JOURNAL OF BACTERIOLOGY, Mar. 1988, p. 1183-1190 Vol. 170, No.

3
0021-9193/88/031183-08$02.00/0
Copyright C) 1988, American Society for Microbiology

Insertion of Foreign DNA into Plasmids from Gram-Positive

Bacteria Induces Formation of High-Molecular-Weight
Plasmid Multimers
A. GRUSS* AND S. D. EHRLICH
Institut Jacques Monod T43, 2 Place Jussieu, 75251 Paris Cedex 05, France
Received 21 September 1987/Accepted 30 November 1987

Plasmids pUB110, pC194, pE194, and pT181 are commonly used as cloning vectors in both Bacillus subtilis
and Staphylococcus aureus. We report that insertion of foreign DNA into any of these plasmids results in the
generation of high-molecular-weight plasmid multimers (HMW) of the recombinant, present as tandem
head-to-tail copies. HMW was detected in wild-type B. subtilis and S. aureus strains. The production of HMW
depended on the nature of the DNA insertion. Inserts of Escherichia coli DNA, e.g., pBR322 or pUC18, resulted
in large amounts of HMW, whereas some inserts of S. aureus DNA of the same size had no effect on plasmid
profile. The generation of HMW depended on the mode of plasmid replication; plasmids which replicate via a
single-stranded DNA intermediate produced HMW upon foreign DNA insertion, whereas plasmid pAMH1,
which does not generate single-stranded DNA, did not generate HMW. We propose that HMW is a product of
impaired termination of rolling-circle replication and that the impairment is due to the nature of the DNA
insertion.

Gram-positive plasmids pUB110, pC194, pE194, and
pT181 are members of a single class of interrelated, small,
multicopy plasmids (25, 29), originally isolated from Staph-
ylococcus aureus (13, 17) and readily established in Bacillus
subtilis (9). Evidence exists that these plasmids replicate by
a rolling-circle mechanism similar to that described previ-
ously for single-stranded (ss) DNA Escherichia coli bacte-
riophages: all have been shown to synthesize ssDNA (36)
and to have distinct plus and minus origins (11). For plas-
mids pT181 (16) and pC194 (10a), it is reported that replica-
tion at the plus origin is initiated at a nick generated by a
plasmid-encoded protein in a double-stranded (ds) DNA
molecule. The plus strand is displaced concomitantly with
DNA synthesis and circularized subsequent to a second
cleavage at the origin; this forms the ssDNA intermediate of
rolling-circle replication. In the absence of a minus origin,
ssDNA is inefficiently converted to dsDNA and is accumu-
lated in the cell (11, 36, 37). The minus origins of pT181,
pC194, and pE194 are active in S. aureus but not in B.
subtilis, while that of pUB110 is active in both (11; unpub-
lished data). These plasmids are retained at high copy
numbers (reported to be between 10 and 55 per cell; 25) in
both S. aureus and B. subtilis. They are segregationally
stable in S. aureus (25), but only pUB110 is stable in B.
subtilis (26).
Often, recombinant plasmids do not retain the features of
the parental plasmid. Insertion of foreign sequences may
decrease the copy number and result in segregational insta-
bility, as reported for pUB110-related plasmids in B. subtilis
by Bron and Luxen (5). For these and a number of other
gram-positive plasmids, we observe that insertion of certain
DNA fragments, e.g., from E. coli, results in a shift in
plasmid distribution from principally monomeric (for wild
type) to principally multimeric (for hybrids) form. The
formation of high-molecular-weight (HMW) multimers ap-
pears to be a function of the mode of replication of the
plasmid; it is observed for plasmids which replicate via an
*
Corresponding author.
1183
ssDNA intermediate but not for a plasmid which does not. It
is notable that commonly constructed shuttle vectors de-
signed to replicate in both B. subtilis and E. coli are present
in B. subtilis in predominantly HMW form.
MATERIALS AND METHODS
Strains used in this study were B. subtilis SB202 trpC2
tyrAl aroB2 hisH2 (this laboratory) and 8G-5 his trp tyr ade
ura nic rib (S. Bron), both wild type for recombination and
replication functions, and GSY908 agrC4 hisAl recE4 (C.
Anagnostopolous) and E. coli JM105 A(lac-pro) thi rpsL
sbcB15 endA hsdR4 (F' traD36 proAB lacIqZAMJ5) (J.
Messing). Plasmids used in this study are presented in Table
1. Construction of pHV1601 and pHV160li is described in
Results and in the legend to Fig. 5. Plasmids were introduced
by standard transformation methods into E. coli or B.
subtilis (22). Strain SB202 was the B. subtilis host for all
plasmid constructions except plasmids pHV1601 and
pHV1601i, which were constructed in E. coli. Strain 8G-5
was transformed with plasmid pHV1020 encoding the pC194
replication protein (Rep) (lOa). Strain 8G-5 with pHV1020
and strain 8G-5 without pHV1020 were used as recipients for
pHV1601 and p1601i. In vitro plasmid constructions were
made according to published procedures (19). When appro-
priate, antibiotics were added at concentrations of 5 p,g/ml
for tetracycline, chloramphenicol, or kanamycin, 0.5 ,ug/ml
for erythromycin, and 75 p,g/ml for ampicillin. Agarose gels,
0.6%, were prepared in Tris-acetate buffer according to
published procedures (19) and electrophoresed at 2 to 3
V/cm overnight. Southern blot hybridizations (SBH) were
performed by published methods (35); an Amersham kit was
used for nick translations, which were performed according
to the supplier's instructions. Whole-cell minilysates were
prepared by published procedures (28). Plasmid DNA was
prepared by published methods (3). Total DNA was pre-
pared by extraction of whole-cell lysates with phenol, fol-
lowed by precipitation with ethanol and treatment of the
nucleic acids with RNase; phenol extractions were per-
1184 GRUSS AND EHRLICH J. BACTERIOL.

