Proc. Natl. Acad. Sci. USA Vol. 73, No. 6, pp. 2151-2155, June 1976 Microbiology Fusion of bacterial protoplasts (Bacillus subtilis/diploid /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 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 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 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 consists after 10 min of gentle shaking at 420, but exposure to lysozyme essentially of a 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. Support for this interpretation will now be otrophic strains (Mu8u5u5 and GSY1104, see Table 1) were sought in various ways in the sections to follow. unsuccessful. In order to avoid possible contaminants, a rif- Fusion experiments subjected to alternative marker amycin-resistant marker was introduced into each of these selections strains by transformation, so that subsequent work would be done under the protection of the antibiotic. When triply aux- The progeny obtained on 10 different selection media from SI otrophic strains (Si and S3, see Table 1), are used in fusion ex- X Sa fusion in three independent experiments appear in Table periments, selection can be instituted not only for true proto- 3. After PEG treatment and wall regeneration, they may con- trophs, on rifamycin-containing minimal medium, but also for tain a few true prototrophs appearing on minimal medium what we will call here "partial phototrophs", on the same me- (SDR), but they generally contain much larger numbers of dium supplemented with two growth factors, of which one is partial prototrophs which are revealed when the medium is required by each parent. When selection was against all six supplemented with two growth factors. The yield of fusion was but when two of products detected is dependent upon not only the number but markers, no prototrophic growth noted, also the particular pair of markers involved in the selection. Each selected colony implies the acquisition of some combi- nation of the wild-type genes derived from both parents. Pre- sumably these could arise only from fusion of the parents, be- cause a known fusion-enhancing agent significantly increases their number, they are only obtained when protoplasts-not cells-are mixed, and they are readily produced and developed in the constant presence of DNase. The widely varying yields with different selection media indicate that we are not, in any case, dealing with the total number of diploids produced. In all or most cases, the diploid phase must have lasted a rather short time and must have soon been succeeded by appearance of a t rpC particular one or a few of the many possible haploid recombi- FIG. 1. Chromosomal map of B. subtilis. The map is drawn to nants. The pattern of particular recombinants obtained is then scale according to ref. 11. The loci mentioned are those in which the markers used are located. 0 and T stand for origin and terminus of t It is pleasant to acknowledge that Dr. M. Fox first suggested to us the replication. Starting from 0 the two arrows in opposite directions use of PEG as a possible fusion-enhancing agent, as early as June, indicate that replication is bidirectional. 1974. Downloaded by guest on September 26, 2021 Microbiology: Schaeffer et al. Proc. Natl. Acad. Sci. USA 73 (1976) 2153

Table 2. Factors influencing the frequency Table 3. Frequency of partial and complete prototrophs of the prototrophic clones obtained derived from 5, x S3 fusion after selection for various sets of four parental markers PEG concentration, % Selective Supple- medium used 0 15 40 mentation No. of colonies obtained* No. of of minimal crossovers SDR* 0 0 8 medium Exp. 1 Exp. 2 Exp. 3 requiredt SMODRt 2 1 90 STUDRt 0 5 None 4 1 4 4R MO 148 76 133 2 SI X S3 cross carried out as described under Materials and Meth- MU 50 8 30 2 ods. The completely or partially prototrophic colonies obtained MA 87 20 76 2 were from 2 X 107 total protoplasts initially plated. TO 93 68 173 2 * Mean from 10 similar plates. TU 7 2 4 4R t Mean from five similar plates. The symbols for media indicate TA 20 1 2 4R SDR supplemented with methionine and tryptophan (MO) or threonine and uracil (TU). LO 63 158 2 LU 0 6 4R the quantitative expression of the more or less discrete tempo- LA 1 11 4R rary confrontation of segments apparently representing the Abbreviations: M, methionine; o, tryptophan; U, uracil; A, entirety of both parental chromosomes. adenine; T, threonine; L, leucine. Whether or not "whole chromosome" recombinations can * Number of colonies from 2 X 107 mixed Si x S3 protoplasts after add to the understanding of recombination in bacteria, they replication on various selective media. In all three experiments can-in an organism already genetically mapped by other shown, all control plates inoculated with only one parental strain means (11)-be coordinated with the presumed genetic map. remained devoid of colonies. t For each selective medium used, the minimal number of cross- To show this, other strains of B. subtilis bearing the same overs required for production of a single haploid recombinant markers as Si and S3 were constructed