A bifunctional plasmid carrying the recA gene of E. coli

Summary To investigate the effect of an active, plasmid-carried recA gene on the stability and/or the expression of plasmid genes in different genetic backgrounds, we have constructed a bifunctional plasmid (able to replicate in Escherichia coli and in Bacillus subtilis). Chimeric plasmids were obtained by inserting pC194 (Ehrlich 1977) into pDR1453 (Sancar and Rupp 1979). pDR1453 is a 12.9 Kbp plasmid constructed by inserting an E. coli chromosome fragment carrying the recA gene into pBR322. The expected bifunctional recombinant (pMR22/1) (15.7 Kbp) was easily obtained but surprisingly the Cm resistance was expressed only at a very low level in E. coli (as compared, for example, to pHV14, pHV15). We attribute this effect to the presence of multiple recA genes in the cell. On the contrary, Cm^r E. coli transformants bear a recombinant plasmid (pMR22/n) containing tandemly repeated copies of pC194 in equilibrium with excised free pC194. Such amplification has never been observed in a Rec^- background and is therefore mediated by the recA genes. Growth of these clones in the absence of Cm causes the loss of the extra copies, yielding a plasmid with a single copy of pC194, indistingishable from pMR22/1. Interestingly, we have observed that deletions occur at high frequency in pC194, which drastically increase Cm^r in E. coli containing plasmids with a single copy of pC194. Two types of such deletions were detected: (a) large 1050 bp deletions covering about onethird of pC194 and (b) small 120–150 bp deletions (near the MspI site) in the region containing the replicative functions of pC194 (Horinouchi and Weisblum 1982). Both types of deletion render the recombinant plasmid unable to replicate in B. subtilis. pM22/1 replicates, although with a low copy-number, and is stable in B. subtilis wild type; the recA gene of E. coli does not complement any of the rec ^- mutations of B. subtilis. A strong instability, mainly of the E. coli and pBR322 sequences, was observed in many dna and rec mutants of B. subtilis yielding smaller plasmid with a much higher copy-number.     

High-frequency transformation ofLactobacillus casei with plasmid pHY300PLK by electroporation

To optimize the conditions for transformation ofLactobacillus casei ATCC 27092 cells with plasmid pHY300PLK, a shuttle vector forEscherichia coli andBacillus subtilis, by electroporation, we investigated the effects of the electrical parameters (voltage and resistance), the concentration of plasmid DNA, the cell age and density, the electroporation buffer, and other factors. Under optimal conditions of 2.0 kV, 100 ohm, and 25F, a transformation efficiency as high as 1.4×10⁷ transformants per g of plasmid DNA was obtained, with a survival rate of about 50%.L. casei YIT 9021, one of the PL-1 phage mutants of the ATCC 27092 strain, was also transformed with the same plasmid under optimal conditions. The transformants were confirmed to harbor the same intact plasmid molecules by agarose gel electrophoretic analysis.     

Recombination-dependent recircularization of linearized pBR322 plasmid DNA following transformation of Escherichia coli

Summary Monomeric pBR322 DNA that had been linearized at its unique SalI site transformed wild-type Escherichia coli with 10² to 10³ times less efficiency than CCC plasmid DNA. Dose-response experiments indicated that a single linear plasmid molecule was sufficient to produce a transformant. Transformation with linearized pBR322 DNA was reduced 10 to 40 fold in recA ^–, recBC ^– or recF ^– backgrounds. In contrast, transformation with CCC DNA was unaffected by the rec status of the host. Transformation with linear pBR322 DNA was increased 3-fold in a DNA ligase-overproducing (lop11) mutant and decreased to a similar degree by transient inactivation of ligase in a ligts7 mutant.A proportion (ranging from about 9% in the wild-type to 42% in a recBC, lop11 mutant) of the transformants obtained with SalI-linearized pBR322 monomeric DNA contained deleted plasmids. Deletion rates were generally higher in rec ^– strains. Dephosphorylation of the termini on linear DNA or the creation of blunt-ended pBR322 molecules (by end-filling the SalI 5 protrusions or by cleavage with PvuII) decreased the transformation frequencywhilst increasing the deletion rate.Linear pBR322 dimeric DNA gave transformation frequencies in recA ⁺ and recA ^– strains that were reduced only 3 to 7 fold respectively relative to frequencies obtained with dimeric CCC DNA. Furthermore, in contrast to transformation with linear monomeric DNA, deletions were not observed.We propose that the majority of transformants arise, not by simple intracellular reannealing and ligation of the two cohesive SelI-termini of a linear molecule, but by intramolecular recombination. Deleted plasmids could be generated therefore during recyclization caused by recombination between short directly repeated sequences within a pBR322 monomer. We suggest that perfectly recircularized monomeric pBR322 molecules, which are found in the majority of transformants, arise primarily by intramolecular recombinational resolution of head-to-tail linear pBR322 dimers. Such linear oligomeric forms are created during preparation of linearized plasmid DNA by annealing of the SalI cohesive termini and constitute a variable proportion of the total molecules present.     

