs的活性.结论:成功克隆了漆酶基因lac2,并首次实现用pGAP启动子在P.pastorris中组成型表达漆酶,为用P.Pastoris规模化生产漆酶奠定了基础.