eat SERGE, REL FEE EN RN NR NET NARIE PY TRO TOM: Sa ue % k i ts See & JuURNAL OF BACTERIOLOGY. Oct. 1981. p. 91-100 4191 -9193,/81 / 100091-10802.00,°0 ATT Vol. 148, No. 1 Distribution of Bacteriophage ¢3T Homologous Deoxyribonucleic Acid Seque ncees in Bacillus subtilis 168, Related Bacteriophages, and Other Bacillus Species IWONA T. STROYNOWSKI} Department of Genetics, Stanford University School of Medicine, Stanford, California 94305 Received 10 October 1980/Accepted 12 June 1981 The Bacillus subtilis 168 chromosome was found to share extensive homology with the genome of bacteriophage o3T. At least three different regions of the bacterial genome hydridized to ribonucleic acid complementary to ¢3T deoxyri- bonucleic acid (DNA). The thymidylate the sequences adjacent to it were sho synthetase gene, thyA, of B. subtilis and wn to be homologous to the region in the 63T DNA containing the phage-encoded thymidylate synthetase gene, thyP3. SP£, a temperate bacteriophage known chromosome, was to be integrated into the B. subtilis 168 demonstrated to be closely related to 93T. Other regions of the bacterial genome were also found to hybridize to the ¢3T probe. The nature and location of these sequences in the bact identified. It was shown, however, that erial and phage chromosomes were not they were not homologous to either the thyP3 gene or the DNA surrounding the thyP3 gene. The chromosomes of other Bacillus species were also screened for the presence of ¢3T homologous se- quences, and the thyP3 gene was localized in the linear genomes of phages o3T and p11 by heteroduplex mapping. It is suggested that the presence of sequences of phage origin in t he B. subtilis 168 chromosome might contribute to the restructuring and evolution of the viral and bacterial DNAs. In general, Bacillus species contain a substan- tial number of temperate phages, and many of the strains are known to be polylysogens. They so harbor defective phages and genes which are expressed when the cells are subjected to mitomycin C or UV light (1, 11, 20, 25, 33). In most cases, the biological function of the inducible genes is not known, nor is it known why they have bzen retaized in the bacterial chromosomes during evolution. The defective phages and cryptic genes have recently been the subject of intensive study. Some of the functions expressed in the induced cells include modifica- tion methylases and DNA repair enzymes (13, 38). In certain instances, the bacterial lysogens are known to benefit from functional comple- mentation by phage genes. In particular, an in- teresting case.is the host-temperate phage rela- tionship observed between Bacillus subtilis and two of its temperate phages, 63T and p11 (5, 35). These phages carry structural genes for thymi- dylate synthetases, which can complement a thymine deficiency in their host. These genes are designated thyP3 and thyPI1, respectively (12). The phages integrate into the B. subtilis . +Present address: Department of Biological Sciences, Stanford University, Stanford, CA 94305. chromosome at sites different from the two loci, thyA and thyB, encoding bacterial thymidylate synthetases (39). The thyP genes are expressed during lysogeny (35). They can also be intro- duced into B. subtilis by DNA-mediated trans- formation, which does not require the successful integration of the entire prophage (39). Thus, lysogenic infection and DNA-mediated transfor- mation appear to utilize different pathways for uptake of genes into the cells. As a preliminary step in the exploration of the mechanism by which the thyP3 gene transforms bacterial aux- otrophs, the B. subtilis 168 chromosome was analyzed for the presence of DNA sequences homologous to the ¢3T genome. The relation- ship between the thyP3 gene of ¢3T and the thyP11 gene of p11 was also examined. To gen- eralize my observations, the genomes of several other Bacillus species were screened for the presence of