[ 1 8] R E G U L A T E D P R O M O T E R FOR H I G H - L E V E L EXPRESSION 201

integrate nonreplicative plasmids into the chromosome by homologous

recombination, and a number of vectors have been designed for this
purpose. These integrated plasmids are replicated by the chromosomal
replication machinery, and are very stable.
Expression vectors for secretion of proteins that is based on the B.
amyloliquefaciens alkaline protease (subtilisin) promoter and signal se-
quence are discussed in [18]. These vectors have been successfully used to
express both prokaryotic and eukaryotic proteins. The vectors are reason-
ably simple to use, and constructions can be made in either E. cull or B.
subtilis. This expression system would be appropriate for the expression of
most prokaryotic secreted proteins, and is also worth considering for eu-
karyotic proteins which can be demonstrated to be reasonably stable in the
culture media of the B. subtilis host strain.
A series of vectors which allow the regulated expression of proteins in
B. subtilis is described in [19]. Although primarily designed for intracellu-
lar expression, this system might be also utilized for secreted proteins. I am
not aware of any proteins which have been successfully expressed in B.
subtilis but not in E. coli, and I suspect that E. coli will be the first choice of
most investigators. However, there has not been a detailed comparison of
the two organisms as hosts, and the vectors described here can be used in
either organism, making such comparisons easy to do. This system should
also be useful to researchers working with B. subtilis who wish to overpro-
duce proteins in this organism.
A brief description of two plasmids which can be used in one step to
insertionally inactivate a gene in B. subtilis and place that same gene under
the control of a tightly regulated promoter is presented in [20]. These
plasmids would be of use for studies of gene regulation and expression in B.
subtilis.

[ 18] R e g u l a t e d P r o m o t e r f o r H i g h - L e v e l E x p r e s s i o n o f
Heterologous Genes in Bacillus subtilis
B y S T U A R T F . J. L E G R I C E

Introduction
A desirable feature of prokaryotic vector design is the ability to control
tightly expression of a gene whose product might be detrimental to the
host. In Escherichia coli, the most commonly employed systems have
incorporated transcriptional control mediated through either the lac, i trpfl
H. A. de Boer, L. J. Comstock, and M. Vasser, Proc. NatL Acad. Sci. U.S.A. 80, 21 (1983).
2 B. P. Nichols and C. Yanofsky, this series, Vol. 101, p. 155.

Copyright 1990by AcademicPress,Inc.

METHODS IN ENZYMOLOGY, VOL. 185 All rightsof reproductionin any formreserved.
202 EXPRESSION XN B. subtilis [ 18]

or bacteriophage 2 C1 repressors. 3 When Bacillus subtilis is considered as a

host for high-level expression of heterologous genes, utilization of similar
regulatory elements would likewise be advantageous. Until recently, pro-
gress in this area was hampered by the lack of well-characterized gram-pos-
itive repressible systems. In light of this, an alternative would be to dissect
the control elements of a gram-negative system and optimize these for
utilization in a gram-positive host. This approach was first accomplished
by Yansura and Henner, 4 who demonstrated inducible gene expression in
B. subtilis mediated via modified E. coli lac regulatory elements.
During our preliminary studies with gram-positive expression vectors,
we discovered that promoters of the E. coli bacteriophage T5 were effi-
ciently recognized by the gram-positive transcriptional machinery.5,6 One
such promoter, PN25,7 had previously been fused to a synthetic lac operator
fragment, generating the regulatable promoter PN25/0, and successfully uti-
lized in an E. coli expression system,s We therefore assumed that insertion
of this regulatable promoter and a functional lacI gene into a gram-positive
vector would generate an inducible expression system for B. subtilis. This
article describes two expression systems displaying these features. The first
of these is a single plasmid pREP9 which contains both a regulatable
promoter and a lacI gene modified for expression in B. subtilis. The second
system comprises a B. subtilis strain which constitutively expresses the E.
coli lac repressor from a derivative of the Staphylococcus aureus plasmid
pE 194.9 This latter strain (designated BR: BL1) can be transformed with a
compatible expression vector containing the regulatable promoter. In ei-
ther the single-or dual-plasmid system, gene expression is tightly controlled
and can be rapidly derepressed by addition of the inducer isopropyl-fl-D-
thiogalactopyranoside (IPTG). The usefulness of these systems in high-
level production of heterologous proteins is illustrated in this article. Fur-
thermore, the utilization of the dual-plasmid system to produce an
enzymaticaUy active heterologous protein is exemplified by the production
of human immunodeficiency virus, type 1 (HIV-l) reverse transcriptase in
B. subtilis.

