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.     

蜡样芽孢杆菌ATCC14579核黄素操纵子的克隆及在枯草芽孢杆菌中的表达

利用BLAST从B．cereus ATCC14579的基因组中找到一段与枯草芽孢杆茵核黄素操纵子具有较高相似性的4．6kb大小的基因组DNA片段,该片段中含有完整的核黄素操纵子。该操纵子结构基因的编码产物的氨基酸序列与枯草芽孢杆菌核黄素操纵子相应结构基因的编码产物的氨基酸序列具有99％的同源性。该片段被克隆到大肠杆茵一枯草芽孢杆茵穿梭载体pHP13M中。表达分析的结果表明B．cereus ATCC14579核黄素操纵子可在大肠杆茵和枯草芽孢杆菌中表达。利用PCR方法用来自枯草杆菌的sac B基因的启动子替换B．cereus ATCC14579核黄素操纵子原有的启动子使其更好表达。替换启动子后的核黄素操纵子在本文使用的发酵条件下有较好的表达,核黄素产量从39．5mg／L增加到61．7mg/L.     

通过信号肽筛选优化耐高温α-淀粉酶在枯草芽孢杆菌中的分泌

以大肠-枯草穿梭载体p MA5质粒为基本骨架,以来源于嗜热脂肪地芽孢杆菌Geobacillus stearothermophilus NUB3621的耐高温α-淀粉酶基因为目标基因,利用POE-PCR法,成功构建针对淀粉酶的信号肽筛选载体。从枯草芽孢杆菌168基因组中扩增得到46个信号肽,利用POE-PCR法,使46个信号肽分别与线性化的筛选载体形成对应的multimer产物,直接转化枯草芽孢杆菌1A751,得到含不同信号肽的重组菌株。发酵结果显示,除了5个与淀粉酶适配性很低的信号肽,其它信号肽均有不同的引导淀粉酶细胞外分泌的能力,其中bgls引导淀粉酶细胞外分泌的能力最强,上清酶活的峰值达1 393.3 U/m L。     

The gut fungus Basidiobolus ranarum has a large genome and different copy numbers of putatively functionally redundant elongation factor genes

Fungal genomes range in size from 2.3 Mb for the microsporidian Encephalitozoon intestinalis up to 8000 Mb for Entomophaga aulicae, with a mean genome size of 37 Mb. Basidiobolus, a common inhabitant of vertebrate guts, is distantly related to all other fungi, and is unique in possessing both EF-1α and EFL genes. Using DNA sequencing and a quantitative PCR approach, we estimated a haploid genome size for Basidiobolus at 350 Mb. However, based on allelic variation, the nuclear genome is at least diploid, leading us to believe that the final genome size is at least 700 Mb. We also found that EFL was in three times the copy number of its putatively functionally overlapping paralog EF-1α. This suggests that gene or genome duplication may be an important feature of B. ranarum evolution, and also suggests that B. ranarum may have mechanisms in place that favor the preservation of functionally overlapping genes.     

Insertion of eukaryotic DNA into the Bacillus subtilis genome by means of a temperature-sensitive plasmid vector

A hybrid temperature-sensitive plasmid capable of integration into the Bacillus subtilis genome was constructed. By using this vector, we inserted a 3.2-kb fragment of eukaryotic DNA (wheat 'Chinese Spring') into the bacterial genome. The fragment of wheat DNA was stably retained and replicated as a part of the bacterial genome. The position of the integrated plasmid in the B. subtilis genome was mapped, as was the site in wheat DNA insert on plasmid at which the integration occurred.     

An SfiI restriction map of the Bacillus subtilis 168 genome

A restriction map of 24 SfiI (GGCCN4/NGGCC) restriction fragments has been constructed for the Bacillus subtilis genome. The combined sizes of the fragments indicate a genome size of approx. 4.2 Mb. The SfiI fragments range in size from 7-730 kb. Genetic markers have been located on 19 of the SfiI fragments, and 69 genetic markers have been assigned to the SfiI restriction map.     

