Distribution of Bacteriophage +3T Homologous
Deoxyribonucleic Acid Sequences in BaciZZus subtihs 168,
Related Bacteriophages, and Other BaciZZus Species

IiVONA T. STROYNOWSKIt

Department of Genetics, Stanford Unkersity School of Medicine, Stanford, California 94305

Received 10 October 1980/Accepted 12 June 1981

The Bacillus subtilis 166 chromosome was found to share extensive homology
with the genome of bacteriophage +3T. At least three different regions of the
bacterial genome hvdridized to ribonucleic acid complementary to +3T deoxyri-
bonucleic acid (DN.1). The thymidylate synthetase gene, thyA, of B. subtilis and
the sequences adjacent to it were shown to be homologous to the region in the
+3T DSA containing the phage-encoded thymidylate synthetase gene, thyP3.
SP,f?, a temperate bacteriophage known to be integrated into the B. subtilis 168
chromosome, was demonstrated to be closelv related to 03T. Other regions of the
bacterial genome were also found to hybridize to the +3T probe. The nature and
location of these sequences in the bacterial and phage chromosomes were not
identified. It was shown, however, that they were not homologous to either the
th~P3 gene or the DXA surrounding the thyP3 gene. The chromosomes of other
Bncillus species were also screened for the presence of +3T homologous se-
quences, and the th~P3 gene was localized in the linear genomes of phages 43T
and pll by heteroduplex mapping. It is suggested that the presence of sequences
of phage origin in the B. subtilis 168 chromosome might contribute to the
restructuring and evolution of the viral and bacterial DNAs.

In general, Bacillus species contain a substan-
rial number of temperate phages, and many of
the straim are known to be polylysogens. They
also harbor defective phages and genes which
e.re esprejsed when the ceh 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 know-n, nor is it known
v;hJ the>- haFe b:en retaired 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 DXA repair enzymes (13,
36). 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 obsemed between Bacillus subtilis and
tivo of its temperate phages. &T and pll (5,35).
These phages c~_structursl genes for thymi-
dylare synthetases, which can complement a
thymine deficiency in their host. These genes
are designated thyP3 and th>Pll, respectively
(12). The phages integrate into the I?. subtilis

91

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 (p3T and the
thyPll gene of pll 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 Bsche-
richia coli strain W5543 (hsdR hsdM leu thi thy rpsL
trp tonB2) or 1V5545 (hsdR hsdM' thr Ieu thi sup&H
rpsL Zac tonA pro). B. subtilis strains SB168 (trpC2),


92    STROYNOWSKI

J. BACTERIO~.

SB1200 (iltd8 thyB1 citB1 gapA2), and SB591 (IhyA
rhyB) and BaciUus strains SB510, SB511, SB512,
SB513, SB514, SB515, SB519, SB522, SB727, SBi30,
SB734, SD1096, SB1098, SB1099, SBllOO, and SBlllO
were from the Stanford University collection. Strain
SB1207 (leu met thr SPp-) was obtained from S. A.
Zahler, and strain SB1213 (trpC2) is a SB168 deriva-
tive which was cured spontaneously of SPB DNA
sequences but was still resistant to infection by SP/3.
Phage stocks. +3T DNA was prepared after mi-
tomycin C (Sigma Chemical Co.) induction of B. sub-
G/is as outlined below. One colony of an SB168 deriv-
ative strain, cured of SP/J' and lysogenized with phage
&T, was inoculated into 50 ml of L broth supple-
mented with lo-' M M&la. The overnight-grown
culture was diluted 10 times into the same medium
and incubated with shaking at 37oC until early log
phase (40 Klett units). At this stage, 0.5 pg of mito-
mycin C per ml was added, and the cells were incu-
bated at 37oC until lysis occurred (2 to 3 h). The
culture was briefly centrifuged (20 min at 8,000 rpm)
to remove bacterial debris. The lysate was filtered,
incubated with DNase (5 pg/ml) for 20 min at 37"C,
and heated for 5 min at 60oC 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,ooO rpm. Further purification of
the phage was obtained by centrifugation in a step
CsCl gradient for 12 h at 37,OOO rpm. The pooled
fractions from several gradients were dialyzed against
3 liters of 10 mM Tris-I 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 IO mM Tris-1 mM EDTA, pH 8.5, and stored
in 4oC. The phage preparation was also examined by
electron microscopy to verify that it did not contain
phage particles with a morphology different from that
of +3T.

