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.     

Transformation of Mucor circinelloides with Autoreplicative Vectors Containing Homologous and Heterologous ARS Elements and the Dominant Cbx r Carboxine-Resistance Gene

Mucor circinelloides transformants prototrophic to leucine and resistant to carboxine (Leu⁺ Cbx^r) have been obtained by treatment of protoplasts with plasmid constructs containing homologous leuA gene and adjacent autonomously replicating sequences (ARS) element combined with the Cbx^r(carboxine-resistance) gene of Ustilago maydis and ARS sequences from this basidiomycete (plasmid pGG37) or from the 2 μ plasmid of Saccharomyces cerevisiae (plasmid pGG43). The presence in the same plasmid molecule of the M. circinelloides leuA gene and adjacent ARS element together with heterologous ARS elements produced an increase in the transformation frequency of about 65–120%. The presence of autoreplicating plasmid molecules in the transformants was demonstrated by mitotic stability experiments, by Southern analysis, and by the rescue of plasmids from transformed bacterial cells.     

Isolation of a recombination deficient Agrobacterium tumefaciens mutant.

Summary The isolation of a recombination deficient (Rec^-) strain of Agrobacterium tumefaciens is described. Strain LBA 4011 was mutagenized with nitrosoguanidine and after segregation 18,000 colonies were replica plated and UV irradiated. Twentytwo UV sensitive strains were isolated and tested for methylmethanesulphonate (MMS) sensitivity. Six of these strains were more MMS-sensitive than LBA 4011. A Ti plasmid that was genetically marked with Tn 1 (Cb^R) was introduced in these strains and the rescue of the Cb^R marker during superinfection with an incompatible cointegrate plasmid Ti::R 702 was determined. One strain exhibited a large reduction in rescue frequency. It is concluded that the latter strain was recombination deficient. This property did not influence the induction of plant tumours.This paper forms part of a Ph.D. Thesis submitted at Leiden University by the first author     

Plasmid rescue - a tool for reproducible recovery of genes from transfected mammalian cells?

Summary The efficient rescue of plasmids containing the thymidine kinase gene (tk) of Herpes simplex virus type I from genetically transformed mouse cells by transformation of bacteria is described. Rescued plasmids contain insertions of calf DNA used as a carrier in the transfection but usually lack portions of plasmid DNA. Deletions generally concern the region spanning from around the PvuII site of pBR322 to within the tetracycline resistance coding sequence, whereas the extent of tk sequence deletion varies, depending on the site of its integration (BamHI or PvuII) into the plasmid. Modelling the rescue process by transformation of bacteria with a mixture of original plasmids and sheared mouse cell DNA clearly demonstrates that deletions are caused by the presence of the mammalian DNA and they probably occur during re-transformation of bacteria before the onset of tetracycline gene expression. Plasmids lacking the Tc^r region are reproducibly rescuable without deletion. Methods for reproducible re-isolation of transferred genes from mammalian cells are discussed.     

Marker Rescue Studies of the Transfer of Recombinant DNA to Streptococcus gordonii In Vitro, in Foods and Gnotobiotic Rats

A plasmid marker rescue system based on restoration of the nptII gene was established in Streptococcus gordonii to study the transfer of bacterial and transgenic plant DNA by transformation. In vitro studies revealed that the marker rescue efficiency depends on the type of donor DNA. Plasmid and chromosomal DNA of bacteria as well as DNA of transgenic potatoes were transferred with efficiencies ranging from 8.1 × 10⁻⁶ to 5.8 × 10⁻⁷ transformants per nptII gene. Using a 792-bp amplification product of nptII the efficiency was strongly decreased (9.8 × 10⁻⁹). In blood sausage, marker rescue using plasmid DNA was detectable (7.9 × 10⁻¹⁰), whereas in milk heat-inactivated horse serum (HHS) had to be added to obtain an efficiency of 2.7 × 10⁻¹¹. No marker rescue was detected in extracts of transgenic potatoes despite addition of HHS. In vivo transformation of S. gordonii LTH 5597 was studied in monoassociated rats by using plasmid DNA. No marker rescue could be detected in vivo, although transformation was detected in the presence of saliva and fecal samples supplemented with HHS. It was also shown that plasmid DNA persists in rat saliva permitting transformation for up to 6 h of incubation. It is suggested that the lack of marker rescue is due to the absence of competence-stimulating factors such as serum proteins in rat saliva.     

