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.     

Overproduction and secretion of Bacillus circulans endo-β-1,3-1,4-glucanase gene (bglBC1) in B. subtilis and B. megaterium

A gene coding for endo--1,3-1,4-glucanase (lichenase) containing a recombinant plasmid, pLL200K, was transferred from Bacillus circulans into a new shuttle plasmid, pLLS920, by ligating linearized DNAs of pLL200K and pUB110. B. subtilis RM125 and B. megaterium ATCC14945 transformed with pLLS920 produced the endo--1,3-1,4-glucanase. The enzyme was produced during active growth with maximum activity. The B. subtilis (pLLS920) enzyme was 83 times (8522 mU ml^–1) more active than that of the gene donor cells (103 mU ml^–1). The B. megaterium (pLLS920) enzyme was 7 times (735 mU ml^–1) more active than that of the gene donor cells. While E. coli secreted only about 10% of the produced enzyme, B. subtilis excreted the enzyme completely into the medium and B. megaterium by about 98%. The plasmid pLLS920 was stable in B. megaterium (98%), and in B. subtilis (51%) but not in E. coli (29%).     

Marker rescue transformation by linear plasmid DNA in Bacillus subtilis

Although plasmid-free Bacillus subtilis cannot be transformed for markers carried by linear or nicked plasmid DNA, a resident plasmid can rescue a marker on such damaged DNA under certain conditions. Linearized chimeric plasmid DNA has been used to transform cultures carrying a resident plasmid which is homologous with a portion of the donor. This system has revealed the following properties of the marker rescue process: (1) It is recE dependent. (2) It requires the presence in the resident plasmid of sequences which are homologous to the donor. (3) When the selected marker is on a nonhomologous segment it must be flanked by segments which are homologous to the resident plasmid. (4) The efficiency of rescue varies in a regular way with the position of the linearizing cut. (5) Marker rescue is first order with respect to DNA concentration. These properties and other data are interpreted as providing a strong indication that marker rescue occurs by recombination, although an alternative explanation involving recE-dependent recircularization of the donor plasmid has not been eliminated. Our results also suggest that if the major pathway of marker rescue is by recombination, an average of 0.15 Mdal (single strand) must be removed from each donor DNA molecule or otherwise rendered unavailable for recombination and that the exchange frequency during transformational recombination is approximately 0.2 to 0.5 Mdal⁻¹.     

Cloning and expression of the gene for neutral protease of Bacillus amyloliquefaciens in Bacillus subtilis

A Bacillus amyloliquefaciens neutral protease gene was cloned and expressed in Bacillus subtilis.The chromosomal DNA of B. amyloliquefaciens strain F was partially digested with restriction endonuclease Sau3AI, and 2 to 9 kb fragments isolated were ligated into the BamHI site of plasmid pUB110. Then, B. subtilis strain 1A289 was transformed with the hybrid plasmids by the method of protoplast transformation and kanamycin-resistant transformants were screened for the formation of large halo on a casein plate. A transformant that produced a large amount of an extracellular neutral protease harbored a plasmid, designated as pNP150, which contained a 1.7 kb insert.The secreted neutral protease of the transformant was found to be indistinguishable from that of DNA donor strain B. amyloliquefaciens by double immunodiffusion test and SDS-polyacrylamide gel electrophoresis.The amount of the neutral protease activity excreted into culture medium by the B. subtilis transformed with pNP150 was about 50-fold higher than that secreted by B. amyloliquefaciens. The production of the neutral protease in the transformant was partially repressed by addition of glucose to the medium.     

Construction of a colony bank of E. coli containing hybrid plasmids representative of the Bacillus subtilis 168 genome. Expression of functions harbored by the recombinant plasmids in B. subtilis.

