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Proc. Nati. Acad. Sci. USA
Vol. 81, pp. 439-443, January 1984 Biochemistry
Use of the Escherichia coli lac repressor and operator to control
gene expression in Bacillus subtilis
(hybrid promoter/isopropyl 13-D-thiogalactoside induction)
DANIEL G. YANSURA*^ AND^ DENNIS J. HENNERt
Departments of *Vaccine Development and^ tMolecular^ Biology, Genentech, 460 Point San Bruno^ Boulevard,^ South San Francisco, CA 94080
Communicated by Mark^ Ptashne, October^ 11, 1983
ABSTRACT The Escherichia coli lac operator has been
placed on^ the^ 3'^ side of^ the promoter for the^ penicillinase gene
of Bacillus licheniformis, creating a^ hybrid promoter control-
lable by the E.^ coli lac repressor. The E.^ coli^ lac^ repressor gene
has been placed under the^ control of^ a^ promoter and ribosome-
binding site that allows expression in Bacillus subtilis. When
the penicillinase gene that contains the lac operator is ex-
pressed in B. subtilis on a plasmid that also produces the lac
repressor, the expression of the penicillinase gene can be mod-
ulated by isopropyl 13-D-thiogalactoside (IPTG), an^ inducer^ of
the lac operon in E.^ coli. A^ similar system was^ constructed
from a promoter of the B.^ subtilis phage SPO-1^ and the leuko-
cyte interferon A gene, which allowed the controlled expres-
sion of interferon in B. subtilis. These two examples show that
a functional control system can be introduced into B. subtilis
from E. coli.
The ability to regulate transcription of a gene in Escherichia coli by using a^ number^ of^ easily controllable promoter sys-
tems has^ been^ important for^ the^ development of^ foreign pro-
tein expression systems as^ well^ as^ for^ the^ study of^ expres- sion of native^ E.^ coli proteins. The^ lac^ repressor operator system is one such system. Repression of lac^ operon tran- scription is a result of binding of the^ lac^ repressor to^ the DNA sequence comprising the lac operator, thus^ preventing RNA polymerase from binding to the promoter (1). Induc- tion occurs through the binding of an inducer to the lac re-
pressor, which^ causes^ a^ conformational^ shift in^ the^ repres-
sor, decreasing the^ affinity of^ the^ repressor for^ the^ operator
(1). In Bacillus subtilis (or other^ Gram-positive organisms), no analogous system has been described. The^ only transcrip-
tionally controlled promoters that have been characterized
use a different mechanism. Gene regulation in these systems
is controlled by a factors, which are proteins that bind to the
RNA polymerase and determine the recognition site for
RNA initiation (2). Such systems cannot be used for the con-
trolled expression of another gene easily.
To take advantage of the desirable characteristics of an
operon whose transcription is easily induced, we have trans-
ferred the regulatory elements of the lac operon into B. sub-
tilis. We report here that the lac operator-repressor control
system can^ be^ transferred into B. subtilis and that the lac
repressor and operator function as transcriptional regulatory
elements in this microorganism. The penicillinase gene of
Bacillus licheniformis system was used as a model system.
In this system, the expression of penicillinase is modulated by isopropyl f3-D-thiogalactoside (IPTG), an inducer of the
lac operon in E. coli. To further illustrate the use of this
transferred (^) control system, we demonstrated IPTG-modulat-
ed expression of leukocyte interferon in B. subtilis.
MATERIALS AND METHODS
Strains and Plasmids. E. coli strain MM294 (F- supE
endAl thi-J^ hsdR4) was^ used for^ all constructions unless oth-
erwise indicated (3). E. coli strain 3300 (Hfr thi-1 lacI
relAl spoTi X-) was obtained from the E. coli Genetic Stock
Center (CGSC 808). B.^ subtilis^ strain 1168^ (trpC2) was^ pro-
vided by James Hoch.^ Plasmids^ pBR322 (4), pBS42 (5),
pUB110 (6), and pBSA105 (7) were used for construction of
the derivative plasmids described here.