3 M. Rosenberg, Y.-S. Ho, and A. Shatzman, this series, Vol. 101, p. 123.
4 D. G. Yansura and D. J. Henner, Proc. Natl. Acad. Sci. U.S.A. 81,439 (1984).
5 U. Peschke, V. Beuck, H. Bujard, R. Gentz, and S. F. J. Le Grice, J. Mol. Biol. 186, 547
(1985).
6 S. F. J. Le Grice, R. Gentz, U. Peschke, D. Stfiber, V. Beuck, and H. Bujard, in "Bacillus
Molecular Genetics and Biotechnology Applications" (A. T. Ganesan and J. A. Hoch,
eds.), p. 433. Academic Press, New York, 1986.
7 R. Gentz and H. Bujard, J. Bacteriol. 10, 70 (1985).
s D. Stiiber, I. Ibrahimi, D. Cutler, B. Dobberstein, and H. Bujard, E M B O J . 3, 3143 (1984).
9 S. Horinouchi and B. W,eisblum, J. Bacteriol. 150, 804 (1982).
[ 1 8] REGULATED PROMOTER FOR HIGH-LEVEL EXPRESSION 203

Elements of a Lac-Based Inducible System for Bacillus subtilis

Promoter/lac Operator Element

Stiaber et aL s illustrated that the E. coli bacteriophage T5 promoter
PN25, when fused to a synthetic lac operator sequence, allowed inducible
gene expression in E. coli. Our own work with gram-positive promoters
suggested that an AT-rich region in the immediate upstream vicinity of the
canonical - 35 hexamer, as found in the B. subtilis chromosomal veg and
bacteriophage SPO1 promoters, ~ might influence promoter strength. A
similar AT-rich region is present at the same position of E. coli bacterio-
phage T5 promoters: and we subsequently showed that T5 promoters
could be efficiently recognized by the vegetative RVA polymerase of B.
subtilis: ,6 Thus, the T5 promoter/lac operator element Pros/o, previously
designed for high-level gene expression in E. coli, conveniently served as
one component of a lac-based inducible system for B. subtilis. The DNA
sequence and relevant features of this promoter/operator element used in
our vectors are illustrated in Fig. 1.

lac Repressor Functional in Bacillus subtilis

The second component of a lac-based inducible system is a lac repres-
sor gene capable of functioning in B. subtilis. Since neither the lacI pro-
moter nor the ribosome binding site was expected to be utilized in B.
subtilis, it was necessary to replace these with equivalent gram-positive
regulatory elements. This was carried out in a stepwise manner as pre-
sented in Fig. 2. We made use of a l a d gene containing a HindIII site in the
immediate vicinity of the initiator methionine codon to remove the origi-
nal promoter and ribosome binding site. The l a d coding region was ini-
tially placed under the transcriptional control of the relatively weak B.
subtilis promoter Pv,~i)2:3 Subsequently, we introduced a synthetic ribo-
some binding site, adapted for gram-positive utilization, between the veglI
promoter and the initiator methionine of the l a d gene. Finally, this modi-
fied lacI cassette was tailored with a transcriptional terminator (TI) ~4 to

to C. P. Moran III, N. Lang, S. F. J. Le Grice, G. Lee, M. Stephens, A. L. Sonenshein, J. Pero,

and R. L. Losick, Mol. Gen. Genet. 186, 339 (1982).
t t M. D. Barkley and S. Bourgeois, in "The Operon" (J. H. Miller and W. S. Reznikoff, eds.),

p. 177. Cold Spring Harbor Monograph Series, Cold Spring Harbor, New York, 1978.
t2 S. F. J. Le Grice and A. L. Sonenshein, J. Mol. Biol. 162, 551 (1982).
13 S. F. J. Le Grice, C.-C. Shih, F. Whipple, and A. L. Sonenshein, Mol. Gen. Genet. 204, 229
(1986).
t4 j. Brosius, T. J. Doll, D. D. Sleeter, and H. F. Noller, J. Mol. Biol. 148, 107 (1981).
204 EXPRESSION IN B. subtilis [ 18]

A:T BOX -35 -10 OPERATOR

TZATAAAAAATTTATTTG[ TTTCA6GAAAATTTTTCT6TATAATA(3ATTEAAA~T6T6ASC6GATAACAATT TSAA
AGTATTTTTTAAATAAAEGAAASTCCTTTTAAAAAfACATATTATETAAGTTTAACACTCOCETATT(]TTAAACTT
- 50 -40 -30 -20 -10 I +'IO +20

RNA POLYMERASE
BINDING SITE
FIG. 1. DNA sequence and pertinent features of the regulatable E. coli bacteriophage T5
promoter-lac operator element P~25/0 employed in our vectors for gene expression in B.
subtilis. The sequence from - 50 to + 26 is indicated. In addition to the canonical hexamers
around - 1 0 (Pribnow box) and - 3 5 , the A :T box spanning - 3 5 to - 5 0 , which possibly
contributes to the utilization of this E. coli bacteriophage promoter in B. subtilis, is overlined.
A "core" lac operator sequence" (i.e., lac repressor binding site) is fused in the vicinity of the
transcription initiation site.

