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)     

Application of protoplast technology to CRISPR/Cas9 mutagenesis: from single‐cell mutation detection to mutant plant regeneration

Plant protoplasts are useful for assessing the efficiency of clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR‐associated protein 9 (Cas9) mutagenesis. We improved the process of protoplast isolation and transfection of several plant species. We also developed a method to isolate and regenerate single mutagenized Nicotianna tabacum protoplasts into mature plants. Following transfection of protoplasts with constructs encoding Cas9 and sgRNAs, target gene DNA could be amplified for further analysis to determine mutagenesis efficiency. We investigated N. tabacum protoplasts and derived regenerated plants for targeted mutagenesis of the phytoene desaturase (NtPDS) gene. Genotyping of albino regenerants indicated that all four NtPDS alleles were mutated in amphidiploid tobacco, and no Cas9 DNA could be detected in most regenerated plants.     

Nuclear genomic composition of asymmetric fusion products between irradiated transgenic Solanum brevidens and S. tuberosum: limited elimination of donor chromosomes and polyploidization of the recipient genome

Summary The production of asymmetric somatic hybrid calli after fusion between gamma-irradiated protoplasts from transgenic Solanum brevidens and protoplasts from S. tuberosum are reported. Transgenic (kanamycin-resistant, GUS-positive) S. brevidens plants and hairy root clones were obtained after transformation with Agrobacterium tumefaciens LBA 1060 (pRi1855) (pBI121) and LBA 4404 (pRAL4404) (pBI121), and A. rhizogenes LBA 9402 (pRi1855) (pBI121), respectively. Leaf protoplasts isolated from the transgenic plants or root protoplasts from the hairy root clones were fused with S. tuberosum leaf protoplasts, and several calli were selected on kanamycin-containing medium. The relative nuclear DNA content of the hybrid calli was measured by flow cytometry (FCM), and the percentages of DNA of the S. brevidens and S. tuberosum genomes in the calli were determined by dot blot analysis using species-specific DNA probes. Chromosome-specific restriction fragment length polymorphism (RFLP) markers were used to investigate the elimination of specific S. brevidens chromosomes in the hybrids. The combined data on FCM, dot blot and RFLP analysis revealed that 18–62% of the S. brevidens DNA was eliminated in the hybrid calli and that the RFLP marker for chromosome 7 was absent in seven out of ten calli. The absence of RFLP markers for chromosomes 5 and 11 hardly ever occurred. In most of the hybrids the ploidy level of the S. tuberosum genome had increased considerably.     

Efficient transformation ofMicromonospora melanosporea protoplasts byStreptomyces plasmid

An efficient and relatively simple procedure forMicromonospora melanosporea protoplast preparation and transformation is described. Transformation ofM. melanosporea protoplast by theStreptomyces plasmid pIJ702 was optimized by altering parameters affecting the formation, regeneration, and transformation of protoplasts. Improvement of regeneration medium resulted in relatively quick growth of transformants (only 7 days). As a result of these experiments we describe a new transformation method that has routinely yielded 10⁶ transformants/µg plasmid DNA.     

Development of a multifunctional and efficient conjugal plasmid for use in Streptomyces spp

A plasmid, pGB112, has recently been developed to transfer DNA from Escherichia coli to Streptomyces spp via conjugation. This technique made use of (A) E. coli replicon, (B) ampicillin (amp) resistance gene for selection in E. coli and thiostrepton (tsr) resistance gene for selection in Streptomyces, (C) a fragment of SCP2* replicon, (D) a 2.6 kb fragment of tra-cassette which consists of pIJ101 transfer gene (tra) and two ermE promoters, (E) a 0.8 kb fragment of oriT of (IncP) RK2. The results showed that this plasmid was able to transfer plasmid DNA from E. coli to Streptomyces coelicolor via conjugation, and that it could also transfer DNA between Streptomyces strains. Since this plasmid has both pBR322 and SCP2* replicons, it may provide a novel and useful method for genetic operation in E. coli and Streptomyces.An erratum to this article can be found at     

Efficient transformation of Arabidopsis thaliana using direct gene transfer to protoplasts

Summary Direct gene transfer has proved to be an efficient transformation method for arabidopsis thaliana, a member of the Brassicaceae. Transgenic Arabidopsis plants resistant to hygromycin B have been regenerated from mesophyll protoplasts treated with polyethylene glycol and plasmid DNA carrying the hygromycin phosphotransferase (HPT) gene under the control of the 35 S promoter of cauliflower mosaic virus. The transformation procedure reproducibly yields transformants at frequencies of approximately 1×10^-4 (based on the number of protoplasts treated) or 5% (based on the number of regenerating calli). DNA from plants regenerated from hygromycin resistant colonies was analysed by Southern blot hybridization demonstrating that the foreign gene is stably integrated into the plant chromosome. Genetic analysis of several hygromycin resistant plants showed that the HPT gene is transmitted to the progeny. Transformation experiments performed with a selectable and a non-selectable gene on separate plasmids resulted in a co-transformation rate of functionally active copies in about 25% of the transformants analysed. Hence this approach can be used to introduce non-selectable genes into the Arabidopsis genome.     

