Ploidy of Bacillus subtilis exfusants: the haploid nature of cells forming colonies with biparental or prototrophic phenotypes

Abstract

To investigate the relationship between DNA content and cell volume, we have attempted to repeat the construction of stable BaciUus subtilis diploid cells through protoplast fusion. Colonies with a biparental phenotype and those with a prototrophic phenotype were identified among exfusants of a cross between two polyauxotrophic strains. The ploiay of cells constituting such colonies was assessed by protoplast self-fusion, determination of the DNA to dry weight ratio of exponentially growing cells, and by quantitative DNA-DNA hybridization. Within the precision of these methods, all colonies were found to consist of haploid cells. A previously described noncomplementing diploid was also found to be haploid. Therefore, the genetic evidence in favour of diploidy, based on continuing segregation of cells with a parental or recombinant phenotype, cannot be accounted for except by the maintenance of such cells as a minority population in mixed colonies through cross-feeding. Reconstruction experiments with mixtures of whole parental cells confirm that biparental colonies are indeed mixed colonies which arise either by sticking of parental cells or through coincidence, i.e. their plating within a distance of about 0.4 mm. The previously reported experimental results can be accounted for in the light of our results.

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

“…The other possibility is that the BsuM enzyme remained in the cytoplasmic portion of the donor R + M + cell in the fusant and that, upon division of the fusants, various quantities of the enzyme were distributed to the progeny cells depending on where the cell division took place. It has been demonstrated that L‐form colonies of B. subtilis arise among those of the bacillary form after PEG‐induced cell fusion (Hauser & Karamata, ), which indicates that cell division can occur while the fusant is still without the cell wall. As the L‐form B. subtilis cell divides by an extrusion–resolution mechanism that is independent of FtsZ (Leaver et al ., ), it is possible that a similar mechanism was involved in the division of the fused cells that were produced by random collision between the donor and the recipient protoplasts.…”

Characterization of the chromosomes of Bacillus subtilis merodiploid strains by quantitative DNA-DNA hybridization

“…In the absence of any selective pressure, additional DNA is often lost, as if the cell tends to restore a normal D N hlvolume ratio, which apparently ensures the highest growth rate. For instance, it was recently shown that Bacilhs ~b t i l i s cells are unable to accommodate two complete chromosomes ; multiple chromosomal copies, present in fused protoplasts, are segregated in separate cells at the time of cell wall regeneration (Hauser & Karamata, 1992). Therefore, assuming that a cell tends to restore an optimal DNA/volume ratio, it is conceivable that diploidy of a given region could be compensated by deletion(s) in other, possibly non-essential, regions of the chromosome.…”