A quantitative analysis of shotgun cloning in Bacillus subtilis protoplasts

Summary We used the Escherichia coli-Bacillus subtilis shuttle vector pHP13, which carries the replication functions of the cryptic B. subtilis plasmid pTA1060, to study the effects of BsuM restriction, plasmid size and DNA concentration on the efficiency of shotgun cloning of heterologous E. coli DNA in B. subtilis protoplasts. In a restriction-deficient strain, clones were obtained with low frequency (19% of the transformants contained a recombinant plasmid) and large inserts (>6 kb) were relatively rare (12% of the clones contained inserts in the range of 6–9 kb). The efficiency of shotgun cloning was severely reduced in restricting protoplasts: the class of large inserts (>6 kb) was under-represented in the clone bank (4% of the clones contained inserts in the range of 6–6.1 kb). Furthermore, BsuM restriction caused structural instability of some recombinant plasmids. Transformation of protoplasts with individual recombinant plasmids showed that plasmid size and transforming activity were negatively correlated. The size effect was most extreme with cut and religated plasmid DNA. The yield of clones was independent of the DNA concentration during transformation. It is therefore unlikely that clones were not detected because of simultaneous uptake of more than one plasmid. It is concluded that shotgun cloning in B. subtilis protoplasts is inferior to that in competent cells.     

The first high frequency of recombination-like conjugal transfer from an integrated origin of transfer sequence in Bacillus subtilis 168

Bacillus subtilis 168 was developed as a genome vector to manipulate large DNA fragments. The system is based on the inherent natural transformation (TF) activity. However, DNA size transferred by TF is limited up to approximately 100 kb. A conjugal transfer system capable of transferring DNA fragments considerably larger than those transferred by TF was developed. A well-defined oriT¹¹⁰ sequence and a cognate relaxase gene from the pUB110 plasmid were inserted into the xkdE gene of the B. subtilis genome. Transfer of antibiotic resistance markers distant from the oriT¹¹⁰ locus to the recipient B. subtilis occurred only in the presence of pLS20, a helper plasmid that provides a type IV secretion system. Marker transmission was consistent with the orientation of oriT¹¹⁰ and required a recA-proficient recipient. The first conjugal transfer system of genomic DNA should provide a valuable alternative genetic tool for editing the B. subtilis genome.     

Cloning and expression in Escherichia coli of two DNA fragments from Bacillus sphaericus encoding mosquito-larvicidal activity

A library of Bacillus sphaericus 1593 DNA was constructed in Escherichia coli using pBR322 as vector and screened for clones expressing larvicidal activity against Culex mosquito larvae. Two larvicidal clones were identified and their plasmids characterized by restriction mapping. pAS233 and pAS377 contained inserts of 8.6 and 15 kb which were reduced by subcloning to 3.6 and 4.3 kb, respectively. A peptide of 29 kDa was the single product detected by maxicell expression of pAS377PT, a plasmid subcloned from pAS377. No insert-encoded peptide could be detected for pAS233HA, a subclone of pAS233, although maxicells containing this plasmid encoded larvicidal activity. The insert of pAS377PT was transcribed from a vector promoter whereas the insert of pAS233HA was transcribed from its own promoter and hence its expression in B. subtilis was possible. The insert was ligated to a shuttle vector yielding pSVI which was then used to transform B. subtilis. Recombinant E. coli and B. subtilis clones showed equivalent larvicidal activity of 1–10 μg cell protein per ml. Larvicidal activity was observed during vegetative growth for recombinant B. subtilis even though B. sphaericus 1593 synthesizes its mosquito-toxin only during sporulation.     

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.     

The effect of restriction on shotgun cloning and plasmid stability in Bacillus subtilis Marburg

Summary Using the bifunctional cloning vehicle pHP13, which carries the replication functions of the cryptic Bacillus subtilis plasmid pTA1060, the effects of BsuM restriction on the efficiency of shotgun cloning of heterologous Escherichia coli DNA were studied. In a restriction-deficient but modification-proficient mutant of B. subtilis, clones were obtained at a high frequency, comparable to frequencies normally obtained in E. coli (10⁴ clones per g target DNA). Large inserts were relatively abundant (26% of the clones contained inserts in the range of 6 to 15 kb), which resulted in a high average insert length (3.6 kb). In the restriction-proficient B. subtilis strain, the class of large inserts was underrepresented. Transformation of B. subtilis with E. coli-derived individual recombinant plasmids was affected by BsuM restriction in two ways. First, the transforming activities of recombinant plasmids carrying inserts larger than 4 kb, were, in comparison with the vector pHP13, reduced to varying degrees in the restricting host. The levels of the reduction increased with insert length, resulting in a 7800-fold reduction for the largest plasmid used (pC23; insert length 16 kb). Second, more than 80% of the pC23 transformants in the restricting strain contained a deleted plasmid. In the non-restricting strain, the transforming activities of the plasmids were fairly constant as a function of insert length (in the range of 0–16 kb), and no structural instability was observed. It is concluded that for shotgun cloning in B. subtilis, the use of restriction-deficient strains is highly preferable. Evidence is presented that in addition to XhoI other sequences are involved in BsuM restriction. It is postulated that AsuII sites are additional target sites for BsuM restriction.     

