Vol. 173, No. 17JOURNAL OF BACTERIOLOGY, Sept. 1991, p. 5280-5289
0021-9193/91/175280-10$02.00/0
Copyright © 1991, American Society for Microbiology
Characterization and Comparative Sequence Analysis of Replication
Origins from Three Large Bacillus thuringiensis Plasmids
JAMES A. BAUM* AND M. PEARCE GILBERT
Ecogen Inc., 2005 Cabot Boulevard West, Langhorne, Pennsylvania 19047-1810
Received 27 February 1991/Accepted 24 June 1991
The replication origins of three large Bacillus thuringiensis plasmids, derived from B. thuringiensis HD263
subsp. kurstaki, have been cloned in Escheyichia coli and sequenced. The replication origins, designated oni 43,
oni 44, and ori 60, were isolated from plasmids of 43, 44, and 60 MDa, respectively. Each cloned replication
origin exhibits incompatibility with the resident B. thuyingiensis plasmid from which it was derived.
Recombinant plasmids containing the three replication origins varied in their ability to transform strains of B.
thuringiensis, Bacillus megaterium, and BaciUus subtilis. Analysis of the derived nucleotide and amino acid
sequences indicates that the replication origins are nonhomologous, implying independent derivations. No
significant homology was found to published sequences of replication origins derived from the single-stranded
DNA plasmids of gram-positive bacteria, and shuttle vectors containing the three replication origins do not
appear to generate single-stranded DNA intermediates in B. thuringiensis. The replication origin regions of the
large plasmids are each characterized by a single open reading frame whose product is essential for replication
in B. thuringiensis. The putative replication protein of ori 60 exhibits partial homology to the RepA protein of
the Bacillus stearothermophilus plasmid pTB19. The putative replication protein of ori 43 exhibits weak but
extensive homology to the replication proteins of several streptococcal plasmids, including the open reading
frame E replication protein of the conjugative plasmid pAMI31. The nucleotide sequence of oni 44 and the
amino acid sequence of its putative replication protein appear to be nonhomologous to other published
replication origin sequences.
The gram-positive soil bacterium Bacillus thuringiensis is
well known for its insecticidal activity against a variety of
lepidopteran, dipteran, and coleopteran pests (17). The
entomocidal activity of this bacterium is due to a family of
insecticidal crystal proteins (ICPs), often referred to as
delta-endotoxins, that exert their effect after ingestion and
subsequent solubilization in the insect midgut (23). The ICPs
are encoded by genes that are typically located on large
(>30-MDa) plasmids (7, 19) but are believed to be found
occasionally on the chromosome (22). Numerous ICP genes
have been cloned, and their encoded products have been
assessed for insecticidal activity (17). An efficient transfor-
mation system that uses electroporation has been developed
for B. thuringiensis (3, 21, 25, 27, 33), stimulating interest in
the regulation of ICP gene expression and the genetic
manipulation of insecticidal activity.
Central to this research effort has been the development of
stable cloning vectors for B. thuringiensis, some of which
employ replication origins derived from resident B. thurin-
giensis plasmids (1, 21). Previously, we described the clon-
ing of seven replication origins derived from resident plas-
mids of B. thuringiensis HD263 and HD73 (subsp. kurstaki)
and the subsequent construction of shuttle vectors based on
replication origins ori 43, ori 44, and ori 60, derived from the
43-, 44-, and 60-MDa plasmids, respectively, of strain
HD263 (1). These and similar cloning vectors can be useful
tools for studying the regulation of ICP gene expression and
the molecular basis of insecticidal activity in B. thuringien-
sis. Moreover, the cloned replication origins provide an
opportunity to study the evolution and distribution of plas-
mid incompatibility groups in B. thuringiensis, the mecha-
nism(s) of plasmid replication and copy number control in B.
* Corresponding author.
thuringiensis, and the relatedness of these replication origins
to those derived from plasmids of other gram-positive bac-
teria. In this report, we describe our initial characterization
of these three plasmid replication origins.