TABLE 1. Plasmids used

Plasmid Size (kb) Description Reference
pUBllO 4.5 S. aureus plasmid Kmr 20
pC194 2.9 S. aureus plasmid Cmr 12
pBR322 4.3 E. coli plasmid Ampr, Tcr 4
pUC18 2.7 E. coli plasmid Ampr 40
pLB2 3.6 Hybrid plasmid composed of the replicon and Kmr gene of pUBllO 5
(1,788-4,377 bp) and the Cmr gene of pC194 (973-2,004 bp)a
pLB2 1-C 4.9 1.3-kb insert of E. coli DNA in BgIll site of pLB2 S
pLB2 3-C 7.8 4.2-kb insert of E. coli DNA in BglII site of pLB2 5
pLBS 5.9 2.7-kb insert of pBR322 DNA (2,066-4,363/23 bp) into EcoRI site of S
LB2; a bifunctional replicon
pLB5 1-C 7.2 1.3-kb insert of E. coli DNA in BglII site of pLB5; total E. coli insert 5
size, 4.0 kb
pLB5 3-C 10.1 4.2-kb insert of E. coli DNA in BglII site of pLB5; total E. coli insert 5
size, 6.9 kb
pHV33 7.3 pC194 and pBR322 linearized and joined at unique HindIl sites 27
pHV1601 9.1 pAMP, HindIII(D,F)::ori pC194::pUC18::pE194 TaqI(B) (Emr), This work
contains active pAM,1 replicon (Fig. 5A)
pHV160li 9.1 Inversion of pDl PvuII fragments, inactive pAMBl replicon; This work
replication proceeds via pC194 origin complemented in trans
pHV1020 5.5 pC194 Rep protein gene inserted into pUBllO 10a
pUB11OBg::pUC18b 7.2 pUBllO, BglII, and pUC18, BamHI, linearized and joined at unique This work
sites
pUB11OF::pUC18 7.2 pUBllO, FnuDII, and pUC18, HincII, linearized and joined at unique This work
sites
pUB11OP::pUC18 7.2 pUBllO, PvuIl, and pUC18, HincII, linearized and joined at unique This work
sites
pUB11OB::pUC18 7.2 pUBilO and pUC18 linearized and joined at unique BamHI sites This work
pUB11OE1::pUC18 7.2 pUBllO and pUC18 linearized and joined at unique EcoRI sites, This work
pUBllO Rep adjacent to pUC18 Ampr
pUB11OE2::pUC18 7.2 pUBllO and pUC18 linearized and joined at unique EcoRI sites, This work
pUBllO Rep distal to pUC18 Ampr
pUB11OB::pBR322 8.9 pUBllO and pBR322 linearized and joined at unique BamHI sites This work
pUB11OE::pBR322 8.9 pUBllO and pBR322 linearized and joined at unique EcoRI sites This work
pUB11OBg::erm 6.7 2.2-kb insert of pI258 DNA encoding erythromycin resistance, This work
inserted in BglII site of pUBllO
pUB11OB::erm 6.7 2.2-kb insert of p1258 DNA encoding erythromycin resistance, This work
inserted in BamHI site of pUBllO
pUB11OE::erm 7.5 3.0-kb insert of p1258 DNA encoding erythromycin resistance, This work
inserted in EcoRI site of pUBllO
a Map positions are given according to published sequences (12, 20). b Plasmid nomenclature used here is simplified to indicate the plasmid vector and the
position into which DNA is inserted. (See Fig. 2A for DNA insertions into pUBllO.)
1 2 3 4 5 6
formed with gentle mixing and extraction with a wide pipette
to avoid DNA breakage.