T4 phage was prepared as described by Kim and
Davidson (15). Phage pll 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, Calif., 1976). The pronase treatment
was omitted. Closed circular plasmid DNAs were iso-
lated from cleared lysates, prepared according to Cle-
well and Hetinski (3). DNA was centrifuged in ethid-
ium bromide-c&urn chloride gradients (23). Ethidium
bromide was removed by chromatography on Dowex
A6 5OW-XB. The DNA was separated from oligori-
bonucleotides by gel filtration on Bio-Gel A-l!jM. The
plasmid DNA was extracted three times with phenol,
ethanol precipitated, and dialyzed against IO mM
Tris-1 mM EDTA, pH 8.5.

Transformation bioassay in B. subtilis. B. sub-
Mis cells were brought to competence as described by
Harris-Warrick (Ph.D. thesis, 1976). The competent
cells were concentrated IO-fold by centrifugation, sus-

pended in 5% glycerol and frozen in liquid nitmet,
Thawed competent cells were diluted IO-fold in c&r.
ma! medium supplemented with 20 mM MgCl,. ar,+
DNA was added in various final concentrations (0.1 ti,
1 pg/ml). The mixture was incubated for 60 min i1
37oC with gentle shaking and then plated on se!cc:i~e
media (Harris-Warrick, Ph.D. thesis, 1976).
Restriction enzyme cleavage of DNA. Res:fic.
tion enzyme digestions were according to the speci6.
cations recommended by the vendors of the enzhaies.
BamHI, SmnI, PatI, B&D, Sir/I, HpaI, and Hind111
enzymes were purchased from New England Biot&.
Inc.; EcoRI was purchased from Miles Laboratories,
Inc. A two- to threefold enzyme excess was used mu.
tinely for most of the experiments. Reactions were
terminated by heating the -sampIes at 65oC for 7 mm.
Agarose gel electrophoresis. Electrophoresis of
DNA was done in 0.7% agarose horizontal slab gels I 1-1
by 20 by 0.3 cm) at 60 V overnight or at 175 V for 3 to
4 h. Paper wicks connected the gel to tanks containing
0.089 M Tris-O.002 M EDT&O.089 M borate, pH 8.5.
The gels contained 1 pg of ethidium bromide per ml.
DSA was visualized by U\' illumination 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/S). 1Ieasurements of the molec-
ular weights of the DNA molecules were made relative
to o3T DNA cleaved by EcoRI (7) and SPPl DS.4
cleaved by EcoRI (10).

Nucleic acid hybridization. Hybridization exper-
iments were carried out ssentially as described b\
Southern (29). The h>-bridizations were done at 3iiC
in a solution containing 50% formamide, 0.75 Al Sac!.
and 0.075 M sodium citrare. "P-labeled complemen-
tary RSA (cRNA) was us.& as the probe. It ws
synthesized on plasmid and chromosomal DNA tem-
plates in a reaction mixture containing 50 to 100 ,ul of
[u-~`P]GTP (1 mCi/mll, 0.5 mM each ATP, CTP, and
UTP, 2 pg of DNA, 40 mM Tris (pH 8),10 mM MgCb.
10 mM B-mercaptoethanol, and E. coli core RN.4
polmerase in a total volume of LOO pl. RNA polm-
erase was a gift from D. Brurlag. The reaction u-as
carried out at 37oC for 3 h. DBase was then added to
20 pg/ml together with carrier RNA. The reaction was
terminated by phenol extraction. [3'P]cRNA was sep-
arated from unincorporated nucleoside triphosphates
by Sephadex G-56 chromatography. A total of 5 x IO"
to 5 x 10" cpm of J'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 Nters were blotted dry and autoradiographed
at -90oC. using Du Pont Crone-x 4 or Kodak XR-5 X-
ray film and Du Pont Crones Lightning Plus intensi-
fying screens.

