JOURNAL OF BACTERIOLOGY, Mar. 1978, p. 1246-1253 0021 -9193/78/0133-1246$02.00/0 Copyright ©1978 American Society for Microbiology Vol. 133, No. 3 Printed in U.S.A. Interspecies Transformation in Bacillus: Mechanism of Heterologous Intergenote Transformation RONALD M. HARRIS-WARRICKt* anp JOSHUA LEDERBERG Department of Genetics, Kennedy Laboratory of Molecular Medicine, Stanford University Medical School, Stanford, California 94305 Received for publication 11 October 1977 Bacillus subtilis-Bacillus globigti hybrids were made by integration of the B. globigiit aromatic region (aroB to aroE) as an intergenote in the B. subtillis chromosome. Transformation of the heterologous intergenote by B. subtillis DNA (or vice versa) occurred at about 10% of the frequency of homologous transformations by hybrid donors into the same region. Heterologous intergenote crosses were unusually sensitive to shear fragmentations of donor DNA to sizes less than 30 x 10° to 40 x 10° daltons. In all cases, the entire intergenote was transferred en bloc. Homologous transformation of intergenote markers by B. globigiti DNA was not unusually shear sensitive, and linkage was normal for markers in the intergenote. A model is proposed in which efficient heterologous intergenote transformation occurs by recognition and base pairing of homologous DNA sequences on both flanks of the intergenote. In our previous paper (7), we constructed hy- brid strains of Bacillus subtilis carrying an ex- tended sequence of heterologous DNA (called an intergenote [4]) from B. globigii. From an analysis of transformations using these hybrids, we concluded that enzymatic resvriction is not a barrier to interspecies transformation in this sys- tem. Rather, sequence nonhomology is the ma- jor barrier to the efficient incorporation and expression of B. globigii DNA in B. subtilis recipients, In this paper, we report the results of more detailed studies on the mechanisms of transfor- mation by foreign B. globigitit DNA sequences carried on an intergenote in B. subtilis and of incorporation of B. subtilis DNA into the B. globigii intergenote in hybrid recipients. In both situations, the nonhomologous sequences to be transformed are surrounded on both flanks by DNA that is homologous between donor and recipient. We have tested the importance of these homologous flanks in the transformation of the intergenote by a study of the DNA con- centration dependence of the transformation as well as its dependence on the size of the donor molecule. Measurements of the cotransfer linked markers in the intergenote transformation were also done. These results demonstrate that high- efficiency integration of the intergenote requires cotransfer of DNA sequences that are homolo- gous to the recipient chromosome on both flanks of the intergenote sequence. + Present address: Department of Neurobiology, Harvard Medical School, Boston, MA 02115. MATERIALS AND METHODS Materials and methods were the same as in the previous paper (7). RESULTS Effect of DNA concentration on transfor- mations with hybrids as recipients. In the accompanying paper (7), we isolated B. globigit- B. subtilis hybrids (called globimar hybrids) in which the B. globigii aromatic region from aroB* to aroE* had been integrated as an inter- genote in the B. subtilis chromosome. B. globigii DNA could transform this region with the same efficiency as B. subtilis DNA, representing a 10°- fold increase in the interspecies efficiency of transformation (ET). If the basis for this in- crease is the presence of sequence homology for the B. globigii donor, one would expect a con- comitant decrease in B. subtilis donor efficiency for the same region. This was not seen; however, the B. subtilis donor carried sequences that were homologous to the recipient chromosome adja- cent to the heterologous intergenote region. It is possible that a subset of B. subtilis donor mo- lelcules is capable of transforming the heterolo- gous intergenote due to integration via these adjacent sequences. The aforementioned experiments were per- formed at saturating DNA concentrations (4 to 8 pg/ml). A further study was made of the effect of varying DNA concentration on intergenote crosses. Hybrid recipients 76-2 and 76-3 were transformed by B. globigii (SB512) and B. sub- tilis (SB19) donor DNA. Intergenote markers 1246 VoL. 133, 1978 (trp, or tyr,) and the B. subtilis marker (lys.) were selected. The relationship between DNA concentration and ET is shown in Fig. 1. With decreasing DNA concentrations, the ET values rose for intergenote markers and fell for the B. subtilis marker. For example, at 0.01 »g/ml, B. globigii DNA transformed the homologous in- tergenote trp, and tyr, markers 8 and 12 times more efficiently than did B. subtilis DNA, while being unable to transform the heterologous B. subtilis marker, lys, at a measurable frequency. In Fig. 2, the intergenote/B. subtilis marker transformation ratio is calculated as a function of DNA concentration. In the B. globigii x hybrid crosses, SB512 x 76-2 and SB512 x 76-3, these ratios represent the ratio of homologous to heterologous transformation efficiency; they rose with decreasing DNA concentration until no lys,* transformants could be obtained. By using B. subtilis DNA in the crosses SB19 x 76- 2 and SB19 x 76-3, the intergenote/homogenote ratio represents the ratio of heterologous to ho- mologous transformation efficiency; as expected, these values fell with decreasing DNA concen- tration. As a control, also shown in Fig. 2, the trpC/lys and tryA/lys ratios were calculated for the entirely homologous cross, SB19 = SB1023; these did not change with DNA concentration. 10-5 410-6 L 1 1 i 10 1 0.1 0.01 0.001 DNA CONCENTRATION (ug/ml) Fic. 1. DNA concentration dependence of effi- ciency of transformation of intergenote and B. subtilis homogenote markers by B. globigii. Hybrid recipients 76-2 and 76-3 were transformed by B. globigti (SB512) and B. subtilis (SB19) DNA at different concentra- tions. For each marker, the ET (ratio of B. globigti to B. subtilis transformants per milliliter) was calcu- lated. The subscript for each marker refers to the origin of the marker in the recipient. Scales for the markers are as indicated. Recipients and markers: (@) 76-2, trp,* selected; (O) 76-2, lys.* selected; (A) 76-3, tyr, selected; (A) 76-3, lys,* selected. HETEROLOGOUS GENOTE TRANSFORMATION 1247 6 310 1 F T T T ve ae ee O-0 RATIO ren £3 RATIO 10 1 0.1 0.01 0.001 DNA CONCENTRATION (jig/ml) Fic. 2. Intergenote/homogenote transformation ratio: dependence on DNA concentration. Hybrid recipients 76-2 and 76-3 were transformed for inter- genote and homogenote markers by SB512 and SB19 DNA at different concentrations. The frequency of transformation was calculated, and the ratio of in- tergenote to homogenote marker transformants was derived. As a control, the homologous ratios were calculated in the cross SB19 X 1023. Crosses: (a—A) SBI9 X 76-2, trpg/lysg; (&---&) SB512 x 76- 2, trp,/lys.; (@—@) SB19 x 76-3, tyr,/lys.; (@-- -®) SB512 x 76-3, tyr,/lys,; (A—A) SB19 x SB1023, trpC./lys,;;5 (O—O) SB19 x SB1023, tyrA,/lys. The subscript for each marker refers to the origin of the marker in the recipient. Scales for the markers are as indicated. For both donors, then, the ability to transform a heterologous DNA sequence was more de- pendent upon high DNA concentration than was the ability to transform a homologous sequence. This could be explained by the need for DNA molecules of a particular size or genetic compo- sition to effect the heterologous cross; the like- lihood of finding this subset of molecules would decrease with decreasing DNA concentration. Transformation with globimar hybrid as donor. In the above experiments, B. subtilis DNA continued to retain a significant transfor- mation efficiency for the heterologous B. globi- gli intergenote, even at very low DNA concen- trations. As stated above, this can be explained by the presence of homologous flanks adjacent to the heterologous intergenote, which are pres- ent on a subset of the B. subtilis donor mole- cules. An analogous mechanism could also op- erate in transformation from a hybrid donor into a homogenotic B. subtilis recipient. Thus, inter- genote transformation efficiencies should be comparable in B. subtilis x globimar hybrid and globimar hybrid x B. subtilis crosses. To test this hypothesis, 7>H-labeled DNA was 1248 HARRIS-WARRICK AND LEDERBERG extracted from the globimar hybrid strain 135-1, a thymine-requiring derivative of SB1112, and the B. subtilis strain SB1070, a thyA thyB deriv- ative of SB19. As recipients, closely related strains were used: SB863, a B. subtilis homogen- ote carrying (among other markers) trpC