不同培养时期的大肠杆菌和枯草芽孢杆菌间发生的跨属自然遗传转化

携带穿梭质粒的大肠杆菌与作为受体的枯草芽孢杆菌分别培养至不同生长阶段混合均匀后静置40min,涂布选择性平板,37℃培养30h后得到一定数目的转化子,DNaseⅠ敏感实验证实质粒是通过自然遗传转化而非其它形式发生转移。实验发现大肠杆菌可以在特定生长时期向胞外分泌DNA,并且在对数期具有最高的提供质粒的能力,而生长后期的细胞因为体系中DNase量的增加转化频率下降。进一步的研究发现枯草芽孢杆菌在营养丰富的LB培养基中也具有与基本培养基中相当的转化能力,并且在对数生长前期具有较高的转化频率。     

Transfer of transformation and conjugation plasmids from Escherichia coli to Bacillus subtilis and Azospirillum brasilense]

The conjugative transfer of RP4 plasmid from Escherichia coli to Azospirillum brasilense was detected after introduction and subsequent incubation of these microorganisms in soil. The plasmid transfer via transformation from Escherichia coli to Bacillus subtilis was observed in case both bacteria were growing together in sand containing sucrose solution. The possible reason for low frequency interspecies plasmid transformation under conditions close to natural habitats is poor survival of "domesticated" rather than wild type Bacillus subtilis strains and lack of competence state in this case.     

携带苏云金芽胞杆菌cry1Ac10基因重组杆状病毒的构建及其杀虫活性

用Bac-to-Bac系统,构建了包含极晚期基因ph启动子驱动的带有全长苏云金芽胞杆菌cry1Ac10基因和完整多角体基因的重组质粒pFCP,用该重组质粒感染昆虫Sf9细胞,得到了带有多角体和能够表达cry1Ac10基因的重组杆状病毒vFcph,并在昆虫细胞中表达了Cry1Ac10蛋白。同时构建了含cry1Ac10的穿梭载体．pHTC,并分别转化大肠杆菌、枯草杆菌和苏云金杆菌晶体缺陷型菌株,结果表明此三种工程菌均表达了分子量为133．3kDa的原毒素蛋白,其中在苏云金芽胞杆菌中的表达量最高。生物测定表明重组杆状病毒vFcph的表达产物具有杀虫活性,能增加杆状病毒力,加快杆状病毒杀虫速度,说明利用杆状病毒极晚期基因启动子驱动苏云金芽胞杆菌杀虫晶体蛋白表达,从而改善杆状病毒的杀虫特性是可行的。     

Intraspecific and interspecific transmission of plasmid and chromosomal DNA during natural transformation in Bacillus subtilis and Escherichia coli

The transmission of plasmids from Escherichia coli to Bacillus subtilis cells was studied in the course of natural transformation in a mixed liquid culture of the two bacteria. The frequency of the transmission was 2-3 times lower than between B. subtilis cells under the same conditions.     

Cj0011c, a periplasmic single- and double-stranded DNA-binding protein, contributes to natural transformation in Campylobacter jejuni

Campylobacter jejuni is an important bacterial pathogen causing gastroenteritis in humans. C. jejuni is capable of natural transformation, which is considered a major mechanism mediating horizontal gene transfer and generating genetic diversity. Despite recent efforts to elucidate the transformation mechanisms of C. jejuni, the process of DNA binding and uptake in this organism is still not well understood. In this study, we report a previously unrecognized DNA-binding protein (Cj0011c) in C. jejuni that contributes to natural transformation. Cj0011c is a small protein (79 amino acids) with a partial sequence homology to the C-terminal region of ComEA in Bacillus subtilis. Cj0011c bound to both single- and double-stranded DNA. The DNA-binding activity of Cj0011c was demonstrated with a variety of DNAs prepared from C. jejuni or Escherichia coli, suggesting that the DNA binding of Cj0011c is not sequence dependent. Deletion of the cj0011c gene from C. jejuni resulted in 10- to 50-fold reductions in the natural transformation frequency. Different from the B. subtilis ComEA, which is an integral membrane protein, Cj0011c is localized in the periplasmic space of C. jejuni. These results indicate that Cj0011c functions as a periplasmic DNA receptor contributing to the natural transformation of C. jejuni.     