Transformation of Escherichia coli by a specific DNA restriction fragment.

Summary Specific transformation of a rifampicin sensitive strain of Escherichia coli to rifampicin resistance has been performed by a single, defined DNA restriction fragment carrying the genetic information for the subunit of E. coli RNA polymerase. In this transformation the transforming genetic character has been substituted for the corresponding recipient gene locus by recombination. The value of the described transformation system for locating genetic markers on DNA restriction fragments is discussed in comparison to previously reported in vitro systems.     

Electroporation of Bradyrhizobium japonicum

Summary Electroporation offers a fast, efficient and reproducible way to introduce DNA into bacteria. We have successfully used this technique to transform two commercially important strains of Bradyrhizobium japonicum, the nitrogen-fixing soybean symbiont. Initially, electroporation conditions were optimized using plasmid DNA which had been prepared from the same B. japonicum strain into which the{imDNA was to b}e transformed. Efficiencies of 10⁵-10⁶ transformants/g DNA were obtained for strains USDA 110 and 61A152 with ready-to-use frozen cells. Successful electroporation of B. japonicum with plasmid DNA prepared from Escherichia coli varied with the E. coli strain from which the plasmid was purified. The highest transformation efficiencies (10⁴ transformants/g DNA) were obtained using DNA prepared from a dcm ^– dam ^– strain of E. coli. This suggests that routine isolation of DNA from an E. coli strain incapable of DNA modification should help in increasing transformation efficiencies for other strains of bacteria where DNA restriction appears to be a significant obstacle to successful transformation. We have also monitored the rate of spontaneous mutation in electroporated cells and saw no significant difference in the frequency of streptomycin resistance for electroporated cells compared to control cells.     

The use of plasmid DNA to probe DNA repair functions in the yeast Saccharomyces cerevisiae

Summary The survival of plasmid YRp12 treated in vitro with ultraviolet- or -radiation, or with restriction endonucleases, has been used to investigate in vivo RAD gene activity in Saccharomyces cerevisiae. Yields of pyrmidine dimers or single and double strand breaks in plasmid DNA were assayed by physical methods. The biological effects of these damages were assayed by transformation of wild-type cells and rad mutants from each of the major groups of radiosensitive mutants. After UV-irradiation plasmid survival depended qualitatively on the same host functions that are needed for cellular survival. After -irradiation no such correspondence was found. Apart from a RAD52-dependent stimulation of transformation efficiency at low doses, other host repair functions had little effect. Stimulation of transformation corresponded with the production of double- but not single-strand breaks in plasmid sequences homologous with the yeast genome and may be linked with a transient increase in mitotic stability.More generally these data also show that transformation events using the LiCl protocol may entail the uptake of a very low number of plasmid molecules per cell over a 10-fold range of DNA concentrations.     

A stable and efficient transformation system for Butyrivibrio fibrisolvens OB156

A 9.5-kb shuttle vector capable of replication and selection in both Escherichia coli and Butyrivibrio fibrisolvens was constructed. Plasmid pUC118 provided replication functions and ampicillin resistance selection in E. coli. In B. fibrisolvens, replication was controlled by the native plasmid pRJF1 from strain OB156, and selectability was provided by a 3.5-kb fragment of plasmid pAM1 containing the erythromycin resistance gene. Optimum conditions for transformation were 15 kV/cm, 2 h recovery, and plating in an agar overlay on medium containing 10 g erythromycin/ml. Maximum efficiency was 1.1×10⁵ transformants per g plasmid DNA (average 3×10⁴), and restriction mechanisms reduced efficiency by a factor of 2×10². Nonselective growth for 200 generations gave no measurable loss of plasmid.     

High efficiency transformation of Salmonella typhimurium and Salmonella typhi by electroporation

Summary Salmonella typhimurium and S. typhi were transformd with high efficiency by electroporation. Transformation efficiencies of up to 10¹⁰ transformants per g of pBR322 were obtained. In contrast to chemical transformation methods, neither the smooth lipopolysaccharide of S. typhimurium nor the Vi capsular polysaccharide of S. typhi greatly affected transformation efficiency. The introduction of a galE mutation slightly improved transformation efficiency in S. typhimurium (< tenfold) while the Vi antigen of S. typhi had no detectable effect. The transformation efficiency of S. typhimurium with DNA derived from Escherichia coli was increased greatly by the removal of the hsd restriction system (100-fold). Under these conditions electroporation can be used for the routine and direct transformation of Salmonella strains with partially purified (alkaline lysis) plasmid DNA from E. coli.     

