Genetic interactions in Streptomyces rimosus mediated by conjugation and by protoplast fusion

The development of a protoplast fusion technique for oxytetracycline-producing Streptomyces rimosus strains, and its evaluation for the application for a breeding programme, has been described. Treatment of S. rimosus protoplasts with 40% (w/v) PEG 1550 for 30 min gave optimal numbers of recombinants ranging from 1 to 10% of the total progeny. Therefore, by comparison with conjugation, protoplast fusion increased the frequency of recombination by two to three orders of magnitude. The proportion of multiple crossover classes amongst recombinants was higher, by a factor of ten, after protoplast fusion (13.3%) than after conjugation (1.5%). Participation of less frequent complementary genotype doubled from 9.0% in conjugation to 17.9% in protoplast fusion. Overall, this suggested that the opportunities for crossing over in a fusion of S. rimosus protoplasts were spatially and/or temporally extended leading to a loosening of linkage with a near-random assortment of genotypes in a cross. However, by minimizing the multiple crossover classes and calculating allele frequency gradients, it was shown that the protoplast fusion technique allows arrangement of genetic markers on the S. rimosus chromosome. These are ideal characteristics for the recombination of divergent lines in a strain improvement programme.     

Plasmid transfer and genetic recombination by protoplast fusion in staphylococci.

The experimental conditions for plasmid transfer and genetic recombination in Staphylococcus aureus and some coagulase-negative staphylococci by protoplast fusion are described. Protoplasts were prepared by treatment with lysostaphin and lysozyme in a buffered medium with 0.7 to 0.8 M sucrose. Regeneration of cell walls was accomplished on a hypertonic agar medium containing succinate and bovine serum albumin. Transfer of plasmids occurred after treatment of the protoplast mixtures with polyethylene glycol (molecular weight, 6,000) not only between strains of the same species but also between parents of different species, although at approximately 100 times lower frequency in the latter case. Recombination of the chromosomal genes in fused protoplasts required simultaneous treatment of the mixed protoplasts with polyethylene glycol and CaCl2. A method was developed for isolation of recombinants after fusion between mutants of S. areus carrying unselectable markers. Antibiotic resistance plasmids were introduced into the parental strains and used as primary markers to detect protoplast fusion. Chromosomal recombinants were found among the clones with both parental plasmids at a high frequency. The method appears to have simple applications in the construction of strains with multiple mutant characters.     

Organellar segregation,rearrangement and recombination in protoplast fusion-derived Brassica oleracea calli

Summary Cauliflower protoplasts were fused to determine the effect of protoplast source and pretreatment on organellar segregation in fusion products. Mitochondrial and chloroplast type were determined for over 250 calli from eight fusions between iodoacetate-treated or -irradiated leaf or hypocotyl protoplasts with fertile or Ogura cytoplasms. Organelles in fusion-derived calli were identified with five mitochondrial probes and one chloroplast probe. Mitochondrial and chloroplast segregation were independent but biased. Most calli had B. oleracea chloroplasts, but more calli had Ogura mitochondria than B. oleracea ones. Neither protoplast source nor pretreatment alone affected organelle segregation. However, iodoacetate treatment of hypocotyl protoplasts reduced their mitochondrial contribution to the fusion products although it did not affect chloroplast segregation. Over half of the calli had mitochondrial genomes distinct from those of either fusion partner; many of these contained the complete mitochondrial genome of one partner along with some mitochondrial DNA from the other. Out of 258 calli, 83 showed evidence of mitochondrial recombination, most commonly by formation of a novel 11-kb PstI fragment near the atp9 region.     

Absence of mitochondrial and chloroplast DNA recombinations in Brassica napus plants regenerated from protoplasts,protoplast fusions and anther culture

Summary Over 400 Brassica napus plants regenerated from individual protoplasts, from protoplast fusions and from anther culture were analysed for chloroplast and mitochondrial genome rearrangements by restriction fragment length polymorphisms. None were detected, attesting to the fidelity of the tissue culture procedures employed. In the majority of protoplast fusion products, the cytoplasmic organelles had completely sorted out at the callus stage but three regenerated plants possessed mixed parental populations of mitochondrial genomes and one regenerant contained mixed chloroplast genomes. In all four examples, the cytoplasmic genome sorted out in planta in favor of one parental type which was faithfully maternally transmitted to progeny.     