sequences homologous to the thyP3 gene. MATERIALS AND METHODS Bacterial strains. Plasmids pFT23, -33, -401, and -502 (7) and pFT thyP3 were propagated in Fsche- richia coli strain W5543 (hsdR hsdM leu thi thy rpsL trp tonB2) or W5545 (hsdR hsdM* thr leu thi sup 44 rpsL lac tonA pro). B. subtilis strains SB168 (t7pC2), 91 $B1200 (ilvA 92 STROYNOWSKI 8 thyB! citBl gapA2), and SB591 (thyA thyB) and Bacillus strains SB510, SB511, SB512, SB513, SB514, SB515, SB519, SB522, 8B727, SB730, SB734, SD1096, SB1098, SB1099, SB1 100, and SB1110 were from the Stanford University collection. Strain $B1207 (leu met thr SP£°) was obtained from S. A. Zahler, and strain SB1213 (trpC2) is a SBI168 deriva- tive which w sequences but as cured spontaneously of SP8 DNA was still resistant to infection by SPZ. Phage stocks. 63T DNA was prepared after mi- fomycin C (Sigma Chemical Co.) induction of B. sub- tilis as outline d below. One colony of an SB168 deriv- ative strain, cured of SP8 and lysogenized with phage $3T, was inoculated into 50 ml of L broth supple- mented with 10° M MnCh. The overnight-grown culture was diluted 10 times into the same medium and incubated phase (40 Klett units). At this with shaking at 37°C until early log Stage, 0.5 pg of mito- mycin C per ml was added, and the cells were incu- bated at 37°C until lysis occurred (2 to 3h). The culture was briefly centrifuged (20 min at 8,000 rpm) to remove bac incubated with terial debris. The lysate was filtered, DNase (5 ug/ml) for 20 min at 37°C, and heated for 5 min at 60°C to inactivate the enzyme. The phage were pelleted by overnight centrifugation at 8,000 rpm in a Sorvall centrifuge. The pellet was suspended in 10 mM Tris-1 mM EDTA, pH 8.5. The phage-containing solution was adjusted to a density of 1.50 (refractive index = 1.3810) with CsCl and centri- fuged for 40 h at 48,000 rpm. Further purification of the phage was obtained by centrifugation in a step CsCl gradient for 12 h at 37,000 rpm. The pooled fractions from several gradients were dialyzed against 3 liters of 10 m M Tris-1 mM EDTA, pH 8.5, DNA extracted with 1% sodium lauryl sulfate for 30 min at room temperature, and then extracted three times with phenol. The phage DNA was extensively dialyzed against 10 mM Tris-1 mM EDTA, pH 8.5, and stored in 4°C. The pha ge preparation was also examined by electron microscopy to verify that it did not contain phage particles with a morphology different from that of 43T, T4 phage was Prepared as described by Kim and Davidson (15). Phage p11 DNA was a generous gift from J. C. Orrego. Bacterial DNA and plasmid DNA preparation, Bacterial DNAs were prepared by a modification of the method of Klotz and Zimmer (16) as described by R. M. Harris-Warrick (Ph.D. thesis, Stanford Univer- sity, Stanford, C was omitted. Closed circular plasmid DNAs were iso- lated from cleared lysates, prepared according to Cle- well and Helinski (3), DNA was centrifuged in ethid- ium bromide-cesium chloride gradients (23). Ethidium bromide was rem A6 50W-XB. The DNA was separated from oligori- bonucleotides by gel filtration on Bio-Gel A-15M. The plasmid DNA was extracted three times with phenol, ethanol precipita Tris-1 mM EDTA, PH 8.5. Transformation bioassay in B. subtilis. B. sub- tilis cells were brought to competence as described by Harris-Warrick (Ph.D. thesis, 1976). The competent cells were concent alif., 1976). The pronase treatment oved by chromatography on Dowex ted, and dialyzed against 10 mM rated 10-fold by centrifugation, sus- J. BAcTERI0, pended in 5% glycerol, and frozen in liquid nit Thawed competent cells were diluted 10-fold in mini. ma! medium supplemented with 20 mM MgCl. ans DNA was added in various final concentrations (0.1 r,, 1 ug/ml). The mixture was incubated for 69 min at 37°C with gentle shaking and then plated on Selective media (Harris-Warrick, Ph.D. thesis, 1976). Restriction enzyme cleavage of DNA. Restric. tion enzyme