Sequential Insertion of Multiple I-SceI Recognition Sites at Designed Loci of the Bacillus subtilis 168 Genome

Bacillus subtilis 168 is the only bacterium-based host serving for the cloning of giant DNA above 1.000 kbp. As rapid verification of the genome structure is crucial during the cloning process, six of 18-base sequence recognized by endonuclease I-SceI were sequentially created in the B. subtilis 168 genome. The established method and materials should be of use for other B. subtilis derivatives.     

An inducible recA expression Bacillus subtilis genome vector for stable manipulation of large DNA fragments

Background

The Bacillus subtilis genome (BGM) vector is a novel cloning system based on the natural competence that enables B. subtilis to import extracellular DNA fragments into the cell and incorporate the recombinogenic DNA into the genome vector by homologous recombination. The BGM vector system has several attractive properties, such as a megabase cloning capacity, stable propagation of cloned DNA inserts, and various modification strategies using RecA-mediated homologous recombination. However, the endogenous RecA activity may cause undesirable recombination, as has been observed in yeast artificial chromosome systems. In this study, we developed a novel BGM vector system of an inducible recA expression BGM vector (iREX), in which the expression of recA can be controlled by xylose in the medium.

Results

We constructed the iREX system by introducing the xylose-inducible recA expression cassette followed by the targeted deletion of the endogenous recA. Western blot analysis showed that the expression of recA was strictly controlled by xylose in the medium. In the absence of xylose, recA was not expressed in the iREX, and the RecA-mediated recombination reactions were greatly suppressed. By contrast, the addition of xylose successfully induced RecA expression, which enabled the iREX to exploit the same capacities of transformation and gene modifications observed with the conventional BGM vector. In addition, an evaluation of the stability of the cloned DNA insert demonstrated that the DNA fragments containing homologous sequences were more stably maintained in the iREX by suppressing undesirable homologous recombination.

Conclusions

We developed a novel BGM vector with inducible recA expression system, iREX, which enables us to manipulate large DNA fragments more stably than the conventional BGM vector by suppressing undesirable recombination. In addition, we demonstrate that the iREX can be applied to handling the DNA, which has several homologous sequences, such as multiple-reporter expression cassettes. Thus, the iREX expands the utility of the BGM vector as a platform for engineering large DNA fragments.

Electronic supplementary material

The online version of this article (doi:10.1186/s12864-015-1425-4) contains supplementary material, which is available to authorized users.     

Construction and characterization of the IGF Arabidopsis BAC library

A bacterial artificial chromosome (BAC) library has been established for Arabidopsis thaliana (ecotype Col-0) covering about seven haploid nuclear genome equivalents. This library, called the Institut für Genbiologische Forschung (IGF) BAC library, consists of 10?752 recombinant clones carrying inserts (generated by partial EcoRI digestion) of an average size of about 100?kb in a modified BAC vector, pBeloBAC-Kan. Hybridization with organellar DNA and nuclear repetitive DNA elements revealed the presence of 1.1% clones with mitochondrial DNA, 0.2% clones with plastid DNA, 3.2% clones with the 180?bp paracentromeric repeat, 1.6% clones with 5S rDNA, and 10.8% clones with the 18S-25S rDNA repeat. With its extensive genome coverage, its rather uniformly sized inserts (80?kb?<85% <120?kb) and low contamination with organellar DNA, this library provides an excellent resource for A. thaliana genomic mapping, map-based gene cloning, and genome sequencing.     

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.     

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.     

Molecular characterization of the genome of Bacillus subtilis (natto) bacteriophage PM1, a phage associated with disruption of food production

“Natto”, regarded as a traditional food, is made by fermenting boiled soybeans with Bacillus subtilis (natto), which is a natto-producing strain related to B. subtilis. Natto production is disrupted by bacteriophage infection of B. subtilis (natto); thus, it is necessary to control bacteriophage infection. A bacteriophage of B. subtilis (natto), PM1, was isolated during interrupted natto production in a factory. As PM1 was shown to have a long non-contractile tail in a morphological study, it was believed to belong to the family Siphoviridae. The genome of PM1 was shown to be a linear double-stranded DNA of approximately 50 kb. Based on the results of studies using restriction endonucleases, PM1 DNA was found to be circularly permuted, similar to bacteriophage DNA without definite ends (e.g. bacteriophage T4). The nucleotide sequence of a 1.1 kb segment of PM1 was determined and used to design a PCR assay. A 0.5 kb product was amplified from eight of ten bacteriophage isolates that infect B. subtilis (natto), and the nucleotide sequences of the PCR-amplified products were identical to those of PM1, suggesting that PM1-related bacteriophages are the most prevalent infectious agents associated with the disruption of natto production. The PCR method might be useful to detect PM1-related bacteriophages and will help to control bacteriophage infection.     

