Cloning vector system forNocardia spp.

An electro-transformation system has been developed forNocardia asteroides andNocardia corallina by using aNocardia-Escherichia coli shuttle vector. The shuttle vector, named pCY104, was constructed by joining a 2.5-kb crypticN. asteroides plasmid pCY101 with theE. coli plasmid pIJ4625. The resistance genes for kanamycin, chloramphenicol, and thiostrepton on plasmid pCY104 were expressed inN. asteroides andN. corallina. The transformation method was optimized forN. asteroides, and transformation efficiency of 8×10⁴ transformants per g plasmid DNA was achieved routinely.     

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.     

pIJ101, a multi-copy broad host-range Streptomyces plasmid: Functional analysis and development of DNA cloning vectors

Summary Streptomyces lividans ISP 5434 contains four small high copy number plasmids: pIJ101 (8.9 kb), pIJ102 (4.0 kb), pIJ103 (3.9 kb) and pIJ104 (4.9 kb). The three smaller species appear to be naturally occurring deletion variants of pIJ101. pIJ101 and its in vivo and in vitro derivatives were studied after transformation into S. lividans 66.pIJ101 was found to be self-transmissible by conjugation, to elicit lethal zygosis and to promote chromosomal recombination at high frequency in both S. lividans 66 and S. coelicolor A3(2). A restriction endonuclease cleavage map of pIJ101 was constructed for 11 endonucleases; sites for five others were lacking. Many variants of pIJ101 were constructed in vitro by inserting DNA fragments determining resistance to neomycin, thiostrepton or viomycin, and having BamHI termini, into MboI or BclI sites on the plasmid, sometimes with deletion of segments of plasmid DNA. The physical maps of these plasmids were related to their phenotypes in respect of lethal zygosis and transfer properties. In vivo recombination tests between pairs of variant plasmids were also done. These physical and genetic studies indicated that determinants of conjugal transfer occupy less than 2.1 kb of the plasmid. A second segment is required for spread of the plasmid within a plasmid-free culture to produce the normal lethal zygosis phenotype: insertion of foreign DNA in this region caused a marked reduction in the diameter of lethal zygosis zones. The minimum replicon was deduced to be 2.1 kb or less in size; adjacent to this region is a 0.5 kb segment which may be required for stable inheritance of the plasmid. The copy number of several derivatives of pIJ101 in S. lividans 66 was between 40 and 300 per chromosome and appeared to vary with the age or physiological state of the culture. pIJ101 derivatives have a wide host range within the genus Streptomyces: 13 out of 18 strains, of diverse species, were successfully transformed.Knowledge of dispensable DNA segments and the availability of restriction sites for the insertion of DNA, deduced from the properties of plasmids carrying the E. coli plasmid pACYC184 introduced at various sites, was used in the construction of several derivatives of pIJ101 suitable as DNA cloning vectors. These were mostly designed to be non-conjugative and to carry pairs of resistance genes for selection. They include a bifunctional shuttle vector for E. coli and Streptomyces; a Streptomyces viomycin resistance gene of this plasmid is expressed in both hosts.     

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.     

Cloning and expression of Candida albicans ADE2 and proteinase genes on a replicative plasmid in C. albicans and in Saccharomyces cerevisiae.

Summary A plasmid vector (denoted pRC2312) was constructed, which replicates autonomously in Escherichia coli, Saccharomyces cerevisiae and Candida albicans. It contains LEU2, URA3 and an autonomously replicating sequence (ARS) from C. albicans for selection and replication in yeasts, and bla (ampicillin resistance) and ori for selection and replication in E. coli. S. cerevisiae AH22 (Leu^–) was transformed by pRC2312 to Leu^– at a frequency of 1.41 × 10⁵ colonies per g DNA. Transformation of C. albicans SGY-243 (Ura-) to Ura⁺ with pRC2312 resulted in smaller transformant colonies at a frequency of 5.42 × 10³ per g DNA where the plasmid replicated autonomously in transformed cells, and larger transformant colonies at a frequency of 32 per g DNA, in which plasmid integrated into the genome. Plasmid copy number in yeasts was determined by a DNA hybridization method and was estimated to be 15±3 per haploid genome in S. cerevisiae and 2–3 per genome in C. albicans replicative transformants. Multiple tandem integration occurred in integrative transformants and copy number of the integrated sequence was estimated to be 7–12 per diploid genome. The C. albicans ADE2 gene was ligated into plasmid pRC2312 and the construct transformed Ade^– strains of both C. albicans and S. cerevisiae to Ade⁺. The vector pRC2312 was also used to clone a fragment of C. albicans genomic DNA containing an aspartic proteinase gene. C. albicans transformants harboring this plasmid showed a two-fold increase in aspartic proteinase activity. However S. cerevisiae transformants showed no such increase in proteinase activity, suggesting the gene was not expressed in S. cerevisiae.     