digestions were according to the specifi. cations recommended by the vendors of the enzvines, BamHI, Smal, Psél, Bgill, SalI, Hpal, and Hind enzymes were purchased from New England Biolabs Inc.; EcoRI was purchased from Miles Laboratories, Ofer Inc. A two- to threefold enzyme excess was used roy. tinely for most of the experiments. Reactions Were terminated by heating the samples at 65°C for 7 min, Agarose gel electrophoresis. Electrophoresis of DNA was done in 0.74 agarose horizontal slab gels (14 by 20 by 0.3 cm) at 60 V overnight or at 175 V for 3 to 4h. Paper wicks connected the gel to tanks containi 0.089 M Tris-0.002 M EDTA-0.089 M borate, pH &.3, The gels contained 1 Hg of ethidium bromide per ml. ng DNA was visualized by UV lumination of the gels, and the fluorescent bands were photographed by using a yellow filter (Kodak no. 9 Wratten filter) and Pola. roid film (type 55P/N). Measurements of the molec. ular weights of the DNA molecules were made relative to 63T DNA cleaved by EcoRI (7) and SPP1 DNA cleaved by EcoRI (10). Nucleic acid hybridization. Hybridization exper- iments were carried out essentially as described by Southern (29). The hybridizations were done at 37°C in a solution containing 50% formamide, 0.75 M NaCl. and 0.075 M sodium citrate. “P-labeled complemen. tary RNA (cRNA) was used as the probe. It was synthesized on plasmid and chromosomal DNA tem. plates in a reaction mixture containing 50 to 100 ul of [a-*P]GTP (1 mCi/ml), 0.5 mM each ATP, CTP, and UTP, 2 yg of DNA, 40 mM Tris (pH 8), 10 mM MgCh, 10 mM &-mercaptoethanol, and E. coli core RNA polymerase in a total volume of 100 pl. RNA polym- erase was a gift from D. Brutlag. The reaction was 20 g/ml together with carrier RNA. The reaction was terminated by phenol extraction. [°P]cRNA was sep- arated from unincorporated nucleoside triphosphates by Sephadex G-50 chromatography. A total of 5 x 10° to 5 x 10° epm of “P-labeled cRNA probe was used per 2 ml of hybridization solution containing a nitro- cellulose filter (8 by 2 cm). After hybridization, the washed filters were blotted dry and autoradiographed at —90°C, using Du Pont Cronex 4 or Kodak XR-5 X.- ray film and Du Pont Cronex Lightning Plus intensi- fying screens, Electron microscopy. Preparation and spreading of heteroduplex molecules was done by the method of Davis et al. (4), except that the molecules were spread on water. No DNA extraction on o3T or p11 phage was performed before using the phage in heteroduplex experiments. The photographs nification of x4,500. The lengths of molecules were determined by measuring projections of 35-mm nega- tives on the surface of a Hewlett-Packard 9864 A digitizer interfaced with a Hewlett-Packard 9810A cal. were taken at a mag- cleave we of J Ligati hat 2 Vol. re ELA EC Se 148. 1981 -igtor. The projections were measured at a total sification of 135,000. All measurements were relative to a standard DNA molecule, pSC10l, ted on the same grid. The molecular weight of andard pSC101 DNA used in all the calculations 3233.8 megadaltons (Mdal) (28). Cloning of EcoR1* fragments. A 3.5-ug amount pFT401 was incubated with 2.5 mM MgChk, 10 mM tris (pH 8.5), and 10 U of EcoRI enzyme (Miles aboratories) at 37°C in a total volume of 50 pl. samples of 10 pl each were removed after 1, 3, 7.5, and >3h, and the reactions were terminated by heating the amples to 65°C for 7 min, A 0.5-2z amount of EcoRI*- “eaved pFT401 from each sample was mixed with 0.2 “zof EcoRI -cleaved pMB9 in a ioral volume of 70 pl. Ligation was carried out at 10 zg of DNA per ml for 1 > at 20°C, using T4 ligase and the buffer described by made oun Sgaramella et al. (26). E. coli W5-443 competent cells were prepared as described elsewhere (27). The transformants (Thy” Tc") were selected on aa slates (Spizizen minimal medium [30] supplemented aith 25 ug each of the common amino acids per ml, gucose, 20 ug of tetracycline per ml, and agar). RESULTS