Transformation of CW-15 Mutant Cells of Chlamydomonas reinhardtii Dang. with pCTVHyg Plasmid

A pCTVHyg plasmid was constructed in a unicellular green alga Chlamydomonas reinhardtii Dang. by using the hygromycin phosphotransferase gene (hpt) as a selectable marker and the Escherichia coli transposon Tn5 under the early SV40 viral gene promoter. CW-15 mutant cells devoid of cell walls were transformed by electroporation in an electric field of 1 kV/cm and a pulse duration of 2 ms. A suspension density of 10⁶ cell/ml and the mid-logarithmic growth phase were the optimum conditions for transformation, producing up to 10³ hygromycin-resistant (Hyg^R) clones per 10⁶ Hyg^R recipient cells. Exogenous DNA integrated in the nuclear genome of C. reinhardtii was steadily inherited in subsequent generations within at least a 8-month period; however, the Hyg^R trait manifestation was not stable. The comparative analysis of frequencies in codon usage in hpt and in the nuclear genes of C. reinhardtii significantly excluded the possibility that the bias in codon usage was the primary factor affecting foreign gene expression. The advantages of using theCW-15 mutant and the described selection system are discussed in the context of heterologous transformation of C. reinhardtii.     

Introduction of plasmid pC194 into Bacillus thuringiensis by protoplast transformation and plasmid transfer

The Staphylococcus aureus plasmid pC194 which codes for resistance to chloramphenicol was introduced into six Bacillus thuringiensis strains representing five varieties by protoplast transformation. Six other varieties could not be transformed. pC194 could be identified in transformed strains as autonomous plasmid. The transformed clones contained in addition a new extrachromosomal element of somewhat lower electrophoretic mobility hybridizing with pC194, and pC194 in multimeric forms. pC194 was also transferred from one B. thuringiensis variety to another and from Bacillus thuringiensis to Bacillus subtilis and vice versa by a conjugation-like process, requiring close cell-to-cell contact.Non-standard abbreviations BSA bovine serum albumin - CAT chloramphenicol acetyltransferase - Cm^R chloramphenicol resistant - PAB Penassay broth - SDS sodiumdodecylsulfate - Tc^R tetracycline resistant     

The Transformation of the Unicellular Alga Chlamydomonas reinhardtii by Electroporation

The cell wall–lacking mutant CW-15 of the unicellular green alga Chlamydomonas reinhardtii was transformed by electroporation using plasmid pCTVHyg, which was constructed with the hygromycin phosphotransferase genehpt as the selective marker and the Tn₅ transposon of Escherichia coli under the control of the virus SV40 early gene promoter. Under optimal conditions (10⁶ mid-exponential cells/ml; electric field strength 1 kV/cm; and pulse length 2 ms), the transformation yielded 10³ Hyg^R transformants per 10⁶ recipient cells. The exogenous DNA integrated into the nuclear genome of Ch. reinhardtii was persistently inherited through more than 350 cell generations. The advantages of this system for the transformation ofCh. reinhardtii with heterologous genes are discussed.     

Characterization of vaccinia virus DNA replication mutants with lesions in the D5 gene

The vaccinia virus D5 gene encodes a 90 kDa early protein that is essential for viral DNA replication. In this report we map and explore the phenotypes of the temperature sensitive mutants bearing lesions in this gene:ts17,ts24,ts69, (WR strain) andts6389 (IHD strain). Viral DNA synthesis was virtually undetectable during non-permissive infections performed withts17, and incorporation of³H-thymidine ceased rapidly when cultures were shifted to the non-permissive temperature in the midst of replication. The D5 protein may therefore be involved in DNA synthesis at the replication fork. The lesions of the four mutants were localized within the D5orf by marker rescue, and the single nucleotide changes responsible for thets phenotype of the three WR mutants were identified. Unexpectedly, the three alleles with N-terminal mutations were impaired in marker rescue when homologous recombination with small (<2 kb), intragenic DNA fragments at 39.5°C was required. This deficiency was not due to degradation of transfected DNA under non-permissive conditions. Efficient marker rescue could be restored by incubation at the permissive temperature for a brief period after transfection, suggesting a requirement for functional D5 in genome/plasmid recombination. Marker rescue under non-permissive conditions could alternatively be restored by co-transfection of unlinked but contiguous DNA sequences.     

DNA repair in Proteus mirabilis. VI. Plasmid (R46-) mediated recovery and UV mutagenesis.