Factors affecting recombinant frequency in protoplast fusions of Streptomyces coelicolor.

The optimum concentration of polyethylene glycol 1000 (PEG) for the production of recombinants through protoplast fusion in Streptomyces coelicolor was about 50% (w/v). The addition of 14% (v/v) dimethyl sulphoxide to the fusion mixture enhanced recombination frequencies, but only at sub-optimal PEG concentrations. After treatment of protoplasts with 50% PEG for 1 min, the frequency of recombinants in a multi-factor 'cross' sometimes exceeded 20% of the total progeny. The frequency of recombinants in the progeny could be significantly enhanced by ultraviolet irradiation of the parental protoplast suspensions immediately before fusion.     

Genetic analysis of intraspecies recombinant formation by protoplast fusion with three species of Streptomyces

Abstract Protoplast fusion was shown to produce high frequencies of recombinant progeny in intraspecies crosses with auxotrophic mutants of Streptomyces canescens, Streptomyces griseus and Streptomyces limosus . The fused protoplasts were regenerated on non-selective media and the progeny spores subsequently analysed on selective media to allow detection of all possible genotypes. Prototrophic recombinants arose with frequencies of between 1% and 8%. All 4 possible genotypes were recovered in a series of 2-factor crosses and 6 of the 8 possible genotypes were detected in a 3-factor cross. In spite of attempts to equalise the ratios of parental protoplasts in the fusion mixture, there were noticeable deviations from unity in the ratios of parental genotypes in the progeny.     

Polyethylene glycol-induced fusion of heat-inactivated and living protoplasts of Bacillus megaterium.

Protoplasts of Bacillus megaterium, incubated at 50 degrees C for 120 min, lost the ability to revert to bacillary form. Such heat-inactivated protoplasts, however, produced recombinants when fused by polyethylene glycol treatment with normal protoplasts. Although this differential inactivation effect is not yet fully reproducible, reciprocal inactivations of the parental protoplasts in genetic crosses have clearly shown that for protoplast fusion (i) either of the parents may serve as the viable recipient for markers coming from the heated parental protoplasts, and (ii) either of the parents may be rendered nonviable and yet, when fused with a viable partner, contribute to formation of a recombinant. Heat inactivation seems to provide a way to counterselect when few markers are available and one of the parents is prototrophic.     

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.     

Nonparental progeny resulting from protoplast fusion inTrichoderma in the absence of parasexuality

The purpose of this study was to characterize a number of progeny from intra- and interstrain protoplast fusion within the genusTrichoderma. We wished to determine whether parasexuality or other genetic mechanisms occur in these fungi. When two different auxotrophs of the same strain were fused, rapidly growing prototrophic progeny were obtained in high frequencies. When single spore isolates of these strains were prepared, equal numbers of strains indistinguishable from the two parental auxotrophic strains were obtained, even though 10⁹–10¹⁰ conidia were tested per strain. Thus, progeny from intrastrain fusions all appeared to be balanced heterokaryons, and no evidence of recombination between the two parental strains was obtained. When 16 separate interstrain fusions were conducted, very different results were obtained, regardless of whether fusions were within or between species. Following interstrain fusions, presumptive somatic hybrids developed very slowly and in low numbers as compared with hybrids from intrastrain fusions. Most were weakly prototrophic. These slow-growing progeny were unstable and sectors developed from them. Such sectors themselves were unstable and gave rise to other progeny. Usually sectors were more strongly prototrophic and more rapid growing than the original progeny strain. Sectoring gave rise to a very wide range of morphotypes. Most of these morphotype variants were stable through conidiation; thus, these types did not occur as a consequence of heterokaryosis. Isozyme analysis was conducted on over 1000 progeny strains. Nearly all progeny were identical to one or the other parental isozyme phenotypes. A few progeny, when tested as soon as possible after fusion, exhibited the isozyme phenotypes of both parents, but such biparental banding patterns were rapidly lost upon subsequent reculturing. Isozyme banding patterns of multimeric enzymes never gave band patterns indicative of heterokaryosis or heterozygosis. Banding patterns indicative of heterozygous diploids or recombinants were never detected. Despite the extreme variation in morphotype and nutritional requirements among progeny, isozyme banding patterns of derived progeny from any fusion were invariably identical to one or the other parental strains. From these results, we conclude that protoplast fusion in the genusTrichoderma gives rise to great variability, but that the classical parasexual cycle is not required for variation to occur.     

