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.     

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.     

Relative Contribution of the Cell Wall, Cytoplasmic Membrane, and Cytoplasm to the Gram-Positive Characteristic of Bacillus megaterium.

Bartholomew, J. W. (University of Southern California, Los Angeles), and Thomas Cromwell. Relative contribution of the cell wall, cytoplasmic membrane, and cytoplasm to the gram-positive characteristic of Bacillus megaterium. J. Bacteriol. 90:643-647. 1965.-A comparison of the roles of the cell wall, cytoplasmic membrane, and cytoplasmic components revealed that the intact cell wall was the dominant contributor to the gram-positive state. Protoplasts of Bacillus megaterium were confirmed as being gram-negative, as reported by Gerhardt et al. The "gram-positive protoplast" report of Amano et al. was shown to be a laboratory-produced artifact, resulting from the comparison of smears made from saline suspensions of Escherichia coli cells with smears made from formalin-sucrose suspensions of B. megaterium protoplasts.     

枯草芽胞杆菌基因组混组方法

基因组混组作为一种育种方法,通过循环原生质体融合等手段,使得不同菌株来源的基因组能够得到充分重组,增加将正向突变整合到同一重组子中的机会。使用4株带有4种不同标记的枯草芽胞杆菌亲本为初始菌株,通过循环转化、循环转导或循环原生质体融合的手段进行基因组混组,统计后代中非亲本类型占整个群体的比例,以衡量基因组混组的效果。分别经过5轮循环原生质体融合、循环转化或者循环转导,结果显示,重组程度较高者在后代群体中的比例较低,带有4种标记的后代未出现,带有3种标记的后代最高分别为4.53×10?4、1.64×10?4、4.47×10?3,明显低于文献报道的天蓝色链霉菌中同样实验的结果:带4种和3种标记的后代分别占2.5%、17%。对比上述实验的结果和文献报道的天蓝色链霉菌、乳杆菌基因组混组的结果,并结合计算机模拟循环融合过程,分析后认为:要达到较充分的基因组混组,需要有能够实现微生物细胞间高频重组的操作技术作为基础,重组频率应该不低于10?3～10?2数量级。     

Enhancement of Lignin Degradation in Streptomyces spp. by Protoplast Fusion

Protoplast fusion was investigated as a technique for genetically manipulating two lignin-degrading Streptomyces strains, Streptomyces viridosporus T7A and Streptomyces setonii 75Vi2. Four of 19 recombinants tested showed enhanced production of acid-precipitable polymeric lignin (APPL), producing 155 to 264% more APPL from corn stover lignocellulose than was produced by the wild-type S. viridosporus T7A. APPLs are lignin degradation intermediates known to be potentially valuable chemical products produced by bioconversion of lignin with Streptomyces spp. The prospects of utilizing protoplast fusion to construct APPL-overproducing Streptomyces strains was considered especially promising.     

Transfer of plasmids among bacilli

Plasmids were transferred from Staphylococcus aureus to Bacillus subtilis and between B. subtilis and several other species of Bacillus by protoplast fusion and regeneration. The plasmids replicate and express themselves normally in all bacilli examined.     

Plasmid-mediated transformation in Bacillus megaterium.

A transformation system was developed for Bacillus megaterium by using antibiotic resistance plasmid deoxyribonucleic acid molecules derived from Staphylococcus aureus and Bacillus cereus. Lysozyme-generated protoplasts of B. megaterium allowed uptake of plasmid deoxyribonucleic acid in the presence of polyethylene glycol. Transformants expressed the antibiotic resistance determinants present on the plasmid deoxyribonucleic acid, and reisolated plasmid deoxyribonucleic acid yielded restriction endonuclease digestion patterns identical to those of the donor deoxyribonucleic acid.     

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.     

Genetic analysis of the actinomycin-producing determinants (plasmid) in Streptomyces parvulus using the protoplast fusion technique

Doubly auxotrophic, actinomycin-producing and nonproducing strains of Streptomyces parvulus were crossed by protoplast fusion. A strong bias toward the act+ character was noted in all recombinant classes obtained. Analysis of four nutritional markers revealed that they are ordered on a circular linkage map as follows: ura-lys-met-rib-(ura). This sequence resulted in the lowest frequency (2.4-3.4%) of quadruple crossover (QCO) recombinants. Inclusion of the property of actinomycin production in this sequence resulted in a much more greater minimum frequency of QCO recombinants (3-4 times higher). Moreover, the location of the act character minimizing the QCO frequency varied from cross to cross despite the fact that fusion of the act- strains did not yield act+ recombinants. It is concluded that actinomycin synthesis is determined (or controlled) by an episomic factor(s).     

Interspecific recombinants of Bacillus thuringiensis x Bacillus cereus

The possibility of interspecies recombination was shown by using protoplast fusion method. The Bacillus thuringiensis var. galleriae strain 48S Thi Nic Gua Rifr Strr and 56R Gua Rifr, and also Bac. cereus carrying the plasmid pBC16 responsible for resistance to tetracycline (150 mcg/ml) were used. Recombinants were selected on the medium containing rifampicin and tetracycline. They were shown to combine the properties of both parents. The majority of recombinants were resistant to phages Tg4 and Td15 and represented the mean level of sensitivity to phages Tg12, Tg13 and Td14. Examination of the plasmid profiles of recombinants revealed that their resistance to tetracycline was due to the plasmid with mobility analogous to pBC16. It was concluded that the protoplast fusion method can be used to obtain recombinants between relatively remote species of microorganisms.     