Construction of chimeric plasmids con- taining 63T DNA sequences. Most of the recombinant plasmids (pFT23, pFT24, pFT33, pPT401, and pFT502) used in this work were constructed previously in our laboratory. Figure la summarizes their molecular structures. The -himeras contain different but overlapping $3T DNA sequences joined to E. colt plasmid psC101 or pMB9. A DNA segment of 63T origin 's common to all of the recombinant plasmids and complements the thyA thymine deficiency sutation in E. coli. It it also able to transform Thy” auxotrophs of B. subtilis to prototrophy. Therefore, this segment of 63T DNA is assumed to contain the thyP3 gene. To achieve higher purification of this specific gene, it was necessary to eliminate nonessential DNA sequences flank- ing the thyP3 gene. This was accomplished by subcloning of EcoR1* fragments (22) of pFT401 in the EcoRI site of plasmid pBR322. Figure 1b shows the structure of the resulting chimeric plasmid, pFT thyP3. The molecular size of the EcoRI DNA insert carried by pFT thyP3 is 0.57 Medal. Sequences homologous to 63T DNA in the B. subtilis chromosome. Structural anal- yses of DNA sequence relationships were per- formed by using the technique of CRNA-DNA hybridization coupled with restriction enzyme analysis (29). At least three different regions of the B. subtilis chromosome capable of hybrid- wing 63T cRNA were identified. DNA homology of the bacterial thyA and phage thy P3 genes was demonstrated by hybrid- ation of CRNA prepared on pFT thyP3 to the po eee re Sas PHAGE o3T DNA SEQUENCES IN B. SUBTILIS 93 a} "I pFT 24 A Sen ae pFT 23 cB A a _ > pFT 25 AE Bc O a _O __ ,, —>/. 0 pFT 33 A Bc D Bo pF T 502 pFT 403 A b) 1,077" pFT thy °3 thy P3 1 1 L L 0 2 4 6 8 10 42 MOLECULAR WEIGHT {x 108) Fic. 1. Molecular structure of chimeric plasmids. All DNAs are aligned from the BamHI site located in the vectors. The vector components of the hybrids are represented by heavy lines: B, pSC101; & RSF2122; ©, pBR322. The inserts are shown as thin lines. There is, in all cases, an EcoRI site at the boundary between the vectors and inserts. Additional EcoRI sites are denoted as arrows. The direction of the arrows indicates the orientation of the inserts. (a) Diagrams of plasmids described earlier (7) but used in this work. The sizes (in megadaltons) of the seg- ments are (A) 4.5, (B} 0.67, (C) 0.24, (D) 0.9, (E) 9.9, (A’) 2.1. A’ is homologous to left part of segment A. (b) Diagram of the chimeric plasmid constructed by cloning the EcoRI* fragment of pFT401 in the pBR322 vector. The size of pFT thyP3 insert is 0.57 Mdal. EcoRI -cleaved fragments of B. subtilis DNA which are associated with thyA biological activ- ity. The data showing partial purification of B. subtilis segments containing thyA and thyB genes are presented in the accompanying paper, where it is shown that wild-type B. subtilis contains two EcoRI fragments associated with Thy* activity (32). The thyA* gene resides on an EcoRI fragment larger than 10 Mdal, whereas the thyB* gene is carried on a 4.2-Mdal EcoRI fragment. RNA complemen‘ary to pFT thyP3 hybridized to only one band of the B. subtilis EcoRI restriction digest (Fig. 2). The molecular weight of the DNA in this band corresponded to the segment containing thyA biological activity. The size of the DNA insert in pFT thyP3 was only 0.57 Mdal (~950 base pairs), and therefore it contained very little, if any, of the DNA se- quences other than the thyP3 gene. Thus, it is assumed that the observed hybridization occurs between the phage and bacterial thy genes. Con- trol experiments involving similar hybridizations between cRNA transcribed from the plasmid vector pBR322 gave negative results. To construct a physical map of the thyA re- gion, B. subtilis DNA was cleaved with different restriction enzymes and then hybridized to P®. labeled cRNA prepared from several of the re- combinant plasmids. Only the HindIII enzyme 94 STROYNOWSKI