Visualization of protoplast fusion and quantitation of recombination in fused protoplasts of auxotrophic strains of Escherichia coli

Protoplast fusion has been used to combine genes from different organisms to create strains with desired properties. A recently developed variant on this approach, genome shuffling, involves generation of a genetically heterogeneous population of a single organism, followed by recursive protoplast fusion to allow recombination of mutations within the fused protoplasts. These are powerful techniques for engineering of microbial strains for desirable industrial properties. However, there is a prevailing opinion that it will be difficult to use these methods for engineering of Gram-negative bacteria because the outer membrane makes protoplast fusion more difficult. Here we describe the successful use of protoplast fusion in Escherichia coli. Using two auxotrophic strains of E. coli, we obtained prototrophic strains by recombination in fused protoplasts at frequencies of 0.05-0.7% based on the number of protoplasts subjected to fusion. This frequency is three-four orders of magnitude better than those previously reported for recombination in fused protoplasts of Gram-negative bacteria such as E. coli and Providencia alcalifaciens.     

Experiments with Bacillus thuringiensis protoplasts. II. Study of the potential interspecies fusion of bacterial protoplasts: Bacillus thuringiensis and Bacillus megaterium

In the present study the possibility of obtaining interspecific bacterial hybrids by polyethylene glycol-assisted fusion of protoplasts from Bacillus thuringiensis and Bac. megaterium has been examined. Electron microscopic and genetic data allow to confirm with great probability that cytological fusion takes place. However, genetic analysis revealed that neither of methods applied for protoplast fusion gave stable recombinants. Apparently, it is due to the lack of recombination or the death of recombinants that follows the functioning of the cell correction system. The mechanism of protoplast fusion and its most important steps are also studied in the present work.     

Variety of Hybrid Characters among Recombinants Obtained by Interspecific Protoplast Fusion in Streptomycetes

To discover whether the protoplast fusion method is useful or not for interspecific breeding, some methods were devised, and the appearance of various hybrids with different characters and the change of antibiotic activities in the recombinants obtained by the protoplast fusion were investigated. The purification of protoplasts, the choice of parental natural characters as selection markers, and the adoption of a replica method for selecting all types of recombinants were devised and used for these experiments. Protoplast fusion was done between S. griseus KCC S-0644 and each strain of 5 species that were clearly different species from S. griseus, in addition to being streptomycin sensitive (SM^s) and capable of L-arabinose utilization for growth (Ara⁺). Recombinants (SM^r, Ara⁺) obtained by protoplast fusion displayed a great variety of hybrids in their taxonomic characters, e.g., 21 recombinant strains obtained by the cross between S. griseus and S. griseoruber consisted of 14 types of hybrids. Antibiotic productivity was examined in all recombinants obtained. Although both parental species produced their respective antibiotics, 60% of the recombinants did not produce any antibiotic and 24% produced different antibiotics from those of their parents. Among those recombinants, it was also found that the distribution of the productivity of each antibiotic among the recombinants was entirely different from that of the allelo-character in each taxonomic feature.     

Further studies on recombination in diploid clones from Bacillus subtilis protoplast fusion

Summary Diploid prototrophs were obtained from protoplast fusion of Bacillus subtilis strains. They are unstable but upon further cultivation they stabilize retaining diploidy but are genetically inactive. It has been suggested that recombination between the parental chomosomes is involved in the production of stable prototrophs and recombinants. In this work the occurrence of this recombination was searched for by determining genetic linkages in transformation experiments. In prototrophs two alleles: hisH2 and trpE8 carried originally on each parental chromosome, were shown to be 48% co-transformable in a stable clone whereas they were only cotransformed in 10% of the unstable colonies. For Trp^- recombinants (the most frequent type of a Leu^- Met^- Thr^- x Ade^- Ura^- Trp^- fusion pair) lysed protoplasts were used as donor DNA for the transformations. High values of co-transfer for Ura⁺ Met⁺ were obtained. These results confirm the occurrence of recombination in stable diploid clones, prototrophs or recombinants.     