by successive transfor- with the requisite wild-type markers is given, together with the mation steps (see Table 1). Similar fusion experiments, and the symbol R if one of the crossovers would have to be in the short selection of partial and complete prototrophs, led to the results interval between metB5 and trpC7. presented in Table 4. There, and in the last column of Table 3, are indicated the minimal number of crossovers assumed from termined by replica plating. Results of such determinations the genetic map (Fig. 1) to be necessary to produce recombi- appear in Table 5. nants able to grow in the particular selective medium. It is seen that the two parental and the two recombinant types The pairs of strains were constructed so that the six loci in- are usually encountered, but that any one of these four types volved would require different levels of recombination to may predominate in the different cases. Once again, in each produce prototrophs. In the pair S6 and S7, wild-type genes of the five selections considered in Table 5, reference to the alternate with mutant markers around the chromosome in each chromosomal map leads to predictions that are in good agree- strain; recombination in each of six intervals are required to ment with the experimental data. These data by themselves obtain contiguous wild-type markers. On the other hand, in the strongly suggest that the PEG-induced prototrophs represent strains S8 and S9, the three wild-type and mutant markers are various haploid recombinants. contiguous in each strain, so that any two crossovers between these sets would produce prototrophs or partial prototrophs. Absence of segregation of auxotrophs from reisolated In SI X S3 crosses (Table 3), only recombinants requiring a prototrophs minimum of either 2- or 4-crossovers, including one possibly If some of the partial prototrophs observed still remained dip- rare crossover, could be selected. The numbers of 2-crossover loid, it would be expected that on further growth in nonselective recombinants were fairly homogeneous, and on the average, medium they would give rise to auxotrophic segregants, of 25 times higher than the average number of 4-crossover re- parental or recombinant types. For this reason, a rather ex- combinants. In an S8 X S9 cross (Table 4), only 2-crossover re- tensive search was made for such segregants among progeny combinants can be selected. The yields of most partially pro- from several carefully reisolated clones of prototrophs or partial totrophic types were higher than that of true prototrophs, al- prototrophs. though only by factors averaging 1.7 (range: 1 to 2.4). Even in The clones were grown for 10 generations in broth, and (in the more complex case of an S6 X S7 cross (Table 4) there was order to reveal any possible segregation brought about by spore a strong tendency for a higher yield of progeny when the formation), some of them were allowed to sporulate. In the minimal number of required crossovers was lower. The assumed results appearing in Table 6, more than one half of the ultimate relative rarity of the crossovers between the loci trpC and metB colonies tested were from heated spores. In spite of this, not a was also borne out, but one other class, selected on factors uracil single auxotrophic colony could be detected. + methionine, was also unexpectedly of relatively low fre- quency. Fusion involving lysogenic strains In conclusion, the relative yields of the various partial and An inducible prophage, entering by conjugation an Escherichia complete prototrophs selected correspond with the known ge- coli cell devoid of phage repressor, is induced and cell netic map of the several parent organisms. drastically reduces the yield of recombinants. This zygotic in- Segregation of unselected markers among partial duction is due to the fact that no cytoplasm, containing prototrophs phage-repressor, is entering the recipient cell at conjugation. If by contrast cell fusion allows for a free mixing of the cy- The requirements of the partial prototrophs for the two growth toplasms, no prophage induction, and no drastic reduction in factors that were present during their selection have been de- the yield of prototrophs are likely to take place when protoplasts Downloaded by guest on September 26, 2021 2154 Microbiology: Schaeffer et al. Proc. Natl. Acad. Sci. USA 73 (1976)

Table 4. Pattern of complete and partial prototrophs obtained from parental protoplasts bearing mutant markers in alternating (A) or in clustered (B) arrangements Fusion A, S6 x S? Fusion B, Ss X S9 Selective No. of No. of Selective No. of No. of medium colonies crossovers medium colonies crossovers Minimal 31 6R Minimal 75 2 UL 192 2R TO 114 2 UA 52 4R TM 125 2 UM 149 4 TU 181 2 OL 357 4 LO 78 2 OA 362 2 LM 91 2 OM 248 4 LU 165 2 TL 189 4R AO 143 2 TA 154 4R AM 120 2 TM 280 2 AU 133 2 Standard fusion experiments with the strains indicated. Single parental protoplasts were, in all cases, carried through the same procedure, and in each ofthe many selective media, each parental type gave no colonies whatever. Yield is from 2 x 107 mixed protoplasts.