cleaved this region internally (Fig. 3). The dif- ference in the hybridization patterns observed in Fig. 3a and 3b is due to the different lengths of the probes used in these two experiments. It follows that the B. subtilis chromosome contains one or more other genes which have homology to ¢3T sequences. This gene (or genes) was linked to thyP3 in the phage and to thyA in B. subtilis, as evidenced by the unsuccessful at- hee — 310 Fic. 2. Hybridization of cRNA pFT thyP3 to EcoRI-digested B. subtilis 168 DNA. Two micro- grams of SB168 DNA was digested with EcoRI en- zyme, electrophoresed through a 0.7% agarose gel, fransferred to a nitrocellulose filter (29), and hybrid- ized to ’P-.labeled RNA complementary to plasmid PFT thyP3. The conditions of hybridization are de- scribed in Materials and Methods. Sizes of the stained DNA bands are expressed in megadaltons. Sh to » J. Bacteri: tempts to separate them by digestion with the following enzymes: EcoRI, BamHI, Smal, Ps Bgl, Safl, and Hpal (data not shown). Th: size of the restriction fragment that contains the thyA region was 7.8 Mdal for PstI, 9.4 Me: for Bgl, 6.9 Mdal for Hpal, and more than ite Mdal for EcoRI, BamHI, Sali, and Smal. Dou. ble digestion of B. subtilis 168 DNA with EcoR} and HindIII enzymes, followed by hybridizatic, with cRNA pFTS02, was compatible with the physical map of the thyP3 homologous regi shown in Fig. 4. In addition to the Segment associated with the OG Fic. 3. Hybridization of (a) CRNA pFT tks P3 and (6) cRNA pFT502 to HindllI-cleaved DN.43- SBISS (channel A) and SB1207 (channel Bj. The sizes of the stained bands are expressed in megadaltons. 1.6 1.8 eae na, HindIll Hind II Hind HI region of the B. subtilis epresents sequences that hybridize to ‘thyP3 DNA (0.57 Mdaij. The than thyP3 or pMBS vector. eet anal ga aap tr caer acy pear scarier: a 148, 1981 PHAGE 63T DNA SEQUENCES IN B. SUBTILIS 95 VoL. : & iL tAvA biological activity, several other EcoRI pb fragments of chromosomes from laboratory a -trains derived from B. subtilis 168 (30) (except al. 10 ae i Seb — >10 Fic. 6. Hybridization of cRNA $3T to EcoRI- cleaved SB168 DNA. The arrows indicate the posi- tions of bands that appear on the autoradiogram upon longer exposure. These bands can also be de- tected in SPB” strains SB1207 and SB1213 (see Fig. 7). ee the size of the phage genome which has a com- Zt 62 plexity similar to the $3T genome. SPf con- i % tained sequences complementary to pF T502, but it did not carry the thyP3 gene. This is revealed a a by the lack of its hybridization with cRNA pFT ead ap <— 1.25 thyP3. = = Finally, it was shown that the B. subtilis chro- : mosome contained yet another region or regions ws homologous to the ¢3T genome. The EcoRI oe fragments of the DNA from two strains inde- eo pendently cured of SP@ prophage (SB1207 and - . $B1213) hybridized to cRNA transcribed from Fis. 5. Hybridization of cRNA pFT502 to Ecorr. the $3T DNA (Fig. 7 and Table 1). The hybrid- cleaved B. subtilis DNAs: (A) SB168; (B) SB1207; zation pattern was limited to four EcoRI bands and {C) SB1207 lysogenized with SPB. Sizes of instead of the large number of bands observed in the SP lysogens. The largest of these bands oe ‘tained bunds are expressed in megadaltons. 96 STROYNOWSKI TABLE 1. Sequences homologous to $3T DNA in the B. subtilis chromosome EcoRI fragments of B. EcoRI fragments of B. sub- subtilis SB1207 and (lis 168 hybridizing g3T S40 (SPB-) hybridizing anne 63T cRNA? sera Size (Mdaly Tybridiza gs intl) Hybridiza- +++ >10 +4 5.6 ++ +44 52.06 “If strong (+++) bands are counted as doublets, the total size of the hybridizing fragments is 70.4 Mdal. ion to the bands listed above, EcoRI fragments of 1.45 and >10 Mdal appeared on the autoradiogram upon longer exposure. +, weak. *The total size of the bands other than the SPZ- Specific or thyA region DNA is 8.35 Mdal. ++, Strong; +, weak. (>10 Mdal) corresponded to the thyA