利用枯草杆菌碱性蛋白酶E基因的信号顺序构建分泌表达载体

本文利用枯草杆菌碱性蛋白酶基因Ｅ（ａｐｒＥ）,对建立枯草杆菌分泌表达的载体－宿主系统作了探讨。首先用大肠杆菌－枯草杆菌穿梭的启动子克隆质粒ｐＧＫＶ２１０对ａｐｒＥ的启动子功能进行检测,发现用Ｅ．ｃｏｌｉ作为研究ａｐｒＥ表达信号的“中间宿主”是可行的;然后在穿梭质ｃｏｌｉ作为研究,ａｐｒＥ表达信号的“中间宿主”是可行的;然后在穿梭质粒,进而构建了基于ａｐｒＥ启动子和信号顺序的分泌表达载体ｐＳＰ１和ｐ     

Easy cloning of mini-Tn10 insertions from the Bacillus subtilis chromosome.

Delivery vectors for mini-Tn10 transposons function in Bacillus subtilis (M. A. Petit, C. Bruand, L. Janniére, and S. D. Ehrlich, J. Bacteriol. 172:6736-6740, 1990). Using this system, we identified a new gene (sytA) whose inactivation affected regulation of genes of sucrose metabolism. For cloning the sytA::Tn10 insertion in Escherichia coli, we developed a methodology similar to that commonly used for B. subtilis Tn917 insertions. We constructed a plasmid which can be used to insert (by in vivo recombination) a ColE1 origin linked to a spectinomycin resistance gene (ori-spc element) into mini-Tn10 transposons inserted into the B. subtilis chromosome. DNA extracted from a sytA::Tn10::ori-spc transformant was cut with restriction enzymes that do not cut into the Tn10::ori-spc sequence; plasmids containing the sytA::Tn10 insertion were cloned by self-ligation, followed by transformation of E. coli. To obtain the wild-type sytA region, one of these plasmids was ligated with an E. coli-B. subtilis shuttle vector conferring erythromycin resistance, and the hybrid was used to transform the wild-type B. subtilis strain. Erythromycin-resistant transformants, detected as spectinomycin sensitive, resulted from conversion of the insertion mutation by the resident wild-type locus. The shuttle plasmid containing the wild-type locus could then be recovered in E. coli.     

A new cloning system for Bacillus subtilis comprising elements of phage, plasmid and transposon vectors

A new cloning system for Bacillus subtilis was devised which makes use of a combination of Tn917-containing phage SP beta derivatives and Tn917-containing Escherichia coli-B. subtilis shuttle plasmids. This system allows the initial cloning of genes in single copy, via 'prophage transformation', with a selection for complementation of mutational defects in B. subtilis hosts and permits subsequent transfer of the cloned material by homologous recombination to low-copy and high-copy vectors that replicate in both B. subtilis and E. coli. Because cloned sequences are adjacent to pB322-derived DNA in the recombinant phages, inserts can also be 'rescued' directly from the phage DNA after digestion with appropriate restriction enzymes, circularization of the fragments by ligation and transformation of an E. coli recipient. Two genomic libraries of B. subtilis chromosomal Sau3A-generated partial-digest fragments in the size ranges of 5-8 kb and 8-10 kb were constructed and screened for the complementation of mutations aroI906, cysA14, dal-1, glyB133, metC3, purA16, purB33, thrA5, trpC2 and recE4. In all cases, specialized transducing phages carrying inserts that complemented the selected markers were recovered. Inserts complementing the dal-1 and trpC2 mutations could be transferred from recombinant phages to Tn917-containing plasmids by homologous recombination without in vitro subcloning. Another insert complementing the purB33 mutation was rescued directly into E. coli from a recombinant phage DNA.     

Cloning of genetic material in Bacillus

Plasmid vectors capable for propagation of Bacillus subtilis DNA fragments containing riboflavin genes were constructed. Cloning of rib operon using pUB110 derivatives was performed in recE4 strain by using sequentional rescue of plasmids containing subfragments of the operon. Also, rib operon was cloned on the vectors containing DNA repeats. It was shown that the presence of direct and inverted repeats within plasmids allows to transform B. subtilis cells by monomers of plasmid DNA. Vectors that contained repeated sequences of DNA and ensured efficient cloning of genetic material in B. subtilis recipient cells were constructed. The use of streptococcal plasmid pSM19035 allowed to obtain vectors which were suitable for cloning large DNA fragments (6 MD and even more) in B. subtilis. A model of B. subtilis transformation by various types of plasmid DNA is presented. The model is in agreement with the general conception of chromosomal DNA transformation.     

