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Fusion of Bacterial Protoplasts (Bacillus Subtilis/Diploid Bacteria/Polyethylene Glycol/Chromosome Recombination) PIERRE SCHAEFFER, BRIGITTE CAMI, and ROLLIN D

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Fusion of Bacterial Protoplasts (Bacillus Subtilis/Diploid Bacteria/Polyethylene Glycol/Chromosome Recombination) PIERRE SCHAEFFER, BRIGITTE CAMI, and ROLLIN D Proc. Natl. Acad. Sci. USA Vol. 73, No. 6, pp. 2151-2155, June 1976 Microbiology Fusion of bacterial protoplasts (Bacillus subtilis/diploid bacteria/polyethylene glycol/chromosome recombination) PIERRE SCHAEFFER, BRIGITTE CAMI, AND ROLLIN D. HOTCHKISS* Institut de Microbiologie, Universit6 de Paris-Sud, 91405, Orsay, France Contributed by Rollin D. Hotchkiss, April 7,1976 ABSTRACT Prototrophic Bacillus subtifis cells can be ation itself, with our material, has been rare and unreliable in formed in the presence of DNase as a result of cell fusion oc- such media. Attempting the required selection first, and then curring in mixed populations of protoplasts derived from two proceeding to wall regeneration seemed excluded, because parental strains which are both nutritionally-complementing and polyauxotrophic. No prototrophs ever appear from mixed protoplasts produced by lysozyme treatment do not divide in nonprotoplasted bacteria, or from the auxotrophic parental liquid minimal medium (13). We were, thus, led to carry out protoplasts plated separately. The frequency of prototroph wall regeneration upon the mixed protoplasts first, on a rich formation, which is appreciable only when the mixed proto- hypertonic agar medium (13), and in a second step the selection plasts are exposed to polyethylene glycol treatment, may exceed by replica plating on various deficient agar media. 10-4 ofthe total protoplast population initially present, which is 1 to 4 X 10-3 of those protoplasts which reverted to the MATERIALS AND METHODS bacillary form. It is strongly dependent on the number and chromosomal location of the markers used in the selection of Bacterial Strains and Media. The strains used in fusion the prototrophs, and it is unaffected when either one of the experiments were constructed as described in Table 1. The parental strains bears the phage k105 in the inducible prophage chromosomal location of their markers is given in Fig. 1. Bac- state. No auxotrophic bacteria, parental or otherwise, were teria were first grown in nutrient broth (17), and protoplasted found as segregants from repeatedly isolated prototrophic clones in SMM, the sucrose-magnesium-maleate buffer of Wyrick and growing in a nonselective medium. Unselected markers segre- Rogers (13), to which 5 ,ug/ml of DNase I (Worthington Bio- gate among the selected recombinants. It is concluded that the observed formation of prototrophic bacteria is due to protoplast chem. Corp.) had been added (SMMD). Protoplasts were made fusion, a process which does not induce prophage development, to revert to bacillary forms by plating on RDR, a rich regen- and that the only stable products of the resulting diploid state eration agar medium of high tonicity (13), to which 5 /g/ml are haploid recombinants. each of DNase I and rifamycin (Lepetit Labs, Milano) were added. Prototrophic clones within the film of growth that ap- Hybridization of mammalian somatic cells, which was intro- peared on RDR plates after incubation were selected out by duced 15 years ago (1), is being widely used to study the ex- replica plating onto variously supplemented SDR medium. This pression of differentiated functions (2-4) and the genetics of is a nonhypertonic minimal medium (14), to which 20 ,M human cells in culture (5,6). Fusion of protoplasts from higher MnCl2, 5 gg/ml of DNase, 1 Ag/ml of rifamycin, and 15 g/liter plant cells has also been achieved (7), and in some cases whole of (Difco) agar have been added. It was used as a selection flowering hybrid plants have been regenerated, starting from medium, both unsupplemented (SDR) and supplemented (see fused protoplasts (8). A broad and most promising field has, Table 2). thus, been opened for rationally combining desirable properties Procedure Adopted for Fusion Experiments. Overnight from two sexually incompatible plant lines (9, 10). In this con- precultures of both parental strains in nutrient broth at 300 were text, it seemed surprising that, to our knowledge, no sustained inoculated, before growth ceased, into 20 ml of broth, to give attempt at fusing bacterial protoplasts has been reported. With an initial optical density (OD570) = 0.05. These cultures were such purposes in mind we decided to try fusion of protoplasts incubated with shaking at 370 until an OD of 0.4 was reached. of Bacillus subtilis, a Gram-positive bacterium in which many From each culture, 15 ml were centrifuged, the pellets were nutritional markers are available. Although no conjugation, taken up in 3 ml of SMMD (OD570 = 2, or about 4 X 108 colony mediated by sex factors, has ever been found in this organism, forming units/ml), and lysozyme was added to a concentration a detailed chromosomal map is available (11), constructed from of 200,ug/ml. Complete protoplast