A new protoplast culture system in Daucus carota L. and its applications for mutant selection and transformation

Petiole protoplasts from in vitro-grown carrot plants are a very good alternative to traditionally obtained protoplasts from suspension cultures. High plating and regeneration efficiencies were obtained in most of the breeding lines that were tested. The embedding of the protoplasts in alginate was crucial for initiating cell division and further development. Several streptomycin resistant and chlorophyll-deficient plant lines were selected for using the petiole protoplast system. Maternally inherited streptomycin resistance was demonstrated by sexual crosses. Protoplast fusion of several chlorophyll-deficient lines did not result in complementation, indicating the cytoplasmic nature of the mutations. Petiole protoplasts were used for direct transformation with plasmid DNA pNUNV containing NPTII as a selectable marker. High transformation frequencies (up to 1%) were obtained after PEG treatment of the protoplasts. Kanamycin resistance was shown to be inherited as a single dominant nuclear trait.     

Repeated polyketide synthase modules involved in the biosynthesis of a heptaene macrolide by Streptomyces sp. FR-008

Genes for biosynthesis of a Streptomyces sp. FR-008 heptaene macrolide antibiotic with antifungal and mosquito larvicidal activity were cloned in Escherichia coli using heterologous DNA probes. The cloned genes were implicated in heptaene biosynthiesis by gene replacement. The FR-008 antibiotic contains a 38-membered, poiyketide-derived macrolide ring. Southern hybridization using probes encoding domains of the type i modular erythromycin polyketide synthase (PKS) showed that the Streptomyces sp. FR-008 PKS gene cluster contains repeated sequences spanning c. 105 kb of contiguous DNA; assuming c. 5 kb for each PKS module, this is in striking agreement with the expectation for the 21-step condensation process required for synthesis of the FR-008 carbon chain. The methods developed for transformation and gene replacement in Streptomyces sp. FR-008 make it possible to genetically manipulate polyene macrolide production, and may later lead to the biosynthesis of novel polyene macrolides.     

Conserved Organization of Genes for Biosynthesis of Chlortetracycline in Streptomyces Strains

By genomic Southern blot analysis, the DNA sequences homologous to the gene cluster responsible for biosynthesis of 6-demethylchlortetracycline in Streptomyces aureofaciens NRRL3203 were shown to be highly conserved in independent chlortetracycline- or tetracycline producing Streptomyces strains. By contrast, oxytetracycline-producing Streptomyces strains had no hybridization with the cluster DNA.     

Physical and genetic analysis of IS110, a transposable element of Streptomyces coelicolor A3(2)

Summary On at least three independent occasions a 1.6 kb segment of Streptomyces coelicolor DNA was detected in apparently the same location in an attP-deleted derivative of the temperature phage C31 that carried a selectable viomycin resistance gene. This sequence (termed IS110) allowed integration of the phage (giving viomycin-resistant transductants) at homologous sequences (detected by Southern hybridisation) at several locations in the S. coelicolor genome. The inserted prophages facilitated genetic mapping of two IS110 copies in the chromosomal linkage map. A third copy did not exhibit simple segregation with chromosomal markers, and there appeared to be a frequent DNA rearrangement close to this copy. Some variation in the number of copies of IS110 and their location has taken place in the pedigree of S. coelicolor derivatives. IS110 did not hybridise to any known S. coelicolor plasmid, nor to any of several other IS-like elements previously described in other Streptomyces plasmids or phages. It hybridised strongly to DNA from only a small minority of other Streptomyces species and was absent from S. lividans, a close relative of S. coelicolor.     

The interaction betweenE. coli spheroplasts and plant protoplasts: A proposed procedure to deliver foreign genes into plant cells

Summary E. coli spheroplasts can be used to deliver DNA vectors into plant protoplasts. The use of fluorescent dyes showed that 25–100% of the protoplast population was associated with 1–9 spheroplasts following incubation with several fusogens. Electron microscopy demonstrated spheroplasts attached to protoplasts via a plasma membrane protrusion after high pH/Ca²⁺ treatment, but PEG-high pH/Ca²⁺ promoted endocytosis of spheroplasts into a plasma membrane bounded vesicle. Ultrastructural profiles showed that fusion between spheroplasts and protoplasts did not occur. Immunofluorescence studies detectedE. coli antigens associated with tobacco protoplasts, and after fusogen treatment the antigens were dispersed within the peripheral cytoplasm. The elimination of residual contaminatingE. coli cells from protoplasts was achieved by lysozyme and antibiotic treatment, thus allowing DNA vector assessment in axenic culture.     