and the unlinked AisA marker, and 99-12, a hybrid constructed by transforming the trp, B. globdigii intergenote from 76-2 into SB863. The entire intergenote was transferred in this cross (see below). The advantage of these transformations is that both donors are isogenic at the AisA,* locus, while both recipients are isogenic at hisA,. Thus, any differences in competence of recipi- ents or the physical state of the donor DNA, except the intergenote, will be seen in transfor- mations to hisA*. Standardizing intergenote transformation frequencies relative to hisAt transformation makes it possible to compare intergenote transformation efficiencies between different crosses. The results of these crosses are shown in Table 1. From the transformation fre- quencies, it is clear that globimar hybrid DNA can transform a B. globigti intergenote with higher efficiency than the corresponding B. sub- tilis sequence; when hisA* ET values were nor- malized to 1.0, hybrid 135-1 DNA transformed the trp, intergenote marker in 99-12 with an ET of nearly 13, but transformed the B. subtilis aromatic marker, trpC, in SB863 with an ET of less than 0.1. Transformation frequencies for the trp markers were normalized by dividing by the frequencies obtained for the isogenic AisA, marker; when the ratio for the homologous B. subtilis x B. subtilis cross was arbitrarily set at 100%, both globimar hybrid x B. subtilis and B. subtilis X globimar hybrid crosses yielded trp*t transformants at about 8.5% efficiency, whereas J. BACTERIOL. the homologous hybrid x hybrid cross efficiency was about 110%. Thus, both heterologous inter- genote crosses are achieved at the same effi- ciency, while both homologous crosses occur about ten times more frequently, in keeping with the hypothesis. These experiments have been repeated using different donors (SB19, 76-2, 76- 3) and recipients (SB1023, 76-3, 76-2) with iden- tical results (data not shown); heterologous transformations can be achieved at fairly high frequency (about 10% of homologous frequen- cies) if the heterologous sequences are located on an intergenote surrounded by homologous sequences. Effect of hydrodynamic shear on inter- genote transformation. If effective heterolo- gous intergenote transformation requires con- current cotransformation of adjacent homolo- gous sequences, this type of cross should require longer donor molecules than homologous crosses. To test size as a variable, DNA from SB1070 (B. subtilis homogenote), SB512 (B. globigit homogenote), and 135-1 (globimar hy- brid) was hydrodynamically sheared to increas- ingly smaller sizes. The weight-average molecu- lar weight of each sheared sample was deter- mined from neutral sucrose gradient centrifu- gation, using P22 DNA as a size marker (data not shown). The peak of biological activity (transformants per microgram of DNA) in each gradient lay on the heavy shoulder of the peak of physical activity (counts per minute), so the weight-average molecular weight was calculated from bioassays of the gradients, using homolo- gous recipient markers. It was not possible to test DNAs from these gradients for their ability to transform a heterologous marker; especially at higher shear, the DNA activity was too low TABLE 1. Biological activity of hybrid DNA compared to homogenote DNA Recipi- ent marker selected Donor Recipient trpCs hisA, irpC. hisA, trp, hisA, ip, hisA, SB1070 (B. subtilis) SB863 (B. subtilis) 135-1 (hybrid) SB863 (B. subtilis) SB1070 (B. subtilis) 99-12 (hybrid) 135-1 (hybrid) 99-12 (hybrid) Transfor- mation Transfor- fre- Relative mation Normal- wo fre- ET ized ET? quency activity a ratio (%) quency trp*/ hisA* Li 0.714 100 1.6 (standard) 0.51 0.45 0.085 0.061 8.5 8.4 5.35 1.0 0.075 0.062 8.7 1.2 3.8 50.0 12.9 0.797 112 4.7 3.88 1.0 * Recipient strains were transformed by the donor strains at saturating DNA concentrations (4 ug/ml). The datum is the proportion of recipient cells transformed per 104 exposed. * ET was normalized to AisA,* ET of 1.0, and other values were adjusted accordingly. * Relative activity is calculated from trp*/hisA* ratios and standardized to the homologous cross SB1070 x SB863 as 100%. VoL. 133, 1978 for reliable transformation assays. Instead, sam- ples of sheared and unsheared DNA were tested for their ability to transfer homologous and het- erologous markers at