HFOURNAL OF BacTERIOLucy, Oct. 1981, p. 10L-1U8 0021-9193/81/1001 01 -68302.00/0 . TINT. - io Vol. 148, No. I Integration of the Bacteriophage 463T-Coded Thymidylate Synthetase Gene into the Bacillus subtilis Chromosome IWONA T. STROYNOWSKItT Department of Genetics, Stanford University School of Medicine, Stanford, Catifornia 94305 Received 10 October 1980/Accepted 12 June 1931 Transformation of Bacillus subtilis 168 Thy~ auxotrophs with 43T deoxyri- bonucleic acid (DNA) to thymine independence was found to involve site-specific recombination of 63T DNA sequences with their homologous counterparts in the bacterial chromosome. During the transformation, the phage 63T-encoded thy- midylate synthetase gene, thyP3, was shown to integrate at two genetically distinct sites in the B. subdilis 168 chromosome. The first site was identified to be in the bacterial thymidylate synthetase gene, thyA. The second site was in a prophage (SPB) known to be carried in the host genome. The frequency of the integration of the thyP3 gene at each of the two loci and some of the parameters affecting this frequency were studied. The common origin of the thyP3 and thyA genes and their. molecular evolution are also reported. ‘Transformation of bacteria of a given species by DNA from other organisms has been termed heterologous or interspecies transformation. It occurs in Bacillus subtilis with very low fre- quency, even if the transforming DNA is derived from a closely related species (5, 14). A known exception is transformation of B. subtilis thy- mine auxotrophs to prototrophy by DNA ex- tracted from its temperate bacteriophage, 63T (11). 63T, which carries a structural gene for thymidylate synthetase, designated thyP3 (3), can be propagated equally well on Thy” and Thy* B. subtilis strains. Its DNA transforms B. subtilis alrnost as efficiently as homologous bac- terial DNA (11). The transformation of Thy™ bacteria to the Thy* phenotype does not require the integration of the entire bacteriophage ge- nome into the chromosome (15), and thus it bypasses siraple lysogenization. Young et al. (15) reported that in one B. subtilis transformant, thyP3 integrated in the vicinity of the 43T at- tachment site, which is in a chromosomal region distinct from the location of the two genes en- cading thymidylate synthetases in B. subtilis, thyA and thyB. We have recently shown that the B, subtilis chromosome contains several regions homolo- gous to 63T DNA which might be potentially involved in site-specific recombination with the thyP3 gene (10). To explore this possibility and understand the mechanism underlying transfor- Mation by 63T DNA, several B. subtilis auxo- trophs were transformed to thymine indepen- dence by the thyP3 gene and were analyzed. t Present address: Department of Biological Sciences, Stanford University, Stanford, CA 94305. 101 Two integration sites were identified. In addi- tion, the three thymidylate synthetase genes, thyA, thyB, and thyP3, were examined with the hope of understanding any possible evolutionary relationships among them. Thymidylate synthe- tase genes in B. subtilis are of particular interest since this bacterium is the only known procar- yotic organism that contains two thymidylate synthetases with very similar catalytic activities. Thymidylate synthetase B appears to play a minor role in thymidine nucleotide biosynthesis under physiological conditions in B. subtilis (6). It is unknown why both thy genes have been retained in this species during evolution. Muta- tions in both tAyA and thyB are necessary to create an absolute thymine requirement, and reversion in either one of them is sufficient to suppress thymine auxotrophy (13). The two pro- trophic mutants can, however, be distinguished phenotypically. A thyA thyB* mutant is par- tially resistant to trimethoprim and aminopterin in the presence of thymine. In addition, a thyA thyB* mutant is capable of incorporating exog- enous thymine into DNA and requires thymine or thymidine for growth at 46°C. In contrast, a thyA* thyB mutant is phenotypically like the wild type: sensitive to antifolates, not affected by high temperature, and unable to utilize ex- ogenous thymine (6). This paper presents results suggesting that the phage thyP3 and the bacterial thyA loci derive from a common ancestral gene. MATERIALS AND METHODS Bacterial strains. All of the B. subtilis strains used in this work are derivatives of Spizizen’s B. S26. - ——ae ws re Rr pene rene Tae Ma Celene. 