Impairment of Streptomyces and Micromonospora Differentiation by Dna-Methylase Inhibitors

Abstract

The effect of several inhibitors of DNA methyltransferases has been tested on Streptomyces and Micromonospora cultures. Some of them (5-aza-2′-deoxycytidine, 5-azacytidine and L-ethionine), inhibited sporulation at concentrations that did not significantly affect growth.     

Transfection of protoplasts from Streptomyces lividans 66 with actinophage SH10 DNA

Summary The slightly modified procedure for the transformation of protoplasts of S. coelicolor A3 (2) with SCP2 plasmid DNA and polyethylenglycol (PEG) (Bibb et al., 1978) was extented to infection of protoplasts of S. lividans 66 with actinophage SH10 DNA (Klaus et al., 1979).Maximal yield of transfected protoplasts was obtained at 20% PEG, 3 mM sodium-citrate and 150 mM NaCl final concentrations. The efficiency of transfection was determined to be about 2×10^-8 to 2×10^-7. The average value of competent protoplasts was about 1–2×10^-4 of regenerating protoplasts.In comparison with outgrowing spores infected with phage particles the average burst size of transfected protoplasts was reduced from 100 to 10 pfu/infected cell, the latent period prolonged from 45 min to 120 min and the rise period was not affected.     

Asymmetric somatic hybridization between UV-irradiated <Emphasis Type="Italic">Citrus unshiu</Emphasis> and <Emphasis Type="Italic">C</Emphasis>. <Emphasis Type="Italic">sinensis</Emphasis>: regeneration and characterization of hybrid shoots

In the present paper, attempts were made to explore the possibility of employing ultraviolet (UV) irradiation in citrus asymmetric fusion for transfer of limited amount of favorable traits from a desirable cultivar to a target one. Exposure of Satsuma mandarin (Citrus unshiu Marc.) embryogenic protoplasts to UV at an intensity of 300 μW cm⁻² led to reduced viability, especially under long irradiation duration. The protoplasts could not grow during culture when they were irradiated for over 30 s. Terminal deoxynucleotidyl transferase biotin-dUTP nick end labeling (TUNEL) assay revealed extensive DNA fragmentation in the UV-irradiated protoplasts compared with those without UV treatment. Electrofusion between UV-irradiated protoplasts of Satsuma mandarin (donor) with those of Jincheng (C. sinensis Osbeck, recipient), a local cultivar of superior quality, gave rise to regeneration of several lines of shoots, which failed to root despite enormous endeavors. Ploidy analysis via flow cytometry and chromosome counting showed that four selected shoots were either diploid, triploid or tetraploid. Random amplified polymorphism DNA (RAPD) and amplified fragment length polymorphism (AFLP) confirmed the shoots, irrespective of their ploidy level, as putative somatic hybrids. Cleaved amplified polymorphism sequences (CAPS) demonstrated that the shoots predominantly got their cytoplasmic components, in terms of chloroplast (cp) and mitochondrion DNA, from Jincheng, along with possible recombination of cpDNA in some shoot lines. The current data indicated that UV-based asymmetric fusion could also be employed in citrus somatic hybridization with the intention of creating novel germplasms, which may provide an alternative approach for cultivar improvement.     

Liposome-mediated delivery of DNA to carrot protoplasts

The encapsulation of DNA within liposomes and subsequent fusion of the liposomes with carrot (Daucus carota L.) protoplasts were examined to determine optimum conditions for effective liposome-mediated delivery of DNA to protoplasts. Escherichia coli [³H]DNA could be encapsulated with 50% efficiency using encapsulation volumes as low as 0.5 ml. Incorporation of liposome-encapsulated [³H]DNA by carrot protoplasts increased linearly for 2.5 h, and increasing the ratio of protoplasts to liposomes increased the total amount of radioactive label incorporated within the protoplasts. Liposome-mediated incorporation of [³H]DNA by protoplasts increased over a range of polyethylene glycol concentrations up to 20%, but Ca²⁺ did not increase liposome-mediated incorporation when present in the liposome-protoplast incubation mixture. Optimum incorporation was observed when the pH of the liposome-protoplast incubation medium was decreased to 4.8. Encapsulation experiments using DNA of the plasmid pBR322 indicated that an average of 200–1,000 intact copies of pBR322 were sequestered within each nucleus after liposome delivery.     