Summary A collection of about 2500 clones containing hybrid plasmids representative of nearly the entire genome of B. subtilis 168 was established in E. coli SK1592 by using the poly(dA)·poly(dT) joining method with randomly sheared DNA fragments and plasmid pHV33, a bifunctional vector which can replicate in both E. coli and B. subtilis. Detection of cloned recombinant DNA molecules was based on the insertional inactivation of the Tc gene occurring at the unique BamHI cleavage site present in the vector plasmid.Thirty individual clones of the collection were shown to hybridize specifically with a B. subtilis rRNA probe. CCC-recombinant plasmids extracted from E. coli were pooled in lots of 100 and used to transform auxotrophic mutants of B. subtilis 168. Complementation of these auxotrophic mutations was observed for several markers such as thr, leuA, hisA, glyB and purB. In several cases, markers carried by the recombinant plasmids were lost from the plasmid and integrated into the chromosomal DNA. Loss of genetic markers from the hybrid plasmids did not occur when a rec ^- recipient strain of B. subtilis was used.Abbreviations Ap^R resistance to ampicillin - Tc^R resistance to tetracycline - Cm^R resistance to chloramphenicol - CCC covalently closed circular duplex - Mdal magadalton     

Across Genus Plasmid Transformation Between <Emphasis Type="Italic">Bacillus subtilis</Emphasis> and <Emphasis Type="Italic">Escherichia coli</Emphasis> and the Effect of <Emphasis Type="Italic">Escherichia coli</Emphasis> on the Transforming Ability of Free Plasmid DNA

The results presented in this article show that direct plasmid transfer from Escherichia coli carrying shuttle plasmid to Bacillus subtilis occurred when close contact between the two species was established by mixing E. coli and B. subtilis onto selective agar plates. The data demonstrate that the production of resistant colonies by plasmid transformation through cell contact was DNase I sensitive and dependent on transformable B. subtilis strains. Furthermore, another observation indicated that the E. coli strain is able to affect the transformation capability of B. subtilis. It is assumed that the donor strain is a momentous factor for taking up plasmid DNA. This conclusion is significant in the assessment of both the possibility of intercellular DNA transfer in natural habitats of micro-organisms and the risk of the application of genetically engineered micro-organisms.     

High Frequency Conjugative Mobilization Accomplished by a Natural Bacillus subtilisStrain Carrying a Large Plasmid

Conjugative properties of the strain Bacillus subtiliscarrying a large plasmid approximately 95 kb in size and isolated in Belarus from forest soil were described. The staphylococcal plasmid pUB110 that had previously been introduced into this strain was transferred to recipient cells of the Bacillus subtilis168 strain with a frequency of approximately 10^–2. The transfer occurred with approximately the same frequency both upon donor and recipient cell contact on the surface of membranes and in a liquid medium. The latter fact makes this system suitable as a model for studying conjugative mobilization in bacilli. A large plasmid cannot be transferred to recipients. An optimal temperature for conjugation of donor and recipient cells was 37°C, but conjugation also proceeded at lower temperatures, up to 21°C.     

Cloning of Bacillus subtilis leucina A, B and C genes with Escherichia coli plasmids and expression of the leuC gene in E. coli.

Summary The leucine genes of Bacillus subtilis have been cloned directly from the chromosomal DNA into Escherichia coli leuB cells by selection for the Leu⁺ phenotype using RSF2124 as a vector plasmid. The hybrid plasmid designated RSF2124-B·leu contained a 4.2 megadalton fragment derived from B. subtilis DNA, including the leu genes. The fragment had one site susceptible to EcoRI^* and another site susceptible to BamNI endonuclease. Among the three fragments produced by EcoRI^* and BamNI endonucleases, the 1.2 megadalton fragment had the ability to transform B. subtilis leuA, leuB and leuC auxotrophs to leu ⁺. However, B. subtilis ilvB and ilvC auxotrophs were not rescued even by the whole 4.2 megadalton fragment present in the hybrid plasmid. -Isopropylmalate dehydrogenase (leuB gene product) activity found in E. coli cells containing the hybrid plasmid was about 60% of that in E. coli wild type cells, despite the high copy number (7.8) of the plasmid per chromosome observed.     