Electron microscopy. Preparation and spreading
of heteroduplex molecules WCS done by the method of
Davis et aI. (4). except that the molecules were spread
on water. No DNA extraction on +3T or pll phage
was performed before using the phage in heteroduples
experiments. The photographs were taken at a mag-
nification of ~4,500. The lengthi 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-


3 -8'
) -_ 146. 1961            PHAGE +3T Dh'A SEQUENCES IN B. SC'BTILIS    93

,J~:J~. The projections were meaxued at a tota!
,,,;7lficarion of X135,ooO. All me%urements were
,-& relative to a standard II?;.\ molecule, pSC101,
r,Ju,lted on the same grid. The molecular weight of
:...+ <Ian&d pSClO1 DSX used in all the calculations
;i 3,s megadaltons (X1da.l) (%I.

cloning of EcoRI* fragments. X 3.5~pg amount
:pn~O1 was incubated with 2.3 m>I >lgCl:, 10 rnY

T-A> 1pH 6.5). and 10 I.i of EcoRI enzyme (Lliles
j'~ratories) at 37'C in a tozzl volume of 50 4.
+nples of 10 4 each were removed after 1, 3, 7.5, and
;; h. and the reactions were temkared by heating the
L&es to 6joC for 7 min. A O.>;y amount of EcoRI'-
.$:.ed pFT4OI from each sample was rnised with 0.2

c EcoRI-cleaved pMB9 in a w-4 volume of i0 ~1.
1 01
kza:ion ms carried out at 10 pg of DS.4 per d for I
5 i! ~I)`C, using Ta ligase and th.e buffer described by
.;zuamella et al. (26). E. coli iv.%-%3 competent cella
Tire prepared as described elsewhere (X1;).

ne rransformants (Thy' Tcrl Kere --leeted on aa
   - ..

&?!PS ~qxzIi!m minimal medium [SO] supplemented
%j:h 2.5 pg each of the common amino acids per ml,
+-o-q, 30 yg of tetracycline per ml, and a&.

RESULTS

Construction of chimeric plasmids con-
taining 03T DNA sequences. Most of the
recombinant plasmids (pl?I?3, pFT-24, pFT33,
pF'T401, and pFT.502) used in this work were
constructed previously in our laboratory. Figure
i3 summarizes their molecular structures. The
Thimeras contain different but overlapping +3T
DS.4 sequences joined to E. coli plasmid
pSC101 or pLIB9. A DNA segment of +3T origin
s common to all of the recombinant plasmids
and complements the thyA thy-mine deficiency
mnuration in E. coli. It it ak~ able to transform
Thr.- ausotrophs of B. subt2i.s to prototrophy.
Therefore, this segment of c;3T DSX is assumed
10 contain the thyP3 gene. To achieve higher
purification of this specific gene, it was necessary
i0 diminate nonessential DS;X sequences flank-
ing the thyP3 gene. This was accomplished by
subcloning ofEcoR1' fragments (22) of pFT401
in the EcoRI site of plasmid pBR322. Figure lb
shows the structure of the resulting chimer%
p!=mid, plYI' thyP3. The molecular size of the
EroRI DSA insert carried by pFT thyP3 is 0.5i
51dal.

Sequences homologous to 03T DNA in
the B. subti~is chromosome. StructuraI anal-
!.*s of DSA sequence relationships were per-
formed by using the technique of cRKX-DNA
hybridization coupled with restriction enzyme
and!.sis (29). At least three different regions of
ihe E. subtih chromosome capable of hybrid-
!% o3T cRNX were identiried.