Plasmid Constructions. The procedures used for isolation
of plasmid DNA, cleavage of restriction fragments, isolation
of DNA fragments from gels, kinase treatment of DNA frag-
ments, ligation with^ T4^ DNA^ ligase, and transformation of E.
coli and B. subtilis were as described (8). "Primer repair"
reactions were done as described (9). Antibiotic concentra-
tions for selection of transformants were as follows: chlor-
amphenicol, 12.5 (^) tkg/ml; neomycin, 20 and 10 (^) pug/ml (E. coli and B. (^) subtilis, respectively); ampicillin, 20 pug/ml.
Hybrid Promoters. The construction of the hybrid promot-
er of the penicillinase promoter and the lac operator, desig-
nated pac-I, and of the hybrid promoter of the phage SPO-
and the lac operator, designated spac-I, have been described
in detail elsewhere (7). Fig. 1 presents the nucleotide se-
quences of these two hybrid promoters and shows relevant
restriction sites.
Assays. Cultures to be assayed for penicillinase were
grown overnight in L broth containing 0.5% glucose and
chloramphenicol at 10 ,ug/ml in the presence or absence of 1
mM IPTG. Appropriate dilutions of the total cell broth were
made in 0.1 M Na phosphate buffer (pH 7.0) and the amount
of penicillinase enzymatic activity was determined by fol-
lowing the conversion of penicillin to penicilloic acid by a
published procedure (10). Detection of penicillinase activity
on polyvinyl alcohol plates was accomplished as described
Cultures to be assayed for interferon expression were
grown in L broth containing 0.5% glucose and neomycin at
10 ag/ml in the presence or absence of 1 mM IPTG. The
cells were harvested by centrifugation, lysed by treatment
with lysozyme and detergent, and assayed for interferon ac-
tivity using a bioassay as described (5).
RESULTS
Expression of the lac Repressor in B. subtilis. The promoter
and ribosome-binding site of the penicillinase gene of B. Ii-
cheniformis were linked in the plasmid pIQ45 to the E. coli
lac repressor gene so that it might be expressed in B. subtilis
(Fig. 2). The first two amino acids of the two genes are iden-
tical. When the lac repressor gene was digested with Hph I,
then repaired to flush ends with the Klenow fragment of
Abbreviations: IPTG, isopropyl 3-D-thiogalactoside; X-Gal, 5-bro- mo-4-chloro-3-indolyl 3-D-galactoside.
439
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440 Biochemistry: Yansura and Henner
EcoRI GAATTCGGTG GAAACGAGGT CATCATTTCC^ TTCCGAAAAA ACGG7T9 AW TTAAATCTTA CATATGT A
<<<< (^) << lac Op >>>>>> Sau3A met XCTrTCAATT GTGAGCGGAT AACAATTCCG GATCAATCAA ATATTCAAAC GGAGGGAGAC GATTTTGATG
EcoRI GAATTCTACA CAGCCCAGTC CAGACTATTC GGCACTGAAA TTATGGGTGA^ AGTGGTCAAG ACCTCACTAG
GCACCTTAAA AATAGCGCAC CCTGAAGAAG^ ATTTATTTGA^ GGTAGCCCTT^ GCCTACCTAG^ CTTCCAAGAA
AGATATCCTA ACAGCACAAG AGCGGAAAGA TGTTTTGTTC TACATCCAGA ACAACCTCTG^ CTAAAATTCC
<<< <<< lac Op
TGMAAATTT TGCAAAAAGT TGMTGACTTT ATCTACAAGG TGTGGZTAW TGTGTGGAAT^ TGTGAGCGGA
HindIII **** *^ XbaI^ EcoRI^ met
TAACAATTAA GCTTAAGGAG GTGTATCTAG AATTCATG
FIG. 1. Nucleotide sequences of the pac-I (Upper) and spac-I (Lower) promoters. The -35 and -10 regions of the promoters are (^) overlined, probable ribosome-binding^ sites^ are^ indicated^ by^ *s,^ the^ palindromic sequences^ of the^ lac^ operator^ are^ indicated by^ <<^ >>,^ and the^ initiation codons are indicated by^ "met."^ Sequences^ not^ derived^ from the^ parent promoter^ are^ underlined.