RESULTS
-HMW
Plasmid pUBllO with an E. coli DNA insertion generates
HMW. A series of plasmids based on the pUB110 replicon,
10.1- constructed by Bron and Luxen (5), contain different sized
inserts of E. coli DNA. It was reported that, in B. subtilis,
plasmid copy number and segregational stability were in-
78- versely proportional to the size of the insert DNA (5). To see
7.2- whether an unresolved replicative intermediate might be
formed in these plasmids, and thus cause the observed
5.9- 0

4.9- FIG. 1. Effects of insertion of E. coli sequences on plasmid

profile. Whole-cell sheared minilysates of B. subtilis strains were
separated on a 0.6% agarose gel. Gels were analyzed by SBH, using
32P-labeled pUB110 as probe. Sizes, in kilobases, corresponding to
3.6-4' the positions of supercoiled monomeric plasmids are given at left.
Below a prominent monomeric band a faintly hybridizing band of
*1. ssDNA can be seen in most samples. HMW, at right, indicates the
position of nonmonomeric material which hybridizes to the plasmid-
specific probe. Lanes: 1, LB2, parental plasmid with no E. coli
insert; 2, LB2-1C; 3, LB2-3C; 4, LB5; 5, LB5-1C; 6, LB5-3C.
VOL. 170, 1988 HMW PLASMID MONOMER FORMATION 1185

A B C
S E S E S E
1 2 3 4 5 6 7 8 9 10 1 2 34 5 6
A

+orl
HMW I....

91.
_ ..
.f *

Ab
is 00

I" .
**'*-

pUB110
FIG. 2. Effects on plasmid profile of insertions of pBR322, pUC18, or staphylococcal DNA into different positions of plasmid pUBllO. (A)
Map of plasmid pUBllO, indicating positions of identified open reading frames and restriction sites (20) relevent to this study. The plus and
minus origins of replication (11; unpublished data) are also indicated. Positions of insertions of pUC18 (0), pBR322 (0), or staphylococcal
DNA containing the erythromycin resistance (erm) gene of plasmid p1258 (A) are indicated by flags. The erm gene is on a 2.2-kb fragment
for insertions in the BgIII and BamHI sites and on a 3.0-kb fragment for the insertion in the EcoRI site. Flags facing outside the map of pUBllO
correspond to inserts which result in HMW production; flags facing inside correspond to inserts which do not result in HMW production. For
recombinant plasmid nomenclature, see Table 1. (B) Whole-cell lysates of strains containing recombinant plasmids were separated on a 0.6%
agarose gel and analyzed by SBH, using 32P-labeled pUBllO as probe. In every case, two lower hybridizing bands correspond to the
supercoiled and open circular forms of the monomeric plasmid. The position of HMW is indicated. As the vector, pUBllO, contains an
efficient signal for conversion of ssDNA to dsDNA (i.e., the minus origin), no band corresponding to ssDNA is detected in these samples.
B. subtilis strains contained plasmids as follows. Lanes: 1, pUBllO; 2, no plasmid; 3, pUB11OBg::pUC18; 4, pUB11OF::pUC18; 5,
pUB11OP::pUC18; 6, pUB11OB::pUC18; 7, pUB11OE1::pUC18; 8, pUB11OE2::pUC18; 9, pUB11OE::pBR322; 10, pUB11OB::pBR322. (C)
DNA fragments containing the erm gene from staphylococcal plasmid pI258 were cloned in the BglII, BamHI, and EcoRI sites of pUBllO,
for comparison with insertions of pUC18 in the same positions. Whole-cell lysates of strains containing recombinant plasmids were analyzed
by SBH, using 32P-labeled pUBllO as probe. The lower hybridizing band corresponds to the supercoiled monomeric plasmid. Lanes: 1,
pUB11OBg::erm; 2, pUB11OBg::pUC18; 3, pUB11OB::erm; 4, pUB11OB::pUC18; 5, pUB11OE::erm; 6, pUB11OE1::pUC18. S or E indicates
that inserted DNA is derived from S. aureus or E. coli, respectively.

