Genetic recombination by protoplast fusion inNocardia asteroides

Summary Fusion and regeneration of protoplasts ofNocardia asteroides strains ATCC 3318, IMRU W3599 and HIK B971 have been used to study genetic recombination in this species. Protoplasts were produced by treatment with lysozyme, following incubation with glycine. Mutants of ATCC 3318 were grown in peptone yeast extract medium at 32°C prior to protoplast production to maximize protoplast frequency, whereas mutants of IMRU W3599 and HIK B971 were grown in trypticase-soy broth. Glycine concentrations favoring protoplast formation varied from 1.5% to 5% depending on strain. For all strains, protoplast formation was complete 1 h after addition of 5 mg/ml lysozyme. Protoplasts were fused by addition of 50% polyethylene glycol-1000. In general, 25% of the protoplasts could be regenerated. The incidence of recombinant recovery was increased up to 750-fold. The distribution of recombinant phenotypes in matings was similar for protoplast fusion and conventional crosses.     

Targeted gene disruption of the avermectin B O-methyltransferase gene in Streptomyces avermitilis

The biological activity of avermectin B components is superior to that of avermectin A components, which are derived from avermectin B by avermectin B 5-O-methyltransferase. Gene disruption, targeting avermectin B 5-O-methyltransferase gene in Streptomyces avermitilis, was carried out to obtain a strain of avermectin B producer. Phenotype analysis of the mutant with the disrupted O-methyltransferase gene showed that only avermectin B components were produced with a significant increase in production     

Parameters governing bacterial regeneration and genetic recombination after fusion of Bacillus subtilis protoplasts.

Bacterial protoplast fusion, induced by polyethylene glycol, has been made more regular and convenient by further specification and improvement of various steps in the previously used procedure. These have made it possible to obtain regularly 100% regeneration of Bacillus subtilis cells from protoplasts before treatment with polyethylene glycol and yields of 10 to 75% from polyethylene glycol-treated protoplasts. Genetic recombination frequencies do not increase correspondingly. Also, when regeneration is reduced by various experimental conditions, recombination does not decrease in proportion. It is concluded that regeneration of recombinant-forming cells is independently determined and not closely related to the average regeneration for the population. Kinetic studies with varying individual parental or total protoplast concentrations strongly indicate that protoplast collision and contact is not the limiting factor determining the number of genetic recombinants obtained. Recombination approximates a linear, rather than quadratic, function of the total or of the majority protoplast population present, from which it is concluded that fusion events are always adequate to produce substantially more potential recombinants than are registered. The strong effect of the majority/minority ratio upon the number of minority cells that become recombinant is independent of which parent is in excess. This shows in a direct and physiological way that both parents are equivalent partners in their genetic contributions.     

Protoplast fusion — a tool for genetic manipulation and breeding in industrial microorganisms

Protoplasts can be isolated from microbial cells by enzymatic digestion of the cell wall, in the presence of an osmotic stabilizer. Such protoplasts can be induced to fuse in the presence of agents such as the poly (ethylene glycols). When suitably selected auxotrophic strains are used, the fusion products can be recovered by selection on the basis of nutritional complementation. Cultivation of the protoplasts on a hypertonic growth medium induces regeneration of new cell wall material and their subsequent reversion to the normal cell form of the organism. The protoplast fusion technique has been applied sucessfully to both bacterial and fungal systems leading to the recovery of recombinant progeny. In the fungi, the recovery of non-parental segregants from inter-species crosses has also been demonstrated. In assessing the value of the fusion technique, caution may be necessary at this stage in its application to genetic mapping in bacteria. The behaviour of protoplasts, especially with respect to reversion, could be an additional factor that operates during selection, distorting recombination frequencies. However, the fusion technique, in providing a mechanism by which genetic recombination can be readily achieved, should be of great potential in empirical breeding and strain improvement. These aspects are reviewed.     