saturating DNA concen- trations (4 pg/ml). To standardize for variations in the physical states of the three DNA species, transformation values for the sheared DNA were calculated as a percentage of the unsheared val- ues, These values were plotted as a function of molecular weight (Fig. 3). Two globimar hybrid strains, 99-12 (Fig. 3A) and 96-3-1 (Fig. 3B), and two B. subtilis strains, SB1023 (Fig. 3C) and SB863 (Fig. 3D), were used as recipients. For all homologous marker crosses, transformation fre- quencies showed a slow inactivation with shear, worsening at sizes below 10 x 10° daltons. In marked contrast, heterologous markers were much more sensitive to shear; at average sizes above 30 x 10° to 40 x 10° daltons, the heterol- ogous crosses were still efficient, but at shear below 10’ daltons they were selectively inacti- vated, retaining less than 0.1% of normal trans- forming activity after shear to less than 5 x 10° daltons. In Fig 3C and D, the effect of shear on cotransfer of the aromatic region from aroB to tyrA in the homologous crosses, SB1070 x SB1023 and SB1070 x SB863, is also shown: cotransfer of this long homologous sequence (12 x 10° daltons; L. Okun, Ph.D. thesis, Stanford University, Stanford, Calif., 1968) is also very sensitive to shear, though 10-fold less so than the heterologous intergenote cross. Thus, het- erologous intergenote crosses are unusually sen- sitive to shearing of donor molecules below a critical size, as predicted by the hypothesis; this critical size is less than 30 x 10° daltons, since heterologous crosses are still fairly efficient at this size. Cotransfer of linked markers in heterol- ogous intergenote transformation. To test the genetic composition of DNA molecules ca- pable of transforming heterologous intergenotes, the cotransfer frequency for linked markers in heterologous intergenote crosses was deter- mined. For these experiments, globimar hybrid x B. subtilis crosses was performed using 135-1 as donor and SB1023, carrying the linked aro- matic B. subtilis markers aroB, trpC, hisB, and tyrA, which are present in the B. globigii inter- genote of 135-1, as recipient. Transformations were performed at saturating DNA concentra- tions at the four levels of shear described previ- ously, and each marker was selected separately. As a control, cotransfer was also measured with unsheared SB1070 (B. subtilis homogenote) DNA. The results are shown in Table 2. When the heterologous intergenote cross was at- tempted, all intergenote markers were cotrans- ferred with essentially 100% efficiency; the few HETEROLOGOUS GENOTE TRANSFORMATION 1249 colonies isolated that remained auxotrophic for one of the central markers were probably the results of rare multiple crossover events during recombination. The 100% cotransfer of intergen- ote markers occurred at all levels of shear, even when the weight-average molecular weight was less than 5 x 10°; thus, with increasing shear, the transformation frequency of heterologous inter- genote markers was drastically reduced, but the entire intergenote continued to be cotrans- formed in the few remaining transformants. The residual biological activity correlated with the residual level of molecules of size greater than 30 x 10® daltons at each shear level (data not shown). In the homologous control cross, co- transfer of all four markers did not exceed 50% of the transformants tested; normal linkage was seen, with significant numbers of colonies trans- formed for only one or two markers. It is possible that the integenote may possess unusual genetic properties that encourage high cotransfer in any cross. This possibility was elim- inated by measuring the cotransfer of the linked markers trp, and tyr, in the hybrid x hybrid cross 76-2 (trp, tyr,*) X 76-3 (trp,* tyrz); tyr* transformants were tested by replica plating for cotransfer of trp. Less than 50% of tyr,* trans- formants were cotransformed to auxotrophy for tryptophan (Table 3). Table 3 also shows cotransfer values for inter- genote markers in homologous intergenote crosses of the type B. globigii X globimar hybrid. New mutations were introduced into the inter- genote by transformation or N-methyl-N’-nitro- N-nitrosoguanidine mutagenesis and penicillin selection; mutants corresponding in phenotype to the AisB and aroF loci were shown to be located in the intergenote by