Noncomplementing Diploids From Bacillus subtilis

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Aug 1, 1996 - Znstitut de Ginitique et Microbiologie, Universiti Paras-Sud, 91405 Orsay Cedex, France. Manuscript ...... recombination oc- curs at a rate.
Copyright 0 1996 by the Genetics Society of America

Noncomplementing DiploidsFrom Bacillus subtilis Protoplast Fusion: Relationship Between Maintenance of Chromosomal Inactivation and Segregation Capacity Valerie Grandjean, Yolande Hauck, Jacques Le Derout and Luisa Hirschbein Znstitut de Ginitique et Microbiologie, Universiti Paras-Sud, 91405 Orsay Cedex, France

Manuscript received December 7, 1995 Accepted for publication August 1 , 1996 ABSTRACT Fusions of Bacillus subtilh protoplasts fromtwo genetically markedstrains produce noncomplementing heterodiploid bacteria. These noncomplementing diploids (Ncds) carry both parental chromosomes, but only one is expressed. Fusion products of strains polymorphic for NotI restriction sites provide new physical evidence tosupport the conclusion that Ncds are not an artifact of cross feeding orcell adhesion. We show that reversible chromosomal inactivationcan only account for the biparental trait of unstable Ncds. Two types of cells were recovered from the late progeny of unstable Ncds: Ncds with irreversible chromosome silencing (stableNcds) and secondary recombinants that displayeda genomic mosaic NotI profile.Segregants from anunstable Ncd population gave rise to two viablehaploid cell types. By contrast, stableNcds segregated into a population of viable and inviable haploid cells.We propose that the latter are derived from irreversible chromosome silencing.Our results indicate that clonal populations of stable Ncds are heterogenous and suggest that segregation and inactivation are independent parameters.

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EVERSIBLE silencing of all or part of a chromosome has been observed in many organisms (GOYONand FAUGERON 1989; ASSWet al. 1993; LYON 1993). Examples of the importance of the phenomenon during development can be foundin mammals (Xchromosome inactivation in female development; GRANT and CHAPMAN 1988), in insects (inactivation of the paternal set of chromosomes in somatic tissue; CROUSIet al. 1971) and in plants (silencing of foreign genes during the regeneration of transgenic plants; JORGENSEN 1991). Unexpectedly, heritable inactivation of a redundant chromosome also occurs in prokaryotes (reviewed in THALER et al. 1990). The first evidence came from the work of HOTCHKISS and GABOR(1980). These authors observed that polyethyleneglycol (PEG)-induced protoplast fusion of strains of Bacillus subtilis carrying multiple markers produced diploid cells in which one of the two parental chromosomes was randomly silenced. These bacteria, called noncomplementing diploids (Ncds), show thephenotype of only oneparent (HOTCHKISS and GABOR1980). The diploid nature of fusion product was demonstrated by several experiments. Extensive genetic experiments showed that the silent chromosome remained unexpressed until segregation occurred, after which it was reactivated (HOTCHKISS and GABOR1980; SANCHEZ-RIVAS et al. 1982). A diploid fusion product displaying phenotype A, grown in medium selective for parentA, typically yielded cells Corresponding author: Luisa Hirschbein, Institut de C2nttique et Microbiologie, Centre d’Orsay, Biitiment 409,91405 Orsay Cedex, France. E-mail: [email protected] Genetics 144: 871-881 (November, 1996)

of phenotype B at afrequency of 10-2-10-4. Therefore, Ncds characterized by following the inheritance of the biparentaltrait (BP) are unstable (SCHAEFFER and HIRSCHBEIN 1985). Purified DNA from Ncd clones exhibited all the transforming activities expected from a diploid donor (BOHIN et al. 1982). Most importantly, cells recombinant for parentaltraits A and B were identified in the Ncd progeny, indicating that inactive nucleoids could coexist withactive nucleoids in the same cell (GABOR and HOTCHKISS 1983; LEVI-MEYRUEIS et al. 1984). Segregating and recombinant Ncds converted spontaneously to a stable form (SANCHEZ-RIVAS et al. 1982; GUILLEN et al. 1985). The rate and frequency of this conversion depended on the particular clone being analyzed. The stable class of progeny displayed markers from the previously silent chromosome at a frequency of 3%. The great majority of the cells within the regenerated colonies were haploids, displaying only one of the parental phenotypes. Together, theamplification and self-fusion results indicate that,althoughheterodiploids are persistent within the clonal population, only a minority of Ncd cells are present within a so-called stable Ncd colony. DISCUSSION

Instabilitytestshave been used to define Ncds (HOTCHKISSand GABOR1980). Colonies were consid-

ered to be Ncds if, during growth, fusion products of one parental phenotype segregated bacteria that expressed all the genetic markers of the other parent. Also, it was necessary that neither parentgrew on minimum medium and that each had different nutritional requirements Only those colonies showing persistence of this biparental behavior after subcloning in liquid medium were considered to be unstable Ncds. “Sub cloning in liquid medium” meant that patches from putative Ncd colonies were inoculated into rich liquid medium and grown overnight. Suitable dilutions were then spread onto plates, so that