MATERIALS AND METHODS
Bacterial strains and plasmids. Escherichia coli TG1 was
used as the host strain for plasmid and M13 phage propaga-
tion (Amersham Corp.). Table 1 describes the plasmids and
Bacillus strains used. The B. thuringiensis cloning vectors
pEG597, pEG853, and pEG854 (1) were maintained in E. coli
GM2163 (kindly provided by New England BioLabs, Inc.).
B. thuringiensis HD73 and HD263 subsp. kurstaki were
obtained from the collection of Dulmage (11). B. thuringien-
sis HD263-6 is a cured derivative of strain HD263 that lacks
a 44-MDa ICP-encoding plasmid; strain HD263-8 is a cured
derivative of HD263 that lacks ICP-encoding plasmids of 110
and 44 MDa (13). Acrystalliferous (Cry-) B. thuringiensis
HD73-26 is a cured derivative of strain HD73 (13).
DNA sequence analysis. The replication origin fragments
derived from the 43-, 44-, and 60-MDa plasmids of B.
thuringiensis HD263 are contained on plasmids pEG599,
pEG851, and pEG588-14a, respectively (Fig. 1) (1). These
replication origin fragments were subcloned into the M13
vectors mpl8 and mpl9 for DNA sequence analysis. Single-
stranded DNA (ssDNA) templates were sequenced by the
dideoxy-chain termination method (32), using [a-35S]dATP
and the Sequenase DNA sequencing kit (U.S. Biochemical
Corp.). Sequencing primers were synthesized on an Applied
Biosystems 380B DNA synthesizer. Computer-assisted anal-
yses were performed with the Beckman Microgenie pro-
grams (SciSoft, Inc.) for DNA and protein sequence analysis
and the FASTA search program (31).
DNA manipulations. Standard recombinant DNA proce-
5280

REPLICATION ORIGINS FROM B. THURINGIENSIS PLASMIDS 5281
TABLE 1. Bacillus strains and plasmids
Strain or Plasmids Relevent Reference or
plasmid (MDa) characteristics source
B. thuringiensis
HD263 130, 110,a 60,a 11
44a 43, 7.5,
5.4, 5.2, 5.0,
4.9
HD263-6 130, 110,a 60a 13
43, 7.5, 5.2,
5.0, 4.9
HD263-8 130, 60,a 43, 7.5, 13
5.2, 4.9
HD263-8 A60 130, 43, 7.5, 5.2, This report
4.9
HD263-8 A43 130, 60,a 7.5, 5.2, This report
4.9
HD73-26-10 44,a 4.9 13
HD73-26 4.9 13
B. cereus BC569-6 9
B. subtilis BD170 10
B. megaterium 36
VT1660
Plasmids
pEG851 ori 44 1
pEG588-14a ori 60 1
pEG599 ori43 1
pEG597 ori 44 1
pEG853 ori 60 1
pEG854 ori43 1
pEG147 pUC18/pBC16 37
a Contains an ICP gene(s).
dures were performed essentially as described by Maniatis et
al. (26). Restriction endonuclease cleavages leaving 5' or 3'
overhangs were rendered blunt with T4 polymerase (Bio-
Rad Corp.). DNAs were dephosphorylated with calf intesti-
nal alkaline phosphatase (Boehringer Mannheim Corp.).
Plasmids from E. coli were prepared by the small-scale
alkaline lysis procedure of Maniatis et al. (26). B. thurin-
giensis plasmids were resolved on vertical 0.52% agarose
gels, using a modified Eckhardt lysis procedure (14).
Transformation procedures. Transformation of B. thurin-
giensis and Bacillus cereus was performed according to the
electroporation protocol described by Mettus and Macaluso
(28). B. subtilis was transformed according to the procedure
of Gryczan et al. (16), and Bacillus megaterium was trans-
formed according to the procedure of Von Tersch and
Robbins (36). All transformations were performed with
plasmid DNAs prepared from the Dam- Dcm- E. coli host
strain GM2163. Plasmid DNAs prepared from strain
GM2163 are not methylated by the dam and dcm methylases
and can be used to efficiently transform strains of B. thurin-
giensis (24) and B. cereus (2).
