Stabilized non-complementing diploids (Ncd) from fused protoplast products of B. subtilis

Non-complementing diploids (Ncd) displaying the parental phenotype can be selected from polyethylene glycol (PEG)-treated fused polyauxotrophic protoplasts of Bacillus subtilis. These bacteria carry the two parental genomes, but only one of them is phenotypically expressed, the other being replicated but not expressed. Cellular cloning and DNA-DNA in situ hybridization led to the discovery of non-complementing diploid cells which at first sight could have been considered as parental haploids. The new class of stabilized Ncd (10(-7) segregants) can be obtained either directly after the primary fusion event or from segregating Ncd after further growth. The totally inactive chromosome of a stable Ncd can be activated after PEG-induced self fusion. DNA-mediated transformation studies using crude stable Ncd lysates as DNA donors show low frequencies for the genetic markers from the 'silent' chromosome. Contrary to the unstable Ncd situation, however, these frequencies remain low even with purified donor DNA. The differences in the transformation properties of the non-expressed markers are correlated to Ncd clone stability. These facts suggest that chromosome inactivation in PEG-induced fusion involves at least a two-stage process. The first would be reversible and the second irreversible, thus preserving the inactive chromosome state.     

RecE-dependent lysogenic induction in the absence of repressor in Bacillus subtilis non-complementing diploids

The RecE protein of Bacillus subtilis, known to be required for induction of the SOS response and of phi 105 prophage, was shown to be involved in mitomycin C induction of B. subtilis diploid lysogens carrying a silent phi 105 prophage in their unexpressed chromosome. These stable non-complementing diploid lysogens, formed by protoplast fusion and regeneration, did not synthesize repressor, so that the induction observed must have resulted from RecE-dependent activation of the prophage rather than from RecE-dependent inactivation of repressor. Mitomycin C treatment does not induce permanent expression of the silent chromosome, so the activation seems to be temporary, perhaps reflecting the action of an SOS function under RecE control.     

Isolation and characterization of the nucleoid of non-complementing diploids from protoplast fusion in Bacillus subtilis

Summary Nucleoids of non-complementing diploids (Ncd) from protoplast fusion of B. subtilis were isolated. Their purified DNA banded in neutral CsCl gradient as a single unimodal peak of buoyant density 1.711 g/cm³, a value which is similar to that of the DNA purified from the original parental strains, suggesting that methylation of bases is not a significant factor in chromosome inactivation. Nucleoids released from a Ncd clone give two peaks in a sucrose gradient with a characteristic S value for each nucleoid. That is in contrast to nucleoids from the haploid parents whose sedimental patterns show only one peak.Both nucleoid preparations from Ncd strains assayed for transformation activity show the fast sedimenting nucleoid devoid of transformation activity while the slow nucleoid was active in transformation for the alleles carried by the genome which is expressed in vivo. Both nucleoids of the Ncd strains are transcribed in vivo. The RNA associated with the inactive chromosome is synthesized by the RNA polymerase of the active one.This study provides evidence that inactivation of one parental genome in the Ncd strain may be related with the tertiary organization of its DNA.     

The spo0A gene is implicated in the maintenance of non-complementing diploids in Bacillus subtilis

Bacillus subtilis can exist in a diploid state in which two genetically distinct chromosomes co-exist in the same cell and yet only one of them is expressed, thereby determining the phenotype. Such cells are called non-complementing diploids (Ncds). In this study, two types of experiments are reported which indicate that a previously known pleiotropic gene, spo0A, plays a role in the maintaining the diploid state, as follows. (i) When protoplasts of two Spo0A mutant strains were fused, the resulting products continued to segregate cells of both parental phenotypes for many more divisions than had been reported previously. (ii) When a stable Ncd (an Ncd in which the unexpressed markers are not spontaneously activated at a detectable level) harbouring a chloramphenicol acetyltransferase gene on the silent chromosome was transformed with spo0A null alleles the transformants often expressed chloramphenicol acetyltransferase activity. Together these results indicate that the spo0A gene is involved in maintenance of the diploid state in both unstable and stable Ncds.     

