Note: Descriptions are shown in the official language in which they were submitted.
PLASM S FOX FOREIGN GENRE
EXPRESSION IN B. _UBTILIS
BACKGROUND OF THE INVENTION
Considerable interest exists in the application of
genetic engineering techniques for the production of
commercially valuable products such as insulin, human
and animal growth hormones and enzymes. Much of the
work to date has involved use of Escherichia golf as
the host into which foreign genetic material is intro-
duped. Expression of the genetic material in E. golf
results in production of desired products. When combined
with growth of genetically engineered cells in culture,
it permits production of the desired products in common-
Shelley meaningful yields. Unfortunately, use of E.
golf as a host is associated with certain disadvantages.
As a result, alternative hosts, including other bacteria
and yeast, are under investigation.
One particularly promising host for commercial applications
of genetic engineering is Bacillus subtilis. B. subtilis
is a non-pathogenic, gram positive bacterium which is
eaten daily by millions of Japanese as part of a fermented
soybean product. B. subtilis may be the safest bacterium
in which to achieve expression of foreign genes whose
products, e.g. interferon, will be purified and sub-
sequently injected into humans for at least two reasons.
First, B. subtilis is known to be non-pathogenic.
Secondly, _ golf is known to produce endotoxins which
may contaminate genetic products and induce endotoxic
shock in humans.
Direct expression in Bacillus subtilis of a gene origin-
cling in Escherichia golf has been achieved in a single
reported case [Ruin, EM., et at., Gene l0:227-235
(1980)] There the E. golf gene specifying thymidylate
synthetase expressed upon integration into the B. subtilis
--2--
chromosome. However, E. golf genes residing on plasmids
typically do not express at the level of genetic function
in B. subtilis. [Knelt, I., et at., Mol. con. Gent.
162:59-67 (1978)]. The least complex explanation for
the lack of foreign gene expression in B. subtilis is
an absence of correct transcription and/or translation.
In vitro transcription studies have indicated that E.
golf RNA polymers is significantly more efficient in
initiating transcription from an E. golf promoter than
is the B. subtilis RNA polymers [Lee, G., et at.,
Mol. con. Gent. 180:57-65 (1980)]. It was therefore
suspected that inserting an _ golf gene, or other
foreign gene, into a segment of DNA known to be
efficiently transcribed in B. subtilis might permit
functional expression of the foreign DNA.
SUMMARY OF THE INVENTION
A double-stranded DNA plasm id which includes a promoter
DNA sequence not derived from B. subtilis plasm id DNA
and a DNA sequence derived from a B. subtilis plasm id
is useful for introducing into B. subtilis foreign DNA
which includes a gene coding for the production of a
desired product. Preferably, the promoter DNA sequence
is not derived from B. sub-tilis. examples of suitable
sources of the promoter include bacteriophages SPY and
~105, B. pummels plasm id ply and B. licheniformis.
The plasm id may be linear or circular and may additionally
include an inducible gene such as a chloramphenicol
inducible gene coding for synthesis of chioramphenicol
acetyltransferase.
A double-stranded DNA plasm id which includes a promoter
DNA sequence which is not derived from B. subtilis, a
DNA sequence derived from a B. subtilis plasm id and
foreign DNA which includes a gene coding for the
production of a desired product such as a palpated
I
- 3
and which is capable of expression when the plasm id is
introduced into _ subtilis is useful in the production of
the desired products for which the foreign DNA codes.
B. subtilis cells which contain the plasmids are novel.
They may be grown in culture to produce commercially
valuable materials which can then he recovered.
BRIEF DESCRIPTION OF THE DRAWINGS
Fig. 1 shows the restriction endonuclease maps of three
plasmids, ply, ply and ply. The thickened
horizontal line indicates the cloned B. pummels DNA in each
plasm id.
Fig. 2 is a diagram of the insertion and orientation of the
1.7 My E. golf tropic fragment in ply. Orientation of
the fragment was determined from double digests of ply
with Hint II and EcoRI.