102 STROYNOWSKI subtilis 168 (9). SBI65 UrpC2), SB591 (thyA thyB), and SB1141 (¢hyA trp tlvD6) are from the Stanford University collection; SBiz00 (thyB ilA8 citBI gapA2) and SB1207 (leu met thr SPf) were obtained from S. Zahler, and SH1123 (63T lysogen of SB163) was obtained from D. Dean. SB1219 (thyA thyB leu SP8") was constructed by transformation of SB1207 with DNA extracted from SB591, followed by trimeth- oprim selection (12). SB1223 (thyB leu SPR) is a thymine-independent, spontaneous revertant of SB1219. Table } lists other derivatives of SB591. pFT plasmids were propagated in Escherichia coli strain W5443 (AsdR AsdM leu tht thy rspL trp tonB) or W5545 (hsdR hsdM* thr leu thi supE44 rspL lac tonA pro). Their molecular structure is summarized in reference 2, in the accompanying paper (10), and in Table 1. Enzymes and reagents. Restriction enzymes Hindill, BamHI, Bgl, and Heell were purchased from Biolabs Inc. EcoRI was purchased from Miles Laboratories, Inc. The digestions were done according to the specifications recommended by the vendors of the enzymes. Trimethoprim was purchased from Cal- biochem. Other materials and methods are described in the accompanying paper (10). RESULTS Evidence for two integration sites. A se- ries of strains was constructed by transformation of B. subtilis thymine auxotrophs with 3T DNA, with BamHI-cleaved 63T DNA, or with chimeric plasmids pFT23, -24, -25, -33, -34, -401, -451, and -603. The molecular structure and properties of most of these recombinant plas- mids have been described elsewhere (10; Table 1). All chimeras carry overlapping inserts of 63T DNA in different E. coli vectors. The size of the inserts varies from, 2.1 to 6.3 megadaltons SS rt neem a ene eee ear arn see aan SS RS Ne gE J. BACTERIOL, (Mdal). The region of overlap includes the thyP3 gene. . Inthe transformants analyzed, the thyP3 gene integrated at two different sites in the B. subtilis chromosome (Fig. 1). DNA from SB168-derived strains SB1200 (thyA* thyB) and SB591 (thyA thyB) as well as from several thyP3* transform- ants of SB591 was digested with the HindIIl enzyme, electrophoresed in 0.7% agarose gels, and transferred to nitrocellulose filters (8). RNA complementary to pFT thyP3 {described in ref- erence 10) hybridized to a 1.6-Mdal HindIII DNA band of SB1200 (thyA* thyB) in channel A and to a 1.5-Mdal HindIII band of SB591 in channel B. This is consistent with the assump- tion that the Thy” phenotype of SB591 is the result of a small deletion (~0.1 Mdal) in the region of the chromosome containing the thyA locus. The existence of a thyA deletion in SB591 was independently confirmed by the analysis of spontaneous Thy” revertants. SB591 reverted with a frequency 107%, Each of the 30 revertants analyzed behaved phenotypically like thyA thyB* mutants; that is, they were partially re- sistant to trimethoprim in the presence of thy- mine. This is consistent with the assumption that SB591 can revert at thyB but not at the deleted thyA locus. Only a few of the revertants were temperature sensitive like the wild-type thyB (2 of 30). Some were partially temperature resistant (10 of 30). However, the majority were heat resistant. These clones were not studied further. Channels C, E, F, G, and H of Fig. 1 contain HindilI-cleaved DNA of strain SB591 transformed to thymine independence with 63T DNA (SB1150) and chimeric plasmids: pFT34 TABLE 1. Derivatives of SB591 (thyA thyB}* DNA used in Strain Genotype transformation of Comments about transforming thyP3* DNA SB591 SB1149 thyA thyB thyP3* SB1150 thyA thyB thyP3* ¢3T 79.1-Mdal linear phage DNA SB1151 thyA thyB thyP3* pFT23 5.4-Mdal 43T DNA insert in pSC101 (7) SB1152 thyA thyB thyP3* pFT24 4.5-Mdal 