H
b q 1
H

e ~ [acl

FIG. 2. Stepwise manipulation of the E. coil lacIgene for utilization in B. subtilis. The lacI
gene (a), with a HindIII site (H) preceding the coding region, was separated from its own
promoter (Pro) and ribosome binding site (R) by HindIII digestion (b). In Step c, a fusion was
made between the lacl coding region and the B. subtilis promoter P , ~ . A synthetic ribosome
binding site (SR), functional in B. subtilis, was subsequently added between P,~ot and the lacI
coding region (d). The lacI cassette after d is now functional in B. subtilis. Finally, to restrict
transcription from Pv~, to the lacIcassette, a transcriptional terminator (T) was inserted 3' to
the lacI coding sequence (e).
[ 18] REGULATED PROMOTER FOR HIGH-LEVEL EXPRESSION 205

ensure the transcription from P,~oI would be confined to the lacI cassette.
Following these manipulations, we generated a vector, pREP4, from which
the E. coli lac repressor was constitutively expressed in B. subtilis (data not
shown).

Inducible Expression in Bacillus subtilis from Plasmid p R E P 9

The general features of pREP9, a bifunctional shuttle vector mediating
inducible gene expression in either E. coli or B. subtilis, are outlined in Fig.
3A. In the major expression cassette, contained between the XhoI (X)
and XbaI (Xb) sites, the E. coli chloramphenicol acetyltransferase (caO
gene ~5has been connected, via a multicloning site, to a synthetic ribosome
binding site (px). Transcription of this gene is mediated by the promoter/
operator element Pros/0 (P/O). Since the same plasmid constitutively syn-
thesizes the lac repressor, transcription from Pr~25 is repressed in the ab-
sence of IPTG. A transcriptional terminator follows the cat gene to ensure
that high-level transcription from Pro5 does not proceed into the plasmid
replication region, since this might have a deleterious effect on stability.
Controlled CAT expression in B. subtilis from pREP9 can be achieved as
follows.
1. Transformation: Plasmid pREP9 is introduced into competent cul-
tures of B. subtilis BRI51 (trp, met, lys) according to the method of
Contente and Dubnau? 6 Although we predominantly use this method,
protoplast transformation |7 or electroporationts can also be employed.
Recombinant clones are selected on LB agar plates containing 10/zg/ml
kanamycin at 37 .
2. Induction: Recombinant clones containing pREP9 are grown, with
vigorous shaking, in LB medium containing 10 #g/ml kanamycin to mid-
logarithmic phase (A600 -- 0.8). Induction of protein synthesis is accom-
plished by adding IPTG (from a stock solution of 100 mg/ml) to a final
concentration of 400/zg/ml. Samples are removed over the next 4 hr for
analysis.
3. Sample preparation: 200 #1 of culture is removed and centrifuged at
room temperature 12,000 rpm for 1 min. The cell pellets are resuspended
in 20 gl of 15% (w/v) sucrose in 50 m M Tris-HC1, pH 7.2. Four microliters
ofa 5 mg/ml lysozyme solution (in 0.25 M Tris-HC1, pH 7.2) is added, and

m5R. Marcoli, S. Iida, and T. A. Biclde, FEBSLett. 110, 11 (1980).

t6 S. Contente and D. Dubnau, Mol. Gen. Genet. 167, 251 (1979).
m7S. Chang and S. N. Cohen, Mol. Gen. Genet. 168, 111 (1979).
n N. M. Calvin and P. C. Hanawalt, J. Bacteriol. 170, 2796 (1988).
m9H. Towbin, T. Staehelin, and J. Gordon, Proc. Natl. Acad. Sci. U.S.A. 76, 4350 (1979).
 k a n ~ T1