region. The nature and location of the other three seg- ments of 5.6, 1.45, and 1.3 Mdal were not iden- tified. When the P® labeled cRNA was transcribed from a B. subtilis chromosome containing SP# digest of 43T labeled (Table 2). The pattern described in Ta- ble 2 indicates the portions of the 43T genome that were detected by this technique. They cor- responded to the portions of the $3T genome present in the B. subtilis chromosome. Sequences homologous to $3T in other Bacillus species. Table 3 summarizes the re- sults of Screening the chromosomes of several Bacillus species for the presence of thyP3 ho- mologous sequences. In most cases, the presence of the thyP3 homologous gene was accompanied by the presence of the linked ¢3T homologous Sequence (B. subtilis Marburg, B. globigii, B. subtilis niger, and RB. coagulans). B. mycoides J. BactEnig, and 3. pumilus DNAs did not hybridize cRNg pFT thyP3 but showed hybridization with cRNA pFT502, indicating the presence of the gene or genes linked to thyP3. Only B. sudritis Marburg and B. coagulans were found to Con. tain a SP-specific EcoRI band which hybrid. ized to cRNA pFT502 (Fig. 5). Thus, these tg species are assumed to be SP8 lysogens, KH Fic. 7. Hybridization of cRNA $3T to EcoR!. cleaved SB1207 (SPB-). An identical hybridization, pattern was observed by using strain SB1213 DNA. Sizes of stained bands are expressed in megadaltons. TABLE 2. Hybridization of RNA complementary to SB591 (SP8*) to E coRl-cleaved 33T DNA® Band no. Hybridization 1 _ 2 _ 3 10 * EcoRI bands of ¢3T were numbered according to Ehrlich et al. (7). Detection of hybridization to bands er than band 10 was below the resolution of the technique used. +, Hybridization present; —, no hy- bridization. hy- Vor. 148, 1981 TABLE 3. Screening of bact PHAGE 63T DNA SEQUENCES IN B. SUBTILIS 97 erial chromosomes for the presence of the thyP3 gene and linked 63T homologous sequence Hybrid Hy Orica ao Transformation efficiency (no. of transformants/ cRNA pFT EcoR! band" no. of viable cells per pg of DNA)’ Strain thyP3 to EcoRI >10 band,* >10 Maal 1.2 Mdal Thy His Phe Mdal 3B1213 (SPB trp-2) + + - 6x 1074 3x 107+ 3x 107 3B522 (B. subtilis Marburg) + + + 5x 10‘ 3x 10° 3x 10% SB319 (B. megatherium) - - - 3x 1077 5x 107 3B515 (B. polymyxa) - - - SB5l4 (B. subtilis subsp. ter- - - - 1.5 1077 15x 107" minalis) 3B513 (B. cereus) - - - $B512 (B. globigit) + + _ SB311 (B. mycoides) - + _ SB510 (B. subtilis subsp. ni- + + - 107' ger) 3B1110 (B. amyloliquefa- - _ - ciens) SB1100 (B. subtilis subsp. - + - 1.5 x 10° 3x 10° 3x 10% natto) SB1099 (B. coagulans) + + + 8x 10% 6x 10° 6x 10% $B1098 (B. brevis) - _ ~ $B1096 (B. pumilus) - + - 1x 10° 5x 107% 5x 10° SB734 (B. subtilis N) - - - 107 SB734 (B. subtilis H) - - - 3B727 (B. subtilis K) - = - W445 (E. colt K-12) - - - NT NT - NT NT T4 phage “ An EcoRI band 2.5 Mdal from SB727 (B. subtilis K) h probes. +. Hybridization present; —, no hybridization. *The recipient strain in the transformation assays was er of transformants in the assays was significantly higher than the ker tested. The numbers shown are averages of six sets of experiments. NT, efficiency is listed only when the numb number of revertants for the mar Not tested. The DNA extracted from the Bacillus species studied was also tested for its ability to trans- form che B. subtilis thymine auxotroph SB591 to prototrophy. In many cases, the high trans- formation efficiency to the Thy” phenotype was correlated with the presence of the thyP3 ho- mologous sequences in the donor species (B. subtilis niger, B. coagulans, B. subtilis Mar- burg, and SB1213). In the case of DNA from B. globigii, which was found to hybridize the thyP3 probe, the transformation efficiency was below the reversion frequency of the recipient strain. This indicates that the DNA sequences sur- rounding the thy genes in the donor and recipi- ent strains are nonhomologous. An alternative explanation