Targeted isolation of a designated region of the Bacillus subtilis genome by recombinational transfer

A method for positional cloning of the Bacillus subtilis genome was developed. The method requires a set of two small DNA fragments that flank the region to be copied. A 38-kb segment that carries genes ppsABCDE encoding five enzymes for antibiotic plipastatin synthesis and another genome locus as large as 100 kb including one essential gene were examined for positional cloning. The positional cloning vector for ppsABCDE was constructed using a B. subtilis low-copy-number plasmid that faithfully copied the precise length of the 38-kb DNA in vivo via the recombinational transfer system of this bacterium. Structure of the copied DNA was confirmed by restriction enzyme analyses. Furthermore, the unaltered structure of the 38-kb DNA was demonstrated by complementation of a ppsABCDE deletion mutant.     

Far different levels of gene expression provided by an oriented cloning system in Bacillus subtilis and Escherichia coli

A gene expression system for both Bacillus subtilis and Escherichia coli was developed. The expression vector, pHASH102, produces any combination of promoter and open reading frame to be expressed based on the T-extended cloning method. Because the pHASH series vectors are designed to shuttle between the genome and a high copy plasmid in B. subtilis, the expression profiles of copy number dependence can be examined systematically. We demonstrated that vectors with Pr, Pspac, and PS10 promoters are suitable for the overexpression of GFPuv. Moreover, aadK encoding aminoglycoside 6-adenylyltransferase (a streptomycin-resistance gene) of B. subtilis was successfully overexpressed in both B. subtilis and E. coli. These highly expressed GFPuv and aadK genes can be used as a genetic marker for both organisms.     

Serial assembly of Thermus megaplasmid DNA in the genome of Bacillus subtilis 168: a BAC-based domino method applied to DNA with a high GC content

Bacillus subtilis is the only bacterium-based host able to clone giant DNA above 1000 kbp. DNA previously handled by this host was limited to that with GC content similar to or lower than that of the B. subtilis genome. To expand the target DNA range to higher GC content, we tried to clone a pTT27 megaplasmid (257 kbp, 69% of G+C) from Thermus thermophilus. To facilitate the reconstruction process, we subcloned pTT27 in a bacterial artificial chromosome (BAC) vector of Escherichia coli. Owing to the ability of BAC to carry around 100 kbp DNA, only 4 clones were needed to cover the pTT27 and conduct step-by-step assembly in the B. subtilis genome. The full length of 257 kbp was reconstructed through 3 intermediary lengths (108, 153, and 226 kbp), despite an unexpected difficulty in the maintenance of DNA >200 kbp. Retrieval of these four pTT27 segments from the B. subtilis genome by genetic transfer to a plasmid pLS20 was attempted. A stable plasmid clone was obtained only for the 108 and 153 kbp intermediates. The B. subtilis genome was demonstrated to accommodate large DNA with a high GC content, but may be restricted to less than 200 kbp by unidentified mechanisms.     

Karyotype polymorphism and chromosomal rearrangement in populations of the phytopathogenic fungus, Ascochyta rabiei

The fungus Ascochyta rabiei is the causal agent of Ascochyta blight of chickpea and the most serious threat to chickpea production. Little is currently known about the genome size or organization of A. rabiei. Given recent genome sequencing efforts, characterization of the genome at a population scale will provide a framework for genome interpretation and direction of future resequencing efforts. Electrophoretic karyotype profiles of 112 isolates from 21 countries revealed 12–16 chromosomes between 0.9 Mb and 4.6 Mb with an estimated genome size of 23 Mb–34 Mb. Three general karyotype profiles A, B, and C were defined by the arrangement of the largest chromosomes. Approximately one-third of isolates (group A) possessed a chromosome larger than 4.0 Mb that was absent from group B and C isolates. The ribosomal RNA gene (rDNA) cluster was assigned to the largest chromosome in all except four isolates (group C) whose rDNA cluster was located on the second largest chromosome (3.2 Mb). Analysis of progeny from an in vitro sexual cross between two group B isolates revealed one of 16 progeny with an rDNA-encoding chromosome larger than 4.0 Mb similar to group A isolates, even though a chromosome of this size was not present in either parent. No expansion of the rDNA cluster was detected in the progeny, indicating the increase in chromosome size was not due to an expansion in number of rDNA repeats. The karyotype of A. rabiei is relatively conserved when compared with published examples of asexual ascomycetes, but labile with the potential for large scale chromosomal rearrangements during meiosis. The results of this study will allow for the targeted sequencing of specific isolates to determine the molecular mechanisms of karyotype variation within this species.     