Rx .I
AAGAGGAGAAATTAACTAT~IGAG'IGGG~ T['--[
6 T~ GA~ [T~ [A6 CCA/~G[ TTA
Ba.imm
HI Sa___I]l H.~i nI!d
Ps.__._fI

STAINED GEL IMMUNOLOI3 ICAL ANALYSIS

M 1 2 3 4 5 6 ? M 1 2 3 4 5 6 7
97-
66-

45-
-[AT

26- -CAT

14-
FIG. 3. (A) Structure of the bifunctional shuttle vector pREP9, determining regulatable
gene expression in B. subtilis. In the lacI expression cassette [contained within the EcoRI (E)
and XhoI (X) sites], P is the vegII promoter previously described; in the expression cassette
[contained within the XhoI (X) and XbaI (Xb) sites], P/O is the regulatable promoter-
operator dement PN25/O" Both promoters are linked to synthetic ribosome binding sites
(R,R'). The synthetic ribosome binding site R" is followed by a multicloning site for the
restriction endonucleases BamHI (B), SalI (S), PstI (P), and HindIII (H); the DNA sequence
below the plasmid diagram illustrates the reading frame from the ribosome binding site R x
through the multicloning site and is delineated into triplet codons. Gram-positive and -nega-
tive origins of replication are depicted ori+ and ori-, respectively. Genes conferring resist-
ance to kanamycin (pUBl l0 derived) and chloramphenicol (IF.. coli derived mS) are abbre-
viated kan and cat, respectively. Both the lacI and cat genes are flanked by the transcriptional
terminator T1. '4 Additional unique restriction sites in the vector are abbreviated as follows:
Pf, PflMI; Sc, ScaI; K, KpnI (Asp718); Bg, BglII. (B) Regulatable expression of the E. coli cat
gene in B. subtilis strain BR 151 containing plasmid pREP9. In the stained gel analysis, lane l
represents a cell lysate prior to addition of IPTG (i.e., preinduction). Following addition of
IPTG, samples were withdrawn for analysis after 15, 30, 45, 60, 120, and 180 min (lanes 2-7,
[ 18] R E G U L A T E D P R O M O T E R FOR H I G H - L E V E L E X P R E S S I O N 207

the suspension incubated at 37 for 5 min. Thirty six microliters of S D S -

polyacrylamide gel sample buffer is added, and the solution is heated for 10
min at 100 .
4. Electrophoresis: 10 ~1 of the cell lysate is fractionated through dis-
continuous 12.5 (w/v) SDS-polyacrylamide gels containing a 3.3% (w/v)
stacking gel. Following electrophoresis, the gels are stained and destained
according to standard procedures.
The results of such an experiment with pREP9 are presented in Fig. 3B.
Prior to addition of IPTG, no CAT protein was observed on either a
stained gel or by an immunological assay with polyclonal antibodies
against CAT, which is at least an order of magnitude more sensitive than
detection by Coomassie blue staining. The latter result illustrates clearly
that, in the presence of lac repressor, transcription from Pro5 is abolished.
Addition of IPTG to the culture results in immediate derepression and
high-level CAT production. After 2 - 3 hr of induction, CAT represents the
major cellular protein, and levels approaching 15% of the total cell protein
can be achieved.

Introduction of Foreign Genes into pREP9

In pREP9, the E. coli CAT protein is translated from an efficient
synthetic ribosome binding site. A multicloning site in the immediate
downstream vicinity of the initiator methionine residue (Fig. 3A) allows
cloning of entire or fragmented genes. By addition of correctly sized molec-
ular linkers, such genes or gene fragments can be expressed as amino-ter-
minal fusions to CAT, allowing the possibility of detecting the fusion
protein with CAT antibodies. Alternatively, if antibodies to the desired
gene product are available, inserted DNA fragments may be so tailored to
contain a translational stop codon. Since pREP9 is a bifunctional vector, it
can be transformed into an E. coli host, from which large amounts of
starting material for genetic manipulation can be rapidly isolated. Finally,
it is worthwhile making a comment on utilization of the polylinker site of
pREP9. Many researchers attempt to increase the versatility of their ex-
pression systems by adding new unique restriction sites to a polylinker.
However, it is also possible to enhance the versatility of a multiple cloning

respectively).M, Molecularweightmarkers(X 10-3). In the immunologicalanalysis,the same

samples were similarly fractionated by SDS-polyacrylamidegel electrophoresis,then trans-
ferred to nitrocellulose by the method of H. Towbin et al.19Immunological detection was
accomplished using a polyclonalantibodyto chloramphenicol acetyltransferase(CAT) and a
colorimetric assay. Lanes are denoted as in the stained gel. In both the stained gel and
immunological analysis, the migration position of CAT is indicated.
208 EXPRESSION IN B. subtilis [ 18]

site simply by making use of compatible cohesive ends shared by two or

more restriction endonucleases. As an example, the cohesive end produced
by BamHI digestion of pREP9 is compatible with those produced by MboI,
BgllI, or BclI digestion. Similarly, DNA fragments produced by XhoI
digestion can be introduced at the Sail site, and those by Nst] digestion at
the PstI site. Thus, without increasing the length of the polylinker region,
sites for eight different restriction endonucleases can be utilized.