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.     

Fusion of protoplasts in Streptomyces antibioticus

The analysis of Streptomyces antibioticus recombinants isolated under non-selective conditions for stability of inheritance of parental properties demonstrated the existence of non-stable diploids and stable haploid recombinants. Also, genetic analysis of heteroclones isolated on selective medium after plating spores of regenerants was conducted. The results of analysis of haploid recombinant and heteroclones pointed to linkage of genetic loci and the possibility to use the technique of protoplast fusion for establishment of genetic map of S. antibioticus.     

Distribution of Bacillus megaterium QM B1551 plasmids among other B. megaterium strains and Bacillus species

Bacillus megaterium QM B1551 contains seven plasmids. Two are small rolling circle plasmids and five are theta-replicating plasmids with cross-hybridizing replicons that define a new family of very homologous yet compatible theta replicons. Previous sequencing of several of the plasmids has shown genes with high similarity to those on the genomes and plasmids of other Gram-positive bacteria. To test the possible distribution of these plasmids, nine other B. megaterium strains and 20 other Bacillus or related species were tested for the presence of similar replicons, and specific flanking DNA by both hybridization and PCR. The theta replicons were widespread among the B. megaterium strains, and two had one or more of the rolling circle plasmids, but none of the plasmid replicon regions were observed in the other Bacillus or related species. It appears from the data that even though some plasmids carry genes suggesting horizontal transfer, their replicons seem to be unique to B. megaterium, or rarely present in related species.     

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.     

Isolation from haddock tissue of psychrophilic bacteria with maximum growth temperature below 20 degrees C.

Protoplast fusion was investigated as a technique for genetically manipulating two lignin-degrading Streptomyces strains, Streptomyces viridosporus T7A and Streptomyces setonii 75Vi2. Four of 19 recombinants tested showed enhanced production of acid-precipitable polymeric lignin (APPL), producing 155 to 264% more APPL from corn stover lignocellulose than was produced by the wild-type S. viridosporus T7A. APPLs are lignin degradation intermediates known to be potentially valuable chemical products produced by bioconversion of lignin with Streptomyces spp. The prospects of utilizing protoplast fusion to construct APPL-overproducing Streptomyces strains was considered especially promising.     

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.     

新组合菌系SCB329-SCB933利用L-山梨糖发酵产生维生素C前体——2-酮基-L-古龙酸的研究 Ⅰ.新组合菌系SCB329-SCB933的生物学特性

采用紫外照射、化学诱变和原生质融合等方法选育到一株性状更优良的突变株SCB329,并与新筛选的一株芽孢杆菌SCB933搭配组成新的组合菌系。产酸小菌SCB329与其亲本菌株氧化葡萄糖酸杆菌性状相似。伴生大菌SCB933属苏芸金芽孢杆菌（B.thuringiensis）。新组合菌系利用L-山梨糖的发酵液提取后经纸层析,元素分析和红外吸收光谱等项鉴定,其发酵产物确系2-酮基-L-古龙酸,对新组合菌系的生物学特性也进行了研究。     

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.     

Genetic analysis of Bacillus stearothermophilus by protoplast fusion.

Efficient and reliable protoplasting, regeneration, and fusion techniques were established for the prototrophic strain Bacillus stearothermophilus NUB36. Auxotrophic mutants were isolated, and protoplast fusion was used to construct isogenic mutant strains and for chromosomal mapping. Markers were mapped using two-, three-, and four-factor crosses. The order of the markers was hom-1-thr-1-his-1-(gly-1 or gly-2)-pur-1-pur-2. These markers may be analogous to hom, thrA, hisA, glyC, and purA markers on the Bacillus subtilis chromosome. No analogous pur-1 marker has been reported in B. subtilis. The relative order of three of the markers (hom-1-thr-1-gly-1) was independently confirmed by transduction.     

Protoplast dehydration correlated with heat resistance of bacterial spores.

Water content of the protoplast in situ within the fully hydrated dormant bacterial spore was quantified by use of a spore in which the complex of coat and outer (pericortex) membrane was genetically defective or chemically removed, as evidenced by susceptibility of the cortex to lysozyme and by permeability of the periprotoplast integument to glucose. Water content was determined by equilibrium permeability measurement with 3H-labeled water (confirmed by gravimetric measurement) for the entire spore, with 14C-labeled glucose for the integument outside the inner (pericytoplasm) membrane, and by the difference for the protoplast. The method was applied to lysozyme-sensitive spores of Bacillus stearothermophilus, B. subtilis, B. cereus, B. thuringiensis, and B. megaterium (four types). Comparable lysozyme-resistant spores, in which the outer membrane functioned as the primary permeability barrier to glucose, were employed as controls. Heat resistances were expressed as D100 values. Protoplast water content of the lysozyme-sensitive spore types correlated with heat resistance exponentially in two distinct clusters, with the four B. megaterium types in one alignment, and with the four other species types in another. Protoplast water contents of the B. megaterium spore types were sufficiently low (26 to 29%, based on wet protoplast weight) to account almost entirely for their lesser heat resistance. Corresponding values of the other species types were similar or higher (30 to 55%), indicating that these spores depended on factors additional to protoplast dehydration for their much greater heat resistance.