Joint transfer of genetic markers in Bacillus subtilis

Takahashi, I. (McMaster University, Hamilton, Ontario, Canada). Joint transfer of genetic markers in Bacillus subtilis. J. Bacteriol. 91:101-105. 1966.-To compare the processes of genetic incorporation in transduction and transformation in Bacillus subtilis, several groups of linked markers were selected and the degree of linkage was determined by the two means of genetic exchange. Bacteriophage PBS 1 was used in transduction experiments. In all cases, frequencies of joint transfer, as expressed by the cotransfer index or by percentage of joint transfer, were higher in transduction than in transformation. With a pair of closely linked markers, the frequency of joint transduction was only slightly higher than that of joint transformation. On the other hand, a considerably higher degree of linkage was obtained by transduction when loosely linked markers were examined. It appears that the size of donor chromosome transferred by transducing phage particles is much larger than that incorporated by recipient cells in transformation. It is suggested that transduction in B. subtilis may be a useful tool in extending further the linkage groups established by the transformation technique.     

The Size and Continuity of DNA Segments Integrated in Bacillus Transformation

We investigated the size and continuity of DNA segments integrated in Bacillus subtilis transformation. We transformed B. subtilis strain 1A2 toward rifampicin resistance (coded by rpoB) with genomic DNA and with a PCR-amplified 3.4-kb segment of the rpoB gene from several donors. Restriction analysis showed that smaller lengths of donor DNA integrated into the chromosome with transformation by PCR-amplified DNA than by genomic DNA. Nevertheless, integration of very short segments (<2 kb) from large, genomic donor molecules was not a rare event. With PCR-amplified segments as donor DNA, smaller fragments were integrated when there was greater sequence divergence between donor and recipient. There was a large stochastic component to the pattern of recombination. We detected discontinuity in the integration of donor segments within the rpoB gene, probably due to multiple integration events involving a single donor molecule. The transfer of adaptations across Bacillus species may be facilitated by the small sizes of DNA segments integrated in transformation.     

Conversion of Bacillus subtilis 168 to a subtilin producer by competence transformation.

Subtilin is a ribosomally synthesized peptide antibiotic produced by Bacillus subtilis ATCC 6633. B. subtilis 168 was converted to a subtilin producer by competence transformation with chromosomal DNA from B. subtilis ATCC 6633. A chloramphenicol acetyltransferase gene was inserted next to the subtilin structural gene as a selectable marker. The genes that conferred subtilin production were derived from a 40-kb region of the B. subtilis ATCC 6633 chromosome that had flanking homologies to the B. subtilis 168 chromosome. The subtilin produced by the mutant was identical to natural subtilin in its biological activity, chromatographic behavior, amino acid composition, and N-terminal amino acid sequence.     

Spontaneous transformation and its use for genetic mapping in Bacillus subtilis

Using a simple semi-synthetic competence and sporulation medium (CSM), we found evidence that Bacillus subtilis cells transformed in the competence phase can sporulate, indicating that genetic information acquired during the competence phase is inherited by the next generation after germination of the transformed spores. Moreover, the results from mixed cell culture experiments suggest that spontaneous genetic transformation can occur between competent cells and DNA released from lysed cells in the natural environment. We also found evidence that the spontaneous transformation system can be used for genetic mapping in B. subtilis.     

Mapping and cloning of the gene coding for beta-glucanase from various bacilli

Beta-glucanase gene from Bacillus subtilis 168 has been mapped by bacteriophage pBS1 transduction technique between sacA and purA genes. The stimulating effect of pleiotropic mutations pap, amyB and sacUh on beta-glucanase production in Bacillus subtilis and Bacillus amyloliquefaciens has been described. Beta-glucanase gene from Bacillus amyloliquefaciens has been cloned ona Charon 4A vector. Expression of the gene in E. coli cells depended on the orientation of the cloned DNA on a pBR322 vector plasmid. Maximal enzymatic activity was registered in periplasm. Beta-glucanase gene was recloned in Bacillus subtilis cells. Bacillus subtilis strain, harbouring pBG1, produces 500 times more beta-glucanase as compared with the wild type strain of Bacillus subtilis.     

Experimental basis for a stable plasmid, pLS30, to shuttle between Bacillus subtilis species by conjugational transfer

The use of Bacillus subtilis 168 as the initial host for molecular cloning and subsequent delivery of the engineered DNA to other Bacillus hosts appears attractive, and would lead to an efficient DNA manipulation system. However, methods of delivery to other Bacillus species are limited due to their inability to develop natural competence. An alternative, unexplored conjugational transfer method drew our attention and a B. subtilis native plasmid, pLS30, isolated from B. subtilis (natto) strain IAM1168 was characterized for this aim. The nucleotide sequence (6,610 bp) contained the mob gene and its recognition sequence, oriT, that features pLS30 as a mobile plasmid between Bacillus species on conjugational transfer. Plasmid pLS3001, a chimera with a pBR322-based plasmid prepared in Escherichia coli to confer an antibiotic resistance marker, showed apparent mobilizing activity in the pLS20-mediated conjugational transfer system recently established. The rep gene and associated palT1-like sequence common to all other pLS plasmids previously sequenced indicated that pLS30 is a typical rolling circle replicating (RCR) type plasmid. Due to the significant stability of pLS30 in IAM1168, application of a mobile plasmid would allow quick propagation to Bacillus species.     