Dual-Plasmid Repressible System for Bacillus subtilis

Although the single plasmid repressible system afforded by pREP9
functions in B. subtilis, we were concerned that, at the time of entry of the
recombinant plasmid into competent cells, RNA polymerase molecules
might recognize the strong promoter PN25 in preference to P,~]. Under
such circumstances, a situation of transient derepression is possible and
could conceivably have a destabilizing effect. One means of overcoming
this problem would be to develop a strain of B. subtilis which constitu-
tively expressed the lac repressor, and use this as recipient for expression
plasmids containing the regulatable promoter.
The S. aureus plasmid pE194, 9 conferring erythromycin resistance, is
capable of replicating in B. subtilis. We elected to transfer the l a d cassette
of plasmid pREP9 into pE194. Previous reports showed that DNA might
be inserted at the unique PstI site of pE 194 without disturbing either the
replication or erythromycin resistance functions. We therefore excised the
lacI cassette from pREP9 by EcoRI and XhoI digestion, converted the
cohesive ends to blunt ends with DNA polymerase Klenow fragment,
added PstI linkers, and inserted this fragment into the PstI site of pE 194.
The resulting plasmid, designated pBL1 (Fig. 4A), was transformed into B.
subtilis, selecting at 32* (due to the temperature-sensitive replicon of
pE 1942) on 10 #g/ml erythromycin. By this approach, we generated the
recombinant B. subtilis strain BR: BL 1, which constitutively expressed lac
repressor. Since the pE194 replicon contained pBLI and the pUB110
replicon2~ on our shuttle vectors were compatible, it was possible to trans-
form competent BR: BL 1 with any pUB 110-based plasmid containing the
regulatable 1~25/o element, selecting for recombinant clones on 10/tg/ml
erythromycin (encoded on pBL1) and kanamycin (encoded on the
pUB110-based vector) at 32*. As an example, we transformed BR:BL1
with the shuttle vector p602/20, generating the strain BR:BL 1- p602/20
(Fig. 4A). Within p602/20, the E. coli cat gene is replaced with a gene

20 j. Scheer-Abramowitz, T. Gryczan, and D. Dubnau, Plasmid 6, 67 (1981).

2~ S. D. Ehdich, Proc. Natl. Acad. Sci. U.S.A. 74, 1680 (1977).
[ 18] REGULATED PROMOTER FOR HIGH-LEVEL EXPRESSION 209

A P/O R"

h f__r

P ~ __
tacI

cry

M123456

FIG. 4. (A) Dual-plasmid system, pBLI-p602/20, determining inducible synthesis of

mouse DHFR in B. subtilis. Construction of the lacI-contalning plasmid pBLl has been
outlined in the text. In p602/20, the reading frame at the BamHI site connecting the synthetic
ribosome binding site (R ~) to the dhfr coding sequence is the same as for plasmid pREP9, i.e.,
-G-G-A-T-C-C-. Notations and restriction site abbreviations are as in the legend to Fig. 3A.
An additional transcription terminator, to [M. Rosenberg, B. De Crombrugghe, and R.
Musso, Proc. Natl. Acad. Sci. U.S.A. 73, 717 0976)], is present on p602/20 between the dhfr
and cat genes. On pBLI, ery is the erythromycin resistance gene. (B) Inducible DHFR
synthesis in the recombinant B. subtilis strain BR: BLI -p602/20. The induction experiment
was performed as described for pREP9, with the exception that the culture was grown at 32
in L broth containing l0 #g/ml of both erythromycin and kanamycin. Time points 1-5, at
which samples were withdrawn for analysis, represent 0 (i.e., preinduction), 30, 60, 120, and
180 min, respectively. Lane 6 is a lysate of cells following prolonged growth in the absence of
IPTG. The migration position of DHFR is indicated.