high transforma- tion efficiencies using SB512 (B. globigit) DNA. Cotransfer of trp-tyrA, hisB-tyrA, and tyrA- aroE markers in homologous intergenote crosses (Table 3) was similar to that seen in the corre- sponding B. subtilis homogenote crosses (Table 2 and [8]), demonstrating a similar mapping order of the aromatic regions of B. globigii and B. subtilis. DISCUSSION The results shown in Table 1 demonstrate that foreign DNA sequences can be transformed at about 10% of homologous frequencies if they are located on an intergenote surrounded by DNA sequences homologous to the recipient chromosome. This is true for both globimar hy- brid < B. subtilis and B. subtilis x globimar hybrid crosses. Figure 3 demonstrates that the efficiency of these crosses is highly dependent on DNA size; heterologous intergenote transfor- mation was selectively inactivated at sizes below 1250 HARRIS-WARRICK AND LEDERBERG J. BACTERIOL. 100 f po tl piel pe tal 1 0.15 a al, 2 al Ld PERCENT SURVIVAL pol po al 1 Tr 1 2 al | el vr 1 T 4 a" 0.1 TTP pe al Torry wh tl, > -- "6" yh 1 1 1 1 oak 1 t 1 1 50 §640 30020 10 0 >60 50 40 30 0=—20 10 WEIGHT—AVERAGE MOLECULAR WEIGHT (x 106 DALTONS) {INCREASING SHEAR] ————_—_—_——_r Vv Dp o o Fic. 3. Effect of shear on homologous and heterologous intergenote transformation. Hybrid and B. subtilis recipients were transformed with B. subtilis (SB1070), B. globigii (SB512), and hybrid (135-1) DNA sheared to various molecular weights. The frequency of transformation was calculated and expressed as a percentage of the unsheared transformation frequency for each DNA and each marker transformed. The first subscript for selected markers refers to the origin of the marker in the recipient; the arrow points to the origin of the marker in the donor, and hence the marker in the transformed recipient. (A) 99-12 as recipient: (@—®) SB1070 xX 99-12, trp,.. selected; (O—O) SB1070 x 99-12, hisAy.. selected; (&---A) 135-1 x 99-12, trp,, selected; (A--~-A) 135-1 Xx 99-12, hisAy.. selected; (HI) SB512 x 99-12, trp,., selected. (B) 96-3-1 as recipient: (@—®) SB1070 x 96-3-1, tyrys; (@-@) SB1070 X 96-3-1, lys..; (d---M) 135-1 X 96-3-1, tyr,.,: (HH) SB512 X 96-3-1, tyry+. (C) SB1023 as recipient: (@—®) SB1070 x SB1023, hisB,..; (C—O) SB1070 x SB1023, irpC...; (O—O) SB1070 x SB1023, tyrA...; (BHAI) SB1070 x SB1023 (aroB,-tyrA,., coselected; (&-—-A) 135-1 X SB1023, hisB,..; (A---A) 135-1 x SB1023, trpC.., (A---d) 135-1 X SB1023, tyrA,.,. (D) SB863 as recipient: (&---A) 135-1 x SB863, trpC.,; (A---A) 135-1 x SB863, tyrA,.,; (BK--) 135-1 x SB863, hisA,..; (@—®) SG1070 x SB863, trpC....; (O—O) SB1070 x SB863, tyrA,..; (I+) SB1070 x SB863, hisA,..; (O—©) SB1070 < SB863, aroB,-tyrA,.. coselected. 10 x 10° to 20 x 10° daltons. Table 3 shows that genote transformation. A model of heterologous all the measurable markers on the intergenote intergenote transformation is presented in Fig. are cotransferred during any heterologous inter- 4. The recipient is assumed to be a globimar VoL. 133, 1978 HETEROLOGOUS GENOTE TRANSFORMATION 1251 TABLE 2. Cotransfer of linked markers in homologous and heterologous intergenote transformation into SB1023 Pri- No. of Phenotypes found (%)* Donor |Shear®| mary colo- marker tested L121 (1110 | O111 | 1011 |1101 | 1100 {0110 | COLL | 1001 | 0101 | 1000 |0100 |0010 | 0001 135-1 0 aroB* 160 158 2 (99) a) trpCt | 389 | 388 1 (99.7) (0.3) hisB* 300 300 (100) tyrA* 404 404 (100) 135-1 18 trpC* 258 258 (100) AisB* 343 343 (100) tyrA* 212 211 1 | (99.5) (0.5) 135-1 23 trpC* 122 122 (100) hisB* 119 119 (100) tyrA* 139 138 1 (99.3) (0.7) 135-1 27 trpC* 69 67 2 (97.1) {2.9) hisB* 33 33 (100) tyrAt | 133 | 133 (100) I 1070 | 90 |aroBt| 65 | 29 | 2 14 20 (44.6) 1 (3.1) (21.5) (30.8) trpC* 70 33 2 18 1 7 2 8 (47.1) | (2.9) | (25.7) (1.4) | (10) | (2.9) (11.4) hisB* 80 40 4 16 2 15 3 (50) | (5) | (25) | (2.5) (18.8) (3.8) tyrA* 69 21 15 8 1 18 i 1 4 (30.4) (21.7) | (11.6) | (1.4) (26.1) | (1.4) | (1.5) (5.8) * Order of genes in 1111 is aroB-trpC-hisB-tyrA. In the phenotype number, 1 stands for the donor marker, i.e., transformants to prototrophy, and 0 stands for the recipient auxotrophy. Figures refer to number of absolute colonies found of each phenotype; parentheses indicate the percentage of total colonies tested that are of that phenotype. ® Numbers indicate size of hypodermic needle used for