Stable positional cloning of long continuous DNA in the Bacillus subtilis genome vector

Direct cloning of a long continuous genome segment in a Bacillus subtilis genome vector was demonstrated for the first time. Two small DNA fragments had to be installed in the vector prior to cloning. The DNA between these two fragments was cloned via homologous recombination. The efficiency of cloning was estimated using the 3,573-kb genome of a cyanobacterium, Synechocystis sp. PCC 6803. Recombinants were selected using the internal selection system of the Bacillus genome vector or with the antibiotic resistance marker in the cyanobacterial genome. Designated genomic segments as large as 77-kb were cloned by means of a single procedure. Cloning efficiency is affected by the molecular weight of the donor DNA and the size of the DNA to be cloned. The method is suitable for direct target cloning of large-sized DNA.     

Recombinational transfer of 100-kilobase genomic DNA to plasmid in Bacillus subtilis 168

Transformation of Bacillus subtilis by a plasmid requires a circular multimeric form. In contrast, linearized plasmids can be circularized only when homologous sequences are present in the host genome. A recombinational transfer system was constructed with this intrinsic B. subtilis recombinational repair pathway. The vector, pGETS103, a derivative of the theta-type replicating plasmid pTB19 of thermophilic Bacillus, had the full length of Escherichia coli plasmid pBR322. A multimeric form of pGETS103 yielded tetracycline-resistant transformants of B. subtilis. In contrast, linearized pGETS103 gave tetracycline-resistant transformants only when the recipient strain had the pBR322 sequence in the genome. The efficiency and fidelity of the recombinational transfer of DNAs of up to 90 kb are demonstrated.     

A new method for multiple gene inactivations in Bacillus subtilis 168, producing a strain free of selectable markers

This study describes a novel method for repeated gene inactivation in Bacillus subtilis 168. A B. subtilis strain (BS-PS) that is conditionally auxotrophic for lysine was obtained by replacing the PlysA promoter with the Pspac promoter. The homologous recombination integration vector PLC-T was constructed to contain lacI, which encodes a Pspac promoter repressor, and the chloromycetin resistance gene. Target genes were manipulated by generating an insertion sequence with two homologous arms and the target gene in PLC-T to create a specific integrating vector. Integration into the BS-PS chromosome occurred by a single crossover at either of the two homologous arms. The resulting transitional strain (BS-PS-PI) was chloromycetin resistant and lysine auxotrophic and had an unstable genome structure because of the duplication. Excision of lacI and chloromycetin resistance gene was achieved by a second single crossover at the duplication. Recovery of a lysine prototroph functioned as counter-selection and was identified by PCR. In this work, we inactivated nprE and aprE, two protease genes secreted by B. subtilis 168 free of selectable markers.     

Direct cloning of full-length mouse mitochondrial DNA using a Bacillus subtilis genome vector

The complete mouse mitochondrial genome (16.3 kb) was directly cloned into a Bacillus subtilis genome (BGM) vector. Two DNA segments of 2.06 and 2.14 kb that flank the internal 12 kb of the mitochondrial DNA (mtDNA) were subcloned into an Escherichia coli plasmid. Subsequent integration of the plasmid at the cloning locus of the BGM vector yielded a derivative specific for the targeted cloning of the internal 12-kb mtDNA region. The BGM vector took up mtDNA purified from mouse liver and integrated it by homologous recombination at the two preinstalled mtDNA-flanking sequences. The complete cloned mtDNA in the BGM vector was converted to a covalently closed circular (ccc) plasmid form via gene conversion in B. subtilis. The mtDNA carried on this plasmid was then isolated and transferred to E. coli. DNA sequence fidelity and stability through the BGM vector-mediated cloning process were confirmed.     

Cloning of Pf3, a filamentous bacteriophage of Pseudomonas aeruginosa, into the pBD214 vector of Bacillus subtilis.

The genome of Pf3, a filamentous single-stranded DNA bacteriophage of Pseudomonas aeruginosa (a gram-negative organism) was cloned into pBD214, a plasmid cloning vector of Bacillus subtilis (a gram-positive organism). Cloning in the gram-positive organism was done to avoid anticipated lethal effects. The entire Pf3 genome was inserted in each orientation at a unique Bc/I site within a thymidylate synthetase gene (from B. subtilis phage beta 22) on the plasmid. Additional clones were made by inserting EcoRI fragments of Pf3 DNA into a unique EcoRI site within this gene.     