Conjugational transfer system to shuttle giant DNA cloned by Bacillus subtilis genome (BGM) vector

The Bacillus subtilis GenoMe (BGM) vector was designed as a versatile vector for the cloning of giant DNA segments. Cloned DNA in the BGM can be retrieved to a plasmid using our Bacillus recombinational transfer (BReT) method that takes advantage of competent cell transformation. However, delivery of the plasmid to a different B. subtilis strain by the normal transformation method is hampered by DNA size-related inefficiency. Therefore, we designed a novel method, conjugational plasmid-mediated DNA retrieval and transfer (CReT) from the BGM vector, and investigated conjugational transmission to traverse DNA between cells to circumvent the transformation-induced size limitation. pLS20, a 65-kb plasmid capable of conjugational transfer between B. subtilis strains, was modified to retrieve DNA cloned in the BGM vector by homologous recombination during normal culture. As the plasmid copy number was estimated to be 3, the retrieval plasmid was selected using increased numbers of marker genes derived from the retrieved DNA. We applied this method to retrieve Synechocystis genome segments up to 90 kb in length. We observed retrieved plasmid transfers between B. subtilis strains by conjugation in the absence of structural alterations in the DNA fragment. Our observations extend DNA transfer protocols over previously exploited size ranges.     

Cloning and expression of the Escherichia coli recA gene in Bacillus subtilis

By means of homopolymer dG-dC tailing, using PstI linearized pBR327 as vector, we constructed small plasmids containing the entire Escherichia coli recA gene. The 1.8-kb inserts were recloned in the Bacillus subtilis expression vector pPL608 in a B. subtilis recE4 strain. Analysis of plasmid-coded proteins showed expression of the E. coli recA gene both in minicells and whole cells of B. subtilis. Expression was under control of the bacteriophage SP02 promoter, which is part of pPL608. A recA-expressing plasmid completely abolished the transformation deficiency of the recE4 mutant as well as its sensitivity to mitomycin C (MC). The expressed recA gene also restored recombination in other B. subtilis strains lacking the recE gene product. These results indicate a high similarity between the functions of the E. coli RecA and B. subtilis RecE proteins.     

枯草芽孢杆菌在琼脂平板上进行的自然遗传转化

本文对发生在琼脂平板上的枯草芽孢杆菌自然遗传转化进行了初步的研究。结果表明,在相同条件下,该菌在琼脂平板上的自然转化率明显高于传统液体转化,并且转化反应对ＤＮａｓｅ 的抗性增强,通常被认为不能建立感受态的ＬＢ 培养物当涂布到平板上后也很快具有了自然转化的能力,说明在固相物表面进行的转化过程与传统的液体法存在一定的差异。在琼脂平板上,也能观察到具不同遗传标记的菌株间进行的细胞间自然转化。     

Transformation of Bacillus subtilis in α-Amylase Productivity by Deoxyribonucleic Acid from B. subtilis var. amylosacchariticus

Deoxyribonucleic acid (DNA) of Bacillus subtilis var. amylosacchariticus showed almost the same ability as B. subtilis Marburg to induce transfer of several genetic markers in DNA-mediated transformation. DNA-DNA hybridization data also showed an intimate relationship between the two strains. Genetic elements involved in the production of extracellular alpha-amylase (EC 3.2.1.1.) in B. subtilis var. amylosacchariticus were studied by using DNA-mediated transformation. Two Marburg derivatives, NA20(amyR2) and NA20-22(amyR1), produced about 50 and 10 U of alpha-amylase per mg of cells, respectively, whereas B. subtilis var. amylosacchariticus produced as much as 150 U of the enzyme per mg of cells. When B. subtilis var. amylosacchariticus was crossed with strain NA20-22 as recipient, transformants that acquired high alpha-amylase productivity (about 50 U/mg of cells) were obtained. Genetic analysis revealed that a regulator gene (amyR) for alpha-amylase synthesis was found in B. subtilis var. amylosacchariticus, as in the case of B. natto 1212 (amyR2) and B. subtilis Marburg (amyR1). The allele was designated amyR3; it is phenotypically indistinguishable from amyR2, but is readily distinguishable from amyR1. The presence of amyR3 was not sufficient for an organism to render production of an exceptional amount of alpha-amylase. Extra-high alpha-amylase producers could be obtained by crossing B. subtilis var. amylosacchariticus as donor with strain NA20 as recipient. The transformants produced the same or even greater amounts of the enzyme than the donor strain. Results suggest the presence of another gene that is involved in the production of the exceptional amount of alpha-amylase.     