hydrodynamic shearing. TABLE 3. Linkage of markers in hybrid x hybrid and B. globigii x hybrid crosses : 3 No. - hi , Donor Recipient Primary Secondary 0 of col Phenotypes _ Cotrans- marker marker tested 11 10 01 fer 76-2 76-3 tyr,* tp, ==———st=~=«SD 429° 491 0.47 SB512 38-1 tyr,” trp,* 20 13 7 0.66 trp,* tyr,* 60 40 20 SB512 6-5 tyr,* his,* 193 148 45 0.74 his," tyr,* 178 126 52 SB512 Phell tyr,* aroE,* 200 200 0 0.98 aroE,* tyr,* 309 299 10 * 1 and 0 are defined as in Table 2. hybrid. The essential feature of this model is | quence recognition and base pairing of homolo- that efficient integration of the heterologous in- gous sequences on both flanks of the intergenote tergenote is accomplished only through se- (Fig. 4A). These homologous sequences function 1252 HARRIS-WARRICK AND LEDERBERG to locate the corresponding region of the recipi- ent chromosome, align the heterologous se- quence properly, and “splice” the intergenote into the recipient chromosome, holding it in place until the transformed region is rendered homogeneous at a later stage. An expected con- sequence of the requirement for integration of the entire intergenote is 100% linkage of all intergenote markers. The mechanism of conver- sion of the integrated heterologous intergenote to homogeneity is unknown but probably in- volves DNA replication or some form of repair. However, the excision repair mechanism does not seem to be active in gene conversion in Haemophilus (2). Transforming DNA that lacks the entire intergenote and homologous se- quences on both sides is not efficiently inter- grated (Fig. 4B) due to difficulty in base pairing and covalent joining of the nonhomologous (A) RECOGNITION Y x | z | | TOMI AO ROMO OO MM { | | x y’ Z INTEGRATION ON BOTH SIDES [fermi BAND FORMATION x Y z | | ] | | | x y’ 2 TRANSFORMED HOMODUPLEX J. BACTERIOL. end(s) of the donor molecule to the recipient chromosome; even if one end of the molecule is covalently bound to the chromosome, cellular exonucleases will digest it from the free end. Treatment of the DNA to reduce the number of molecules carrying the entire intergenote (such as shear, reduction in DNA concentration, or in vitro cleavage by the B. globigii restriction sys- tem [7]) will thus selectively inactivate heterol- ogous intergenote integration; the surviving mol- ecules should continue to transform recipients with 100% linkage of intergenote markers. A similar mechanism to ours has been dem- onstrated for the transformation of deletion mu- tations in B. subtilis (1; R. Harris-Warrick, un- published data). Deletions represent the ex- treme model for integration of foreign DNA sequences where no sequence homology exists. Integration of the sequence that is deleted in the (B) RECOGNITION z TOOT WOOO GOOD mmo | | | x y z | INTEGRATION ON ONE SIDE ONLY Y | Zz vay Sra TMM WOT NUnOn | | | x y’ Zz DEGRADATION OF SINGLE- STRANDED MATERIAL z | TOMI IIOP MM mo | | | x y’ Zz COVALENT BOND FORMATION Zz | IMM MM 0 Ooo Tod Mo! | | | x y’ 2 TRANSFORMED HOMOOUPLEX Fic. 4. Proposed mechanism of heterologous intergenote transformation. (A) Successful transformation of the intergenote B. subtilis x globimar hybrid cross. The homogeneous B. subtilis donor (—, represented by marker y) transforms the entire B. globigii intergenote (- - -, represented by marker y') as well as homologous B. subitlis sequences (—) on both flanks of the intergenote, represented by markers X and Z. The integrated sequence is rendered homogeneous at a later stage either by gene conversion or semiconservative replication. (B) Unsuccessful intergenote transformation. Here the donor molecule lacks homologous sequences, repre- sented by X, on one side of the intergenote. As a result, the heterologous sequence is not integrated and is digested by single-strand-specific nucleases. VoL. 133, 1978 recipient chromosome also occurs with 100% cotransfer of the deleted markers, again suggest- ing a requirement for homologous sequences on both flanks of the deletion. Bernheimer and co-authors have studied a model whereby the capsular genome is trans- formed heterologously in its entirety by two possible mechanisms. (i) If the transforming DNA molecule contains the entire heterologous capsular genome and sequences homologous with the recipient chromosome on both flanks of it, the heterologous capsular genome is inte- grated at the site