DNA shuttling between plasmid vectors and a genome vector: systematic conversion and preservation of DNA libraries using the Bacillus subtilis genome (BGM) vector

The combined use of the contemporary vector systems, the bacterial artificial chromosome (BAC) vector and the Bacillus subtilis genome (BGM) vector, makes possible the handling of giant-length DNA (above 100 kb). Our newly constructed BGM vector efficiently integrated DNA prepared in the BAC vector. A BAC library comprised of 18 independent clones prepared from mitochondrial DNA (mtDNA) of Arabidopsis thaliana was converted to a parallel BGM library using the new BGM vector. The effectiveness of the combined use of the vector systems was confirmed by the stable recovery of all 18 DNAs as BAC clones from the respective BGM clones. We show that DNA in BGM was stably preserved at room temperature after spore formation of the host B.subtilis. Rapid and stable shuttling between Escherichiacoli and the B. subtilis host, combined with spore-mediated DNA storage, may facilitate the long-term and low-cost preservation and the transportation of DNA resources.     

Insertion of transformation vector DNA into different chromosomal sites of Dictyostelium discoideum as determined by pulse field electrophoresis

Chromosomes of the cellular slime mold Dictyostelium discoideum were fractionated on three pulse field gel electrophoresis systems (pulse field, orthogonal field and C.H.E.F. (Contour-clamped Homogeneous Electric Fields] into a series of 13 bands ranging from 0.1 Mb to over 2 Mb in size. Since this organism has only seven chromosomes (estimated to be 1-10 Mb), and -90 copies of an 88-kilobase linear ribosomal DNA molecule (14% of genome), it was apparent that not all of these bands were whole chromosomes. However these bands were reproducibly obtained with the cell preparation used. They fell into three categories: i) four large poorly resolved DNA molecules (-2 Mb in size) which represent very large fragments or intact chromosomes, ii) eight faint bands ranging from 0.1 Mb to 2 Mb, iii) a prominent band in the apparent size range of about 0.15 Mb. Cloned Fragment V of an EcoR1 digest of the ribosomal DNA, hybridized to the 0.15 Mb band indicating it contained the linear ribosomal DNA. This chromosomal banding pattern was used to examine the stability and location of vector DNA in 16 transformed strains of D. discoideum. Each transformed strain was initially selected on the basis of G418 resistance with an integrating vector containing pBR322 sequences. Eleven transformants still carried pBR322 sequences after more than 60 generations of growth without selection on G418. All four strains transformed with constructs containing regions of the D. discoideum plasmid Ddp1 had lost their pBR322 insert, indicating that integration of Dictyostelium plasmid DNA into chromosomes leads to instability. Orthogonal field electrophoresis of the eleven strains still carrying pBR322 sequences revealed at least seven different integrating sites for the transforming DNA. We conclude that these vectors have many possible sites of integration in the D. discoideum genome.     

Differences in chromosome number and genome rearrangements in the genus Brucella

We have studied the genomic structure and constructed the Spe I, Pac I and I- Ceu I restriction maps of the four biovars of the pathogenic bacterium Brucella suis . B . suis biovar 1 has two chromosomes of 2.1 Mb and 1.15 Mb, similar to those of the other Brucella species: B . melitensis , B . abortus , B . ovis and B . neotomae . Two chromosomes were also observed in the genome of B . suis biovars 2 and 4, but with sizes of 1.85 Mb and 1.35 Mb, whereas only one chromosome with a size of 3.1 Mb was found in B . suis biovar 3. We show that the differences in chromosome size and number can be explained by rearrangements at chromosomal regions containing the three rrn genes. The location and orientation of these genes confirmed that these rearrangements are due to homologous recombination at the rrn loci. This observation allows us to propose a scheme for the evolution of the genus Brucella in which the two chromosome-containing strains can emerge from an hypothetical ancestor with a single chromosome, which is probably similar to that of B . suis biovar 3. As the genus Brucella is certainly monospecific, this is the first time that differences in chromosome number have been observed in strains of the same bacterial species.     

Bottom-up genome assembly using the Bacillus subtilis genome vector

We established a protocol to construct complete recombinant genomes from their small contiguous DNA pieces and obtained the genomes of mouse mitochondrion and rice chloroplast using a B. subtilis genome (BGM) vector. This method allows the design of any recombinant genomes, valuable not only for fundamental research in systems biology and synthetic biology but also for various applications in the life sciences.