43T DNA insert in pSC101 (7) SB1155 thyA thyB thyP3* BamHI ¢3T 36.6-Mdal linear phage DNA SB1162 thyA thyB thyP3* pFT25 6.3-Mdal 43T DNA insert in pSC101 (7) SB1163 thyA thyB thyP3* pFT33 7,2-Mdal 63T DNA insert in pSC101 (7) SB1164 thyA thyB thyP3* pFT24 4.5-Mdal 43T DNA insert in pSC101 (7) SB1165 thyA thyB thyP3* pFT501 2.1-Mdal 63T DNA insert in pMB9 (7) SB1166 thyA thyB thyP3* pFT502 2.1-Mdal 43T DNA insert in pMB9 (7) SB1167 thyA thyB thyP3* pFT401 4.5-Mdal 33T DNA insert in RSF2124 (7) SB1168 thyA thyB thyP3* pFT402 4.5-Mdal 63T DNA insert in RSF2124 (7) SB1169 thyA thyB thyP3* pFT603 4.5-Mdal 63T DNA insert in pML2 (7) $B1170 thyA thyB thyP3* pFT451 2.1-Mdal 3T DNA insert in pSC101 (7) SB1203 thyA thyB thyP3 . SB1204 thyA thyB thyP3 * Most of the strains listed above (SH1150 to SB1170) are Thy* transformants of SB591 obtained with 63T DNA or DNA from 43T-F. coli chimeric plasmids. SB1149 is a 63ST lysogen of SB59t; SB1903 and SB1204 were constructed by lysugenization of SB591 with two different Thy” derivatives of a3T. pet te , et ae teri ee ee ee ET Oe SR Vou. 148, 1981 THYMIDYLATE SYNTHETASE GENES IN B. SUBTILIS 103 -F GH Fic. 1. Integration of the thyP3 gene into two distinct sites of the B. subtilis chromosome. DNA from (A) S§B1200, (B) SB591, (C) SB1150, (D) SB 1155, (E) SB1164, (F) SB1167, (G) SB1169, and (H) SB1170 was cleaved with HindIII enzyme, fractionated in 0.7% agarose gels, and transferred to nitrocetlulose filters. ®Plabeled RNA complementary to pFT thyP3 was used as a probe in this hybridization experiment. Sizes of stained bunds are expressed in megadaltons. (SB1164), pFT401 (SB1167), pFT603 (SB1169), and pFT451 (SB1170). All of these transform- ants hybridized complementary RNA (cRNA) pFT thyP3 to a 1.6-Mdal band which was iden- tical in molecular weight to the band observed in the thyA® strain. A similar result was ob- served in strains transformed by pFT23 (SB1151), pFT24 (SB1152), pFT25 (SB1162), and pFT33 (SB1163) (data not shown). Appar- ently, the transformation reintroduced the amount of DNA that was missing in the SB591 deletion, thus restoring the hybridization pat- tern observed with the wild-type gene. Further support for the integration of the thyP3 gene into the thyA locus was provided by the experiment described in Fig. 2. The DNA restriction fragments containing the ¢AyA gene, thyB gene, and thyP3 gene, which was intro- duced into strain SB591 by transformation with pFT33, were partially purified by agarose gel electrophoresis. To accomplish this, B. subtilis DNA from strain SB1207 (thyA” thyB*), SBI141 (thyA thyB*), SB1200 (thyA* thyB), and SB1163 (thyA thyB thyP3*) was digested with EcoRI restriction endonuclease. DNA sam- ples (2 to 3 pg) were electrophoresed in agarose gel. The gels were sliced into 2-mm slices, the DNA was extracted from each slice, and the extracted DNA was assayed for its Thy* trans- forming activity as described by Harris-Warrick et al. (4). Only two populations of fragment sizes were found to be associated with Thy” trans- forming activity: fractions 8 and 9, containing large DNA fragments (>10 Mdal), and fractions 21 and 22, containing DNA fragments of 4.2 Mdal. In DNA from the thyA* thyB* strain, beth sets of fractions were biologically active. In thyA thyB* DNA, only the 4.2-Mdal fragments could transform to Thy*. In thyA®* thyB or thyA thyB thyP3* DNA, only high-molecular-weight fragments had the Thy* transforming activity. The results indicate that the thyB gene is located onasmaller EcoRI fragment, whereas both thyA and thyP3 are on high-molecular-weight frag- ments which are identical in size (within the limits of resolution of the technique used). This wm, te IE RE RE ee ek eke eM ee et ant 24, Dyn phe ca PT Ly PPG a ey ky Mit ie Fa ee se? “yo Yeh eet 104 STROYNOWSKI ban] seed ss e ~ eo te Fe. | ACIS Oe LO 0 ~ 2 SSL LP wa G-3T Band Ni € MEME Fe 4 yor T y 7 tT T 7 T T T T T tT 80h (a 60h a 40 thy A* thy Bt 4 20b 4 40 TTT TT TT do 20- lt ty aAtyst J 1 i L to L 2 T ~ T tT t T q t T T T 5 60k id - < = 40 + z thy A thy B thy P3 B 20 f 4 < boi oy 4 tt a) SS 3 