Characterization and comparative sequence analysis of replication origins from three large Bacillus thuringiensis plasmids.

The replication origins of three large Bacillus thuringiensis plasmids, derived from B. thuringiensis HD263 subsp. kurstaki, have been cloned in Escherichia coli and sequenced. The replication origins, designated ori 43, ori 44, and ori 60, were isolated from plasmids of 43, 44, and 60 MDa, respectively. Each cloned replication origin exhibits incompatibility with the resident B. thuringiensis plasmid from which it was derived. Recombinant plasmids containing the three replication origins varied in their ability to transform strains of B. thuringiensis, Bacillus megaterium, and Bacillus subtilis. Analysis of the derived nucleotide and amino acid sequences indicates that the replication origins are nonhomologous, implying independent derivations. No significant homology was found to published sequences of replication origins derived from the single-stranded DNA plasmids of gram-positive bacteria, and shuttle vectors containing the three replication origins do not appear to generate single-stranded DNA intermediates in B. thuringiensis. The replication origin regions of the large plasmids are each characterized by a single open reading frame whose product is essential for replication in B. thuringiensis. The putative replication protein of ori 60 exhibits partial homology to the RepA protein of the Bacillus stearothermophilus plasmid pTB19. The putative replication protein of ori 43 exhibits weak but extensive homology to the replication proteins of several streptococcal plasmids, including the open reading frame E replication protein of the conjugative plasmid pAM beta 1. The nucleotide sequence of ori 44 and the amino acid sequence of its putative replication protein appear to be nonhomologous to other published replication origin sequences.     

蛋白激发子PeaT1在枯草芽胞杆菌中的分泌表达及重组菌株提高小麦抗旱和促生的作用

PeaT1是从极细链格孢菌Alternaria tenuissima中分离的一种蛋白激发子,具有促进植物生长和诱导植物产生系统获得抗性的功能,为了实现peaT1基因在枯草芽胞杆菌Bacillus subtilis中的分泌表达,增加其应用途径,从枯草芽胞杆菌基因组DNA中分别扩增获得P43启动子和nprB基因的信号肽序列,并用SOE (Splicing by over lapping extension) 方法与peaT1基因连接,将连接产物克隆到大肠杆菌-枯草芽胞杆菌穿梭表达载体pHY300-PLK上,构建了重组表达载体pHY43N-peaT1。将重组载体转化枯草芽胞杆菌WB800菌株,SDS-PAGE和Western blotting分析证实,在NprB信号肽的引导下,枯草芽胞杆菌成功分泌表达了PeaT1蛋白。构建的重组菌株能够显著增强幼苗抗旱性,提高小麦株高。     

Effects of dnats genes on the replication of plasmids in Bacillus subtilis

An essential region (2.3 kb) for the replication of a low-copy-number plasmid, pBS-2, has been identified and cloned into plasmid pHV60 in Bacillus subtilis. The resultant plasmid, pKW1, and two other plasmids, pC194 (medium copy number) and pTP5 (high copy number), were examined by double radio-labelling and gel electrophoresis to determine which host functions are required for their replication in B. subtilis. Replication of pKW1 requires the functions of most dna genes, in particular dnaB, C, E, F, G and H; pC194 requires only dnaG and H; and pTP5 requires dnaE, F, G and H. Thus dnaG and dnaH are required for the replication of all three plasmids tested, even though each plasmid showed a different spectrum of dependency on other host functions. Because of its greater dependence on host functions and its low copy number, pKW1 should be a useful model with which to investigate the function of host genes in the replication of DNA in B. subtilis. pKW1 should also be a useful shuttle vector for cloning of genes in B. subtilis in cases when high gene dosage might be a problem.     

A Bacillus subtilis plasmid that can be packaged as single-stranded DNA in Escherichia coli: use for oligodeoxynucleotide-directed mutagenesis

A Bacillus subtilis/Escherichia coli shuttle vector was modified to contain the origin of DNA replication of the E. coli filamentous phage f1, in both orientations. Upon superinfection with and f1-related phage of an E. coli strain containing either of the modified vectors, the single-stranded (ss) form of the plasmid was packaged in virions and released to the culture medium. Each of these ss DNAs has been purified from the virions and used as a template for oligodeoxynucleotide-directed mutagenesis. The resulting mutations were demonstrated by DNA sequencing. The capacity of these vectors to be isolated as phage ss DNA from E. coli and to replicate as plasmids in B. subtilis makes them convenient substrates for the production of oligodeoxynucleotide-directed mutations for studies in B. subtilis.     