Photosynthetic performance of regenerated protoplasts from disintegrated cells of <Emphasis Type="Italic">Bryopsis hypnoides</Emphasis> (Chlorophyta)

The photosynthetic performances of regenerated protoplasts of Bryopsis hypnoides, which were incubated in seawater for 1, 6, 12, and 24 h, were studied using chlorophyll (Chl) fluorescence and oxygen measurements. Results showed that for the regenerated protoplasts, the pigment content, the ratios of photosynthetic rate to respiration rate, the maximal photosystem II (PSII) quantum yield (F_v/F_m), and the effective PSII quantum yield (Φ_PSII) decreased gradually along with the regeneration progress, indicated that during 24 h of regeneration there was a remarkable reduction in PSII activity of those newly formed protoplasts. We assumed that during the cultivation progress the regenerated protoplasts had different photosynthetic vigor, with only some of them able to germinate and develop into mature thalli. The above results only reflected the photosynthetic features of the regenerated protoplasts at each time point as a whole, rather than the actual photosynthetic activity of individual aggregations. Further investigation suggested a relationship between the size of regenerated protoplasts and their viability. The results showed that the middle-sized group (diameter 20–60 μm) retained the largest number of protoplasts for 24 h of growth. The changes in F_v/F_m and Φ_PSII of the four groups of differently sized protoplasts (i.e. < 20, 20–60, 60–100, and > 100 μm) revealed that the protoplasts 20–60 μm in diameter had the highest potential activity of the photosynthetic light energy absorption and conversion for several hours.     

Isolation and identification of Streptomyces sp. Act4Zk,a good producer of Staurosporine and some derivatives

In this study, strain Streptomyces sp. Act4Zk was isolated based on a method developed for the isolation of myxobacteria. Due to the low efficiency of the majority of conventional DNA extraction techniques, for molecular identification of the strain Streptomyces sp. Act4Zk, a new technique for DNA extraction of Actinobacteria was developed. In order to explore potential bioactivities of the strain, extracts of the fermented broth culture were prepared by an organic solvent (i.e. ethyl acetate) extraction method using. These ethyl acetate extracts were subjected to HPLC fractionation against standard micro-organisms, followed by LC/MS analysis. Based on morphological, physiological, biochemical and 16S rRNA gene sequence data, strain Streptomyces sp. Act4Zk is likely to be a new species of Streptomyces, close to Streptomyces genecies and Streptomyces roseolilacinus. Antimicrobial assay indicated high antifungal activity as well as antibacterial activity against Mycobacterium smegmatis and Gram-positive bacteria for the new strain. HPLC and LC/MS analyses of the extracts led to the identification of three different compounds and confirmed our hypothesis that the interesting species of the genus Streptomyces being a good producer of staurosporine and some derivatives.     

Transformation ofStreptomyces avermitilis by plasmid DNA

Summary Polyethylene glycol (PEG) efficiently mediated the transformation ofStreptomyces avermitilis protoplasts by plasmid DNA to yield 10⁷ transformants per g of plasmid DNA. Under conditios in which the maximum transformation frequency was observed, the cotransformation frequency exceeded 10%. The number of transformants increased linearly with the amount of DNA and number ofS. avermitilis protoplasts. Relaxed and supercoiled, but not linear DNA transformed protoplasts efficiently. Dimethyl sulfoxide (DMSO)-mediated transformation of protoplasts was 1000-fold less efficient. PEG and, less efficiently, DMSO also mediated the transformation of whole cells ofS. avermitilis by DNA.     

Production of somatic hybrids between Daucus carota L. and Nicotiana tabacum

Protoplasts of a kanamycin-resistant (KR, nuclear genome), streptomycin-resistant (SR, chloroplast genome) and chlorophyll-deficient (A1, nuclear genome) Nicotiana tabacum (KR-SA) cell suspension cultures or X-ray-irradiated mesophyll protoplasts of kanamycin- and streptomycin-resistant green plants (KR-SR) were fused with protoplasts of a cytoplasmic male-sterile (CMS) Daucus carota L. cell suspension cultures by electrofusion. Somatic hybrid plants were selected for kanamycin resistance and the ability to produce chlorophyll. Most of the regenerated plants had a normal D. carota morphology. Callus induced from these plants possessed 23–32 chromosomes, a number lower than the combined chromosome number (66) of the parents, and were resistant to kanamycin, but they segregated for streptomycin resistance, which indicated that N. tabacum chloroplasts had been eliminated. Genomic DNA from several regenerated plants was analyzed by Southern hybridization for the presence of the neomycin phosphotransferase gene (NPTII); all of the plants analyzed were found to contain this gene. Mitochondrial (mt) DNA was analyzed by Southern hybridization of restriction endonuclease digests of mtDNA with two DNA probes, PKT5 and coxII. The results showed that the two plants analyzed possessed the mitochondria of D. carota. These results demonstrate that the regenerated plants are interfamilial somatic hybrids.