i) D 240r + a @ 220- 4 2 2 200 4 180 4 160+ / 4 140 thy A* thy B 4 120+ 4 100 Ff 4 Bor - 60}- + 40h 4 20+ 5 rl foporip it 3 70 20 30 40 50 60 FRACTION NUMBER tL 1 i L 1 1 L I 1 1 i Loot 1 2 3 4 5S 6 7 8 9 10 47 Ja DISTANCE FROM ORIGIN (em) Fic. 2. Partial purification of restriction frag- ments containing the bacterial thyA and thyB genes and the 63T thyP3 gene integrated into the bacterial chromosome. The experimental procedure was as de- scribed elsewhere (5). After electrophoresis, 0.7% aga- rose gels containing EcoRI-cleaved DNA from (a) SB1207, (b) SB1141, (c) SB1163, and (d) SB1200 was cul into 2-mm slices. The DNA was extracted and used to transform SB748 (Thy~) competent cells. The peaks of Thy* transforming activity are shown as a function of gel slice number. The number of trans- formants shown is the number of Thy* clones scored per 10” cells plated. The positions of the 63T EcoRI and BamHI bands (molecular weight standards) are indicated on the top of the figure. was independently verified by testing the phe- notype of the Thy® transformants. As expected, all transformants obtained with DNA from frac- tions 21 and 22 behaved like thyA thyB* mu- J. BacTERIOL, tants; that is, they were temperature sensitive and partially resistant to trimethoprim in the presence of thymine. The transformants ob- tained with DNA from fractions 8 and 9 were like thyA”* thyB mutants; they were resistant to high temperature and sensitive to trimethoprim. These experiments demonstrate that in the case of nine independently constructed thyP3* trans- formants, the phage thymidylate synthetase gene integrated at the tAyA locus. The hybridization pattern in channel D of Fig. 1 indicates that in this case (SB1155, constructed by transformation of SB591 with BamHI- cleaved 63T DNA), thyP3 integrated at a site different from tAhyA. In this channel, a 1.5-Mdal Hindltl band from the SB591 deletion is still visible; in addition, a new 2.1-Mdal band is stained. In this case, the integration of the thyP3 gene occurred at a site on the SP prophage (12) which is known to lysogenize almost all B. sub- tilis 168 strains (Fig. 3). EcoRI-cleaved SB1207 DNA (channel A) hybridized CRNA pFT23 to one band only, the thyA region. The SP£ lyso- gen, SB591, digested with EcoRI (channel B) Fic. 3. BamHI-cleaved 63T DNA recombines with SP£ prophage. EcoRI-cleaved DNA from (A) SB1207, (B) SB591, (C) SB1155, and (D) $3T lyxsogen of SB1207 was transferred by Southern blotting and hybridized with cRNA pFT23. Sizes of the stained bands are expressed in megadaltons. a rt, wht F + ee vee ie ya Vor. 148, 1981 hybridized to this probe at two additional bands (1.85 and 1.25 Mdal). In a strain transformed to thymine independence by BamHlI-cleaved $3T DNA (SBi155 [channel C}}, the two EcoRI SPp-specific bands could not be detected, and instead a new fragment was found. This frag- ment had a size of 4.5 Mdal and was the same as the one observed in an EcoRI -cleaved $3T ly- sogen of SB1207 (channel D). . To confirm that the integration of the thyP3 occurred at a site on SPf prophage, it was shown that SPS phage induced from strain SB1155 converted thymine auxotrophs of B. subtilis to thymine independence upon lysogenization. The test was done by allowing SP phage that were released spontaneously into the medium during the growth of SB1155 to infect a lawn of Thy” Spf” bacteria (SB1219). All SB1219 lysogens selected from the middle of the phage plaques (50 out of 50) were Thy* and had the immunity region of SP8. Frequency of the thyP3 integration at each of the two loci. To measure the relative frequency at which thyP3 DNA integrates at the thyA and SP regions, SB591 was transformed with 63T DNA cleaved by EcoRI, Bglll, and BamHI endonucleases. The size of the 68T DNA segments containing thyP3 genes was estimated to be 4.5 Mdal for EcoRI (2), 4.2 Mdal for Bgill, and 36.6 Mdal for BamHI. Twenty Thy” trans- formants from each experiment were tested. None of the clones transformed by EcoRI or Bgill 3T DNA released SPf phage capable of converting the SB1219 auxotroph to thymine prototrophy. In