coding for mouse dihydrofolate reductase (dhfr22). The procedures for

transformation and analysis of recombinant clones are similar to those
previously described, with the following exceptions: (l) Recipient cells
were competent BR:BL1, which were prepared at 32 in the presence of
l0 gg/ml erythromycin. (2) Following introduction of p602/20, selection
22j. H. Nunberg, R. J. Kaufman, A. C. Y. Chang, S. N. Cohen, and R. T. Schimke, Cell 19,
355 (1980).
210 EXPRESSION IN B. subtilis [ 18]

of recombinant clones was at 32 on 10 #g/ml of both kanamycin and

erythromycin.
Figure 4B illustrates inducible dihydrofolate reductase (DHFR) expres-
sion in the B. subtilis strain BR:BL1 -p602/20. Prior to addition oflPTG,
we observed no DHFR on a stained gel. Addition of IPTG led to rapid
accumulation of DHFR, estimated to be in excess of 10% of the total
cellular protein. Figure 4B also shows DHFR levels in BR:BL 1 -p602/20
following prolonged growth in the absence of IPTG (Fig. 4B, lane 6),
demonstrating that, also in the dual-plasmid system, the PN25/o promoter
element is very tightly regulated in the absence of IPTG.
As indicated in Fig. 4A, a BamHI site lies between the initiator methio-
nine residue and the coding region of dhfr. The reading frame within this
BamHI site is as in pREP9 (i.e., GGA-TCC); thus, by addition of appropri-
ately sized BamHI, BgllI or Bcll linkers to the DNA clone in question, it is
possible to fuse a foreign coding sequence to DHFR. Alternatively, if the
foreign coding sequence provides its own translational stop codon, it can
be expressed individually.

Regulated Expression of Enzymatically Active R e v e r s e

Transcriptase in Bacillus subtilis
To determine whether enzymatically active heterologous protein could
be produced in B. subtilis from our expression systems, we used the
dual-plasmid system to express the HIV-1 reverse transcriptase. As illus-
trated in Fig. 5A, the major portion of the HIV-I polymerase (pol) open
reading frame2a was introduced as a translational fusion to the E. coli cat
gene. This pol fragment encodes a protease, reverse transcriptase, and a
truncated endonuclease (here as a CAT fusion). We used plasmid p602/18
as the vector, which does not contain the lad gene now supplied by
plasmid pBL1. The pol-encoding shuttle vector, pRTL11 (Fig. 5A), was
introduced into strain BR:BLI, selecting for transformants at 32 on LB
agar containing 10 gg/ml erythromycin and kanamycin. When a culture of
the recombinant clone B R : B L I - p R T L 1 1 was induced with IPTG and
immunoreactive polypeptides in the cell lysate analyzed with a pool o f
HIV-1 positive sera, 24 we observed accumulation of a high-molecular-
weight polypeptide (Fig. 5B), indicative of the pol-encoded precursor poly-

23 L. Ratner, W. Haseltine, R. Patarca, K. J. Livak, B. Starich, S. F. Josephs, E. R. Doran,

J. A. Rafalski, E. A. Whitehorn, K. Baumeister, L. Ivanoff, S. R. Petteway, Jr., M. L.
Pearson, J. A. Leutenberger, T. S. Papas, J. Ghrayeb, N. T. Chang, R. C. Gallo, and F.
Wong-Staal, Nature (London) 313, 277 (1985).
24 U. Certa, W. Bannwarth, D. Stiiber, R. Gentz, M. Lanzer, S. F. J. Le Gdce, F. Guillot, I.
Wendler, G. Hunsmann, H. Bujard, and J. Mous, EMBO J. 5, 3051 (1986).
[ 18] REGULATED PROMOTER FOR HIGH-LEVEL EXPRESSION 211

protein. After prolonged induction, the precursor polyprotein disappeared,

and was replaced by polypeptides of molecular mass 66 and 51 kDA. Since
these latter polypeptides correspond in size to the expected size of the
HIV-1 reverse transcriptase polypeptides, the results presented in Fig. 5B
suggest that the HIV-1 pol polyprotein is properly processed in B. subtilis.
Once again, no immunoreactive polypeptides were observed prior to IPTG
induction, showing tight regulation of the N25 promoter. Using a rapid
purification method outlined in more detail elsewhere,2s we isolated HIV-l
reverse transcriptase from strain B R : B L 1 - p R T L 1 1 . The following steps
were used in the purification: (1) lysozyme-Triton X-100 lysis at l0 , (2)
high-speed centrifugation to remove cell debris, (3) overnight dialysis of the
soluble fraction, (4) elimination of nucleic acids by streptomycin sulfate
precipitation, and (5) ion-exchange chromatography on DEAE-Sephacel.
Figure 5C illustrates that, under these conditions, the p66 and p51
reverse transcriptase polypeptides did not bind to the DEAE-Sephacel,
providing a very convenient means of partial purification (approximately
90% of the soluble protein is retained on the column). Finally, Fig. 5D
shows that the partially purified enzyme preparation displayed high levels
of reverse transcriptase activity.