Genetic and molecular events in transformation ofHaemophilus influenzae with plasmid RSF 0885 carrying cloned segments of chromosomal DNA

InHaemophilus influenzae genetic transformation for a plasmid marker is significantly increased when recombinant plasmid RSF 0885 DNA carrying chromosomal DNA segments is used instead of the plasmid DNA alone. Chromosomal DNA by itself, added even a few minutes after the addition of plasmid DNA to competent cells, stopped further uptake of the plasmid DNA. These observations are consistent with the idea that plasmid RSF 0885 contains a ‘degenerate’ version of the required eleven base-pair ‘uptake sequence’ inHaemophilus. The transformation activity of the recombinant plasmid DNA is recoverable after its entry into cells, although the specific biological activity of the re-isolated plasmid DNA is less than that of the parental recombinant plasmid DNA. Therec 1 gene function of the host is necessary for obtaining higher transformation frequencies with recombinant DNA from five different clones. The reduced transformation frequencies seen inrec 1 ^- strain is not all due to a permanent damage to the donor DNA since the recovered recombinant plasmid DNA from such cells can increase the transformation efficiency onrec 1 ⁺ strain.     

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.     

Optimum Conditions for Electric Pulse-mediated Gene Transfer to Bacillus subtilis Cells

It was found that plasmid DNA (pUB 110) can be introduced into not only protoplasts but also intact cells of Bacillus subtilis by electric field pulses. The transformation of, B. subtilis using protoplasts results in an efficiency of 2.5 × 10⁴ transformants per μg of DNA, with a single pulse of 50 jisec with an initial electric field strength of 7kV/cm. Even transformation of intact B. subtilis cells results in a maximum efficiency of 1.5 × 10³ transformants per μg DNA, with a single pulse of 400 μsec with an initial electric field strength of 16kV/cm. The cell survival of protoplasts and intact cells was approximately 100% and 30%, respectively, under the conditions found to be optimal for the transformation process. Plasmid DNA isolated from pUB 110 containing transformants was indistinguishable from authentic preparations of pBU 110 on gel electrophoretic analysis.     

DNA repair and the evolution of transformation IV. DNA damage increases transformation

Natural genetic transformation in the bacterium Bacillus subtilis provides a model system to explore the evolutionary function of sexual recombination. In the present work, we study the response of transformation to UV irradiation using donor DNAs that differ in sequence homology to the recipient's chromosome and in the mechanism of transformation. The four donor DNAs used include homologous-chromosomal-DNA, two plasmids containing a fragment of B. subtilis trp⁺ operon DNA and a plasmid with no sequence homology to the recipient cell's DNA. Transformation frequencies for these DNA molecules increase with increasing levels of DNA damage (UV radiation) to recipient cells, only if their transformation requires homologous recombination (i.e. is recA⁺-dependent). Transformation with non-homologous DNA is independent of the recipient's recombination system and transformation frequencies for it do not respond to increases in UV radiation. The transformation frequency for a selectable marker increases in response to DNA damage more dramatically when the locus is present on small, plasmid-borne, homologous fragments than if it is carried on high molecular weight chromosomal fragments. We also study the kinetics of transformation for the different donor DNAs. Different kinetics are observed for homologous transformation depending on whether the homologous locus is carried on a plasmid or on chromosomal fragments. Chromosomal DNA- and non-homologous-plasmid-DNA-mediated transformation is complete (maximal) within several minutes, while transformation with a plasmid containing homologous DNA is still occurring after an hour. The results indicate that DNA damage directly increases rates of homologous recombination and transformation in B. subtilis. The relevance of these results and recent results of other labs to the evolution of transformation are discussed.     