Isolation and nucleotide sequence of an autonomously replicating sequence (ARS) element functional in Candida albicans and Saccharomyces cerevisiae

Summary An 8.6-kb fragment was isolated from an EcoRI digest of Candida albicans ATCC 10261 genomic DNA which conferred the property of autonomous replication in Saccharomyces cervisiae on the otherwise non-replicative plasmid pMK155 (5.6 kb). The DNA responsible for the replicative function was subcloned as a 1.2-kb fragment onto a non-replicative plasmid (pRC3915) containing the C. albicans URA3 and LEU2 genes to form plasmid pRC3920. This plasmid was capable of autonomous replication in both S. cerevisiae and C. albicans and transformed S. cerevisiae AH22 (leu2 ^–) to Leu⁺ at a frequency of 2.15 × 10³ transformants per pg DNA, and transformed C. albicans SGY-243 (ura3) to Ura⁺ at a frequency of 1.91 × 10³ transformants per g DNA. Sequence analysis of the cloned DNA revealed the presence of two identical regions of eleven base pairs (5TTTTATGTTTT3) which agreed with the consensus of autonomously replicating sequence (ARS) cores functional in S. cerevisiae. In addition there were two 10/11 and numerous 9/11 matches to the core consensus. The two 11/11 matches to the consensus, CaARS1 and CaARS2, were located on opposite strands in a non-coding AT-rich region and were separated by 107 bp. Also present on the C. albicans DNA, 538 by from the ARS cores, was a gene for 5S rRNA which showed sequence homology with several other yeast 5S rRNA genes. A sub-fragment (494 bp) containing the 5S rRNA gene (but not the region containing the ARS cores) hybridized to genomic DNAs from a number of yeast species, including S. cerevisiae, C. tropicalis, C. pseudotropicalis, C. parapsilosis, C. kruseii, C. (Torulopsis) glabrata and Neurospora crassa. The 709-bp ARS element (but not the 5S rRNA gene) was necessary for high-frequency transformation and autonomous plasmid replication in both S. cerevisiae and C. albicans.EMBL/GenBank database accession number: X16634 (5S rRNA)     

Characterization of chromosome and plasmid transformation in Bacillus subtilis using gently lysed protoplasts

Competent cells of Bacillus subtilis were transformed with DNA from gently lysed protoplasts. Significant linkages among markers separated by distances of approximately 2.3% of the total chromosome were found, which have not been detected for conventional transformation. In comparison to previous reports, enhanced plasmid transformation was observed [4.0×10⁷ transformants per g DNA (one transformant per 5×10⁴ molecules added)], when competent cells were transformed with DNA from lysed protoplasts harboring pUB110.     

Optimization of transformation procedures in avermectin high-producing Streptomyces avermitilis

The optimal pH conditions for efficient transformation of protoplasts and intact cells were established in avermectin high-producing mutants, ATCC31780 and L-9. Among all factors tested, protoplast buffer pH was elucidated as the most important factor influencing transformation efficiency. The optimal pH of the protoplast buffer for the regeneration of ATCC31780 was 6.5, and using this condition, 4.5 × 10⁶ transformants per g of pIJ702 were produced. At pH 6.3, the maximal number of L-9 transformants was 1.6 × 10⁵ per g of the same plasmid. However, the protoplasting process decreased avermectin productivity to half or one-sixth of ATCC31780 or L-9, respectively. To avoid the productivity loss, electroporation of intact cells without lysozyme treatment was developed for these mutant strains even though this method was approximately 100-fold less efficient. In this method, the initial pH of culture medium was elucidated as a critical factor and optimized for both transformation efficiency and avermectin productivity.     