Establishment of a gene transfer system for Rhodothermus marinus

Genetic manipulation of Rhodothermus marinus has been hampered by the lack of a selection system for gene transfer. We report construction of a Rhodothermus/Escherichia coli shuttle plasmid, containing the R. marinus trpB gene, based on pUC18 and the cryptic R. marinus plasmid pRM21. A plasmid-less R. marinus recipient strain was selected on the basis of growth characteristics and absence of restriction activity. The shuttle plasmid, pRM100, was successfully introduced into a TrpB^– mutant of the recipient strain using electroporation and was found to transform it to prototrophy. No loss or rearrangement of pRM100 was observed after growth for 80 generations in non-selective medium. The relative copy numbers of pRM100 and of the parental plasmid, pRM21, were determined as 7±1 and 42±4, respectively. The shuttle plasmid was used to optimize an electroporation protocol, and the maximal number of transformants obtained was 4.3±0.7×10⁶ cfu/g DNA at 22.5 kV/cm, 200 and 25 F. Transformation failed, however, after chemical preparation of cells according to several protocols. This is the first report of genetic transformation in the genus Rhodothermus.     

Cloning and expression of β-galactosidase gene from Streptococcus thermophilus in Saccharomyces cerevisiae

Summary The -galactosidase gene ofStreptococcus thermophilus was cloned into plasmid vector, pVT100-U, and used to transform a strain ofEscherichia coli andSaccharomyces cerevisiae. Transformants which expressed -galactosidase activity were obtained in bothE. coli andSaccharomyces cerevisiae, the highest activity found in a yeast recombinant. The expression and thermostability of the cloned -galactosidase genes from different plasmid constructions were compared with the streptococcal -galactosidase. The recombinant protein was equivalent to the specific activity and thermostability ofS. thermophilus.     

Development of a transformation system forTrichoderma longibrachiatum and its use for constructing multicopy transformants for theegl1 gene

An efficient transformation system for the fungusTrichoderma longibrachiatum has been developed. Transformation was obtained both by electroporation and polyethyleneglycol treatment, using a plasmid carrying theEscherichia coli hygromycin B phosphotransferase gene as a dominant selectable marker. The transformation frequency was 0.5 to 5 transformants /g plasmid DNA. Transformation normally occurred by tandem integration of the transforming DNA. A high percentage of the transformants were mitotically unstable. The efficiency of co-transformation was very high (around 90%), and several co-transformants containing multiple copies of theegll gene encoding a -(1,4)-endoglucanase were obtained. Some of them secrete increased levels of endoglucanase to the culture medium. In addition, theE. coli lacZ gene was expressed in an active form under control of theAspergillus nidulans gpdA gene promoter.     

Development of an electro-transformation system for Escherichia coli DH10B

Summary Escherichia coli can be transformed to high efficiencies by subjecting a mixture of cells and DNA to a brief but intense electrical field. Factors that affect the transformation efficiency of E.coli strain DH10B were analysed. Optimal conditions gave an efficiency of 10⁸ to 10⁹ transformants/g DNA with E.coli strains K803 and DH10B, and plasmids pB1221.23 and pBSK+. The use of ligated DNA resulted in 10⁶ transformants/g DNA. Detailed protocols for these systems are given.     

High-frequency electroporation and maintenance of pUC- and pBR-based cloning vectors inPseudomonas stutzeri

A number ofEscherichia coli cloning vectors, based on ColE1-like replicons, were shown to be maintained inPseudomonas stutzeri ATCC 17588. A restrictionless mutant ofP. stutzeri was isolated, and this strain was used to develop an efficient electroporation system. With theE. coli cloning vector pHSG298, transformation frequencies of up to 2×10⁷ transformants/g DNA were achieved. This frequency is comparable to that obtained for CaCl₂-mediated transformation ofE. coli; thus, direct cloning of DNA intoP. stutzeri is feasible. As will be discussed, this may prove useful for cloning DNA from high mol% G+C genera in cases in whichE. coli is not a suitable heterologous cloning host.     

Stable yeast transformation with chimeric plasmids using a 2 micron-circular DNA-less strain as a recipient.

Summary By using two chimeric plasmids containing yeast URA3 gene as a selection marker and 2 m yeast DNA linked to the bacterial plasmid pCR1, a yeast strain devoid of any 2 m DNA sequence was transformed. Recovery in E. coli of plasmids from yeast transformants showed that the 2 m-less strain was able to maintain the chimeric plasmids as autonomous replicons, with very infrequent plasmid recombination. Hybridization experiments gave no evidence for integration of the URA3 DNA sequence in the chromosomal DNA. The transformed clones showed a high stability of the ura⁺ character during vegetative multiplication, even in the absence of selective pressure. The specific activity of orotidine 5 monophosphate decarboxylase (coded by the URA3 gene) was 5 to 10 fold higher than in the wild type.These features should offer new possibilities for cloning with yeast.     