Genetic mapping by means of protoplast fusion in Bacillus subtilis

Summary A new mapping method involving protoplast fusion in Bacillus subtilis is described. Protoplasts from an isogenic standard marker strain containing purA and from a strain containing both purB and the marker, x, to be mapped were fused with polyethylene glycol, and purA ⁺ purB ⁺ fusants were selected. After isolation of single colonies and determination of unselected markers, marker x was mapped between two standard markers. This method was fully applicable to PBS1-resistant strains (e.g., lyt strains). The results obtained by protoplast fusion, conventional transformation and/or lysed protoplast transformation indicated that a lyt strain, Ni15, contained two new autolysin-minus mutations (lyt-151 and lyt-152). The properties of lyt-15 are also discussed.Abbreviations NTG N-methyl-N-nitro-N-nitrosoguanidine - SMM 0.5 M sucrose, 0.02 M MgCl₂, 0.02 M maleate buffer, pH 6.5     

Reconstitution of succinate dehydrogenase in Bacillus subtilis by protoplast fusion

Bacillus subtilis succinate dehydrogenase (SDH) is composed of two unequal subunits designated Fp (Mr, 65,000) and Ip (Mr. 28,000). The enzyme is structurally and functionally complexed to cytochrome b 558 (Mr, 19,000) in the membrane. A total of 21 B. subtilis SDH-negative mutants were isolated. The mutants fall into five phenotypic classes with respect to the presence and localization of the subunits of the SDH-cytochrome b558 complex. One class contains mutants with an inactive membrane-bound complex. Membrane-bound enzymatically active SDH could be reconstituted in fused protoplasts of selected pairs of SDH-negative mutants. Most likely reconstitution is due to the assembly of preformed subunits in the fused cells. On the basis of the reconstitution data, the mutants tested could be divided into three complementation groups. The combined data of the present and previous work indicate that the complementation groups correspond to the structural genes for the three subunits of the membrane-bound SDH-cytochrome b558 complex. A total of 31 SDH-negative mutants of B. subtilis have now been characterized. The respective mutations all map in the citF locus at 255 degrees on the B. subtilis chromosomal map. In the present paper, we have revised the nomenclature for the genetics of SDH in B. subtilis. All mutations which give an SDH-negative phenotype will be called sdh followed by an isolation number. The designation citF will be omitted, and the citF locus will be divided into three genes: sdhA, sdhB, and sdhC. Mutations in sdhA affect cytochrome b558, mutations in sdhB affect Fp, and mutations in sdhC affect Ip.     

Direct selection of complementing diploids from PEG-induced fusion of Bacillus subtilis protoplasts

Summary A minimal medium containing horse serum is described on which Bacillus subtilis protoplasts revert to bacillary forms at high frequency (ca. 30%). Used as a plating medium for a mixture of polyethyleneglycol-treated protoplasts from two complementary polyauxotrophic parental strains, it selects the prototrophic fusion products efficently, and also allows isolation of various auxotrophic recombinants. These prototrophs and recombinants amount respectively to 1% and 10% of the regenerated bacteria.We confirm that two types of prototrophs can be isolated after fusion: stable recombinants and complementing diploids, the latter segregating into various types of recombinants. Based on easily recognized colonial aspects, an approximate estimation of the proportion of the two types becomes possible when a spoOA mutation has been introduced in one of the parents. At least 50% of the prototrophic fusion products are complementing diploids. Incidently, the data also settle a controversy by showing the dominance of spoOA mutations in heterozygotic bacteria.This work was supported by the Centre National de la Recherche Scientifique (Contrat L.A. 136).     

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.     

Electron microscopic study of Bacillus subtilis protoplast fusion.