decrease in copy number, minilysates of strains containing
these plasmids were electrophoresed on an agarose gel and
analyzed by SBH, using 32P-labeled pUB110 as probe. The
autoradiograph is presented in Fig. 1. Vector plasmid pLB2
(composed of the replicative and kanamycin resistance
genes of pUB110 as well as the chloramphenicol resistance
gene of pC194; lane 1) did not contain an E. coli insert and
showed a standard plasmid pattern in which the predominant
plasmid form is a supercoiled monomer. In lanes 2 to 6 are
minilysates of strains containing plasmid pLB2 into which
random fragments of E. coli DNA or pBR322 DNA or both,
ranging in size from 1.3 to 8.6 kilobases (kb), are inserted. A
second prominent hybridizing band (indicated by HMW) is
present in these lanes and migrates in the position of
chromosomal DNA irrespective of the size of the monomeric
plasmid. Plasmids carrying the largest sized inserts also
produce the tnost HMW. ssDNA is also visible in most
samples (Fig. 1).
As the starting plasmid, pLB2, in the series described
above was itself a recombinant plasmid, we also constructed
a series of DNA insertions into wild-type plasmid pUB110.
Either pUC18 or pBR322 sequences were cloned into five
different sites of pUB110 (Fig. 2A). B. subtilis strains con-
taining these recombinants were analyzed for HMW, using
32P-labeled pUB110 as the hybridization probe (Fig. 2B).
Wild-type pUB110O was present in monomeric supercoiled
and open circular forms only (lane 1). However, all recom-
binants produced HMW, irrespective of the site of insertion.
Insertion of pUC18 (2.7 kb) or pBR322 (4.3 kb) sequences
into the EcoRI site generated less HMW relative to mono-
mer form than insertions into other sites (cf. lanes 7, 8, and
9 with 3, 4, 5, 6, and 10). Scanning of autoradiographs of
SBH indicated that as much as 90% of plasmid hybridization
may correspond to HMW. These results show that insertion
of pUC18 and pBR322, as well as other E. coli DNA
sequences (as shown above for pLB2 series), in gram-
positive plasmid pUB110 results in HMW production. No
particular plasmid function need be inactivated, as insertions
of foreign DNA in any position (including the antibiotic
resistance gene) caused HMW production. ssDNA was not
detected in the samples, since the plasmids carried an
efficient minus origin (unpublished results).
1186 GRUSS AND EHRLICH J. BACTERIOL.

A B
2 I 2
o =: Oa0 -0
3- }XX
x
X
- ti.,-
S
- HMW-
HMW-II
0
_a; *"6-I- __
L 4

SC
s. -rSc SS
I1

FIG. 3. HMW is composed of linear plasmid multimers. Total DNA was prepared from B. subtilis strains containing plasmids pUB110 and
pUB11OB::pUC18. (A) DNA samples were treated with either lambda exonuclease (A exo), a 5'-to-3' exonuclease, or exonuclease III (exoIII),
a 3'-to-5' exonuclease. (B) DNA samples were digested with restriction enzymes which recognize no sites (ClaI) or one site (BglII) on each
plasmid. Treated and untreated (UT) samples were analyzed by SBH, using a 32P-labeled pUB110 as probe. SC and L, Supercoiled and linear
monomeric plasmids. Panels: 1, pUB110; 2, pUB11OB::pUC18. Weak hybridization in the region preceding SC (A, panel 1) may be due to
ssDNA generated by exonuclease treatment. Increased hybridization to open circular form in ClaI-treated pUB11OB::pUC18 is probably due
to minor nuclease contamination of the DNA sample.