Inducible Bacillus subtilis System E m p l o y i n g Integrated C o p y of

lacI Gene
Although both the single- and dual-plasmid-inducible systems work
well in B. subtilis, each system has disadvantages. For example, pREP9 is
somewhat large (7.3 kb), and increasing its size via insertion of foreign
DNA could affect plasmid stability. In the dual-plasmid system, the ex-
pression vector is smaller, but recombinant clones must be grown at 32 .
Integration of the lacI gene into the B. subtilis chromosome would over-
come both problems. One consideration in following this approach is to
ensure sufficient levels of lac repressor from an integrated gene. In prelimi-
nary experiments, we have constructed a lacI cassette with a stronger
transcriptional signal. This cassette on a multicopy plasmid produces lac
repressor levels in B. subtilis approaching 5-10% of the total cellular
protein. This cassette was introduced into the B. subtilis chromosome via a
bacteriophage ~b105 cloning vehicle recently constructed by Errington et
aL 26 Bacillus subtilis containing the integrated lacI gene can be trans-
formed with plasmid p602/20 described earlier and again displays induci-
ble DHFR synthesis. (S. F. J. Le Grice, unpublished data).
2s S. F. J. Le Grice, V. Beuck, and J. Mous, Gene 55, 95 (1987).
26 j. Errington, in "Bacillus Molecular Biology and Biotechnology Applications" (A. T.
Ganesan and J. A. Hoch, eds.), p. 217. Academic Press, New York, 1986.
 A

+(\ +++ t
B E
M 2 3 z, 5 6 7 8 9 I 2 3
20O-~:i~+i~i+++++~++~i;+i,++~
i+;,++~++~+~
i:,:~i+i+~'++
,'++
!+!:+ ;i+~~iiii!ii~i~ii!i!i!iiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiii
,'i,'++i+++++ i+
,:i++i~i+i,+~
!i+~ii,+i+~
~i~ii~iiiiiiiiiiiiiiiiiCiiiii i+i++i++ii+iii+i++ii i i i i ++
+ =: ~ ":i=: "~"'::iiiiiiii!ii~iiiiiiii- PP
~ 'iil~!i!i~iii~p66.
-
i ~ i+i+i+i+ ~-p++;~+
i!i!ii~iiii~i! ~i~i~i~!ii~i!~!ii!i~i!,iii!i~~i:-
iiil p 51
4 s-++iii+++i
++i+++++++i
+++++
++++++N!
+ ~++++i+i+++i++!++ii+i iii+i+i~iiiii

20"
Z
O

rY
0 QJ
~ 10

F--

o'.s 11o I'.s o

SAMPLE VOLUME (IJ[)
Fro. 5. (A) Dual-plasmid repressible system, pBL1 -pRTL11, for expression of a trun-
cated HIV-1 pol open reading frame in B. subtilis. Plasmid pBL1 is as before. On plasmid
pRTLI 1, a BglII-XmnI pol fragment was cloned into a related expression vector, p602/19.
The cloning translationally fused the pol endonuclease portion to the E. coli CAT protein
(ENDO/CAT). The other pol-encoded proteins, protease and reverse transcriptase, are abbre-
viated PROT and RT, respectively. (B) Induction of reverse transcriptase from the HIV-I pol
precursor polyprotein in a culture of the B. subtilis strain BR: B L I - p R T L I 1. The culture
was grown and induced at 32 in a similar manner to the BR: BLI -I)602/20 system. Lane I
is the preinduction sample. Lanes 2 - 8 represent samples removed after 15, 30, 45, 60, 120,
180, and 240 min, respectively. An immunological analysis is presented, wherein the source
[ 18] REGULATED PROMOTER FOR HIGH-LEVEL EXPRESSION 2 13

Construction of Inducible Secretion Vectors

Although another section of this volume will deal with secretion vectors
for B. subtilis in considerably more detail, it is nonetheless worthwhile to
mention that the regulatable system outlined here can also be modified to
secrete foreign proteins. In both the single- and dual-plasmid systems
described above we have inserted a cassette containing the Bacillus amylo-
liquifaciens a-amylase signal peptide z7 linked to the mature portion of the
E. coli fl-lactamase gene.28 When cultures of B. subtilis containing the
recombinant plasmids are grown in a minimal medium and induced dur-
ing logarithmic growth, we observe accumulation of mature fl-lactamase in
the culture supernatant.