Summary The expression of plasmid R46-mediated recovery and mutagenic function (s) was studied in P. mirabilis, which is normally either weakly or nonmutable after UV exposure. The plasmid was found to confer on P. mirabilis enhanced UV resistance as well as UV-induced mutability for various types of forward mutations and reversion of the thr273 mutation. The plasmid enhanced survival of UV-irradiated phages in P. mirabilis both in unirradiated host cells and with increased efficiency after UV-exposure of host cells, as is characteristic of UV-inducible phage reactivation. Spontaneous mutability of P. mirabilis harboring R46 was about 2 to 7 times higher than that of cells without plasmid, depending on the marker, repair type, and plating density of the cells used. All of these R46-mediated rescue and mutagenic functions require the rec672⁺ gene function.It is assumed that the plasmid R46 adds functions to P. mirabilis comparable to those deficient in umuC and uvm mutants of E. coli (Kato and Shinoura, 1977; Steinborn, 1978) and that P. mirabilis possesses functions homologous to those controlled in E. coli by the recA ⁺ and lexA ⁺ genes.The significance of plasmid-mediated rescue and mutagenic functions for bacteria which lack the misrepair branch of mutagenesis, is discussed.     

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.     

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.     

Transfer of hygromycin resistance into Brassica napus using total DNA of a transgenic B. nigra line

The successful transfer of a marker gene (hpt gene) from Brassica nigra into B. napus via direct gene transfer was demonstrated. Total DNA was isolated from a hygromycin-resistant callus line, which contained three to five copies of the hpt gene. This line had been produced via direct gene transfer with the hygromycin resistance-conferring plasmid pGL2. The treatment of B. napus protoplasts with genomic DNA of B. nigra (Hyg^R) resulted in relative transformation frequencies of 0.1–0.4%. Similar transformation rates were obtained in direct gene transfer experiments using B. napus protoplasts and plasmid pGL2.     

Characterization of genetic transformation in Streptococcus mutans by using a novel high-efficiency plasmid marker rescue system.

We developed a marker rescue system for study of competence development and genetic transformation in Streptococcus mutans. The system involved the recombinational rescue of a tetracycline resistance (Tcr) determinant by a homologous, inactive locus (Tcs because of a small deletion). Streptococcal cells harboring this in vitro-prepared Tcs construct (pVA1208) were restored to Tcr when plasmid (pVA981) DNA was used as donor material. pVA981 contained the intact streptococcal Tcr locus and was unable to autonomously replicate in streptococci. Marker rescue with this system followed first-order kinetics and occurred at a frequency 8- or 160-fold higher than did transformation with homologous chromosomal or plasmid DNA, respectively. By using the rescue system, we were able to confirm that competence of S. mutans appeared to be inducible. This was indicated by a sequential increase and then decrease in Tcr transformation frequencies during growth in complex medium. Also, donor DNA binding was not sequence specific, since the recovery of Tcr transformants was reduced by increasing the concentrations of heterologous DNA. We investigated the fate of donor DNA and the kinetics of plasmid establishment in the transformation of S. mutans with plasmid DNA. Monomeric plasmid molecules transformed S. mutans as a second-order process, whereas multimeric plasmid DNA and chromosomal markers were recovered as a first-order process. Approximately 50% of the initially bound donor plasmid DNA was found to remain in a trichloroacetic acid-insoluble form. Our results suggested that molecular cloning in S. mutans would be conducted most efficiently by using helper plasmid systems or shuttle vectors and that gene transfer by transformation of S. mutans occurred in a manner similar to that observed in Streptococcus sanguis.     

Transformation of the astaxanthin-producing yeast Phaffia rhodozyma

Summary This paper describes the genetic transformation of the astaxanthin-producing yeast Phaffia rhodozyma with the cloning vector pGH-1. The plasmid replicates autonomously in this yeast, and the selection of transformants was possible by using both, the URA3 marker from Saccharomyces cerevisiae, and the kanamycin resistance (Km^R) determinant from the bacterial transposon Tn903.     

Transformation of Bacillus Protoplasts by Plasmid pTP4 DNA

Polyethylene glycol (PEG)-induced protoplast transformation by plasmid pTP4 DNA encoded chloramphenicol resistance determinant was developed for Bacillus subtilis, B. amyloliquefaciens, B. licheniformis, B. megaterium and B. pumilus. Protoplasts were formed by treatment of cells with lysozyme and the transformation frequencies (transformants per regenerants) were in the range of 1.3 × 10^?2 to 7.1 × 10^?1. Reisolated plasmid DNA prepared from transformants exhibited covalently closed and open circular forms similar to those of the donor DNA. These results indicate that PEG-induced protoplast transformation is an adequate method for plasmid transformation and pTP4 is a useful plasmid as a cloning vector in a wide range of varieties of the genus Bacillus.     