Dx.\ homology of the bacterial thy-4 and
?hage th~P3 genes was demonstrated by hybrid-
IzXion of cRNX prepared on pFT Ih3,P3 to the

b) L-
  ** P-3
  -

FIG. 1, Molecular structure of chimeric plasmids.
All DNAs are aligned from the BarnHI site located
in the vectors. The vector components of the hybrids
are represented by heavy lines: E, pSClO1; @
RSF2122; Kl, pBR322. The inserts are shoun 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. (Q)
Diagrams of plasmids described earlier (7) but used
in this work. The sizes (in megadaitons) of the seg-
ments are (A) 4.5, (B) 0.67, (C) 0.24, (0) 0.9, (E) 0.9,
(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 th.yA 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-Mda.l EcoRI
fragment. RNA complemenkry 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 UzyP3 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 Pas-
labeled cRNA prepared from several of the re-
combinant plasmids. Only the Hind111 enzyme


94    STROYXOWSKI

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. subfilis 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-

- >10

FIG. 2. Hybridization of cRNA pFT thyP3 to
EcoRI-digested B. subtilis 168 DNA. Two micro-
grams of SB168 DNA u,as digested uith EcoRI en-
zyme, electrophoresed through a 0.7% agarose gel,
transferred to a nitrocellulose filter (29), and hybrid-
ized to `"P-labeled RL\`A 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.

               J. Bacx~,,;
tempts to separate them by digestion wirh ttt
following enzymes: EcoRI, BumKI, SmaT, Prti,
&$I, S&I, and HpaI (dara nor &OFIT~). n..,
size of the restriction fragment thar con:ainbj
the thyA region was 7.8 >ldal for PstL 5.4 116~
for BgZII, 6.9 M&J for HpaI, and -more rhzn 1::
Mdal for EcoRI, BamHI, San, and SmaI. DO;.
ble digestion of B. subtilis 165 D?iX Cth Eco&
and Hind111 enzymes, follou-ed by hybridizatic,;
with cRNA pFT.502, was compatible uith the
physical map of the thyP3 homologous regics
shown in Fig. 4.

In addition to the segment ass0cia:e-d with the

a)

b)

A 6     A B

FIG. 3. Hybridization of la) cRS-4 pFT tk~P.3 and
(bj cRN.4 pFT502 to HindHI-cleaved D.Y--<i: SBl55
(channel A) and SBl2Oi (channel Bj. The sire- cf the
stained bands are expressed in megada/fofi.r.

1.6      1.8
--

Hind III  Hind III    Hind III

                      /

FIG. J. Proposed map of HindIII and EcoRI restriction sites in the thy-4 region of the B. ssbtilis
chromosome (SBJZOO). The heavy bar represents sequences that hybridize to thTP3 DXA (0.57 .?idc.& The
hatched bar represents a gene or genes homologous to sequences ofpFT502 other than thTP3 orp.\iBb rector.
Sizes of segments are indicated in megadaltons.


\`()I., 1-C 1981               PHAGE 03T DNA SEQUENCES IN B. SUBTILZS
:J,?.-J biological activity, several other EcoRI
in~gnrrnts of chromosomes from laboratory
.-trains derived from B. subti/Ls 368 (30) (except
.;bl?u; and SB1213; see below) hybridized with
[he following probes: cRN.4 pFT23, cRNA
+T?J, cRNA pFT25, and cRSX pFT302. These
E~,,BI fragments were identified as cleavage             - >lO
i'rOducts of a cr"yptic prophage, SP/3, which is
knolvn to be integrated into the B. subtilis chro-              -
nosome between &A and kcrrtil (40). Figure 5             - 5.6
presents an example of the experiment support-
:ng this conclusion. The EcoRI band of 1.25            &WV
hi&l, which hybridized to cRNA pFT502 in
sBl6.8 (Fig. 5A), was missing in the EcoRI digest
"f srrain SB1207 cured of SPJ3 (Fig. 5B) and
reappeared after this strain was lysogenized with     z;
5Pp (Fig. 32.

.Qproximately 20 bands appeared in the au-
torndiogram shown in Fig. 6 when the RNA
Ienrplementary to the whole 03T genome was
used as a probe in hybridization with the restric-
tion digest of the SPP lysogen. The bands mere
.<rained unequahy, indicating either their uneven
degree of homology with the probe or their
presence in more than equimolar amounts. The
.sm of their sizes was 52 Mdal (or 70.4 Mdal if
strongly hybridizing bands are counted as dou-
blets; see Table 1). This estimate c.orresponds to

.