Concluding R e m a r k s
Rather than give explicit details of vector construction, I have at-
tempted to give a brief description of the systems available, together with
their application. The single- and dual-plasmid systems presented here
have been constructed so that entire expression cassettes can be mobilized.
For example, the modified lacI cassette can be removed from pREP9 by
EcoRI and XhoI digestion, or from the plasmid pBL1 by PstI digestion.
Similarly, the cassette conferring inducible CAT expression can be excised
from pREP9 by XhoI and XbaI digestion. This flexibility in the expression
vectors should permit those interested to modify further or produce their
own gram-positive expression systems. This could be especially worthwhile
with the lacI cassette, which could be stably integrated into the B. subtilis
chromosome in an alternative manner to that described here.
27 I. Palva, R. F. Petterson, N. Kalkkinen, P. Lehtovaara, M. Servas, H. S6derlund, K.
Takkinen, and L. K~iari~inen, Gene 15, 43 (1981).
2s j. T. Kadonaga, P. E. Gautier, D. R. Strauss, A. D. Charles, M. D. Edge, and J. R. Knowles,
J. Biol. Chem 259, 2149 (1984).
29 p. Dhaese, C. Hussey, and M. Van Montagu, Gene32, 181 (1984).
30 M. S. Osbourne, R. J. Craig, and D. Rothstein, J. Bacteriol. 163, 1101 (1985).

of antibody was a pool of HIV-1-positive sera. PP refers to the pol precursor polyprotein.
Lane 9 contains a sample of purified p66/p51 HIV-I reverse transcriptase (RT). (C) Partial
purification of HIV- l reverse transcriptase, produced from the B. subtilis recombinant strain
BR: BL 1 - pRTL 11 by DEAE-Sephacel ion-exchange chromatography. An immunological
analysis is presented, using again pooled HIV-l-positive sera as antibody source. Lane l,
DEAE-Sephacel flow-through material (i.e., non-DEAE-Sephaeel-binding proteins); lane 2,
proteins eluted with a buffer containing 0.2 M NaCl; lane 3, purified p66/p51 HIV-I reverse
transcriptase. (D) Activity profile for HIV-I reverse transcriptase isolated from B. subtilis
strain BR: BLI-pRTL l 1. The assay here, performed on the DEAE-Sephacel-purified enzyme,
is based on the ability of reverse transcriptase to incorporate [32p]dGTP into polynucleotide,
using a poly(rC)oligo(dG) template-primer system. 25
214 EXPRESSIONIN B. subtilis [ 19]

In the systems presented, foreign genes are expressed during logarith-

mic growth, taking advantage of the observation that the E. coli bacterio-
phage T5 promoter Pro5 is recognized by the vegetative (tr55) RNA poly-
merase of B. subtilis. Since there is rapid interchange of a factors in B.
subtilis at later growth stages, 3t it is unlikely that PN2Sand P,~aI are recog-
nized by the alternative forms of RNA polymerase. If it were necessary to
produce foreign proteins at later growth stages, the system described here
could be modified by replacing the vegII and N25 promoters with counter-
parts utilized by other forms of B. subtilis RNA polymerase. Alternatively,
these growth-stage-specific promoters could be placed in the immediate 5'
vicinity of Pv~i and Pms/o. Such a "twin" promoter would ensure constant
synthesis of lac repressor. Furthermore, Deutchle et al. have recently dem-
onstrated that the lac operator sequence can be separated from the pro-
moter and still prevent RNA polymerase from entering into productive
transcription. 32 Thus, transcription from a growth-stage-specific promoter
placed 5' to the Pros/0 element would still be controlled by the lac operator.
The plasmids and strains described in this article, together with com-
plete nucleotide sequence and restriction maps, can be made available
upon request.

Acknowledgments
I wish to thank Ursula Peschke and Verena Beuck for their assistance in early expression
vector studies. The gifts of various expression cassettes from D. Stuber, R. G. Gentz, and J.
Knowles are also acknowledged, as well as helpful comments on the manuscript from Jan
Mous and Oktavian Shatz. Finally, I wish to thank Professor H. Bujard, whose excellent work
on T5 promoters and belief in B. subtilis were instrumental in generation of the early
expression systems.

3mR. Losick and J. Pero, Cell 25, 585 (198 I).

32 U. Deutchle, R. Gentz, and H. Bujard, Proc. Natl. Acad. Sci. U.S.A. 83, 4134 (1986).

[19] S y s t e m f o r S e c r e t i o n o f H e t e r o l o g o u s P r o t e i n s in
Bacillus subtilis
B y VASANTHA N A G A R A J A N

Introduction
Secretion of heterologous proteins from Bacillus subtilis has several
attractive properties. The secreted protein is usually soluble and active.
Most importantly, secreted proteins from B. subtilis are located in the
growth medium, in contrast to Escherichia coli, where the majority of

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