Not all DNA insertions into pUB110 cause HMW produc-
tion. DNA fragments containing the erythromycin resistance
gene derived from staphylococcal plasmid p1258 were in-
serted in three different positions of pUB110, the BglII site,
the BamHI site, or the EcoRI site (Fig. 2A). Resultant
recombinants were compared for HMW production with
constructions in which E. coli plasmid pUC18 was inserted
into the same positions (Fig. 2C). Only those plasmids which
contained E. coli DNA insertions produced significant
amounts of HMW (Fig. 2C, lanes labaled E); little or no
HMW was generated by the staphylococcal DNA insertion
(Fig. 2C, lanes labeled S). However, inserts of random S.
aureus chromosomal DNA into pUB110 gave HMW, al-
though generally less than E. coli inserts (data not shown).
We have noted that the pUB110-based plasmid construc-
tions described here are segregationally stable (data not
shown). This is in contrast to the results of Bron and Luxen
(5), which indicate that DNA insertion results in a marked
decrease in plasmid stability. As previously mentioned, the
constructions of Bron and Luxen are based on pLB2, a
deletion derivative and recombinant of pUB110. Apparently,
the alterations introduced in pLB2 were sufficient to cause
stability differences which cannot be attributed solely to the
insertion of foreign sequences.
Recombinants using pT181, pC194, or pE194 replicons
generate HMW; HMW is also generated in S. aureus. Plas-
mids pT181, pC194, and pE194 all produce ssDNA, and
presumably, like pUB110, all replicate by a rolling-circle
mechanism (16, 36). pBR322 was joined at convenient sites
to these plasmids, and the recombinants were introduced in
B. subtilis. A pE194::pBR322 recombinant was also estab-
lished in S. aureus. HMW formation was determined by
SBH, using 32P-labeled pBR322 as the probe. In all strains
carrying recombinant plasmids, HMW was present in high
proportions with respect to monomeric plasmid DNA (data
not shown). Plasmid profiles were similar in B. subtilis and
S. aureus. These results demonstrate that HMW is produced
by plasmids which replicate via an ssDNA intermediate, and
its formation is not strictly dependent on a particular host.
HMW is composed of linear plasmid multimers present as
head-to-tail tandem repeats. To determine the structure of
HMW, whole-cell DNA was prepared from strains contain-
ing pUB110 or HMW producer pUB11OB::pUC18 and
treated with lambda exonuclease or exonuclease III. The
former degrades linear dsDNA from the 5' end (30); the
latter degrades it from the 3' end (31). After treatment, DNA
was separated on an agarose gel and analyzed by SBH, using
32P-labeled pUB110 as probe. HMW was totally degraded by
either exonuclease, while circular plasmid forms remained
largely intact (Fig. 3A). These results indicate that in HMW
there is at least one dsDNA end accessible to exonucleolytic
degradation. In this experiment, low levels of HMW were
VOL. 170, 1988
A
B bination (15) and can be complemented by the recA function
rec E
1 2
3 4 5 6
HMW-
HMW-
sc- 0
Sc-
FIG. 4. HMW is not formed by generalizecJ recombination. (A)
Whole-cell lysates of wild-type and recE4 strainis containing plasmid
pUB11OB::pUC18 were separated by agarose gel electrophoresis
and subjected to SBH analysis, using 32P-labeleed pUB110 as probe.
Position of HMW is indicated. (B) Total DNA was prepared from
strains containing plasmid pUB11OB::pUC18 or pHV33 or both.
Each plasmid generates HMW (lanes 1 and 2, i
were treated with BamHI', which generates two bards pUBl.OB::
for
pUC18 and a single band for pHV33, and analybzed by SBHp Shown
here are whole-cell DNA samples. Lanes: 1, p1 UB11OB::pUC18, un-
treated; 2, pHV33, untreated; 3, pUB11OB::pUC'18, BamHI; 4 and 5,
pHV33 plus pUB11OB::pUC18, BamHI; 6, p HV33, BamHI. The
restriction pattern generated by each of the biiplasmid strains con-
tains only those bands which correspond to olne of the two homo-
plasmid strains. SC, Supercoiled.
also detected in a strain containing iwild-type plasmid
pUB110.
Whole-cell DNA was also treated withh restriction endo-
nucleases which recognize either no sites (ClaI) or one site
(BglII) within the plasmids. DNA patterns were analyzed by
agarose gels and SBH (Fig. 3B). For DN[A samples treated
by a restriction endonuclease not recognLizing the plasmid,
HMW remained in essentially the same position as in the