A counter-selectable marker for genetic transformation of the yeast Schwanniomyces alluvius

We report here a counter-selectable marker system for genetic transformation of the yeast Schwanniomyces alluvius, based on the complementation of uracil auxotrophs defective in either orotidine-5′-phosphate decarboxylase (URA3) or orotidine-5′-pyrophosphatase (URA5). Uracil auxotrophs of S. alluvius were obtained by ethyl methanesulphonate mutagenesis and complemented using the ura3 gene from S. cerevisiae. A␣transformation frequency of approximately 10⁴/μg DNA was obtained, which is tenfold higher than results described in earlier reports. Transformants were analysed by Southern blot hybridisation and were found to be mitotically stable. The extrachromosomal nature of the transforming DNA was confirmed by Southern hybridisation and plasmid rescue. The rescued plasmid DNA had a restriction pattern identical to that of the parent plasmid. Received: 19 August 1996 / Received last revision: 30 April 1997 / Accepted: 4 May 1997     

R-plasmid effects on bacterial multiplication and survival

The multiplication of Escherichia coli C containing either the plasmid R46 or its non-selftransmissible derivative was studied in the presence or absence of the isogenic R^– parent strain. Neither plasmid conferred any detectable effect on the host's ability to multiply. Similarly under conditions of prolonged incubation neither plasmid conferred a disadvantage on its host when the bacteria were grown in pure culture. However, when the incubation of mixed R⁺/R^– cultures was prolonged, the possession of either R-plasmid resulted in small but reproducible differences which favoured the R^– strain.     

Plasmid transformation of naturally competent Acinetobacter calcoaceticus in non-sterile soil extract and groundwater

The natural transformation of Acinetobacter calcoaceticus BD413 (trp E27) was characterized with respect to features that might be important for a possible gene transfer by extracellular DNA in natural environments. Transformation of competent cells with chromosomal DNA (marker trp ⁺) occurred in aqueous solutions of single divalent cations. Uptake of DNA into the DNase I-resistant state but not the binding of DNA to cells was strongly stimulated by divalent cations. An increase of transformation of nearly 3 orders of magnitude was obtained as a response to the presence of 0.25 mM Ca²⁺. With CaCl₂ solutions the transformation frequencies approached the highest values obtained under standard broth conditions, followed by MnCl₂ and MgCl₂. It is concluded that transformation requires divalent cations. DNA competition experiments showed that A. calcoaceticus does not discriminate between homologous and heterologous DNA. Furthermore, circular plasmid DNA competed with chromosomal DNA fragments and vice versa. The equally efficient transformation with plasmid pKT210 isolated from A. calcoaceticus or Escherichia coli indicated absence of DNA restriction in transformation. High efficiency plasmid transformation was obtained in samples of non-sterile natural groundwater and in non-sterile extracts of fresh and air-dried soil. Heat-treatment (10 min, 80°C) of the non-sterile liquid samples increased transformation only in the dried soil extract, probably by inactivation of DNases. The results presented suggest that competent cells of A. calcoaceticus can take up free high molecular weight DNA including plasmids of any source in natural environments such as soil, sediment or groundwater.     

Transformation of Chlamydomonas reinhardtii CW-15 with the Hygromycin Phosphotransferase Gene as a Selectable Marker

To transform Chlamydomonas reinhardtiiDang. cells, plasmid pCTVHyg was constructed with the use of theEscherichia colihygromycin phosphotransferase gene (hpt) controlled by the SV40 early promoter. Cells of the CW-15 mutant strain were transformed by electroporation, with the yield reaching 10³ hygromycin-resistant (Hyg^R) clones per 10⁶ recipient cells. The exogenous DNA integrated in the Ch. reinhardtii nuclear genome showed stable transmission for approximately 350 cell generations, while hygromycin resistance was expressed as an unstable character. Codon usage was compared for the hptgene and Ch. reinhardtiinuclear genes. The results testified that codon usage bias, which is characteristic of Ch. reinhardtii, is not the major factor affecting foreign gene expression. The advantages of the selective system for studying Ch. reinhardtii transformation with heterologous genes are discussed.