A BAC Library Constructed to the Rice Cultivar

"Minghui 63" is the restorer line for a number of the most important commercial rice hybrids varieties in China. To facilitate long-term commitment in genetic analysis and molecular cloning of the superior genes in the genome of "Minghui 63", the authors have constructed a largeinsert genomic DNA library using the bacterial artificial chromosome (BAC) cloning vector (pBe- loBAC 11). Size fractionated Hind m digest of genomic DNA was ligated to the BAC vector, and the ligation mixture was used to transform the bacterial strain DH10B. A total of over 26 000 clones were obtained with the average insert size of about 150 kb, ranging from 90 to 240 kb. These clones thus represent 9 x rice haploid genome equivalents. The library is now being used for physical mapping of several genomic regions for map-based gene cloning.     

Construction of hybrid plasmid vectors for cloning inEscherichia coli,Bacillus subtilis,and the cyanobacteriumAnacystis nidulans

Two hybrid plasmids capable of acting as shuttle cloning vectors inAnacystis nidulans andBacillus subtilis were constructed by in vitro ligation. One construct, pMG202, consists of theB. subtilis vector pNN101 and the endogenous cyanobacterial plasmid pUH24. This 14.6 kb plasmid confers chloramphenicol resistance in both hosts and tetracycline resistance inB. subtilis. A second vector, pMG101, consists of pNN101 linked to theA. nidulans-Escherichia coli chimeric plasmid pCB4 and is 12.9 kb in size. The pCB4 portion of the vector enables pMG101 to replicate in the third host,E. coli, and confers ampicillin resistance in this bacterium as well as inA. nidulans. Both plasmids possess identical uniqueStu I sites which permit insertional inactivation of the chloramphenicol resistance gene; and, in addition, identical uniqueXho I sites are present on both vectors. Each vector also has a third unique site:Sma I on pMG101 andXba I on pMG202.     

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.     

Fast genome editing in Bacillus subtilis

Bacillus subtilis is a model organism for Gram‐positive bacteria and widely used in the study of cellular functions and processes including protein secretion, sporulation, and signal transduction. It is also an important industrial host for the production of proteins and chemicals. Generally, genome editing of B. subtilis often needs the construction of integration vectors in Escherichia coli, linearizing the constructed plasmids, and subsequent transformation of the linear deoxyribonucleic acid via natural competence or electroporation. In this work, we examined the feasibility to directly transform and integrate B. subtilis using linear deoxyribonucleic acid from Gibson assembly without the need for cloning in E. coli. Linear deoxyribonucleic acid of 8–10 kb showed the highest transformation efficiency which was similar to that of using linearized plasmids constructed in E. coli. This method shortens the overall process from 1 week to 1 day and allows the integration of multiple genes in one step, providing a simple and fast method for genome editing in B. subtilis.     

Complete genome sequence of the industrial bacterium Bacillus licheniformis and comparisons with closely related Bacillus species

Background

Bacillus licheniformis is a Gram-positive, spore-forming soil bacterium that is used in the biotechnology industry to manufacture enzymes, antibiotics, biochemicals and consumer products. This species is closely related to the well studied model organism Bacillus subtilis, and produces an assortment of extracellular enzymes that may contribute to nutrient cycling in nature.

Results

We determined the complete nucleotide sequence of the B. licheniformis ATCC 14580 genome which comprises a circular chromosome of 4,222,336 base-pairs (bp) containing 4,208 predicted protein-coding genes with an average size of 873 bp, seven rRNA operons, and 72 tRNA genes. The B. licheniformis chromosome contains large regions that are colinear with the genomes of B. subtilis and Bacillus halodurans, and approximately 80% of the predicted B. licheniformis coding sequences have B. subtilis orthologs.

Conclusions

Despite the unmistakable organizational similarities between the B. licheniformis and B. subtilis genomes, there are notable differences in the numbers and locations of prophages, transposable elements and a number of extracellular enzymes and secondary metabolic pathway operons that distinguish these species. Differences include a region of more than 80 kilobases (kb) that comprises a cluster of polyketide synthase genes and a second operon of 38 kb encoding plipastatin synthase enzymes that are absent in the B. licheniformis genome. The availability of a completed genome sequence for B. licheniformis should facilitate the design and construction of improved industrial strains and allow for comparative genomics and evolutionary studies within this group of Bacillaceae.     

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.     

Bacillus subtilis genome project: cloning and sequencing of the 97 kb region from 325° to 333deg;

In the framework of the European project aimed at the sequencing of the Bacillus subtilis genome the DNA region located between gerB (314°) and sacXV (333°) was assigned to the Institut Pasteur. In this paper we describe the cloning and sequencing of a segment of 97 kb of contiguous DNA. Ninety-two open reading frames were predicted to encode putative proteins among which only forty-two were found to display significant similarities to known proteins present in databanks, e.g. amino acid permeases, proteins involved in cell wall or antibiotic biosynthesis, various regulatory proteins, proteins of several dehydrogenase families and enzymes II of the phosphotransferase system involved in sugar transport. Additional experiments led to the identification of the products of new B. subtilis genes, e.g. galactokinase and an operon involved in thiamine biosynthesis.