- 1.45
- 1.3

A B c

           . 1.: , _
       `-     em

Fro. 5. Hybridization of cRK;-l pFT502 to EcoRI-
~~WCC~ B. subtilis DLVAs: (A) SBZM; (B) SBI207;
and K') SBZ207 lysogenized with SPj3. Sizes of
..`uined bands are expressed in megadulfons.

FIG. 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 SP/ strains SB1207 and SBI.213 (see Fig.
7J.

the size of the phage genome which has a com-
plexity similar to the +3T genome. SP,G con-
tamed sequences complementary to pFT502, but
it did not carry the thy.?`3 gene. This is revealed
by the lack of its hybridization with cRNA pFT
thyp3.

Finally, it was shown that the B. subtilis chro-
mosome contained yet another region or regions
homologous to the +3T genome. The EcoRI
fragments of the DNA from two strains inde-
pendently cured of SPP prophage (SB1207 and
SB1213) hybridized to cRNA transcribed from
the 93T DNA (Fig. 7 and Table 1). The hybrid-
ization pattern was limited to four EcoRI bands
instead of the large number of bands observed
in the SPB lysogens. The largest of these bands


96    STROYNOWSKI

TABLE 1. Sequences homologous lo 632 DNA in
     the B. subtilis chromosome

EcoRl fragments of B. sub-   EcoRI fragments of B.
lilis 168 hybridizing +3T     subtilis SB1207 and

     cRNA"       SBl213 (SPfl-) hybridizing
               @T cRNAb

Size (hldal)  Hybridiza-          Hybridiza-
           tion     Size (Mdal)     tion

7.9
5.6
4.7
4.1
4.
3.6
3.1
2.8
2.6
2.3
2.05
1.85
1.7

+++      >lO        ++
+          5.6      ++
+++
+
+
+
+++
+
+++
+
+
+
+

1.3        +
1.25       i-
1.15       +
0.9        +
0.63       +
0.53       +
52.06

1.45      +
1.3      ++

" If strong (++i) bands are counted as doublets,
the total size of the hybridizing fragments is 70.4 Mdal.
In addition to the bands listed above, EcoRI fragments
of 1.45 and >lO Mdal appeared on the autoradiogram
upon longer exposure. +, weak.
*The total size of the bands other than the SPP-
specific or thyA region DNA is 8.35 Mdal. ++, Strong;
+, weak

(>I0 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 P3*-labeled cRNA was transcribed
from a B. subtilis chromosome containing SPj3
prophage (SB581) and hybridized to an EcoRI
digest of +3T DNA, not all of the bands were
labeled (Table 2). The pattern described in Ta-
ble 2 indicates the portions of the +3T genome
that were detected by this technique. They cor-
responded to the portions of the #3T genome
present in the 3. 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 43T homologous
sequence (B. subtilis Marburg, B. globigii, B.
subtilis niger, and B. cougufans). B. mycoides

and B . pumilus DNAs did not hybridize cRS+.\
pFT thyP3 but showed hybridization !virh
cRNA pFT502, indicating the presence of &
gene or genes linked to thyP3. Only B. subtiIi,q
Marburg and 8. coagulans were found to CC,>-
tain a SPP-specific EcoRI band which h-h&
ized to cRNA pFTjD2 (Fig. 5). Thus, these t;r-0
species are assumed to be SPP lysogens.

- >lO

- 5.6

:1

FIG. 7. Hybridization of cRNA +3T to EcoRl-
cleaved SB1207 (SP/3-). An identical h@idizatiol;
pattern was observed by using strain SBI213 D-V.4
Sizes of stained bands are expressed in megadalton.+.

TABLE 2. Hybridization of RNA compiementaq to
SB591 (SPb+) to EcoRI-cleaved +.3T D.%`A"
     Band no.         Hybridization
       1
      2
       3
       4                  +
      5ab                +
      6                  +
       7                  +
       8                  ol-

       9
      10                  +
n EcoRI bands of p3T were numbered according to
Ehrlich et al. (7). Detection of hybridization to bands
smaller than band 10 was below the resolution of the

technique used. +, Hybridization present; -, no hy-
bridization.