of the recipient capsular ge- nome by a classical exchange mechanism. (ii) If the transforming molecule contains sequences homologous with the recipient chromosome on only one flank of the heterologous capsular ge- nome, it is integrated ectopically by insertion at a site different from the resident capsular ge- nome; the result of this addition is a binary strain carrying both capsular genomes, although only one may be expressed phenotypically. Our results involving interspecies transformation in Bacillus suggest that only the first kind of trans- formation can occur and that DNA molecules lacking sequences homologous to the recipient on one or both flanks of the heterologous inter- genote are unable to transform the recipient. The reasons for this discrepancy are unknown, although it is likely that the increased genetic distance between B. globigii and B. subtilis compared to that between the different capsular types in Pneumococcus is a contributing factor. Our model and data conflict with the results of Biswas and Ravin (5, 9); working with strep- tococcal-pneumococcal transformations, they did not find increased linkage between evolu- tionarily conserved antibiotic resistance markers in heterologous intergenote crosses. The discrep- ancy could be due to differences in heterologous transformation in conserved and nonconserved regions of the chromosome. In conserved re- gions, interspecies homology is high enough that recognition and integration may occur with short lengths of foreign donor DNA, and homol- ogous nonforeign sequences may not be needed to facilitate recognition. The “linked” markers in the reported pneumococcal-streptococcal sys- tem may not even be located on a single contin- uous intergenote; identical linkage is seen when both intergenote markers are introduced simul- taneously and when each marker is introduced independently in separate transformations (5). In nonconserved regions, DNA homology be- tween two species is so low that effective inte- gration is very rare; the presence of homologous nonforeign sequences adjacent to the continuous intergenote will have a significant effect on rec- ognition capability. Alternatively, the discrep- ancy in our results could be due to species-spe- HETEROLOGOUS GENOTE TRANSFORMATION 1253 cific differences in the mechanism of heterolo- gous DNA integration. We do not know the size of the B. globigii intergenote in our globimar hybrid strains. It extends across the entire aromatic region from aroB to aroE; the order of mapping of our markers in the intergenote (Table 3) suggests that the B. globigii sequence is organized simi- larly and should be the same length as the aromatic region of B. subtilis. Using EcoRI- cleaved B. subtilis DNA segments, the trpE- aroE linkage group (lacking only aroB, which is closely linked to trpE [8]) has been shown to lie on a segment of 12.5 x 10° daltons (6). Estimates of the aroB-tyrA linkage group based on shear sensitivity and cotransfer data yield similar val- ues (L. Okun, Ph.D. thesis, Stanford University, Stanford, Calif., 1968). A minimal estimate of the intergenote size, then, would be slightly greater than these values, or 13 x 10° to 15 x 10° daltons. ACKNOWLEDGMENTS The expert technical assistance of P. Ruiz, J. Jennings, and P. Evans is gratefully acknowledged. This study was supported by grants GM-00295 from the National Institute of General Medical Sciences, AI-5160 from the National Institute of Allergy and Infectious Diseases, CA- 16896 from the National Cancer Institute, and NGR-05-020- 004 from the National Aeronautics and Space Administration, and by an award from the Center for Interaction, Houston, Texas. LITERATURE CITED 1, Adams, A. 1972. Transformation and transduction of a large deletion mutation in Bacillus subtilis. Mol. Gen. Genet. 118:311-322, 2. Beattie, K. L., and J. K. Setlow. 1970. Transformation between Haemaphilus influenzae and Haemophilus parainfluenzae. J. Bacteriol. 104:390-400. 3. Bernheimer, H. P., and I. E. Wermundsen. 1972. Ho- mology in capsular transformation reactions in Pnew- mococcus. Mol. Gen. Genet. 116:68-83. 4. Bernheimer, H. P., I. E. Wermundsen, and R. Aus- trian. 1967. Qualitative differences in the behavior of pneumococcal deoxyribonucleic acids transforming to the same capsular type. J. 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