Fig. 3 shows induction by chloramphenicol of InGPS specified
by ply. BD224 (ply) was grown to the middle of
the exponential growth phase in Mix OH containing 100 go per
ml of tryptophan. The culture was split and 50 ml samples
were withdrawn from each. A sub inhibitory concentration of
Cur (0.1 ~ug/ml) was added to one culture, and during
subsequent growth, 50 ml samples were periodically
withdrawn. Cells in each sample were harvested, processed
and assayed for InGPS as described in Hook, SO. and
Crawford, IMP., J. Bacterial. 116:685-693 (1973).
Fig. 4 is a diagram of pPL608-TRl. Orientation of the Sty
fragment was deduced from Sty and Bgl II double digests.
--'I--
DETAILED DESCRIPTION OF THE INVENTION
Plasmids have been created which are useful for the
introduction into Bacillus subtilis of foreign DNA
whose nucleic acid sequence includes one or more genes
encoding the information necessary for production of
desired products, particularly palpated or partially
palpated products such as insulin, Thomson, growth
hormones, enzymes, antibodies and the various interferon.
These plasmids ale double-stranded DNA molecules which
include a promoter DNA sequence which is not derived
from B. subtilis plasm id DNA and a DNA sequence which
is derived from a B. subtilis plasm id. These plasmids
may be linear or circular. However, when used for
bacterial transformation, they will generally be circular.
The promoter DNA sequence is preferably not derived
from B. subtilis chromosomal DNA, but rather from another
source. Numerous suitable sources are available, including
the bacteriophages SPY and ~105, the B. pummels plasm id
ply and B. licheniformis. The origin of the DNA from
a B. subtilis plasm id may likewise vary widely, any B.
subtilis strain being capable of functioning as a source
of the plasm id DNA. One example of a suitable source
is the B. subtilis plasm id pub.
-
These plasmids may additionally include an inducible gene such as a drug inducible gene to facilitate selection
of cells carrying the plasm id and increased gene expression
in the cells so selected. Illustrative of such inducible
genes are a chloramphenicol-inducible gene coding for
synthesis of chloramphenicol acetyltransferase. One
such gene may be obtained from B. pummels NIB 8600.
Additional plasmids have been constructed which are
useful for effecting expression in Bacillus subtilis of
DNA which does not naturally occur in B. subtilis and
which has a nucleic acid sequence which includes one or
I
--5--
more genes associated with the production of desired
products or the expression of useful properties. These
plasmids are double-stranded, deoxyribonucleic acid
molecules which include a promoter DNA sequence which
is not derived from B. subtilis plasm id DNA, a DNA
sequence derived from a B. subtilis plasm id and foreign
DNA having a sequence coding for the production of a
desired product. These plasmids may be linear or circular,
although they are generally circular when used in bacterial
transformation.
The promoter DNA sequence is preferably not derived
from B. subtilis chromosomal deoxyribonucleic acid, but
from a source other than B. subtilis. Although numerous
_
sources are available, suitable sources include bacteria-
phases SPY and ~105, the B. pummels plasm id ply and
B. licheniformis. Of these, a promoter obtained from
SPY is presently preferred.
The DNA derived from a _ subtilis plasm id may be obtained
from any such plasm id At present, the preferred source
is the B. subtilis plasm id pueblo.
Foreign DNA which includes a gene or genes associated
with production of a desired product may be obtained
from any appropriate source or may be chemically Cynthia-
sized. Examples of suitable foreign DNA sequences
- include mammalian genes coding for interferon and genes
coding for insulin, enzymes, growth hormones, viral
antigens and the like.
The plasmids may also include an inducible gene such as
a chloramphenicol-inducible gene coding for the synthesis
of chloramphenicol acetyltransferase.
fly
Methods for identifying, recovering and purifying the
various DNA segments which are portions of the plasmids are
known to those skilled in the art as are methods for
ligating the segments, transforming bacterial cells, cloning
and recovering products synthesized. Accordingly, the
methods will only be described by reference to specific
embodiments of the invention set forth hereinafter.