untreated samples (panel 2, compare laines UT and Cla).
This indicates that HMW is not intersper-sed within foreign
sequences. When samples are digested with an enzyme
recognizing one site in the plasmid, all hyb)ridizing material is
present as a single band corresponding to the size of the
linear monomer (see BglII-treated sample ~s).
Taken together, these results suggest t hat HMW is com-
posed of tandem head-to-tail linear plasmi d multimers. How-
ever, as there is some shearing during DNJA preparation, we
cannot rule out the existence of another structure, e.g., a
a-shaped molecule or a large circular concatamer, in the
intact cells.
Homologous recombination does not g enerate HMW. B.
HMW PLASMID MONOMER FORMATION 1187
subtilis recE4 strains are defective in intermolecular recom-
of E. coli (8). HMW production was compared for plasmid
pUB11OB::pUC18 in a recE4 versus a wild-type strain. A
representative experiment is shown in Fig. 4A. Less HMW
was produced in the recE4 host. To test the possibility that
HMW is a product of iterative "single crossing-over" ho-
mologous recombination, wild-type B. subtilis strains were
constructed, containing two plasmids which both produce
HMW and share 2.3-kb homology within their E. coli inserts
(pUB11O::pUC18 and pHV33; Fig. 4B, lanes 1 and 2). If
HMW was predominantly produced through recombination,
then "mixed" linear multimers composed of combinations
of both plasmids should be present in the cell. To determine
whether such HMW was present, whole-cell DNA was
prepared from mono- and biplasmid strains and treated with
a restriction endonuclease that could be used to distinguish
the two component plasmids (BamHI, cutting pHV33 once
and pUB11O::pUC18 twice). SBH analysis of restriction
patterns (using a probe which recognized both plasmids) is
shown in Fig. 4B, lanes 3 to 6. HMW recombinants would be
detected by the presence of bands in the biplasmid strain not
present in either of the monoplasmid strains. We found no
such bands (cf. monoplasmid strains, lanes 3 and 6, and
biplasmid strain, lanes 4 and 5) indicating that interplasmid
recombinants are not present in HMW. These results sug-
gest that intermolecular recombination is not a major mech-
anism by which HMW is formed.
HMW production depends on the replication mode of the
plasmid. pAM31 is a 26.5-kb low-copy plasmid, isolated
from Streptococcus faecalis (6). Deletion derivatives of
pAMP1 are maintained in B. subtilis. These plasmids do not
produce ssDNA (L. Janniere and S. D. Ehrlich, submitted
for publication), and it is therefore likely that they do not
replicate by the mechanism characteristic of other plasmids
studied above. We observed that a pAMP1 derivative with
an insert of pBR322 does not produce HMW (data not
shown), suggesting that the mode of replication may play a
role in HMW generation. To show directly that the mode of
replication is the determining factor, hybrid plasmid
pHV1601 was constructed, comprising the replication region
of pAMP1, the replication origin of pC194, the erythromycin
resistance gene of pE194, and pUC18 sequences (as shown
schematically in Fig. 5A). This plasmid should be able to
replicate either via an ssDNA intermediate (if the pC194
origin is active) or without such an intermediate (if the
pAMP1 origin is active). pHV1601 was introduced into a B.
subtilis strain either alone or together with plasmid
pHV1020, which carries the cloned pC194 replication pro-
tein (Rep) (lOa). Figure SB and C shows agarose gel and
SBH analyses of mono- and bi-plasmid strains. Whereas
plasmid pHV1601 alone did not produce HMW, it produced
significant amounts of HMW in the presence of pC194 Rep
(Fig. 5B). Activation of the pC194 origin of pHV1601 in trans
was confirmed by the detection of plasmid ssDNA upon
further exposure of SBH filters (data not shown). Thus,
HMW formation is contingent upon the activity of the pC194
origin. These results indicate that HMW is generated only
when a plasmid replicates via an ssDNA intermediate.
This conclusion was confirmed with plasmid pHV1601i, in
which the pAMP1 replication region was inactivated by
inversion of the PvuII fragments of pHV1601 (Fig. 5A); this
ensured that replication initiated exclusively at the pC194
origin. As expected, pl601i, which could be established only
in the strain containing active pC194 Rep, produced HMW
(Fig. SC).
1188 GRUSS AND EHRLICH J. BACTERIOL.