contrast, all (20 of 20) of the BamHI digested 63T DNA-transformed clones were lysogenic for arecombinant SP8/¢3T Thy* transducing phage. This suggests that the thyP3 gene integrates preferentially into the SP re- gion when the transforming DNA is BamHl- cleaved 63T DNA and into other loci (presum- ably thyA) when the transforming DNA is EcoRI- or Baglli-digested ¢3T DNA. This suggests that the probability of integra- tion of the thyP3 gene at each of the two loci depends on the extent of homology shared by the transforming DNA and the recipient region of the chromosome. It can be predicted from this assumption that the transformation effi- ciency of uncleaved high-molecular-weight ¢3T DNA would be greatly reduced if the recipient chromosome were cured of SPf. That this in- deed was the case js shown in Table 2. 63T DNA transformed an SPf* strain (SB1219) with an efficiency approximately 20-fold lower than that of an SP@* strain (SB591). A strain that was doubly lysogenic for ¢3T and SP? and which thus contained twice the homology of SB591 THYMIDYLATE SYNTHETASE GENES IN B. SUBTILIS 105 TaBLE 2. Transformation efficiency of small and large restriction fragments of o3T in B. subtilts strains Transformation efficiency . > Recipient Thy” EcoRT Belll : thy genes $3T 4 “in nero (79.1 (42 Mudal) 7 Mdal) Maal) thyAthyB200.2~=*O*XNTONT SB1203 (thy, SP8, ¢3T) thyP3 SB591 (thy, thyA thyB 100 0.5 0.6 SP£) SB1219 (thyA) = thyA thyB 5° (05 0.5 “Defined as the number of Thy” transformants obtained with 63T DNA divided by the number of Thy* transformants obtained with SB163 DNA. Transformation efficiency of un- cleaved $3T DNA in the SB591 recipient was arbitrarily designated as 100%, and the results were standardized accord- ingly. The transformation assays were performed at limiting DNA concentrations, and the numbers shown are the average of five experiments. The competence level (defined as the number of Thy® transformants obtained with S5B168 DNA divided by the number of viable transformants) was 0.2% (SB1219), 0.1% (SB591), and 0.005% (SB1203). SB1203 carries a thymineless derivative of 63T in the SB591 background. The regions homologous to the transforming DNA in the recipient strain are indicated in parentheses. was transformed by 43T DNA at twice the effi- ciency of a single lysogen. The transformation efficiency of the small EcoRI and Bgl o3T fragments was the same in both strains, indicat- ing that the probability of crossing over between these segments and their homologous counter- parts in SPP is low. Comparison of the thyA and thyP3 genes. The two thymidylate synthetase genes are re- lated at the molecular level. This was shown by the hybridization technique and by the ability of thyP3 DNA to recombine with thyA se- quences. Since neither of these methods is quan- titative, they do not exclude the possibility that thyP3 is also related to the bacterial thyB gene. Therefore, the phenotypic expression of all three genes was tested as an alternative measure of their relatedness. The two thymidylate synthe- tase genes of B. subtilis can be phenotypically distinguished. The phenotype of the thyP3 gene was tested by growing #3T lysogens and B. subtilis strains transformed with thyP3 on aa plates (see Materials and Methods) containing trimethoprim (5 pg/ml) and thymine (50 pg/ml) or on aa plates at 37 and 48°C (Table 3). It was found that thyP3* strains consistently resem- bled thyA* strains. The phenotypic expression of thyP3 was not affected by its chromosomal location or 43T lysogeny. When hybridization experiments were per- formed on Haell-digested B. subtifis DNA, us- 106 STROYNOWSKI TaRLe 3. Phenotypic expression of the thyA, thsB, and thy P genes in B. subtilis” J. BACTERIOL. I 2 Straj Pheno- aa + . aa + 4 ae 5 6 ‘ 8 Scrain thy genes type TRM(5).TRM 10) aa t TRM a, L, aa, L : + Thy + Thy TRY 15) (10) 4° 48°C 87FC BFC (50) (50) SB1223 thyA* thyB Thy” - - - - + + + + SBil4l thyA thyB* Thy* + - ~ ~ ~ + + + SBi68 thyA* thyB* Thy* - ~ - _ + + + + SB591 ss thyA thyB Thy” + + - - - + - + SB1149 thyA thyB thyP3* Thy" - ~ - - + + + + SB1123 