which proposes the existence ofa B. subtilis restriction system that degrades B. glo- bigii DNA was ruled out by the results of Harris- Warrick (Ph.D. thesis, 1976). A few strains (B. megatherium, B. subtilis subsp. terminalis, B. subtilis subsp. natto, and B. pumilus) did not hybridize to the thyP3 probe, but their DNA ybridized both cRNA pFT thyP3 and cRNA pFT502 SB748 (thyA thyB his-2 aro-2). Transformation still transformed the B. subtilis 168 thymine auxotroph to prototrophy. This might have been due to transformation with the thyB locus which was contained in the donor strains. Another possibility is that the thyA genes in the donor strains diverged considerably from the B. sub- tilis thyA sequence and therefore were not de- tected under the hybridization conditions used. Neither E. coli nor T4 phage DNA was able to transform B. subtilis strain SB591 to Thy* or hybridize to thyP3 sequences. These results demonstrate specificity of the Southern hybrid- ization technique and transformation assay used in these studies. Mapping of the thyP11 gene in pll. The thyP3 gene has been mapped in the 63T genome by heteroduplex analysis of hybrids formed be- tween the chimeric plasmid pFT25 and phage DNA (7). In view of the known similarities be- tween $3T and p11 (5), I decided to use the same technique to localize thyP11 in pll. The pFT24 plasmid containing thyP3 of the 63T phage was 2 Soraes ae St (C) 4.7; (D) 34.8; (E, 98 STROYNOWSKI linearized with the BamHI restriction enzyme. After denaturation, this plasmid DNA was an- nealed to the complementary single strands of the p11 DNA (Fig. 8). A double-stranded region of homology with a size of 3.0 + 0.2 Mdal was found at a distance of 50 to 54% of the total length from one of the termini of the p11 DNA molecule. The standard error of this measure- ment was 3% of the fractional Pll length. The double-stran ded region found in the heterodu- plex of p11 with Segment A of plasmid pFT24 was not perfectly homologous. Two small loops ed. The right part of segment A (1.5 Mdal) did not hybridize to the pll DNA. This indicates that the DNA sequences sur- rounding the thyP gene in the ¢3T and ell phages are different. Figure 8 shows also the localization of the thy P3 gene in the o3T ge- nome. The thyP3 gene was situated at a distance NUMBER OF MOLECULES J. BACTERI01, 44 to 48% from one of the 63T ends, Statistica error of this measurement was 2% of the frac. tional $3T length. DISCUSSION The results described here provide evidence for the existence of extensive DNA homologies between the chromosomes of B. subtilis 168 ang its temperate bacteriophage 43T, Three distinct regions of the bacterial chromosome were iden. tified that were capable of hybridizing RNA complementary to the o8T genome, One of these regions contains the structural gene for thymidylate synthetase A and was shown to be homologous to the phage-encoded thyP3 gene. In addition, another homologous DNA Sequence (equal to or less than 2.15 Mdal) was located next to thyA in B. subtilis and next to thyP3 in $3T. The nature of this other gene un o8 53 so DO 2s Q 79.1 d) D 8 E AT \ec 73.1 es thy P3 Lo J I 1 =a 0 20 40 ) 41.1. Fifteen molecules BamHI linearized PFT25 DNA. Segment B represents the region of homology between ¢3T and pFT25, The sizes of heteroduplex regions are with the structure represented b (in megadaltons): (A) 0.2; (B) 6.3; y this diagram were measured, e ES ER A age eg Vou. 148, 1981 (or genes) was not identified. No markers closely linked to thyA or thyP3 have been reported in B. subtilis of 63T. In other systems, such as T4 phage, DNA sequences close to the structural gene for thymidylate synthetase code for pro- reins involved in DNA metabolism (37). In con- trast, the E. coli or Salmonella typhimurium thyA locus maps between the lys and arg loci, away from other genes involved in DNA synthe- sis (7). The second region of extensive homology be- tween the