PROTOPLAST FUSION: A NOVEL APPROACH TO ORGANELLE GENETICS IN HIGHER PLANTS

In the majority of higher plants there is maternal inheritance of cytoplasmic organelles and, as a consequence, there are few opportunities for the study of the effects on plant phenotype of having cytoplasm initially containing organelles of both parents. Now the availability of somatic plant protoplasts, which can be fused together and suitably cultured to produce somatic hybrid plants, is enabling the effects of such hybrid cytoplasms to be investigated in higher plants exhibiting maternal inheritance. A very wide range of cytoplasmic genetic diversity, including mitochondrial and chloroplast recombinants, can be produced by such somatic hybridizations, and a theoretical model is presented to show the origins of this wide range of cytoplasmic diversity. Cybrids produced by such protoplast fusions have been shown to be of importance in plant breeding especially in relation to transfer of cytoplasmic male sterility and herbicide resistance. Protoplast fusion, including the fusion of gametic and somatic protoplasts, is also enabling the study of the inheritance of cytoplasmic controlled traits in higher plants.     

Genetic recombination in Micromonospora rosaria by protoplast fusion.

Auxotrophic strains of Micromonospora rosaria were isolated by N-methyl-N'-nitro-N'-nitrosoguanidine mutagenesis and used in intraspecific recombination by protoplast fusion. High-frequency fusion of protoplasts of M. rosaria strains was induced by polyethylene glycol (molecular weight, 1,000) (PEG 1,000). The optimum concentration of PEG 1,000 for fusion of M. rosaria was 50% (wt/vol). PEG 4,000 was slightly better than PEG 1,000 at concentrations lower than 50% (wt/vol). The recombinant frequency did not increase after treatment with PEG 1,000 (50% [wt/vol]) for longer than 20 min. Under these conditions, fusion with many auxotrophic strains of M. rosaria resulted in a high frequency of formation of true recombinants (sometimes more than 10%). Additionally, when ros (rosamicin nonproducing) strains were crossed by protoplast fusion; about 5% of the resultant prototrophic recombinants were shown to have the ros+ (rosamicin producing) characteristic restored. Rosamicin production by M. rosaria colonies was clearly distinguished by the broth overlay method. The results of fusion experiments between ros and ros+ strains indicated that either the chromosomal mutation or pleiotrophic effect of some auxotrophic markers is involved.     

Hybridization of Candida albicans through fusion of protoplasts

Protoplasts of complementing auxotrophs of Candida albicans can fuse in the presence of polyethylene glycol and generate prototrophic cells. The yields of prototrophs from fusion mixtures depend greatly on the particular combinations of auxotrophies involved but not on other features of the strain backgrounds of protoplasts. The initial cellular products of fusions isolated on selective media are heterokaryons which replicate slowly but also segregate single parental nuclei into blastospores in high frequency. Karyogamy within heterokaryons produces hybrid nuclei which, on segregation, give rise to rapidly growing, uninucleate substrains. Analyses of the substrains show that hybrid nuclei either stabilize as diploid or undergo random loss of chromosomes to stabilize at various levels of aneuploidy prior to segregation. Chromosome losses and radiation induced mitotic crossing-over can effect recombination for parental auxotrophic markers in hybrids; patterns of recombination for ade^r and arg markers provide the first documented example of chromosomal linkage in C. albicans. Thus, protoplast fusions offer opportunities otherwise unavailable for applying the incisive tools of genetic recombination to analysis of this important, asexual yeast.     

Genetic recombination in Micromonospora rosaria by protoplast fusion

Auxotrophic strains of Micromonospora rosaria were isolated by N-methyl-N'-nitro-N'-nitrosoguanidine mutagenesis and used in intraspecific recombination by protoplast fusion. High-frequency fusion of protoplasts of M. rosaria strains was induced by polyethylene glycol (molecular weight, 1,000) (PEG 1,000). The optimum concentration of PEG 1,000 for fusion of M. rosaria was 50% (wt/vol). PEG 4,000 was slightly better than PEG 1,000 at concentrations lower than 50% (wt/vol). The recombinant frequency did not increase after treatment with PEG 1,000 (50% [wt/vol]) for longer than 20 min. Under these conditions, fusion with many auxotrophic strains of M. rosaria resulted in a high frequency of formation of true recombinants (sometimes more than 10%). Additionally, when ros (rosamicin nonproducing) strains were crossed by protoplast fusion; about 5% of the resultant prototrophic recombinants were shown to have the ros+ (rosamicin producing) characteristic restored. Rosamicin production by M. rosaria colonies was clearly distinguished by the broth overlay method. The results of fusion experiments between ros and ros+ strains indicated that either the chromosomal mutation or pleiotrophic effect of some auxotrophic markers is involved.     