A B C

1 2 3 1 23
1

I
I
F

I
I 7 HMW-

'pA

pHVI601

pHV1O20
ori QCl94

FIG. 5. Activation of HMW production by rolling-circle replication. (A) Map of plasmid pHV1601, which contains pAM,1 sequences
(open bar) including the entire minimal replication region of pAMP1 (wide open bar), 55 base pairs which constitute the origin of pC194,
pUC18 sequences (filled bar), and the gene encoding Emr from pE194. The direction of potential replication from the pC194 origin is indicated
by an arrow. pHV160li is derived from pHV1601 by inversion of the PvuII fragments; this plasmid is unable to replicate via the pAMP1
replicon. Plasmids were constructed in E. coli and subsequently established in B. subtilis wild type, for pHV1601, or the same strain
containing plasmid pHV1020, which supplies pC194 Rep, for pHV1601 and pHV1601i. Whole-cell minilysates were analyzed by agarose gel
electrophoresis (B) and SBH (C), using 32P-labeled pUC18 as probe. Plasmid pHV1020 shares no homology with pHV1601 and thus does not
hybridize with this probe. The copy number of pHV1601 in the host devoid of pHV1020 is higher than that of plasmids in the host containing
pHV1020; a lighter exposure of this lane is presented so that the plasmid pattern is visible. Lanes: 1, pHV1601; 2, pHV1601 and pHV1020;
lane 3, pHV160li and pHV1020. Positions of plasmids are indicated. HMW is produced by pHV1601 and pHV160li when the pC194 origin
is complemented in trans by pC194 Rep.