Somatic hybrids between Brassica juncea (L). Czern. and Diplotaxis harra (Forsk.) Boiss and the generation of backcross progenies

An attempt to transfer genes from droughttolerant Diplotaxis harra, a wild relative of Brassica species, to an elite oil-yielding cultivar, B-85, of mustard (Brassica juncea) was made through protoplast fusion, as the two plant systems are sexually incompatible. By following the standard protocol for PEG-mediated protoplast fusion followed by high pH, high Ca⁺⁺, DMSO treatment and appropriate cell-culture technique, 16 presumptive somatic hybrid plants could be regenerated. Chromosomal analysis of four such somatic hybrids revealed that three of them were asymmetric. Analysis of morphological characters, meiotic chromosomes, and esterase isoenzyme pattern revealed that all the somatic hybrids were different from each other. Furthermore four chromosomes of each genome could undergo homoeologous pairing at meiosis indicating the possibilities for genetic recombination and chromosomal rearrangements. Irregular distribution of chromosomes at anaphase-II at meiosis has been a consistent feature of these plants. Eventually, pollen of all the somatic hybrids showed complete infertility preventing the recovery of any selfed seed. Nevertheless, ovule fertility of one somatic hybrid was not totally impaired as it had set some seeds upon backcrossing with the B. juncea parent. The esterase isoenzyme banding pattern of 24 individual progeny plants of this backcross provided evidence for their recombinant nature. It was thus confirmed that a transfer of genetic traits from Diplotaxis harra to B. juncea had indeed taken place. Furthermore, it was conceptualised that a transfer of alien genes through the protoplast-fusion technique is primarily possible in situations where meiotic pairing of the chromosomes of the two participating genomes generates recombinant gametocytes which can pass through subsequent filial generations.     

Avermectin: biochemical and molecular basis of its biosynthesis and regulation

Avermectin and its analogues, produced by Streptomyces avermitilis, are major commercial antiparasitic agents in the field of animal health, agriculture, and human infections. They are 16-membered pentacyclic lactone compounds derived from polyketide and linked to a disaccharide of the methylated deoxysugar l-oleandrose. Labeling studies, analyses of the biosynthetically blocked mutants, and the identification of the avermectin gene cluster allows characterization of most of the biosynthetic pathway. Recent completion of S. avermitilis genome sequencing is also expected to help in revealing the precise biosynthetic sequence and the complicated regulatory mechanism for avermectin biosynthesis, which has been long-awaited to be elucidated. The well characterized avermectin biosynthetic pathway and availability of S. avermitilis genome information in combination with the recent development of combinatorial biosynthesis should allow us to redesign more potent avermectin analogues and to engineer S. avermitilis as a more efficient host for the production of important commercial analogues.     

Construction of ivermectin producer by domain swaps of avermectin polyketide synthase in Streptomyces avermitilis

Ivermectin, 22, 23-dihydroavermectin B1, is commercially important in human, veterinary medicine, and pesticides. It is currently synthesized by chemical reduction of the double bond between C22 and C23 of avermectins B1, which are a mixture of B1a (>80%) and B1b (<20%) produced by fermentation of Streptomyces avermitilis. The cost of ivermectin is much higher than that of avermectins B1 owing to the necessity of region-specific hydrogenation at C22–C23 of avermectins B1 with rhodium chloride as the catalyst for producing ivermectin. Here we report that ivermectin can be produced directly by fermentation of recombinant strains constructed through targeted genetic engineering of the avermectin polyketide synthase (PKS) in S. avermitilis Olm73-12, which produces only avermectins B and not avermectins A and oligomycin. The DNA region encoding the dehydratase (DH) and ketoreductase (KR) domains of module 2 from the avermectin PKS in S. avermitilis Olm73-12 was replaced by the DNA fragment encoding the DH, enoylreductase, and KR domains from module 4 of the pikromycin PKS of Streptomyces venezuelae ATCC 15439 using a gene replacement vector pXL211. Twenty-seven of mutants were found to produce a small amount of 22, 23-dihydroavermectin B1a and avermectin B1a and B2a by high performance liquid chromatography and liquid chromatography mass spectrometry analysis. This study might provide a route to the low-cost production of ivermectin by fermentation.     