Certain of the experimental details which follow were
reported in Williams, DIM., Devil, EDGY. and Levitt, P.S.,
J. Bacteriology, 146:1162-1165, June 1981, and show the
state of the art.
DNA preceding structural genes contains regulatory sites
essential to gene expression, including sequences necessary
for initiation of transcription and translation. Selective
expression of genes involved in the developmental process of
bacterial sporulation may be partially controlled by the
occurrence of sporulation-specific regulatory sequences that
participate in modulating sporulation gene expression. A
direct approach to isolate regulatory sequences from DNA was
provided by the development of plasm id vectors [Clowns, ARC
Bacterial. Rev. 36:361-405 (1972)] useful for cloning DNA
segments that act as promoters and translation initiation
signals in Escherichia golf [An, G., and Frozen JO J.
Bacterial. 140:400-410 (1979); Casadaban, MY et at., J.
Bacterial. 143:971 980 (1980); and Casadaban, M. and Cohen,
SUN., J. Mol. Blot. 138:179-207 (1980)]. The potential value
of similar cloning vectors for the Bacillus subtilis system
prompted screening for a structural gene that was
potentially capable of expression in B. subtilis, but
appeared to lack those regulatory controls needed for
expression. A description follows of the development
I
--7--
of a plasm id cloning vector that permits direct selection
for cloned fragments of DNA which are necessary to the
expression of a chloramphenicol acetyltransferase gene
in B. subtilis.
Plasm id ply was generated by cloning into the 3-
megadalton, neomycin-resistance plasm id pueblo [Gryczan,
TO et at., J. Bacterial. 134:318-323 (1978); Sedate,
Y., et at., J. Bacterial. 141:1178-1182 (1980~] a 1.44-
My, EcoRI-generated fragment from DNA isolated from a
chloramphenicol-resistant (Char) derivative of Bacillus
pummels NIB 8600, using methods previously described.
[Levitt, P.S. and Keg gins, KIM., Methods Enzymol.
68:342-357 (1979)]. ply (shown in Fig. 1) confers
on host cells, e.g., BR151,resistance to both neomycin
(10 gel and Cur.
Plasm id ply is a derivative of plus in which the
orientation of the Char fragment was reversed by EcoRI
cleavage and ligation. BRIE harboring either pow
or ply grew in Puns broth (PUB; Disco Laboratories)
containing up to 200 go of Cur per ml (higher levels
were not tested). Cells harboring both plasmids produced
chloramphenicol acetyltransferase (Table I). BR151(pPL531)
or BRl51(pPL600) grown on 5 I of Cur per ml produced
approximately 10-fold higher levels of chloramphenicol
acetyltransferase than cells grown in the absence of
the drug (Table I). Deletion of the Sty fragment
(~0.55 My) from ply generated plasm id ply (Fig. l;
Table I). BR151(pPL601) was incapable of growth in PUB
containing 25 go of Cur per ml, although normal growth
was observed in 5 go of Cur per my and sparse growth
was observed in 10 I of the drug per ml. The specific
activity of chloramphenicol acetyltransferase in
BR151(pPL601) grown in PUB containing 5 go of Cur per ml
was 20-fold lower than the activity produced by BRIE
(ply) (Table I). Reversal of the orientation of the
a
C~LoRA~p~ENIcoL RESISTANCE PROPERTIES
. Chlora~nenia~ ethyl-
Plod Insert Do wit Swallower of erupt transfer go sup act Sax Oh b
( 106~
cry or
pP1531 0.04 0.52 200
10pÆ.600 0.09 0~96 2û~
pull Wool 0~03 S
purl 0~04 0~71 ~00
~03 (Allah 0~04 5
S041 1~3 Decor BLUE 0~334~37 2C~
ply ~54 En pP~10 0~03 0~2~3100
OWE 0~21 Eke* oil ~;P02 0~214~02 kiwi
F$~613 0.15 ITS oil
B EYE o . 63 21 !110
pP~614 1.0 EKE of
En. 0.34 0.68 100
Jo .
method ox arrowhead [arrowhead. PLY, Anal it owe (1976)].
b. Prowl 107 pld~cont~ cells prune grin in Pi
ode 5 lug Shea For ml were inlaid into 2 ml of PUB canonry Cur
30 at 5, I 25, 50, 100 or 2~0 ~g/TDl. Lncuba~n ceded or 20 hocus at
which time cx~nOE~ratiC~n of Cur allowing Goethe I remrded.