thyA* thyB* thyP3* Thy” - - - - + + + + SB1151 thyA thyB thyP3* Thy* ~ - 7 - + + + + SB1152 thyA thyB thyP3* — Thy* - - - - + + + + SB1162 thyA thyB thyP3* Thy” - - - _ + + + + SB11635 thyA thyB thyP3* Thy” _ - - ~ + +: + + SB1165 thyA thyB thyP3* Thy” - - - - + + + + SB1167 thyA thyB thyP3* Thy* - - - - + + + + SBI168 thyA thyB thyP3* Thy” - - - - + + + + SB1155 thyA thyB thyP3* Thy* - - - - + + + + SB1i203 thyA thyB thyP3 Thy” + + - - - + ~ + SB1204 thyA thyB thyP3 Thy + + - - - + - + * The growth of bacterial strains in trimethoprim (TRM) was scored in the presence and absence of thymine (Thy) added to aa plates (Spizizen salts [9] supplemented with glucose, agar, and 20 pg each of the common amino acids/ml). TRM and Thy concentrations are indicated in the parentheses (in micrograms per milliliter). The resistance to high temperature was monitored at 48°C on aa plates and as a control on I plates at permissive and nonpermissive temperatures. Strains $B1149, SB1151, SB1152, SB1162, SB1165, $B1167, SB1168, and SB1155 were constructed by lysogenization of SB591 with a ¢3T DNA or by transformation of SB591 with DNA from different pFT plasmids. $B1203 and $B1204 were constructed by lysogenization of SB591 with different thymineless mutants of o3T. SB1123 is a g3T lysogen in the SB168 background. ing cRNA pFT thyP3 as a probe, a DNA se- quence difference between thyP3 and thyA was found (Fig. 4). In wild-type DNA (SB168, SD 1207), thyA was located on an Haell fragment of 3.7 Mdal. In strain SB591, the deletion had apparently re- _ moved one of the HaelI sites and fused the thyA region to another segment. The thyA gene was now found on a larger band with a size of 7.9 Mdal. In this case, integration of the thyP3 gene at the thyA locus (SB1162) did not restore the hybridization pattern of the wild-type DNA. It is argued, therefore, that thyP3 gene does not contain the Haell recognition sequence, whereas the thyA region does. This result suggests that some nucleotide change at the molecular level has occurred between the two genes during ev- olution. DISCUSSION Recent results from this laboratory establish that 43T shares extensive homology with the B. subtilis chromosome (10). At least three differ- ent regions of the bacterial genome were shown to be capable of hybridizing RNA complemen- tary to d63T. These regions include: (i) the thyA region which was found to be homologous to the thyP3 region in 437; (it) SPf, which is a cryptic temperate bacteriophage of B. subtilis strain 168 and which was demonstrated to be a close rela- tive of 63T (although the SP8 genome does not carry the thy gene, it does contain sequences surrounding thyP3 in 63T); and (iii) other re- gions of the B. subtilis chromosome. The nature and location of these sequences in the bacterial or phage chromosome were not identified. It was shown, however, that they are not homologous to the thyP3 gene or the DNA surrounding the thyP3 gene in 63T. This investigation demonstrates that the mechanism of transformation of B. subtilis Thy” strains to thymine prototrophy by ¢3T DNA involves site-specific recombination of $3T se- quences with their homologous counterparts in the bacterial chromosome. As a result of this recombination, the ‘hyP3 gene of 63T is inte- grated into the bacterial DNA. Two genetically distinct integration sites were identified: the bac- terial thy.4 locus and a site on the SP8 prophage. The attachment site for SPf prophage lies be- tween ilvA and kauA (16). The integration of the thyP3 DNA at the thyA locus presumably involves crossing over of the two structural thy- midylate synthetase genes. Since sequences ho- mologous to the thyP3 gene were not detected in SP, the insertion of the thyP3 into SPB prophage occurs, most likely, by recombination of the sequences surrounding thyP3 gene with VoL. 148, 1981 Fic. 4. DNA sequence differences between the thyA and thyP3 genes. Haell-cleaved DNA from (A} SB168, (B) SB1207, (C) SB591, and (D) SB1162 was transferred to nitrocellulose filters and hybridized with CRNA pFT thyP3. Sizes of the stained bunds are