DNA polymerase I, the nucleotides for^ the^ first^ two^ codons
of the gene were removed. Subsequent digestion with BstEII
produces a DNA fragment containing the amino-terminal
portion of the lac repressor gene but lacking the first two
codons. A^ DNA^ fragment containing^ the^ promoter,^ ribo-
Bsf Ell Hp/
some-binding site, and the first two codons of the penicillin-
ase gene was created using a "primer repair" reaction, fol-
lowed by digestion with EcoRI. These two fragments were
ligated into^ the^ previously constructed^ plasmid^ pIQ2,^ which
already contained the carboxyl-terminal portion of^ the^ lac
BSA42 subclone
Sam H^ 1, Pst 650bp fragment denature primer 5
It Ppcn
BomHl EcoRl
K
partial Hphl, PolI
Bst Ell
525 Dp rr
8st Ell BstEll EcoRlI Alu BomHl, Pol EcoRI (^) (Bom H l)
pIQ
pcn ti Pst
| Klenow Poll
Eco RI
ragmenT EcoRI blunt
blunt Hph I, Pol I
lacI
EcoRl (^) IstPpcn Ell Bst Ell EcoRI BstEII EcoRI pIQ45 (^) lacI
BomHl
FIG. 2. Construction of pIQ45. Construction of the^ lac^ repressor gene under the^ control of the^ penicillinase promoter and^ the^ ribosome- binding site^ is^ diagrammed.^ Plasmid^ pHiQ6,^ containing^ the lac^ repressor,^ and^ plasmid pBS42,^ containing^ the^ pBR322^ and^ pUB110^ origins^ of replication and^ the^ pC194^ chloramphenicol^ acetyltransferase^ gene,^ have been^ described^ (5,^ 11).^ The^ carboxyl portion^ of the^ lac^ repressor gene was subcloned into pBS42 in a three-part ligation, creating pIQ2. Fragment 1 used^ in^ the^ construction^ was^ simply^ a^ convenient^ EcoRI/BstEII restriction fragment whose BstEII^ site^ had^ the^ same^ sequence as^ the^ BstEII^ site in the^ lac^ repressor.^ This^ allowed^ the^ BstEII^ site^ to^ be conserved for the^ subsequent construction.^ Plasmid^ pIQ2 was^ constructed^ in^ strain^ MM294,^ with^ selection for^ chloramphenicol^ resistance. Plasmid pIQ45 was^ constructed^ by ligating^ three^ fragments,^ one^ containing^ the^ penicillinase promoter,^ ribosome-binding^ site, and^ first^ two amino acids of the coding sequence, a second containing the^ amino terminus^ of^ the^ lac^ repressor gene (minus the^ first two^ amino^ acids), and^ a third containing the pBS42 vector and the^ carboxyl portion of the^ lac^ repressor gene. The^ derivation of the^ three^ fragments^ is^ shown in^ the figure. The^ pBSA42^ subclone^ contains^ the^ promoter^ and amino^ terminus of the^ penicillinase^ gene^ as^ described^ (7).^ The^ sequence^ of^ the^ primer used for the primer repair reaction was^ 5'^ T-T-T-C-A-T-C-A-A-A-A.^ Plasmid^ pIQ45 was^ constructed^ in^ strain^3300 (lac^ I-),^ with^ selection^ for chloramphenicol resistance on^ X-Gal^ indicator^ plates. Ppcn, penicillinase promoter; pcn,^ penicillinase-encoding^ region;^ lac^ I,^ lac^ repressor coding region.