RNA recombination of murine coronaviruses: recombination between fusion-positive mouse hepatitis virus A59 and fusion-negative mouse hepatitis virus 2.

It has previously been shown that the murine coronavirus mouse hepatitis virus (MHV) undergoes RNA recombination at a relatively high frequency in both tissue culture and infected animals. Thus far, all of the recombination sites had been localized at the 5' half of the RNA genome. We have now performed a cross between MHV-2, a fusion-negative murine coronavirus, and a temperature-sensitive mutant of the A59 strain of MHV, which is fusion positive at the permissive temperature. By selecting fusion-positive viruses at the nonpermissive temperature, we isolated several recombinants containing multiple crossovers in a single genome. Some of the recombinants became fusion negative during the plaque purification. The fusion ability of the recombinants parallels the presence or absence of the A59 genomic sequences encoding peplomers. Several of the recombinants have crossovers within 3' end genes which encode viral structural proteins, N and E1. These recombination sites were not specifically selected with the selection markers used. This finding, together with results of previous recombination studies, indicates that RNA recombination can occur almost anywhere from the 5' end to the 3' end along the entire genome. The data also show that the replacement of A59 genetic sequences at the 5' end of gene C, which encodes the peplomer protein, with the fusion-negative MHV-2 sequences do not affect the fusion ability of the recombinant viruses. Thus, the crucial determinant for the fusion-inducing capability appears to reside in the more carboxyl portion of the peplomer protein.     

Genetic Analysis of Fusion Recombinants in Bacillus Subtilis: Function of the Rece Gene

Bacillus subtilis protoplast fusion allows the study of the genetic recombination of an entire procaryotic genome. Protoplasts from bacterial strains marked genetically by chromosomal mutations were fused using polyethylene glycol and the regenerated cells analyzed. Recombinants represent 19.3% of heterozygotic cells; they are haploids. Individual characterization of clones show a unique particular phenotype in each colony suggesting that recombination takes place immediately after fusion, probably before the first cellular division. Recombination occurs in the whole chromosome; in one-third of the cases both reciprocal recombinants could be shown in the colony. The genetic interval that includes the chromosome replication origin shows the highest recombination level. Our results suggest that the RecE protein accounts for most of the fused protoplast recombination; however, some "replication origin-specific" recombination events were independent of the recE gene product.     

Rapid chloroplast segregation and recombination of mitochondrial DNA in Brassica cybrids

Summary Brassica cybrids were obtained after fusing protoplasts of fertile and cytoplasmic male sterile (CMS) B. napus lines carrying the original b. napus, and the Ogura Raphanus sativus cytoplasms, respectively. Iodoacetate treatment of the fertile line and X-irradiation of the CMS line prevented colony formation from the parental protoplasts. Colony formation, however, was obtained after protoplast fusion. Hybrid cytoplasm formation was studied in 0.5 g to 5.0 g calli grown from a fused protoplast after an estimated 19 to 22 cell divisions. Chloroplasts and mitochondria were identified in the calli by hybridizing appropriate DNA probes to total cellular DNA. Out of the 42 clones studied 37 were confirmed as cybrids. Chloroplast segregation was complete at the time of the study. Chloroplasts in all of the cybrid clones were found to derive from the fertile parent. Mitochondrial DNA (mtDNA) segregation was complete in some but not all of the clones. In the cybrids, mtDNA was different from the parental plants. Physical mapping revealed recombination in a region which is not normally involved in the formation of subgenomic mtDNA circles. The role of treatments used to facilitate the recovery of cybrids, and of organelle compatibility in hybrid cytoplasm formation is discussed.