Electrotransformation of three pathovars of Xanthomonas campestris

Procedures for electrotransformation have been adapted for three pathovars of Xanthomonas campestris: campestris, vesicatoria and manihotis. Three differently sized plasmids (51, 9.3 and 3.3 kb) at different concentrations (10, 30 and 50 ng/sample) and different field strengths (10, 12, 14 and 18 kV/cm) were used. The efficiency of transformation was dependent on the recipient strain and the plasmid introduced. In general, a field strength of 14 kV/cm as well as a concentration of 30 ng plasmid DNA/sample seemed to be adequate for most conditions. Only X. campestris pv. vesicatoria strain 479 required a higher field strength for better efficiency. The plasmid size was inversely related to the efficiency of transformation; up to 1.6 × 10¹⁰, 5.3 × 10⁷ and 3.7 × 10⁵ transformants/g DNA were obtained using the pXG31 (3.3 kb), pUFR027 (9.3 kb) and RP4 (51 kb) plasmids, respectively.     

Transformation of protoplasts ofCellulomonas flavigena

Summary Protoplasts ofCellulomonas flavigena (Cm^s) were transformed with plasmid pC194. Transformation frequency was 2.72×10^–3 in MR-1 regeneration medium with 2 g/ml chloramphenicol. Transformation conditions are described.     

High frequency transformation of Alcaligenes eutrophus producing poly-β-hydroxybutyric acid by electroporation

Summary A strain of Alcaligenes eutrophus producing poly--hydroxybutyric acid was successfully transformed by the electroporation. The plasmid used was a broad host range plasmid pKT230 conferring kanamycin resistance. The optimum yield of transformant was 0.8×10²/g DNA when 50 l competent cells at 10¹⁰/ml were pulsed by 11.5 kV/cm for 5 ms with 1 g DNA. Plasmid DNA in the A. eutrophus transformant was stably maintained as a monomeric structure.     

Development of an intergeneric conjugal transfer system for rimocidin‐producing Streptomyces rimosus

Aims: To develop an intergeneric conjugation system for rimocidin‐producing Streptomyces rimosus. Methods and Results: High efficiencies of conjugation [10^?2–10^?3 transconjugants/recipient colony forming units (CFU)] were obtained when spores of S. rimosus were heat treated at 40°C for 10 min prior to mixing with E. coli ET12567(pUZ8002/pIJ8600) as donor. Mycelium from liquid grown cultures of S. rimosus could also be used as recipient instead of spores, with 24‐h cultures giving optimal results. TSA (Oxoid) medium containing 10 m mol l^?1 MgCl₂ was the preferred medium for conjugation. Southern hybridization was used to confirm that transconjugants of S. rimosus contained a single copy of pIJ8600 integrated at a unique chromosomal attachment site (attB). The transconjugants exhibited a high stability of plasmid integration and showed strong expression of green fluorescent protein when using pIJ8655 as the conjugative vector. Conclusion: Intergeneric conjugation between E. coli and S. rimosus was achieved at high efficiency using both spores and mycelium. Significance and Impact of the Study: The conjugation system developed provides a convenient gene expression system for S. rimosus R7 and will enable the genetic manipulation of the rimocidin gene cluster.     

Optimized transformation by electroporation ofLactobacillus plantarum strains with plasmid vectors

Summary Whole cell transformation ofLactobacillus plantarum CCM 1904 by electroporation was optimized. Pulse duration and electric field strength were shown to be important parameters: the optimum conditions were 12.5 kV/cm, a time-constant of 10 ms for an exponential decay waveform and 6.7 kV/cm applied during 2.5 ms for a square waveform. Transformation efficiency was increased if cells were cultivated on medium containing sorbitol and harvested during their early exponential growth phase: 8 × 10^–4 transformants/g pGK12 DNA per viable cell were obtained, with a survival rate of 10%–30% Cryotreatment by several freeze-and-thaw cycles decreased transformant yields. Transformation efficiency with different plasmids was studied and plasmid pGK12 was found to transformL. plantarum the most efficiently. Transformation by electroporation ofL. plantarum is strain dependent. The best results were obtained withL. plantarum NCIB 7220, giving 5 × 10⁶ transformants/gmg plasmid pGK12 DNA.     

Genetic transformation of Clostridium botulinum hall a by electroporation

Summary A method was developed for the introduction of plasmids into Clostridium botulinum by electroporation. A 4.4 kb plasmid vector, pGK12, which contains genes for resistance to erythromycin (Em^r) and chloramphenicol (Cm^r) was electroporated into C. botulinum type A (Hall A). The highest transformation efficiency was obtained using midlog phase cells, 10% PEG 8000 as the electroporation solution, and 2.5 kV field strength. The transformation efficiency was highest (10³ transformants/g of DNA) when 1 g of plasmid DNA and 4 × 10⁸ CFU/ml of recipient cells were used. Plasmid DNA recovered from the transformants was indistinguishable from that introduced on the basis of restriction enzyme digestion and agarose gel electrophoresis.     