formation was usually seen transduction and transformation data. The cell wall consists after 10 min of gentle shaking at 420, but exposure to lysozyme essentially of a peptidoglycan layer, easily removed by lyso- was continued for 20 more minutes. Samples (0.1 ml) of each zyme treatment, and regenerated under osmotic protection (12, suspension were then plated on ordinary nutrient agar. The 13). An important element in the choice of this material was the plates usually remained sterile, and indicated that the frequency absence of an outer membrane, a potential obstacle to cyto- of osmotic shock resistant forms was below 2.5 X 10-8. plasmic membrane contact and fusion. One milliliter samples from each of the two suspensions were Assuming that fusion occurs between protoplasts from two mixed in a third tube, the three tubes were centrifuged, and polyauxotrophic strains, the appearance of prototrophic clones each pellet was resuspended in 0.2 ml of SMMD. To one tube, would presumably require wall regeneration and selection of 1.8 ml of a 40% (wt/vol) solution of polyethylene glycol (PEG)t the prototrophs. Plating the mixed protoplasts on an hypertonic in SMM was added and the suspension immediately homoge- minimal medium efficiently supporting these two processes has nized by shaking. After a 1 min exposure to PEG, either at 200 been tried repeatedly, with only occasional success. Regener- or at 00, several 0.05 ml samples were spread on the surface of Abbreviations: PEG, polyethylene glycol; SMM, sucrose-magne- duplicate RDR plates and used to make 10-1 and 10-2 dilutions sium-maleate buffer; RDR, rich regeneration agar medium; SDR, in SMMD, from which, in turn, further reversion plates and also nonhypertonic minimal media; SMMD, sucrose-magnesium-maleate buffer containing 5 ,g/ml DNase. tThe molecular weight is not critical; PEG 6000 from Merck was * Present address: The Rockefeller University, New York, N.Y. 10021. usually used. 2151 Downloaded by guest on September 26, 2021 2152 Microbiology: Schaeffer et al. Proc. Natl. Acad. Sci. USA 73 (1976) Table 1. Derivation of the parental strains used Strains constructed by transformation Recipient DNA donor Phenotypic changes S, (rfm-486 metB5 leu-8 thr-5) Mu8u5u5* MO21t Rfmr S3 (rfm-486 purB34 ura-1 trpC7) GSY1104t MO21t Rfmr S, (rfm-486 ura-1 trpC7 thr-5) S3 Si Ade+ Thr- S7 (rfm-486 purB34 metB5 leu-8) S1 S3 Thr+ Ade Ss (rfm-486 purB34 leu-8 thr-5) S1 S3 Met+ Ade- S9 (rfm-486 ura-1 metB5 trpC7) S3 S1 Adel MetF In the transformation experiments (14) excess DNA (5 ,ug/ml) was used whenever double transformants were wanted. Rfmr refers to rifamycin resistance. * This strain is metB5 leu-8 thr-.5 (15). t This strain is rfm-486 trpC2 leu-2 (16). T Supplied by C. Anagnostopoulos, this strain is purB34 ura-I trpC7. further dilutions were prepared. The three pellets were pro- these markers were not selected against, a very few partial cessed and plated in succession, so that prolonged exposure to prototrophs did grow out, even in the presence of DNase. Not PEG, which can diminish the number of prototrophs, was all combinations of two growth factors seemed to be effective avoided. After 48 hr of incubation at 370, the most heavily in- (see the first column of Table 2), but no conclusions could be oculated RDR plates were replicated with a velvet surface on drawn from such low numbers of colonies. SDR plates, or SDR plates carrying various limited combina- Recognition of fusion products became possible only when tions of the six growth factors. The prototrophic or partially a PEG treatment was appliedt, in awareness of its usefulness prototrophic colonies were counted after at least 72 hr of in- in the fusion of plant protoplasts (18). The treatment turned out cubation at 37'. An unexplained crowding effect was noted, to be effective (Table 2), but only when PEG concentrations i.e., 3 to 10 times higher counts of prototrophs per ml were approaching 40% were used. Since control plates bearing obtained from RDR plates inoculated with 2 X 106 protoplasts PEG-treated protoplasts from only one parent in every case or less than from plates that received the highest inoculum (2 remained sterile, it appeared that prototroph production did X 107 protoplasts). Regeneration frequency for the parental occur as a result of protoplast fusion, but was very rare in the strains themselves varied from 0.3 to 20%, such extreme values absence of an appropriate fusion-enhancing treatment, or when being rarely observed. Within this range, this variation did not a large number of markers were involved. An important notion greatly influence the numbers of prototrophs obtained on a already suggested by these observations was that the prototro- given selection medium. phic growth observed is more likely to result from postfusional genetic recombination, than from divisions of the initially RESULTS created heterodiploid cells; appearance of stable diploid pro- Attempts to produce prototrophic clones by fusion occurring totrophs should be independent of the growth factors supplied spontaneously in mixtures of protoplasts from two triply aux- during selection.
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