Screening of spontaneous and induced mutants in Streptomyces avermitilis enhances avermectin production

Because of the loss of productivity in industrial strains (as a consequence of genetic instability), the selection of spontaneous and induced mutants in Streptomyces might generate enhanced producers of bioactive compounds. In this work, a spontaneously high producing mutant of Streptomyces avermitilis, strain 267/2H, was isolated. This mutant produced 8.2 times more avermectin B₁ than the wild type and it was treated with methyl methanesulphonate (MMS) in order to obtain better avermectin producers. One mutant, strain IPT-85, produced about 16 times more avermectin than the wild-type strain ATCC 31267 and twice as much as the parental strain 267/2H. Reversion studies showed that avermectin production by the IPT-85 mutant was unstable and required constant selection to maintain high levels of avermectin B₁ production. Upon a second MMS treatment of IPT-85, a new avermectin-aglycone-producing mutant, strain IPT 85-62, was isolated. Received: 2 March 1999 / Received revision: 16 June 1999 / Accepted: 27 June 1999     

Induction and Characterization of Artificial Diploids from the Haploid Yeast Torulaspora delbrueckii

The yeast Torulaspora delbrueckii, which propagates as a haploid, was made into a diploid by treatment with dimethyl sulfoxide (DMSO) on the regeneration of protoplasts. The diploid state was stably inherited; the cell volume was three times that of the parent strain and the cellular DNA content was two times that of the parental strain. No essential difference was found between diploids induced by DMSO and those formed through intraspecific protoplast fusion. The diploid strains sporulated fairly well, with their cells converting directly into asci. Random spore analysis revealed that diploids induced through protoplast fusion gave rise to auxotrophic segregants (haploids) with the parental genetic marker or to segregants formed by recombination, while diploids induced by DMSO from a doubly auxotrophic parent gave rise to no recombinant, indicating that it was chromosomally homoallelic in nature. The magnesium level in the protoplast regeneration medium was found to be an important factor for inducing diploid formation. At 0.2 mM magnesium diploids appeared even in the absence of DMSO, while at 2 mM magnesium diploids never appeared unless DMSO was added to the regeneration medium. Evidence is provided that the diploids induced by DMSO or a low magnesium level are due to direct diploidization but not protoplast fusion. UV light irradiation of intact cells (without protoplasts), 10% of which survived, also produced diploids among this surviving population. From these results we conclude that the perturbation of protoplast regeneration or of cell division by the treatments mentioned above somehow induced direct diploidization of T. delbrueckii.     

Characterization of a regulatory gene, <Emphasis Type="Italic">aveR</Emphasis>, for the biosynthesis of avermectin in <Emphasis Type="Italic">Streptomyces avermitilis</Emphasis>

Avermectin is an important macrocyclic polyketide produced by Streptomyces avermitilis and widely used as an anthelmintic agent in the medical, veterinary, and agricultural fields. The avermectin biosynthetic gene cluster contains aveR, which belongs to the LAL-family of regulatory genes. In this study, aveR was inactivated by gene replacement in the chromosome of S. avermitilis, resulting in the complete loss of avermectin production. The aveR mutant was unable to convert an avermectin intermediate to any avermectin derivatives, and complementation by intact aveR and its proper upstream region restored avermectin production in the mutant, suggesting that AveR is a positive regulator controlling the expression of both polyketide biosynthetic genes and postpolyketide modification genes in avermectin biosynthesis. Despite the general concept that an increased amount of a positive pathway-specific regulator leads to higher production, a higher amount of aveR resulted in complete loss of avermectin, indicating that there is a maximum threshold concentration of aveR for the production of avermectin.     

Impact of the stringency of cell selection on plastid segregation in protoplast fusion-derived Nicotiana regenerates