of lysogenic and phage-sensitive bacteria are fused. In order When one of the three growth factors required by each of to verify this point, use has been made of 4105, a temperate the parental lines was supplied during the selection of the fusion phage of B. subtilis 168. Si and S3 were lysogenized, and in- products-i.e., when two of the six markers were not selected ducibility of their derivatives was tested with mitomycin as against-the following observations were made: (a) higher described (19). No clearcut reduction in the yield of partial yields were generally obtained than when complete prototrophs prototrophs was observed, when one of the parental strains was were selected for, and (b) not all combinations of the two growth lysogenic (Table 7). factors added were equally effective in increasing the yield. (This was true with the three pairs of parental strains used, DISCUSSION which all bore the same six markers, but in different combi- nations.) Confronted with the known chromosomal map of the When mixed together, lysozyme-induced protoplasts of two organism (Fig. 1), these data clearly establish that in a given triply auxotrophic strains of B. subtilis were shown to produce, fusion experiment the yields observed on 10 different selection after regeneration of cell wall, clones of prototrophic bacteria media are an inverse function of the minimal number of the which do not form from similarly treated protoplasts of the crossovers required to allow growth on the various selection single parental strains. Since this appearance of prototrophs was media (Tables 3 and 4). Therefore, for given rates of fusion and not observed from mixtures of nonprotoplasted bacteria, and regeneration, the yields observed are largely determined by the was unaffected in the continued presence of DNase, it is be- frequency of the recombination events required to allow growth lieved to result from protoplast fusion. This conclusion is further on the medium being used. Such events as chance association supported by the observation that prototroph formation is un- of cells of the two parental lines at the time colonies are ini- affected when one of the parents is supplied as an inducible tiated, appear to be relatively uniform. On the other hand, the lysogenic strain, and also by the fact that it is increased sub- concurrence of a second pair of required crossovers with a re- stantially when the mixed protoplasts are treated with poly- quired first pair is commonly greater than 10-1, so it is clear that ethylene glycol, an effective, apparently universal, fusion- parental chromosomes are, at some stage, liable to multiple enhancing agent (18, 20-22). Similar conclusions were reached exchanges, once being liable to any exchange. simultaneously by Fodor and Alfoldi, working on Bacillus The precise cellular mechanism of genome-to-genome in- megaterium (23). We thank these workers for the benefit of teraction is not of course defined, but it appears to be unre- free exchange of information as the two investigations, com- stricted in completeness. In this connection, in a study of ex- menced independently, have proceeded to the stage of these perimentally attempted fusion of bacterial protoplasts, collateral publications. Lederberg and St. Clair (24) indicate that -protoplasts Table 5. Segregation of unselected markers among partial prototrophs from one S, x S3 fusion experiment Number of colonies having indicated constitution Unselected S, parental type (-/+) S3 parental type (+/-) +/+ Recombinant -/-Recombinant markers S % % S met/trp 20 78 0 2 met!ura 74 3 2 21 met/pur 60 1 2 38 thr/thr 0 84 2 14 thr/pur 27 16 55 2 Colonies obtained on the variously supplemented selection media were transferred and set in rows on plates of their original selection medium, containing two growth factors. After overnight incubation, these plates were replicated on minimal medium and on medium containing either growth factor or both. Downloaded by guest on September 26, 2021 Microbiology: Schaeffer et al. Proc. Natl. Acad. Sci. USA 73 (1976) 2155

Table 6. Stability of prototrophic clones during growth in nonselective medium Medium in which originally Number of reisolated Total no. of ultimate Maximal frequency of isolated and finally retested clones tested colonies tested* auxotrophic subcolonies Minimal 4 1122 <9 x 10-4 Minimal + TO 4 1781 <6 X 10-4 Minimal + MO 3 3072 <3 x 10-4 Several original potentially-prototrophic clones derived from Si X S3 fusion upon selective media were reisolated twice as clones from the same media, then grown for 10 generations in nonselective broth, and retested for possible regain of auxotrophic markers derived from the original parents. See Table 3 for abbreviations. * The tested colonies were derived in approximately equal numbers from the reisolated clones within each group. Not one auxotrophic colony was recovered in any case. The upper limits of possible auxotrophs per clone would therefore be somewhat higher than the limits shown. of Escherichia coli show only such rare unidirectional chro- plate, frequencies as high as 2 X 10-4 (0.4% of those protoplasts mosome