DISCUSSION co-workers reported the presence of linear multimeric

We report here that plasmids pUB110, pT181, pC194, and
pE194 into which certain foreign DNA is inserted appear in
HMW form. Estimations by scanning films of SBH show
that as much as 90% of plasmid DNA may be present as
HMW. We observe that (i) only those plasmids which
replicate by a rolling-circle type mechanism with ssDNA
intermediates generate HMW upon DNA insertion (a plas-
mid with no ssDNA replicative intermediate does not pro-
duce HMW) (ii) the nature of the inserted DNA determines
whether HMW is generated or not. Plasmids containing
inserts of E. coli DNA produce HMW, whereas plasmids
containing other inserts of the same approximate size
showed variable amounts of HMW. As little as 500 base
pairs of inserted DNA was observed to result in HMW
formation (data not shown); however, as the size of the
insert is increased, the proportion of HMW is also increased.
The site of insertion is not the determining factor in HMW
production.
Although less HMW is produced by the same plasmid in a
recE4 background, generalized recombination does not ac-
count for the generation of HMW. The presence of 2.3 kb of
homology among two HMW-producing plasmids in a hete-
roplasmid strain does not result in any detectable recombi-
nant formation. It has been reported that strains carrying
recE mutations show enhanced degradation of cellular DNA
in response to UV (32). If nucleases are more readily
induced, it may be that the amounts of DNA structures such
as HMW are decreased by degradation in this strain.
Several previous reports have shown that host factor
alterations can induce production of HMW. Kornberg and
(X174 in vitro (2, 33). This phage also replicates by a
rolling-circle mechanism with an ssDNA intermediate (10).
By altering the concentrations of replication components
such as ssDNA-binding protein, XX174 replication products
shifted from predominantly supercoiled ds monomer (plus a
monomeric ssDNA intermediate) to long linear phage mul-
timers extending from a duplex phage circle (2). It was
proposed that, in limiting amounts of ssDNA-binding pro-
tein, termination of replication could be sterically hindered
by increased nonspecific primosome binding to displaced
ssDNA. In other in vitro experiments with XX174, HMW
was also generated during conversion of ssDNA to dsDNA,
presumably by primosome-catalyzed strand displacement,
which allowed rolling-circle replication to occur (21).
HMW has been identified for plasmids in recBC sbcB
mutants of E. coli (7, 34). Their production was shown to be
independent of the replicon (ColEl-like, OriC, mini-F, and
pSC101 plasmids were used). It was proposed that random
nicks on the plasmid could initiate aberrant rolling-circle
replication, and as there is no termination signal for replica-
tion, linear multimers are formed. In a wild-type host,
RecBC enzyme would degrade an unprotected displaced
end, whereas in the mutant host such structures remain
intact. HMW formation was recently described in B. subtilis
add mutants, which lack a function corresponding to exonu-
clease V (RecBCD enzyme) of E. coli (38). It is likely that
HMW is generated independently of the mode of plasmid
replication in B. subtilis add host mutants as well.
It has also been reported for wild-type plasmid pT181 that
linear plasmid multimers are formed upon phage infection of
VOL. 170, 1988
S. aureus and that this process was dependent upon plasmid
replication (24). It was proposed that a phage product is
inhibitory for plasmid termination, thus allowing multimers
to form.
The observation reported here differs in three features
from the previous ones: (i) HMW is produced in a wild-type
host; (ii) HMW production is contingent upon the- mecha-
nism by which the plasmid replicates; and (iii) HMW pro-
duction is dependent on the presence of certain DNA
insertions. The first two characteristics may be related, as
follows. As HMW is present in a wild-type host, it is
presumably immune to exonucleolytic degradation by host
enzymes. Replication of plasmids studied here (with the
exception of pAMfi1) is thought to generate a displaced
strand with a free 5' end, which must be immune to degra-
dation by host enzymes. For plasmid pT181, there is evi-
dence that the 5' end is protected by covalent attachment of
the plasmid-encoded RepC protein (16). Protection of the 5'
free end of HMW generated during replication readily ex-
plains why nuclease inactivation (as in E. coli recBCD sbcB)
need not be imposed for plasmids which replicate via an
ssDNA intermediate. We would expect that insert-induced
HMW formation occurs in E. coli in a similar manner, by
replicons such as 4X174, which utilize a rolling-circle repli-
cation mechanism. This possibility is presently being exam-
ined.
The HMW production can occasionally be detected in a
wild-type plasmid (cf. Fig. 3), but it is greatly enhanced by
certain DNA inserts. It is presently not obvious how an
insert could interfere with the termination of DNA replica-
tion. Different amounts of HMW resulting from different
insertions indicated that DNA "quality" may vary from one
sequence to another. For example, the inserted pBR322
sequences are rich in potential secondary structure and G+C
content compared with the vector plasmids used here. As
DNA is rendered single stranded in the course of replication,
secondary structures are likely to form. Such DNA struc-
tures may exhibit altered binding of host proteins that affect
replication. Following the lead from in vitro experiments (2,
33), one could speculate that regions of DNA with extensive
secondary structure may have decreased binding capacity
for ssDNA-binding protein and that resulting small exposed
hairpin structures could allow nonspecific primosome bind-
ing, which could in turn sterically inhibit the termination of
replication.
DNA secondary structures may have more complex ef-
fects on plasmid replication by acting as pause sites (14, 39).
Pausing of the replication fork possibly could enhance its
susceptibility to displacement and subsequent invasion of a
homologous molecule. If this occurs, replication could con-
tinue on the second molecule by a nonterminated rolling-
circle mechanism to generate HMW of the second plasmid.
Such an event may not have been detectable by our assay for
generalized recombination (see Fig. 4B). A similar recombi-
nation mechanism was proposed for initiation of late repli-
cation of phage T4 (18) and for the generation of plasmid
multimers transduced by B. subtilis phage SPP1 (1). In
addition, plasmid replication was shown to stimulate recom-
bination (23). Possible effects of secondary structure on
HMW formation are currently being examined.
ACKNOWLEDGMENTS
We thank Benedicte Michel for suggestions and critical reading of
the manuscript, Hein te Riele for frequent discussions of the work,
and Marie-Francoise Gros and Laurent Jannibre for providing
plasmid constructions and results prior to publication.
HMW PLASMID MONOMER FORMATION 1189
This work was supported by a fellowship grant from INRA to
A.G. and grants from the Commission of the European Communi-
ties (BAP-0141-F) and the Ministere de la Recherche et de l'En-
seignement Superieur (510070).
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