When protoplasts derived from sporulating cells of Bacillus subtilis were fused by exposure to polyethylene glycol (PEG) and fixed immediately thereafter, protoplasts with two enclosed prespores could be seen by electron microscope. The number of fusion events was greatly increased, and multiply fused protoplasts appeared, when the PEG-treated suspension was diluted in hypertonic broth and reincubated before fixation. This post-PEG incubation effect is taken to indicate a fusion mechanism of two steps: a short, PEG-dependent step of membrane activation, followed by a slow, metabolism-requiring step completing fusion. When prespore-bearing protoplasts from two genetically different strains were mixed and fused, the extent of fusion could also be followed by counting clones of recombinant bacteria. Maximal from the start, their number (1% of each parent type protoplast present) was unaffected by post-PEG incubation. Fusion in this case is apparently completed after plating on the wall-regeneration medium. After optimal post-PEG incubation, the majority of the protoplasts were seen to participate in fusion, and the cytological fusion observed, corrected for wall-regeneration frequency, accounted quantitatively for the prototrophic bacteria eventually recovered. These results are in good agreement with those obtained independently by Sanchez-Rivas and Garro (J. Bacteriol. 137:1340--1345, 1979).     

Transfer of DNA coding for cellulases from Cellulomonas species to Bacillus subtilis by protoplast fusion

Summary Cellulose utilising hybrids between Cellulomonas spp. and Bacillus subtilis were isolated after PEG mediated protoplast fusion. These stable hybrids selected using genomic markers harboured DNA from both the parents. Higher CMCase and -glucosidase activities were detected extracellularly in case of one of the hybrids unlike only CMCase in case of Cellulomonas.NCL Communication No. 3542     

Hybrids obtained by protoplast fusion with a salt-tolerant yeast

Summary An industrial strain ofSaccharomyces cerevisiae was fused with an osmotolerant yeast,Debaryomyces hansenii, to obtain hybrids having increased tolerance to elevated salt concentrations. The hybrids were intermediate to parent species in production of ethanol and polyols.This paper is dedicated to Professor Herman Jan Phaff in honor of his 50 years of active research which still continues.     

Effect of Bacillus subtilis BsuM restriction-modification on plasmid transfer by polyethylene glycol-induced protoplast fusion

Polyethylene glycol (PEG)-induced cell fusion is a promising method to transfer larger DNA from one cell to another than conventional genetic DNA transfer systems. The laboratory strain Bacillus subtilis 168 contains a restriction (R) and modification (M) system, BsuM, which recognizes the sequence 5'-CTCGAG-3'. To study whether the BsuM system affects DNA transfer by the PEG-induced cell fusion between R(+)M(+) and R(-)M(-) strains, we examined transfer of plasmids pHV33 and pLS32neo carrying no and eight BsuM sites, respectively. It was shown that although the transfer of pLS32neo but not pHV33 from the R(-)M(-) to R(+)M(+) cells was severely restricted, significant levels of transfer of both plasmids from the R(+)M(+) to R(-)M(-) cells were observed. The latter result shows that the chromosomal DNA in the R(-)M(-) cell used as the recipient partially survived restriction from the donor R(+)M(+) cell, indicating that the BsuM R(-)M(-) strain is useful as a host for accepting DNA from cells carrying a restriction system(s). Two such examples were manifested for plasmid transfer from Bacillus circulans and Bacillus stearothermophilus strains to a BsuM-deficient mutant, B. subtilis RM125.     

Transformation of undomesticated strains of Bacillus subtilis by protoplast electroporation

A rapid method combining the use of protoplasts and electroporation was developed to transform recalcitrant wild strains of Bacillus subtilis. The method described here allows transformation with both replicative and integrative plasmids, as well as with chromosomal DNA, and provides a valuable tool for molecular genetic analysis of interesting Bacillus strains, which are hard to transform by conventional methods.     

Highly bioluminescent Bacillus subtilis obtained through high-level expression of a luxAB fusion gene.

Summary Bioluminescence levels comparable to those achievable in Escherichia coli have yet to be obtained from luxAB expression in gram-positive bacteria. In this communication we describe the gene engineering required to generate a highly bioluminescent derivative of Bacillus subtilis. The combination of a powerful promoter, P _xyn , a fusion derivative of luxAB from Vibrio harveyi and translational coupling have overcome the previously reported limitations in luxAB expression. The implications for highly bioluminescent gram-positive organisms are discussed.     

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.