Summary Vegetative segregation of a mixed plastid population in protoplast fusion-derived cell lines can be directed by a selection favouring the multiplication of one of the parental plastid types. This report defines some of the critical conditions leading to a homogeneous plastid population in cybrid plants generated by protoplast fusion between Nicotiana plumbaginifolia and an albino and streptomycin-resistant N. tabacum plastid mutant. Light (1,500 lx) conferred a strong selective advantage to chloroplasts versus albino plastids, while the lack of this effect in dim light (300 lx) indicated that a sufficient light intensity is essential to the phenomenon. Selection on streptomycin-containing medium in the dark, however, led to the preferential multiplication of resistant plastids. Streptomycin selection of resistant chloroplasts in the light, consequently, results in a plastid selection of doubled stringency. In another experiment a definite, but leaky, selection for chloroplast recombination (selection for greening on streptomycin-containing medium in dim light) was used to reveal various recombination products. Protoplast fusion in fact resulted in cybrid plants showing only simple chimeric segregation of unchanged parental plastids. These results demonstrate the essential requirement for stringent plastid selection, as defined by cell culture conditions, to precede the formation of shoots expected to possess the desired plastid genetic composition.     

Transmission and recombination of mitochondrial genes in Saccharomyces cerevisiae after protoplast fusion

Summary Protoplasts of auxotrophic strains of Saccharomyces cerevisiae of opposite and identical mating types carrying different mitochondrial drug-resistance markers, with both homosexual and heterosexual mitochondrial backgrounds, were induced to fuse by polyethylene glycol. After selective regeneration of prototrophic fusion products, the transmission and recombination frequencies of mitochondrial genes in populations of cells were determined and compared with those obtained in mating processes. The frequencies obtained in the fusion experiments proved very similar to those found in the zygote clones. The behavior of mitochondrial genes was apparently affected neither by nuclear mating type background nor by the method of transfer of mitochondrial genomes (i.e., protoplast fusion or mating), making possible mitochondrial genetic studies by protoplast fusion irrespective of the mating type barrier of yeast strains.     

微生物原生质体融合育种技术及其应用

工业微生物菌种选育在发酵工业中占有重要地位。微生物原生质体融合(microbial protoplast fusion)技术具有重组频率高、受结合型或致育型限制小以及遗传物质传递完整等优点,是微生物育种最常用的方法之一。结合相关研究进展,分析了原生质体融合技术的组成,包括制备、再生、融合的影响因素以及融合子的筛选方法,重点评述了原生质体融合技术应用在微生物育种中的最新进展,以及微生物原生质体融合技术的发展前景。     

Modulation of Serine/Threonine Protein Kinase Activity in Chloramphenicol-Resistant Mutants of Streptomyces avermitilis

A mutation to chloramphenicol resistance (Cml^r) stimulates production of macrolide avermectin in Streptomyces avermitilis; production starts in the early stationary phase. By labeling in vivo, the Cml^r mutation was shown to stimulate phosphorylation of Ser and Thr in several proteins in the same growth phase. Autophosphorylation of active protein kinases (PK) was analyzed in gel after one- or two-dimensional PAGE for the original S. avermitilis strain ATCC 31272, its Cml^r mutant, and a Cml^s revertant. An increase in in vivo phosphorylation was associated with an increase in autophosphorylation of Ser/Thr-PK 41K, 45K, 52K, 62K, and 85K and complete suppression of autophosphorylation of PK 66K. Comparison of the PK molecular weights and pI with the parameters deduced for putative PK encoded by S. avermitilis genes identified the 41K, 45K, 52K, 62K, and 85K proteins as pkn 24, pkn 32, pkn 13, pkn12, and pkn5, respectively. Prenylamine lactate, a modulator of calmodulin-dependent processes, substantially reduced the avermectin production, impaired the Cml resistance, and selectively inhibited Ca²⁺-dependent PK 85K in the Cml^r mutant. It was assumed that PK 85K is involved in regulating the avermectin production.     

Fungal protoplast fusion – a revisit

Protoplast fusion is a non-specific recombination technique used for transfer of cytosolic organelles including genetic material. The process involves cell wall breakdown, regeneration of protoplasts, chemofusion and electrofusion. This review article discusses all the stages involved in fusion of protoplasts and some of the applications of protoplast fusion technique in fungal systems.     