Nucleotide sequence accession numbers. The nucleotide
sequences were submitted to GenBank and assigned acces-
sion numbers M60465 (ori 44), M60475 (ori 60), and M60513
(ori 43).
RESULTS
DNA sequence analysis of the replication origins. The
resident plasmids from which ori 43, ori 44, and ori 60 were
isolated differ with respect to their transferability and their
association with ICP genes. The 43-MDa plasmid of strain
HD263 is transmissible (Tra+) by conjugation but is Cry-
1 kb
cat S ori " plac amp f
.41
pEG851
pEG588-14a
pEG599
E Sp Nd A 9H pTZl9u E
cat SB cr1 60 t pPa
E Sp E Nd H XbS PSpH E
plac 4amp tfBg cr143 Sp, cat
.-
EpTZl8u EE-EHSp PS Xb E E XGBSmKStESmBS
FIG. 1. Linear restriction maps of replication origin clones. The
replication origin fragments ori 43, ori 44, and ori 60 are contained
on an E. coli vector consisting of either pTZ18u or pTZ19u and a cat
gene (1). The maps are opened at the EcoRI site (E) between the
pTZ18u/pTZ19u (light-shaded boxes) and cat gene (solid boxes)
segments. The replication origin regions are designated by open
boxes. Notation: plac, lacZ promoter; f, fl phage replication origin;
amp, P-lactamase gene; cat, chloramphenicol acetyltransferase
gene. Restriction sites: A, AccI; B, BamHI; Bg, BgIII; E, EcoRI; H,
HindIII; K, KpnI; Nd, NdeI; P, PstI; S, Sall; Sm, SmaI; Sp, SphI;
St, SstI; Xb, XbaI.
(lacks an ICP gene). In contrast, the 44-MDa plasmid is both
Tra+ and Cry', while the 60-MDa plasmid is Cry' but
appears to be Tra- (13). The replication origin regions from
these large plasmids were sequenced to provide further
information on their structural organization and relatedness.
Replication origin ori 44 was subcloned into mpl8 and mpl9
as a 2.3-kb HindIII-SalI fragment isolated from pEG851
(Fig. 1). Replication origin ori 60 was subcloned into mpl8 in
both orientations as a 2.3-kb Sall fragment isolated from
pEG588-14a (Fig. 1). Replication origin ori 43 was subcloned
into mpl8 in both orientations as a 2.8-kb XbaI fragment
isolated from pEG599 (Fig. 1). Sequence analysis was initi-
ated by using the M13 universal primer. Subsequently,
sequencing primers were synthesized as needed based on the
derived nucleotide sequences. The complete nucleotide se-
quences of the DNA fragments harboring the replication
origin regions are shown in Fig. 2 to 4.
The replication origin from the 44-MDa plasmid, ori 44
(Fig. 2), is contained on a 2,249-bp DNA fragment charac-
terized by an open reading frame (ORF) of 936 bp (312 amino
acids) and an overall A+T content of 71.4%. The ORF starts
at nucleotide (nt) 797 with a GUG as the probable initiation
codon. A Shine-Dalgarno sequence, GGAGGT, is posi-
tioned 6 nt upstream of the initiation codon. The putative
gene product has a molecular weight of 36,478. The se-
quence also contains segments of A+T-rich DNA (>85%
A+T) which lie outside the ORF (nt 483 to 569, 683 to 728,
and 2018 to 2111). An inverted repeat sequence, designated
a, spans the putative Shine-Dalgarno sequence (Fig. 2). A
direct repeat sequence, designated b, overlaps a portion of
the sequence element a. The ORF is followed by an inverted
repeat sequence (designated c) that could serve as a tran-
scriptional terminator (AG' = -18.0 kcal [ca. -75 kJ]/mol).
The replication origin from the 60-MDa plasmid, ori 60
(Fig. 3), is contained on a 2,290 bp DNA fragment charac-
terized by an ORF of 1,218 bp (406 amino acids) and an
VOL. 173, 1991