bacterial genome and the 63T genome was identified as an SP8 prophage. SP is a temperate, cryptic bacteriophage present in B. subtilis strains derived from Spizizen’s trans- formable strain 168 (31). The discovery by War- ner et al. (36) of a cured strain has permitted the characterization of SP. It is a fairly large phage of complex structure similar morphologically to 63T and p11 (36) but not to PBSX, a defective bacteriophage also known to lysogenize all B. subtilis 168 strains (25). The prophage attach- ment site for SPB lies between ilvA and kauA (40). SP@ does not convert B. subtilis Thy” auxotrophs to prototrophy upon lysogenization. It can, however, carry out a specialized trans- duction of the citK and kawA genes (40). Its mechanism of specialized transduction was pro- posed by Zahler et al. (40) to resemble closely the E. coli phage A dgal system (2). The results described in this communication demonstrate that the phages SBP and ¢$3T are closely related. When RNA complementary to the 63T genome was hybridized to the EcoRI restriction digest of a SP lysogen, more than 20 bands (total of 50 to 80 Mdal) were homologous to the radio- active RNA probe. The restriction fragments which did not cross-hybridize between the two genomes presumably code for the traits that are different in the two phages, such as immunity. In addition, it was shown that SP8 lacks the thyP3 sequence (0.57 Mdal), although the DNA sequence located next to this gene in d3T is still present in the SPB genome. Finally, it was shown that the B. subtilis chro- mosome contains yet another region homolo- gous to 43T which is different from ThyA or SP8 DNA. Two strains independently cured of SPB still hybridized ¢3T probe. Three EcoRI fragments of B. subtilis 168 DNA (molecular sizes of 5.6, 1.45, and 1.3 Mdal) showed homology to sequences other than the thy P3 region of 43T. The nature and chromosomal locations of these sequences were not identified. The presence of 3T homologous sequences scattered at different locations in the B. subtilis chromosome might promote site-specific recom- bination and, in consequence, restructuring and i {AGE 63T DNA SEQUENCES IN B. SUBTILIS 99 evolution of the bacterial and phage chromo- somes. The ability of SPB to recombine with $3T (described in the accompanying paper [32}) supports this hypothesis and suggests that thy-transducing phages such as ¢3T and pli could have been created during recombination events between SP phage and the ¢hyA region of B. subtilis. B. subtilis is not the only organism known to carry multiple sequences of viral origin in its genome. This has been shown to be a common property of many eucaryotic systems. Simian virus 40 (14, 17) and adenovirus (6, 9, 24) se- quences are present in many copies in the ge- nomes of their transformed mammalian hosts. The thymidine kinase gene coded by herpes simplex virus can integrate stably into mouse DNA at many different sites (21). Numerous species of vertebrates also contain endogenous latent RNA tumor viruses present in the cells as proviral DNA copies (8, 18, 28). The distribution and functions of these proviruses in the host genomes are not known at present, nor is it known whether they can promote site-specific recombination of the eucaryotic chromosomes. In view of the complexity and inherent difficul- ties in studying the structure of the mammalian genome, the arrangement and role of cryptic genes and phages in B. subtilis might be of general interest as a model system for under- standing the virus-host relationship. ACKNOWLEDGMENTS I am 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. Winkler, 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 excellent technical assistance. This work was supported Ly Public Health Service precoc- toral fellowship GM 00295 and grant CA 16896 from the National Institutes of Health. LITERATURE CITED 1. 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