An improved beta-galactosidase alpha-complementation system for molecular cloning in Bacillus subtilis

The recently described beta-galactosidase alpha-complementation system for molecular cloning in Bacillus subtilis [Haima et al., Gene 86 (1990) 63-69]was optimized in several ways. First, the efficiency of translation of the lac Z delta M15 gene was improved. Second, the plasmid-borne lacZ delta M15 gene was segregationally stabilized by integration into the B. subtilis chromosome. Third, a new lacZ alpha complementing cloning vector was constructed, containing more unique target sites. It was shown that large heterologous DNA fragments (up to at least 29 kb) could be cloned with lacZ alpha-complementing vectors carrying the replication functions of the cryptic B. subtilis plasmid pTA1060, and that these inserts were structurally stably maintained for at least 100 generations of growth.     

Nucleotide sequence and characterization of the cryptic Bacillus thuringiensis plasmid pGI3 reveal a new family of rolling circle replicons.

The complete nucleotide sequence of plasmid pGI3 from Bacillus thuringiensis subsp. thuringiensis H1.1. was obtained. Although this 11,365-bp molecule contained at least 11 putative open reading frames (ORFs), extensive database searches did not reveal any homologous sequences with the exception of ORF6, which displayed similarity to the largest ORF of pSTK1, a 1,883-bp cryptic plasmid isolated from Bacillus stearothermophilus. Deletion analysis to determine the pGI3 minimal replicon revealed that ORF6 is the rep gene. Replication occurred via a single-stranded DNA (ssDNA) intermediate, as demonstrated by S1 treatment and Southern hybridization in nondenaturating conditions. Interestingly, however, no homology was found between the pGI3 (ORF6) and pSTK1 (ORF3) rep genes and those from other single-stranded DNA plasmids, nor was there any DNA similarity to the double-strand origins of replication characterized so far, indicating that pGI3 and pSTK1 form another, new family of ssDNA plasmids. PCR analysis revealed that the pGI3 rep gene is largely distributed among B. thuringiensis strains but can also be found in B. cereus and B. mycoides strains, albeit at a lower frequency. Finally, segregation experiments performed with B. subtilis and B. thuringiensis showed that the pGI3 derivatives, including the minimal replicon, were segregationally stable at temperatures suitable for B. thuringiensis growth (<43 degrees C).     

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。     

Molecular cloning in Bacillus subtilis of a Bacillus licheniformis gene encoding a thermostable alpha amylase

A resident-plasmid cloning system developed for Bacillus subtilis has been used to isolate recombinant plasmids carrying DNA from Bacillus licheniformis which confer alpha-amylase activity on alpha-amylase-negative mutants of B. subtilis. These plasmids contain a 3550-bp insert at the EcoRI site of the plasmid pBD64. Subcloning various lengths of the B. licheniformis DNA has localised the gene to a 2550-bp BclI fragment. We present evidence that the cloned fragment codes for a B. licheniformis heat-stable alpha-amylase with a temperature optimum of 93 degrees C. The foreign gene is expressed efficiently in B. subtilis and is stably maintained.     

Lactobacillus plantarum and Lactobacillus buchneri as Expression Systems: Evaluation of Different Origins of Replication for the Design of Suitable Shuttle Vectors

The objectives of this study were to establish transformation protocols for Lactobacillus plantarum CD033 and Lactobacillus buchneri CD034, two industrial silage strains and to test the influence of selected origins of replication on plasmid copy number, plasmid stability, and plasmid incompatibility in these strains. Electro-transformation protocols were optimized by examination of the influence of different electroporation solutions and cell wall weakening agents on transformation efficiency. Using Lithium acetate as cell wall weakening agent, we could achieve transformation efficiencies of 8?×?10(4) transformants per 1?μg DNA for L. buchneri CD034 which is to our knowledge the highest described for this species up to now. In order to test feasibility of previously described origins of replication derived from Bacillus subtilis, L. plantarum, Lactococcus lactis, and two novel L. buchneri CD034 plasmids to drive replication in our two selected Lactobacillus strains, six shuttle vectors were constructed. Results indicate that, in terms of stable propagation and high gene copy numbers (up to 238 copies/chromosome), the most suitable origins of replication for the construction of expression vectors for the selected silage strains were the ones derived from the novel L. buchneri CD034 plasmids.     