I-OL. 148, 1981            PHAGE: +3T DNA SEQUENCES IN B. SUBTXLIS    97

TABLE 3. Screening of bacterial chromosomes for the presence of the thyP3 gene and linked @T
                   homologous sequence

Hybridiza-   Hybridization of
tion of    cRNA pFT502 to   Transformation efficiency (no. of tran$formants/
cRNA pFT    EcoRI band"      no. of viable cells per pg of DSA)"
thyP3 to

EcoRI
band." >I0
hldal

>lO
Mdal

1.2 hldal

Thy

His

Phe

,;Blx3 (SP/- Q-2)
sg3~2 (B. subtilis Marburg)
5B315 (B. megatherium)
SB.515 (B. polymyxa)
SBjIl (B. subtilis subsp. Cer.
i-llidis)
SB513 (B. cereus)
SBj12 (B. globigii)
SBjll (B. mycoides)
SBjIO (B. subtilis subsp. ni-
ger)
SF31110 (B. amy!oIiquefu-

ciens)
SBllW (B. snbtilis subsp.

-

       6 x 1O-4    3 x lo-*    3 x lo-'
+       5 x lo-'     3 x lo-'     3 x 1o-4
       3 x 10-T             5 x 10-1

     1.5 x lo-'          I.5 x lo-'

natto)
SBIEEI (B. coagulans)          +       +      +      8 x lO-4    6 x lo-*    6 x 1o-4
SBlO98 (B. brevis)
SB1096 (B. pumilus)                   +           1 x 1o-3    5 x lo-<    5 x lo-'
SBZ.4 (B. subtilis N)                         -      10-'
SBX4 (B. subtilis H)
SBiS (B. subtilis K)
b'.%j (E. coli K-12)                                        NT     NT
`I'1 phage                                             NT     XT
" Xn EroRI band 2.5 Mdal from SBi27 (B. subtilis K) hybridized both cRNA pFT thyP3 and cR?!A pFT502
probes. +. Hybridization present; -, no hybridization.
`The recipient strain in the transformation as-says was SB748 (thyA thyB his-2 w-o-2). Transformation
&ciencv is listed only when the number of transformants in the assays was significantly higher than the
number bf revertants for the marker tested. The numbers shown are averages of six sets of experiments. NT,

Sot tested.

The DSA extracted from the Bacillus species
studied was also tested for its ability to trans-
form ihe B. subtilis thbmine 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 SBl213). In the case of DNA from B.
globigii, which was found to hybridize the thyP3
probe, the transformation efficiency was below
the rel-ersion 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
esplanation which proposes the existence of a B.
s1:btili.s restriction system that degrades B. glo-
b&i DS.1 was ruled out by the results of Harris-
l\`arrick (Ph.D. thesis, 1976). A few strains (B.
megatherium, B. subtilis subsp. terminalis, B.
.~ubtilis subsp. natto, and B. pumilus) did not
hybridize to the thyP3 piobe, but their DNA

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 thyPl1 gene in pll. The
thyP3 gene has been mapped in the +3T genome
by heteroduplex analysis of hybrids formed be-
tween the chimeric plasmid pFT25 and phage
DNA (7). In view of the known sirniIarit,ies be-
tween +3T and pll(5), I decided to use the same
technique to IocaIize thyPl1 in ~11. The pFT21
plasmid containing thyP3 of the 63T phage was


98   STROYNOWSKI

linearized with the BarnHI restriction enzyme.
After denaturation, this plasmid DNA was an-
nealed to the complementary single strands of
the pll 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 pll DNA
molecule. The standard error of this measure-
ment was 3% of the fractional pll length. The
double-stranded region found in the heterodu-
plex of pll with segment A of plasmid pFT24
was not perfectly homologous. Two small loops
could be detected. 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 pll
phages are different. Figure 8 shows also the
localization of the thyP3 gene in the +3T ge-
nome. The thyP3 gene was situated at a distance

J. BACTERIOL.

44 to 48% from one of the +3T ends. Statistical
error of this measurement was 2% of the frac.
tional03T length.