Molecular cloning and expression of a Micromonospora chalcae β-glucosidase encoding gene in Escherichia coli

A Sau3A I genomic library from the actinomycete Micromonospora chalae was constructed in Escherichia coli using the expression vector pUC18. Using the chromogenic substrate 5-bromo-4-chloro-3-indolyl--glucoside (X-glu), a number of positive recombinant colonies were identified. One of those exhibiting the strongest phenotype contained a recombinant plasmid, pANNA1 which harboured a 4.2kb DNA insert. Using restriction endonuclease site mapping and subcloning strategies a 2.3kb DNA fragment encoding the -glucosidase activity was identified. Characterization of the strongly expressed recombinant enzyme demonstrated that it had a dramatically increased thermal stability at 50 °C. The Km values obtained for the recombinant enzyme and that from M. chalcae using the substrate p-nitrophenyl--D-glucoside were 0.19mM and 0.25mM, respectively.     

Stability of cloned Brassica napus chloroplast DNA fragments in the cyanobacterium Anacystis nidulans R2

Summary Brassica napus (cv. Triton) chloroplast (cp) DNA BamHI gragments were inserted into a bacteria-cyanobacteria shuttle vector pCB4. The chloroplast genomic library was screened in Escherichia coli and 28 individual clones, which represent 94% of the total chloroplast genome, were isolated. Cyanobacterium Anacystis nidulans R2 was transformed with each member of the clone bank by selection for ampicillin resistance. A study of transformation efficiency showed dramatic variation (up to 200-fold) among recombinant clones. Furthermore, plasmid DNA reisolated from some cyanobacterial transformants exhibited instability. Variations in transformation efficiency and plasmid instability were shown to be DNA sequence specific. B. napus cpDNA clones were thus classified into three types according to their stability in the cyanobacterial host.     

Electroporation and stable maintenance of plasmid DNAs in a biocontrol strain of Pseudomonas syringae

Transformation efficiencies as high as 10⁷ transformants g^–1 DNA have been previously reported for pseudomonads using electroporation protocols established for E. coli with plasmid DNAs prepared from methylation proficient E. coli hosts. We report here a protocol for electroporation of plasmid DNAs into a biocontrol strain of Pseudomonas syringae which could not be electroporated by standard E. coli methods. Transformation efficiencies of 10⁷ or higher were obtained with DNA recovered from initial P. syringae transformation or with DNA prepared from methylation deficient E. coli. Both plasmids used in this study were stably maintained in the absence of selection for at least 50 generations.     

Genetic transformation of the pathogenic fungus Wangiella dermatitidis

Genetic transformation of Wangiella dermatitidis was studied using three plasmid vectors (pAN7-1, pWU44, and pKK5) and both electroporation and polyethyleneglycol-mediated methods. pAN7-1 contains the E. coli hygromycin B (HmB) phosphotransferase (hph) gene. Expression of the hph gene confers resistance to antibiotic HmB. Selection for resistance, indicative of transformation, resulted in 10–203 HmB-resistant colonies/g pAN7-1 on medium containing 100 g HmB/ml. Strains of W. dermatitidis used in this study have innate sensitivity to HmB at a critical inhibitory concentration of 20–40 g/ml. Vectors pWU44 and pKK5 contain a URA5 gene from Podospora anserina. A ura5 auxotroph of W. dermatitidis was transformed to prototrophy with pWU44 or pKK5 by complementation. Transformation frequencies for these two plasmids were between 17–50 transformants/g vector DNA. Southern blotting analysis and polymerase chain reaction detection of DNA from putative transformants confirmed transformation.     

Transformable mutants of a biopesticide strainStreptomyces griseoviridis K61

Summary The aim of this work was to isolate transformable mutants ofStreptomyces griseoviridis K61 without affecting the secondary metabolism of this strain.S. griseoviridis K61 produces an antifungal aromatic heptaene polyene antibiotic, and is used as a biological control agent. In protoplast transformation experiments using plasmid pIJ702 DNA, the few spontaneous transformants were phenotypically bald and their secondary metabolism was pleiotropically affected. By mutagenizing K61 withN-methyl-N-nitro-N-nitrosoguanidine (MNNG) a highly transformable variant K61-42 was obtained. Protoplasts ofS. griseoviridis K61-42 could be transformed by several model plasmids producing 10⁴–10⁵ transformants/g plasmid DNA. The polyene synthesis of K61-42 was normal, making this strain a useful tool in genetic studies on the mechanism of biopesticide action.