Multicopy plasmids affect replisome positioning in Bacillus subtilis

The DNA replication machinery, various regions of the chromosome, and some plasmids occupy characteristic subcellular positions in bacterial cells. We visualized the location of a multicopy plasmid, pHP13, in living cells of Bacillus subtilis using an array of lac operators and LacI-green fluorescent protein (GFP). In the majority of cells, plasmids appeared to be highly mobile and randomly distributed. In a small fraction of cells, there appeared to be clusters of plasmids located predominantly at or near a cell pole. We also monitored the effects of the presence of multicopy plasmids on the position of DNA polymerase using a fusion of a subunit of DNA polymerase to GFP. Many of the plasmid-containing cells had extra foci of the replisome, and these were often found at uncharacteristic locations in the cell. Some of the replisome foci were dynamic and highly mobile, similar to what was observed for the plasmid. In contrast, replisome foci in plasmid-free cells were relatively stationary. Our results indicate that in B. subtilis, plasmid-associated replisomes are recruited to the subcellular position of the plasmid. Extending this notion to the chromosome, we postulated that the subcellular position of the chromosomally associated replisome is established by the subcellular location of oriC at the time of initiation of replication.     

利用枯草芽孢杆菌ytkA和ywoF基因启动子与信号肽重组分泌表达mpd基因

用大肠杆菌-枯草芽孢杆菌穿梭载体pNW33N和去除了信号肽编码序列的成熟mpd基因构建了穿梭启动子探针pNW33N-mpd。用该探针从质粒pMPDP3和pMPDP29上克隆来自于枯草芽孢杆菌ytkA和ywoF基因上游的启动子功能片段,构建了穿梭表达载体pNYTM和pNYWM。将表达载体pNYTM和pNYWM转入枯草芽孢杆菌1A751获得表达菌株1A751(pNYTM)和1A751(pNYTM),mpd基因在ytkA和ywoF基因的启动子和信号肽的带动下实现了分泌表达且具有天然活性,结果表明ytkA基因的启动子强度强于ywoF基因的启动子。利用ytkA基因的强启动子和nprB基因的分泌型信号肽编码序列构建了新的穿梭分泌表达载体pYNMK,并使mpd基因在枯草芽孢杆菌WB800中得到了更高水平的分泌表达,表达菌株WB800(pYNMK)在培养到第84h时甲基对硫磷水解酶酶活达到最高值为10.40u/mL,是出发菌株邻单胞菌M6表达量的10.8倍,重组表达产物有91.4%分泌在培养基中。     

Characterization of a gram-positive broad-host-range plasmid isolated from Lactobacillus hilgardii

Two plasmids, pLAB1000 and pLAB2000 (3.3 and 9.1 kb, respectively), have been isolated from a grass silage strain of Lactobacillus hilgardii. Both plasmids were cloned in Escherichia coli and characterized through restriction mapping. A 1.6-kb XbaI-SacI fragment of pLAB1000 appeared to be sufficient for autonomous replication in Lactobacillus plantarum and in Bacillus subtilis. Different shuttle vectors for E. coli and gram-positive bacteria were developed using the pLAB1000 plasmid. These could stably be maintained in Lactobacillus, Enterococcus, and Bacillus under selective conditions. Plasmids sharing DNA homologies with pLAB1000 have been observed in different strains of the related species L. plantarum.     

Recombination between compatible plasmids containing homologous segments requires the Bacillus subtilis recE gene product.

Plasmid pSL103 was previously constructed by cloning a Trp fragment (approximately 2.3 X 10(6) daltons) from restriction endonuclease EcoRI-digested chromosome DNA of Bacillus pumilus using the neomycin-resistance plasmid pUB110 (approximately 2.8 X 10(6) daltons) as vector and B. subtilis as transformation recipient. In the present study the EcoRI Trp fragment from pSL103 was transferred in vitro to EcoRI fragments of the Bacillus plasmid pPL576 to determine the ability of the plasmid fragments to replicate in B. subtilis. Endonuclease EcoRI digestion of pPL576 (approximately 28 X 10(6) daltons) generated three fragments having molecular weights of about 13 X 13(6) (the A fragment), 9.5 X 10(6) (B fragment, and 6.5 X 10(6) (C fragment). Trp derivatives of pPL576 fragments capable of autonomous replication in B. subtilis contained the B fragment (e.g., pSL107) or both the B and C fragments (e.g., pSL108). Accordingly, the B fragment of pPL576 contains information essential for autonomous replication. pSL107 and pSL108 are compatible with pUB110. Constructed derivatives of the compatible plasmids pPL576 and pUB110, harboring genetically distinguishable EcoRI-generated Trp fragments cloned from the DNA of a B. pumilus strain, exhibited relatively high frequency recombination for a trpC marker when the plasmid pairs were present in a recombination-proficient strain of B. subtilis. No recombination was detected when the host carried the chromosome mutation recE4. Therefore, the recE4 mutation suppresses recombination between compatible plasmids that contain homologous segments.     