DISCUSSION

The results described here provide evidence
for the existence of extensive DNA homologies
between the chromosomes of B. subtilis 168 and
its temperate bacteriophage +3T. Three distinct
regions of the bacterial chromosome were iden-
tified that were capable of hybridizing RNA
complementary to the +3T genome.

One of these regions contains the structural
gene for thymidylate synthetase A and wss
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

i
thyPl1

d)

I           I           I           I
0                                          1)
          20          40          60          80

          MOLECULAR WEIGHT IMDAL)

FIG. 8. Localization of the thyP gene in pll and +3T. (a) Histogram of the position ofpoint X (see belou
ti shown in relation to the ends of plI DNA. X corresponds to the far left end of the +jlT insert in pFT24 and
pFT25. (b) Diagram of pll Dh'A heteroduplexed with BamHI-linearizedpFT24 D.U. Segments B, C, and D
represent the regions of homology between the two DNA strands. Loops E and F correspond to regions of
nonhomology. The sizes of heteroduplex regions are (in megadultons): (A) 0.2; (B) 1.i3; (C] 0.11; (D) 1.1; (E)
0.13; (F) 0.26; (G) 6.39; (H) 41.1; (I) 38.8. Secenteen molecules with the sfructure represented by this diagram
were analyzed. (c) Histogram of the position of point X is shoun in relation to the ends of +3T D.VA. (d)
Diagram of +3T DNA heterodupiexed with BarnHI-tinearizedpFT25 DNA. Segment B represents the region
of homology between +`T andpFT25. The sizes of heteroduplex regions are (in megadaltons): (A) 0.2; (B) 6.3;
(C) 4.7; (D) 34.8; (E) 41.1. Fifteen molecules with the structure represented by this diagram were measured.


\`OL. 146, 1951             i". iAGE +3T DNA SEQUENCES IN B. SUBTZLIS    99

(or genes) was not identified. No markers closely
Linked to thyA or thyP3 have been reported in
H. subtilis of +3T. In other systems, such as T4
phage, DNA sequences close to the structural
gene for thymidylate synthetase code for pro-
teins involved in DNA metabolism (37). In con-
trast, the E. coli or Salmonella typhimurium
th?A Iocus maps between the lys and arg loci,
awav from other genes involved in DNA synthe-
.cis (1;).

The second region of extensiv*e homology be-
tween the bacterial genome and the +3T genome
spas identified as an SPP prophage. SPP is a
temperate, cryptic bacteriophage present in B.
&tiZis 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 SPP. It is a fairly large phage
of complex structure similar morphologically to
@T and pll (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 ilcA and kauA
(10). SPj? does not convert B. subtilis Thy-
aurotrophs to prototrophy upon lysogenization.
It can, however, carry out a specialized trans-
duction of the citK and hau.4 genes (40). Its
mechanism of specialized transduction was pro-
posed by Zahler et al. (40) to resemble closely
the E. coli phage X dgal system (2). The results
described in this communication demonstrate
that the phages SB/3 and +3T are closely related.
When RNA complementary to the +3T genome
was hybridized to the EcoRI restriction digest
of a SP/3 lysogen, more than 20 bands (total of
50 to SO Mdal) were homologous to the radio-
active RN.4 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 SPP lacks the
th~P3 sequence (0.57 Mdal), although the DNA
sequence located next to this gene in +3T is still
present in the SPB genome.

Finally, it was shown that the B. subtilis duo-
mosome contains yet another region homolo-
gous to 03T which is different from ThyA or
s1'/3 DSA. 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 thyP3 region of +3T.
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

evolution of the bacterial and phage chromo-
somes. The ability of SPP to recombine with
+3T (described in the accompanying paper
[32]) supports this hypothesis and suggests that.
thy-transducing phages such as 43T and pll
could have been created during recombination
events between SPP phage and the thyA 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 (8,18,23). 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.

ACKNOmEDGMEhTS

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 wa; supported t,y Public Health Service prec'oc-
toral fellowship GM 00295 and grant CA 16896 from the
National Institutes of Health.

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i