Optimized transformation by electroporation of Lactococcus lactis IL1403

Summary Rolling circle replication of plasmid pGKV21 in L. lactis was a more stable replication mode than the theta replication of plasmid pIL253. When the plasmid pGKV21 was used to develop and optimize transformation of L. lactis by electroporation, optimum transformation was obtained using dense suspension of late-exponential phase cells subjected to a high voltage (12.5 kV/cm) and a short pulse time (0.5 ms) in the presence of a plasmid DNA. With plasmid vector pGKV21, a transformation frequency of 2.2 × 10⁸ transformants per g of DNA was obtained.     

Transformation of Salmonella typhimurium TA1538 by optimized electroporation

Summary Standard electroporation of Salmonella typhimurium TA1538 by the plasmid pAMH70 yielded 1.3 × 10² transformants/g of plasmid DNA. Three parameters: resistance (U₁), voltage (U₂) and dose of plasmid DNA (U₃) were optimized with two Doehlert designs. Transformation was increased 75 fold. Optimal values for U₁, U₂ and U₃ were 761.0 , 1.6 kV and 96.1 ng respectively. The most important parameters were U₂ and the couple U₁/U₂.     

Selection methods in bacilli for recombinants and transformants of intra- and interspecific fused protoplasts

The intraspecific fusion frequencies obtained with the direct selection method on a semi-synthetic regeneration medium between strains of B. subtilis and B. licheniformis were distribution from 9.9×10^-2 to 4.5×10^-3, which was one or two orders higher than those of interspecific recombinations between B. subtilis and B. licheniformis.The regeneration media were also useful for selecting interspecific transformants from plasmid carrier to non-carrier. This selection could be used as a primary selection method for inter-and intraspecific recombinants obtained by protoplast fusion.Abbreviations NTG N-methyl-N-nitro-N-nitrosoguanidine - Cm chloramphenicol - SMM 0.5 M sucrose-0.02 M MgCl₂-0.02 M maleate buffer, pH 6.5     

Secretory overproduction of Arthrobacter simplex 3-ketosteroid Δ1-dehydrogenase by Streptomyces lividans with a multi-copy shuttle vector

The gene for 3-ketosteroid ¹-dehydrogenase (ksdD) of Arthrobacter simplex was expressed in Streptomyces lividans and the secreted enzyme was overproduced by using a multi-copy shuttle vector composed of pIJ702 and pUC19. Deletional analysis of the recombinant plasmid showed that the entire coding sequence of the ksdD gene was located within a 7-kb segment of the chromosomal DNA obtained from the enzyme-producing strain of A. simplex. When S. lividans carrying the recombinant plasmid was grown in an appropriate medium, the cells produced about 100-fold more 3-ketosteroid ¹-dehydrogenase than the original strain. Although the percentage of enzyme secreted was changed during cultivation, a maximum 55% of the enzyme was secreted into the cultured medium of S. lividans, while A. simplex did not produce the enzyme extracellularly. Secretory overproduction of 3-ketosteroid ¹-dehydrogenase in S. lividans was also identified by sodium dodecyl sulfate/polyacrylamide gel electrophoresis and on native gel, and the enzyme reaction was confirmed by reverse-phase HPLC using 4-androstene-3,17-dione as a substrate.     

Development of Chainia as a host for xylanase gene cloning : Evidence for occurrence of a restriction-modification system

Summary Conditions for genetic transformation of the xylanase-negative (X^–) strain of Chainia with pIJ 702 were optimized. The growth of Chainia at 30°C for 36 – 40 h and addition of geletin (1%) to the medium resulted in the maximum yield of protoplasts and regeneration efficiency. Poor transformation efficiency of Chainia (X^–) protoplasts by native pIJ 702 versus improved efficiency (16 transformants ug^–1 of plasmid DNA) by prior heating of protoplasts at 42°C for 10 min suggests the occurrence of a restriction system in Chainia. Increased transformation efficiency by passage of the plasmid through Chainia together with the altered methylation status of the transformant plasmid presents evidence for the existence of an operative modification system in Chainia. Development of thiostrepton resistance and formation of me1amin pigment in Chainia (X^–) by transformation with pIJ 702 reveal that genes from Streptomyces can be functionally expressed by Chainia (X^–).(NCL Communication No. 6207)