Replication Mechanism and Sequence Analysis of the Replicon of pAW63, a Conjugative Plasmid from Bacillus thuringiensis

A 5.8-kb fragment of the large conjugative plasmid pAW63 from Bacillus thuringiensis subsp. kurstaki HD73 containing all the information for autonomous replication was cloned and sequenced. By deletion analysis, the pAW63 replicon was reduced to a 4.1-kb fragment harboring four open reading frames (ORFs). Rep63A (513 amino acids [aa]), encoded by the largest ORF, displayed strong similarity (40% identity) to the replication proteins from plasmids pAMbeta1, pIP501, and pSM19035, indicating that the pAW63 replicon belongs to the pAMbeta1 family of gram-positive theta-replicating plasmids. This was confirmed by the facts that no single-stranded DNA replication intermediates could be detected and that replication was found to be dependent on host-gene-encoded DNA polymerase I. An 85-bp region downstream of Rep63A was also shown to have strong similarity to the origins of replication of pAMbeta1 and pIP501, and it is suggested that this region contains the bona fide pAW63 ori. The protein encoded by the second large ORF, Rep63B (308 aa), was shown to display similarity to RepB (34% identity over 281 aa) and PrgP (32% identity over 310 aa), involved in copy control of the Enterococcus faecalis plasmids pAD1 and pCF10, respectively. No significant similarity to known proteins or DNA sequences could be detected for the two smallest ORFs. However, the location, size, hydrophilicity, and orientation of ORF6 (107 codons) were analogous to those features of the putative genes repC and prgO, which encode stability functions on plasmids pAD1 and pCF10, respectively. The cloned replicon of plasmid pAW63 was stably maintained in Bacillus subtilis and B. thuringiensis and displayed incompatibility with the native pAW63. Hybridization experiments using the cloned replicon as a probe showed that pAW63 has similarity to large plasmids from other B. thuringiensis subsp. kurstaki strains and to a strain of B. thuringiensis subsp. alesti.     

Characterization, cloning, curing, and distribution in lactic acid bacteria of pLP1, a plasmid from Lactobacillus plantarum CCM 1904 and its use in shuttle vector construction

A small 2.1-kb plasmid called pLP1 was extracted from Lactobacillus plantarum CCM 1904 (ATCC 8014) and cloned into the Escherichia coli pUC19 plasmid. As determined by DNA-DNA Southern hybridization with a pLP1-radioactively labeled probe, other lactic acid bacteria such as L. curvatus, L. sake, Carnobacterium, and Leuconostoc mesenteroides harbor pLP1-related plasmids. Shuttle vectors based on the pLP1 replicon were constructed by inserting the erythromycin-resistance gene from pVA891 into the various pUC19-pLP1 constructions. pLP1-based shuttle vector transformation efficiencies (TE) by electroporation were compared to TE of a broad-host-range plasmid pGK12 in different lactobacilli strains. Expression of the pUC19-pLP1 plasmids in Escherichia coli maxicells showed that pLP1 encodes for a 37,000 MW protein which can act in trans allowing the replication of plasmids in which this protein is truncated. The pLP1-based shuttle vectors producing this protein replicate in lactobacilli and also in Bacillus subtilis. A pLP1-free strain was obtained by incompatibility with a pLP1-based shuttle vector introduced in L. plantarum CCM 1904 by electroporation. The absence of pLP1 has no incidence on the strain phenotype suggesting that pLP1 is not essential for the strain in our laboratory conditions.     

Features of transformation of competent cells and Bacillus subtilis protoplasts by integrative vectors

The effect of structural peculiarities of DNAs from integrative plasmids on the transformation activity was studied. Monomeric forms of the plasmids can only transform B. subtilis competent cells, when plasmid selective marker is inserted into chromosomal fragment within the plasmid. Polymeric forms are needed for efficient transformation. Both single- and double-stranded DNAs of integrative plasmids transform no B.subtilis protoplasts, this being irrespective of plasmid structure.