Cloning and expression of Bacillus subtilis spore genes

Summary Two spore genes, spoOB and spoIIG have been cloned from the B. subtilis genome library, constructed by ligating Sau3A partially digested DNA to the dephosphorylated pHV33 plasmid vector at its BamH1 site.An hybrid plasmid pGsOB2, carrying a 1.7 Kb insert of B. subtilis DNA amplifiable in E. coli was cloned. This recombinant plasmid was capable of transforming the appropriate B. subtilis Rec⁺ and Rec^- recipients to Spo⁺ at very high efficiency. The pGsOB2 was further subcloned and four hybrid plasmids, pGsOB8, pGsOB9, pGsOB10 and pGsOB11 were selected and their restriction enzyme maps established. The four subcloned hybrid plasmids retained their entire transforming activity in both Rec⁺ and Rec^- recipients although two of them carry the insert in an inverse orientation, indicating thus, that the spoOB gene in these plasmids is being transcribed by the B. subtilis RNA polymerase using an internal promotor of the cloned DNA fragment. The adjacent genes spoIVF and pheA, mapped respectively to the right and left of the spoOB locus, that normally show 90% cotransformation, are absent on the cloned DNA fragments. The cloned hybrid plasmids have been expressed in E. coli minicells and it was shown that the spoOB locus encoded a polypeptide of 24 K.We have also cloned the spoIIG gene in two hybrid plasmids, pGsIIG24 and pGsIIG26, carrying respectively inserts of 2 and 3 Kb. From the transforming activity and the endonuclease cleavage maps it was shown that these two hybrid plasmids do not carry the entire spoIIG locus. The use of these plasmids for further cloning of this gene is discussed.     

Restriction of plasmid-mediated transformation in Bacillus subtilis 168.

Summary When plasmids carrying leucine genes of Bacillus subtilis 168 were isolated from a restriction and modification deficient (r^-m^-) strain and used for transformation of a restricting strain B. subtilis 168 leu recE4, the number of transformants was greatly reduced. Transformation of a rec ⁺ strain (transformation by integration of the donor DNA into the chromosome) with the plasmids was not affected irrespective of whether the recipient carried the r⁺ or r^- phenotype. These results show that the plasmid-mediated transformation is subject to the host controlled restriction and suggest that r^-m^- strains should be used for construction of recombinant DNA molecules in B. subtilis 168.     

Simple,fast and high‐efficiency transformation system for directed evolution of cellulase in Bacillus subtilis

Bacillus subtilis can serve as a powerful platform for directed evolution, especially for secretory enzymes. However, cloning and transformation of a DNA mutant library in B. subtilis are not as easy as they are in Escherichia coli. For direct transformation of B. subtilis, here we developed a new protocol based on supercompetent cells prepared from the recombinant B. subtilis strain SCK6 and multimeric plasmids. This new protocol is simple (restriction enzyme‐, phosphatase‐ and ligase‐free), fast (i.e. 1 day) and of high efficiency (i.e. ～10⁷ or ～10⁴ transformants per µg of multimeric plasmid or ligated plasmid DNA respectively). Supercompetent B. subtilis SCK6 cells were prepared by overexpression of the competence master regulator ComK that was induced by adding xylose. The DNA mutant library was generated through a two‐round PCR: (i) the mutagenized DNA fragments were generated by error‐prone PCR and linearized plasmids were made using high‐fidelity PCR, and (ii) the multimeric plasmids were generated based on these two DNA templates by using overlap PCR. Both protein expression level and specific activity of glycoside hydrolase family 5 endoglucanse on regenerated amorphous cellulose were improved through this new system. To our limited knowledge, this study is the first report for enhancing secretory cellulase performance on insoluble cellulose.     

Integration of vector-containing Bacillus subtilis chromosomal DNA by a Campbell-like mechanism

Summary Using plasmid pHV60, which contains a chloramphenicol resistance (Cm^r) gene that is expressed in Bacillus subtilis, a set of transformation-deficient strains of B. subtilis was isolated by insertional mutagenesis. When chromosomal DNA from these mutants was used to transform a transformation-proficient B. subtilis strain, almost all of the Cm^r transformants had the mutant phenotype as expected. However, with a frequency of approximately 3×10^-4 atypical transformants with the wild-type phenotype were produced. Data concerning amplification of the DNA containing the Cm^r marker and duplication of DNA sequences are presented that suggest that these atypical transformants are the result of a Campbell-like integration of the chromosomal DNA containing the integrated plasmid. Transductional mapping showed that in the atypical transformants the vector-containing DNA had a strong tendency to integrate at sites adjacent to the original site of integration, although integration at sites elsewhere on the chromosome was also observed. The production of atypical transformants is explained on the basis of integration of chromosomal DNA by a Campbell-like mechanism. Circularization of vector-containing chromosomal DNA is thought to occur through joining of the extremities of single-stranded DNA molecules by fortuitous base pairing with an independently entered single-stranded DNA molecule.     