BstEll,A/ul par 573bp fragment
Proc. Natl. Acad ScL USA (^81) (1984)
442 Biochemistry: Yansura and Henner
pAIQ
I
EcoRI Xbo^ Barn HI Sau3A
ari orgi^
2
)aa I, PollI
PBS7 (^) EXoRl
I
pUBIIO (BaMHl)^ Sau3A vector BamHI neor
BomrHl, Pol^ I, EcoRI
C/o pcn Ppcn
ECORIEcaRI (^) B6aHI,Ppoc-I PolI Xbal,PolI (BaSnHl) (^) C/oIl, PolI pAIQ120 (^) EcoRl
pSPIF-N
Bm HI, Pol I, EcoRI
EcoRI Bam^ HI, Pol I Eco R Le.^ C/o I, Pol^ I Pspoc-I Ppcn
pLIQ-I lac I
FIG. 5. Construction of pLIQ-1. The vector plasmid, pBS7, is the neomycin-resistance-conferring plasmid pUB110, with the^ pBR322 origin inserted into the unique BamHI site of pUB110 as shown. Plasmid pAIQ120 was constructed by the ligation of a restriction fragment of^ pAIQ that contained the penicillinase gene and the lac repressor gene to the pBS7 vector plasmid by using the restriction sites^ indicated. Plasmid pLIQ-1 was^ derived^ from^ pAIQ120^ by^ replacing^ the^ penicillinase^ gene^ and^ its^ pac-I^ promoter^ with^ the^ human^ leukocyte interferon^ A gene^ under the control of the spac-I promoter. The^ interferon^ gene was^ derived^ from pSPIF-III (5).^ Both^ pAIQ120^ and pLIQ-1 were^ constructed^ in^ strain
MM294 with selection for neomycin resistance. Restriction sites in parentheses were re-created^ by^ the ligation. Pspac-I, spac-I^ promoter; Ppac-
I, pac-I promoter; pcn, penicillinase-encoding region; lac I, lac repressor coding region; LeIF-A,^ leukocyte interferon^ A coding region.
tilis by using hybrid promoters and the lac repressor should
be useful for a wide variety of purposes. It is often desirable
to regulate the expression of a protein and be able to induce
its synthesis rather than rely on constitutive expression be-
cause the expression of high levels^ of proteins can^ have^ dele-
terious effects on growth of the cells (16).^ This^ may be true
for the expression of the B. licheniformis penicillinase gene.
This gene cannot be transformed into B. subtilis on a high-
copy plasmid under the control of either its natural promoter
or the pac-I promoter (unpublished results; ref. 17). Howev-
0 100 _
.'IICn DI
min
FIG. 6. Induction^ curve^ of^ pLIQ-1. B.^ subtilis^ strain^1168 trans-
formed with^ plasmid pLIQ-1 was^ grown in^ liquid culture^ to^ an^ OD6w
of (^) -0.2. The culture was then divided in half and 1 mM IPTG^ was added to one portion (indicated by the^ arrow). Triplicate samples
were removed to assay for interferon^ activity at^ the indicated times.
Values shown are means of^ triplicate determinations^ and have^ been
corrected for differences in^ optical densities^ of the cultures.^ Thus,
the values reported are^ units/ml per optical density unit.^ *, IPTG
added; o, no^ IPTG.
er, when the transcription of the gene was placed under the
control of^ the^ lac^ repressor, as^ on^ plasmid^ pAIQ25,^ the^ gene
could then be transformed into B. subtilis. This^ controllable
system now gives us a potential tool to study the effect of
high levels of expression of the penicillinase gene on B. sub-
tilis. Also, the use of this transcriptionally controlled system
might have advantages^ over^ previously described^ B.^ subtilis
expression systems that^ appear to be^ regulated^ at^ the transla-
tional level (18, 19), as the presence of very strong^ unregulat-
ed promoters can be incompatible with plasmid replication in
E. coli (20) and perhaps in B. subtilis (21).
One exciting feature of this system is^ that^ it offers the po-
tential of regulated expression systems in other^ bacteria^ for
which DNA transfer techniques exist. This sytem should be
transferable to any organisms in which the pBR322 or
pUB110 origins of replication function; alternatively, the
genes could^ easily be moved^ to^ plasmids^ of different^ host
ranges. The^ promoters and^ ribosome-binding^ sites^ used^ to
express the^ lac^ repressor gene and^ to^ construct the^ hybrid
promoters should be functional in^ a^ wide range of both
Gram-positive and Gram-negative organisms. Even if^ new
promoters needed to be created for expression in^ some alter-
native organism, this process should reduce the efforts that
would otherwise be needed to isolate and characterize ap-
propriate gene control systems that are native to that orga-
nism.
We would like to acknowledge Dennis Kleid for encouragement
and useful discussions and Herman de Boer for generously provid-
ing some^ of^ the^ plasmids^ and^ DNA^ fragments^ used in^ the^ construc- tions.
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