Transformation of Bacillus subtilis and Escherichia coli by a hybrid plasmid pCD1.

The gene thyP3 from Bacillus subtilis bacteriophage phi 3T was cloned in the plasmid pMB9. The resulting chimeric plasmid, pCD1, is effective in transforming both Escherichia coli and Bacillus subtilis to thymine prototrophy. The activity of the thyP3 gene product, thymidylate synthetase, was assayed and found to be 9 times greater in a transformed strain of Escherichia coli than in a phi 3T lysogen of Bacillus subtilis. The physical location of restriction sites has been determined for two related plasmids pCD1 and pCD2. Hybridization studies clearly indicate that the plasmid gene responsible for Thy+ transformation is the gene from the bacteriophage phi 3T. The lack of restriction in this transformation process is consistent with our previous studies using bacterial DNA in heterospecific exchanges indicating that the nucleotide sequence surrounding the gene is the dominant factor in determining interspecific transformation.     

Engineered biosynthesis of the peptide antibiotic bacitracin in the surrogate host Bacillus subtilis.

Nonribosomal peptides are processed on multifunctional enzymes called nonribosomal peptide synthetases (NRPSs), whose modular multidomain arrangement allowed the rational design of new peptide products. However, the lack of natural competence and efficient transformation methods for most of nonribosomal peptide producer strains prevented the in vivo manipulation of these biosynthetic gene clusters. In this study, we present methods for the construction of a genetically engineered Bacillus subtilis surrogate host for the integration and heterologous expression of foreign NRPS genes. In the B. subtilis surrogate host, we deleted the resident 26-kilobase srfA gene cluster encoding the surfactin synthetases and subsequently used the same chromosomal location for integration of the entire 49-kilobase bacitracin biosynthetic gene cluster from Bacillus licheniformis by a stepwise homologous recombination method. Synthesis of the branched cyclic peptide antibiotic bacitracin in the engineered B. subtilis strain was achieved at high level, indicating a functional production and proper posttranslational modification of the bacitracin synthetases BacABC, as well as the expression of the associated bacitracin self-resistance genes. This engineered and genetically amenable B. subtilis strain will facilitate the rational design of new bacitracin derivatives.     

Evolution of Natural Transformation: Testing the DNA Repair Hypothesis in Bacillus Subtilis and Haemophilus Influenzae

The hypothesis that the primary function of bacterial transformation is DNA repair was tested in the naturally transformable bacteria Bacillus subtilis and Haemophilus influenzae by determining whether competence for transformation is regulated by DNA damage. Accordingly, DNA damage was induced by mitomycin C and by ultraviolet radiation at doses that efficiently induced a known damage-inducible gene fusion, and the ability of the damaged cultures to transform was monitored. Experiments were carried out both under conditions where cells do not normally become competent and under competence-inducing conditions. No induction or enhancement of competence by damage was seen in either organism. These experiments strongly suggest that the regulation of competence does not involve a response to DNA damage, and thus that explanations other than DNA repair must be sought for the evolutionary functions of natural transformation systems.     

Random mutagenesis by recombinational capture of PCR products in Bacillus subtilis and Acinetobacter calcoaceticus.

We describe a general method for random mutagenesis of cloned genes by error-prone PCR or DNA shuffling that eliminates the need for post-amplification subcloning following each cycle of mutagenesis. This method exploits the highly efficient and recombinogenic nature of DNA uptake during natural transformation in the Gram-positive bacterium Bacillus subtilis and the Gram-negative bacterium Acinetobacter calcoaceticus. Plasmid systems were designed that allow capture of PCR-amplified DNA fragments by marker-replacement recombination with a structurally similar helper plasmid resident in the transformation recipient. This recombination event simultaneously transfers the amplified sequences into the helper plasmid and restores the integrity of a drug resistance gene, thereby affording a direct selection for fragment capture. Although this strategy was sufficiently effective to permit recovery in B. subtilis of up to 10(3) transformants/microgram of PCR product, equivalent plasmid systems were approximately 100 times more efficient in A.calcoaceticus. Acinetobacter calcoaceticus also offers the advantage of essentially constitutive transformation competence in ordinary complex broth, such as LB, in contrast to two-step growth in semi-synthetic media required for optimal transformation of B.subtilis.