Molecular cloning with bifunctional plasmid vectors in Bacillus subtilis

Summary Cloning in Escherichia coli and Bacillus subtilis was carried out using the bifunctional plasmid pDH5060. B. subtilis chromosomal DNA and pDH5060 DNA were digested with either BamHI or SalI, then annealed, ligated, and transformed into E. coli SK2267. Transformants containing sequences ligated into the BamHI or SalI sites in the Tc^r gene of pDH5060 were selected directly using a modification of the fusaric acid technique. The BamHI and SalI clone banks contain about 250 and 140 B. subtilis fragments, respectively, with an average insert size of 8–9 Kbp in the BamHI and 4–5 Kbp in the SalI bank. The inserts ranged in size from 0.3 Kbp to greater than 20 Kbp. The vector used here therefore accepts inserts which are significantly larger than previously reported for other B. subtilis cloning systems. All individual cloned B. subtilis sequences examined were stably propagated in E. coli SK2267. Eight of eighteen B. subtilis auxotrophic markers tested (aroG, gltA, glyB, ilvA, metC, purA, pyrD, and thrA) were transformed to prototrophy with BamHI or SalI clone bank DNA. All or part of the hybrid plasmid DNA recombined at the sites of homology in the chromosome of these Rec⁺ recipients. Loss of sequences from hybrid plasmids was not prevented in a r ^- m ^- recE4 recipient strain of B. subtilis. Although the recE4 background prevented recombination between homologous chromosomal DNA, a variety of cloned fragments were shown to be unstable and undergo deletions of both insert and plasmid sequences. In addition, B. subtilis sequences propagated in E. coli transformed B. subtilis recE4 recipients with a 500-1,000-fold reduced efficiency.     

An efficient transformation method for <Emphasis Type="Italic">Bacillus subtilis</Emphasis> DB104

Bacillus subtilis strains are used for extracellular expression of enzymes (i.e., proteases, lipases, and cellulases) which are often engineered by directed evolution for industrial applications. B. subtilis DB104 represents an attractive directed evolution host since it has a low proteolytic activity and efficient secretion. B. subtilis DB104 is hampered like many other Bacillus strains by insufficient transformation efficiencies (≤10³ transformants/μg DNA). After investigating five physical and chemical transformation protocols, a novel natural competent transformation protocol was established for B. subtilis DB104 by optimizing growth conditions and histidine concentration during competence development, implementing an additional incubation step in the competence development phase and a recovery step during the transformation procedure. In addition, the influence of the amount and size of the transformed plasmid DNA on transformation efficiency was investigated. The natural competence protocol is “easy” in handling and allows for the first time to generate large libraries (1.5 × 10⁵ transformants/μg plasmid DNA) in B. subtilis DB104 without requiring microgram amounts of DNA.     

Inter- and Intraspecies Conjugal Transfer of Different Plasmids in Bacilli

Conjugal transfer of plasmid pUB110 between different strains of bacilli was studied. The plasmid transfer was possible not only between various strains of B. subtilis, but also when many other species of bacilli served as recipients. Conjugation of a donor strain B. subtilis 19 (p19 pUB110) was accompanied by a transfer of plasmid p19 along with plasmid pUB110 to the B. subtilis recipient strains lacking a large plasmid p19. If, like the donor cells, the recipient B. subtilis strain carried plasmid p19, the frequency of conjugation decreased. The small plasmid pBC16 was also capable of conjugative transfer. However, if this plasmid carried the mob gene with an inverted region, the frequency of its transmission dramatically decreased. If the donor strain contained another small plasmid, pV, which also carried the mob gene, the efficiency of transmission was partially restored.     

Soil Strain of Bacillus subtilis Harboring a Large Plasmid That Mediates High-Frequency Conjugal Mobilization

The ability of a soil strain of Bacillus subtilis harboring a large plasmid, p19, to mobilize a small staphylococcal plasmid, pUB110, was studied. The latter plasmid was transferred to the recipient cells of Bacillus subtilis168 at a high frequency (about 10^–2 per recipient cell) both on the filter surface and in liquid medium. Mobilization was initiated 40 min after the beginning of the contact between donor and recipient cells.