expressed in megadaltons. homologous sequences present in SPS, When thyP3 integrates at the SPf site, a recombinant SP8/¢3T phage is created which was shown to carry the immunity region of SPB. The frequency of the integration of the thy P3 gene at each of the two loci and some of the parameters affecting this frequency were stud- ied. It was found that when large o3T fragments (36.6 Mdal; generated by BamHI cleavage of 63T DNA) or intact 3T molecules (79.1 Mdal) were used to transform B. subftlis strains, the integration occurred 95% of the time at the site on SPB prophage. In 5% of the cases, thyP3 integrated at the thyA locus. When the primary integration site was deleted by curing strains of SP, the efficiency of transformation dropped, as expected, to 5%. It is presumed that in these transformants the integration occurred at the secondary thyA site. When small 63T fragments were used (EcoRI, 4.5 Mdal; Bef, 4.2 Mdal), thyP3 did not integrate at the SPf site (none of 20 cases). Instead, the ‘AyP3 was shown to have recombined with the fhyA gene (eight of eight cases). As expected, no reduction in transfor- mation efficiency strains cured for SPf was ob- served for EcoR! and Bgill 631 fragments. These findings are consistent with the assump- tion that the probability of recombination at each of the sites is positively correlated with the amount of homology shared by the transforming THYMIDYLATE SYNTHETASE GENES IN &. SUBTILIS 107 fragment of DNA and the recipient region. This conclusion was further corroborated by the fact that the transformation efficiency by large frag- ments of 63f DNA in strains doubly lysogenic for SPB and Thy” derivatives of 43T is twice that observed for a single lysogen. No conclusive statement can be made about the history of the thyP3 gene. However, evidence presented here and in the accompanying paper (10) suggests that SPf phage acquired a bacte- rial thyA gene by transformation or by recom- bination with homologous DNA sequences and _ became an ancestor of 63T and another related -- phage, pil (1). It can be proposed that 63T (or. . pli) transduced the thyA gene into B. subtilis from another source. The function of the thyP gene in 43T and p11 is not clear. It is expressed _ constitutively in the lysogen, unlike many other genes associated with phage growth. Regardless of the evolutionary origin of the thyP3 gene, it is certain that the thyP3 gene in 63T and the thyA gene of B. subtilis share a common ancestry. This conclusion is based on the studies of their structure (cross-hybridiza- tion and ability to recombine) and phenotypic expression (resistance to trimethoprim in the presence of thymine and to high temperature). At some time, however, molecular evolution has occurred between the two thy genes. This was revealed by the minor differences in nucleotide sequences observed during the studies of their restriction enzyme digests. ACKNOWLEDGMENTS Iam grateful to J. Lederberg, in whose laboratory this work was done, for his continuous support and many helpful sug- gestions. I also thank him, M. Winker, and A. T. Ganesan for critical reading of this manuscript. I am indebted to H. Bursz- tyn-Pettegrew for sharing her unpublished data with me and to P. Evans for excelient technical assistance. This work was supported by Public Health Service predoc- toral fellowship GM-00295 and grant CA-16896 from the Na- tional Institutes of Health. LITERATURE CITED 1. Dean, D. H., J. C. Orrego, K. W. Hutchison, and H. O. Halvorson. 1976. New temperate phage for Bacillus subtilis, p11. J. Virol, 20:509-519. 2. Ehrlich, S. D., H. Bursztyn-Pettegrew, I Stroy- nowski, and J. Lederberg. 1976. Expression of the thymidylate synthetase gene of the Bacillus sudtilis bacteriophage o3T in Escherichia colt. Proc. Natl. . Acad. Sci. U.S.A. 73:4145-4149. 3. Graham, R. S., F. E. Young, and G. A. Wilson. 1977. Effect of site specific endonuclease digestion on the thyP3 gene of bacteriophage o3T and the thyPJ1 gene of bacteriophage pll. Gene 1:169-180. 4. Harris-Warrick, R. M., Y. 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