transfer as is already possible from F+ to F- cells that did regenerate a wall) have been recorded. For obvious themselves, which we would therefore suppose is unidirectional reasons, the frequency of the fusion event itself cannot be de- and incomplete. The evidence in our case is that neither duced from the experiments presented here. crossfeeding of some kind, nor limited chromosome transfer would explain the observations; they are, rather, explained by Our thanks are due to C. Anagnostopoulos and L. Rutberg, who the formation by fusion of protoplasts that must be at least kindly supplied bacterial strains. This work was supported by the Centre National de la Recherche Scientifique (Contract L.A. 136) and diploids. The very large size of the aggregates that are seen to the Fondation pour la Recherche Medicale Francaise. One of us form as soon as PEG is added, may suggest that mostly poly- (R.D.H.) was a visiting Professor of the University of Paris-Sud in 1975. ploids are formed. Aggregation however is not to be confused with fusion, and may be at most a prerequisite. In fact, it largely 1. Barski, G., Sorieul, S. & Cornefert F. (1960) C. R. Acad. Sci. Paris disappears when the PEG is eliminated by centrifugation, 251, 1825-1827. without drastically reducing the final yield of prototrophs. The 2. Harris, H. (1970) Cell Fusion (Clarendon Press, Oxford). mean ploidy level of the fused protoplasts, and the factors on 3. Ephrussi, B. (1972) Hybridization of Somatic Cells (Princeton which it may depend, remain to be determined. University Press, Princeton, N.J.). Whatever this level may be, it is clear that after two reiso- 4. Davidson, R. L. (1973) in Genetic Mechanisms of Development, lations all of the prototrophic bacteria produced by fusion are ed. Ruddle, F. H. (Academic Press, New York), pp. 295-328. haploid recombinants. Already inferred from the data in Tables 5. Ephrussi, B. & Weiss, M. C. (1969) Sci. Am. 220-4,26-35. 6. Ruddle, F. H. & Kucherlapati, R. S. (1974) Sci. Am. 231-1,36-44. 3 and 4, this was confirmed directly when segregation of the 7. Power, J. B., Cummins, S. E. & Cocking, E. C. (1970) Nature 225, unselected markers in partial prototrophs and of auxotrophs 1016-1018. from the reisolated prototrophs were studied (see Tables 5 and 8. Carlson, P. S., Smith, H. H. & Dearing, R. D. (1972) Proc. Natl. 6, respectively). If we think we know the initial (polyploid), and Acad. Sci. USA 69,2292-2294. the final (haploid) states of the fusion-induced formation of 9. Villanueva, J. R., Garcia-Acha, I., Gascon, S. & Uruburu, F. eds. prototrophs, the minimal number of generations required to (1973) in Yeast, Mould and Plant Protoplasts (Academic Press, produce the change remains to be estimated. New York), Part V, pp. 307-344. From the data presented, the highest yield of prototrophs 10. Centre National De La Recherche Scientifique (1973) Proto- observed seems to be 1.8 X 10-5 (Table 4, Fusion A). This is an plastes et fusion de cellules somatiques vegetales, " Colloq. Intn. underestimation, however. The experiments in Tables 3 and CNRS, no. 212 (INRA Publ. 73-1, Paris). 11. Young, F. E. & Wilson, G. A. (1975) in Spores VI, eds. Gerhardt, 4 were aimed at comparing, in different crosses, the yields on P., Costilow, R. N. & Sadoff, H. L. (American Society of Micro- various selection media. Because some of these yields were very ), pp. 596-614. low, large inocula (2 X 107 protoplasts per plate) were used. 12. Landman, 0. E. & Forman, A. (1969) J. Bacteriol. 99,576-589. While such inocula will usually produce higher numbers of 13. Wyrick, P. B. & Rogers, H. J. (1973) J. Bacteriol. 116, 456- prototrophic colonies per plate than smaller ones, they produce 465. a crowding effect, not all prototrophs showing up as colonies. 14. Anagnostopoulos, C. & Spizizen, J. (1961) J. Bacteriol. 81, When in other experiments prototroph frequency was more 741-746. correctly measured by plating only 2 X 106 protoplasts per 15. Yoshikawa, H. & Sueoka, N. (1963), Proc. Natl. Acad. Sci. USA 49,559-566. 16. Sonenshein, A. L., Cami, B., Brevet, J. & Cote, R. (1974) J. Bac- Table 7. Frequency of partial prototrophs from teriol. 120, 253-265. lysogenic inducible parental strains 17. Schaeffer, P., Millet, J. & Aubert, J. P. (1965) Proc. Natl. Acad. Sci. USA 54,704-711. Cell Frequency of 18. Kao, K. N. & Michayluk, M. R. (1974) Planta 115,355-367. regener- partial prototrophs 19. Rutberg, L., Hoch, J. A. & Spizizen, J. (1969) J. Virol. 4, 50- ation (on minimal + 57. Strains crossed % MO agar medium) 20. Ahkong, Q. F., Fischer, D., Tampion, W. & Lucy, J. A. (1975) Nature 253, 194-195. Si X S3 5.2 0.6 x 10-4 21. Ahkong, Q. F., Howell, J. I., Lucy, J. A., Safwat, F., Davey, M. SI (0105) x S3 7.5 1.0 x 10-4 R. & Cocking, E. C. (1975) Nature 255, 66-67. S1 X S3 (0105) 3.5 0.4 x 10-4 22. Vasil, I. K. & Giles, K. L. (1975) Science 190, 680. Si (0105)X S3 q105) 7.0 0.75 x 10-4 23. Fodor, K. & Alfoldi, L. (1976) Proc. Natl. Acad. Sci. USA 73, 2147-2150. See Table 3 for abbreviations. 24. Lederberg, J. & St. Clair, J. (1958) J. Bacteriol. 75, 143-160. Downloaded by guest on September 26, 2021