Diploid state of phenotypically recombinant progeny arising after protoplast fusion in Bacillus subtilis

Summary After fusion of Bacillus subtilis protoplasts the phenotypically recombinant clones isolated, whether immediately or as segregants of complementing diploid clones, have in common the following properties. They appear independently of the recN ⁺ gene, most often as the result of apparently non-reciprocal recombination occurring in genetic intervals encompassing the origin and the terminus of replication. First indicated by reciprocal fusion crosses between 105-lysogenic and 105-sensitive strains, the diploidy of the recombinants was confirmed by studying the transforming activities of their DNA. These experiments establish heterozygosity at eight loci scattered on the chromosome map. By revealing the presence of the rpF ⁺ allele in trpF7 recombinants, the results also strongly suggest that stable phenotypic recombinants may arise by genetic inactivation. Two possible genetic structures for these recombinants are discussed, one implying total inactivation of one recombinant chromosome, the other a segmentary inactivation of one unrecombined chromosome. Whatever the structure, genetic stability is not a reliable sign of haploidy in bacterial clones produced after protoplast fusion.     

Genotyping of somatic hybrids between <Emphasis Type="Italic">Festuca arundinacea</Emphasis> Schreb. and <Emphasis Type="Italic">Triticum aestivum</Emphasis> L.

In order to genotype hybrid genomes of distant asymmetric somatic hybrids, we synthesized hybrid calli and plants via PEG-mediated protoplast fusion between recipient tall fescue (Festuca. arundinacea Schreb.) and donor wheat (Triticum aestivum L.). Seventeen and 25 putative hybrid clones were produced from the fusion combinations I and II, each with the donor wheat protoplast treated by UV light for 30 s and 1 min, respectively. Isozyme and RAPD profiles confirmed that ten hybrid clones were obtained from combination I and 19 from combination II. Out of the 29 hybrids, 12 regenerated hybrid plants with tall fescue phenotype. Composition and methylation-variation of the nuclear and cytoplasmic genomes of some hybrids, either with or without regenerative ability, were compared by genomic in situ hybridization, restriction fragment length polymorphism, and DNA methylation-sensitive amplification polymorphism. Our results indicated that these selected hybrids all contained introgressed nuclear and cytoplasmic DNA as well as obvious methylation variations compared to both parents. However, there were no differences either in nuclear/cytoplasmic DNA or methylation degree between the regenerable and non-regenerable hybrid clones. We conclude that both regeneration complementation and genetic material balance are crucial for hybrid plant regeneration.     

Genetic recombination in Actinoplanes brasiliensis by protoplast fusion.

Protoplast formation, fusion, and cell regeneration have been achieved with mutant strains of Actinoplanes brasiliensis. Three-, four-, and five-factor crosses have shown genetic recombination among the markers, and a five-factor cross is analyzed and discussed. Possibilities of using protoplast fusion for gene mapping and strain improvement are suggested.     

The avermectin-producing Streptomyces avermitilis possesses an inducible valine dehydrogenase

Summary Valine dehydrogenase (VDH) is believed to be absent in Streptomyces avermitilis. In the present study, a VDH (M _r, 72 000) was detected by activity measurement and activity staining on a native-PAGE gel. The enzyme activity was induced by L-valine and repressed by ammonia. VDH activity was found to be significantly lower than L-valine transaminase activity. The results suggest that one active VDH does exist in S. avermitilis, and plays a role in valine catabolism and avermectin biosynthesis.     

“Arabidobrassica”: Chromosomal recombination and morphogenesis in asymmetric intergeneric hybrid cells

A somatic hybrid cell line, cloned from an individual protoplast-fusion product between Arabidopsis thaliana and Brassica campestris, gave rise to formation of numerous plants differing drastically in morphology. Analysis of these various regenerants, all of which originated from one and the same heterokaryon derived from the fusion of two cells, shows the unspecific elimination of chromosomes of both parental species during the callus growth phase. Whereas the parental cells have so far not been sucessfully regenerated into plants, several of their different asymmetric hybrids are capable of morphogenesis. Furthermore, chromosomal analysis indicates extensive recombination. Most of the plants are predoinantly morphologically regular. Abnormalities are mostly limited to the flowers which tend to undergo phyllody. The results demonstrate that remote somatic hybridization may have applications although true amphidiploids may not be obtainable. The transfer of small units of genetic material between distantly related species by protoplast fusion seems to be a more realistic approach than the combination of complete, highly diverse genomes.