I _
insert in ply or deletion of -the Sty fragment from
ply generated ply which, in BRIE, conferred
resistance to 200 go of Cur per ml (Table I).
Since ply did confer high-level Char, it was evident
that an intact structural gene for chloramphenicol
acetyltransferase was present on the cloned EcoRI frog-
mint after the internal Sty deletion was made. Moreover,
the inability of ply to confer high-level Char sup-
gusted that expression in ply was dependent on some
property of the vector plasm id pueblo. For example, if
the Sty deletion removed a promoter region from the
cloned fragment, expression of the gene would be dependent
on a pueblo promoter. Hence, chloramphenicol acetyltrans-
erase activity would only be expressed when the struck
tubal gene was correctly oriented relative to the ox-
vernal promoter. In ply, the gene is expressed and
therefore the orientation would be correct. In ply,
the orientation is incorrect, and the gene is unexpressed
or very weakly expressed.
Cloning Bohemia or MboI fragments of Bacillus licheniformis
DNA into the Bohemia site on ply failed to permit
ply to transform BRIE to Char (selection on tryouts
blood ajar base containing 10 go of Cur per ml). Cloning
Sty fragments of the same DNA into ply allowed
ply to successfully generate numerous Char transform
mints of BRIE, suggesting that inserts left of the
Sty site of ply (shown in Fig. 1) might promote
chloramphenicol acetyltransferase activity. Accordingly,
MU the Bohemia proximal EcoRI site on ply was deleted by
Bohemia and BgIII digestion, ligation, and transformation
of BRIE with selection for Noon. A resulting plasm id
which retained a single EcoRI site was designated
ply (Fig. l; Table I).
EcoRI* or EcoRI activity [Polisky, BY e-t at., Pro.
Neil. Aged. Sue, U.S.A. 72:3310-3314 (1975)] was used
to digest ply, pub, and DNA from B. licheniformis
9945~ and phase ~P02 (~emphill, HUE. and Whitely,
HER., Bacterial Rev. 39:257-315 (1975); Levitt, P.S.,
et at., J. Bacterial. 127:817-828 (1976); Escher, BUM.,
et at, J. Viral. 28:395-402 (1978); and Yenned, Y.,
et at., Gene 7:51-68 ~1979)]. Approximately 1 go of
EcoRI*- or EcoRI-digested DNA was mixed with 1 go of
EcoRI-digested ply in 100 lo The ligated DNA
(approximately 2 go was shaken with 3 X 108 competent
[Boil, OF and Wilson, GUY., J. Bacterial. 94:562-570
(1967)] _ subtilis cells for 1 hour and plated directly
onto tryouts blood ajar base containing 10 go of Cur
per ml. About 200 to 600 Char transform ants were
obtained per go of input DNA. Each of more than 200
Char transform ants was neomycin resistant, and each of
a total of 83 examined by the single colony lysis-gel
electrophoresis procedure contained plasm id having a
molecular weight greater than ply. A derivative of
I ply containing an EcoRI* or an EcoRI fragment cloned
from each of the above DNA species was further examined
ply is the designation for a ply derivative con-
twining a 1.3-megadalton EcoRI* fragment of pub DNA.
ply was cloned and maintained in BRIAR, a Russ
derivative of Brollies, because of the homology between
the cloned fragment and a portion of ply. ply
conferred on BRIAR the ability to grow in 200 go of
Cur per ml, and -the cells produced a high level of
chloramphenicol acetyltransferase (Table I). Removal
of the insert regenerated a plasm id indistinguishable
from ply in size and Cur sensitivity. One of these
derivatives of ply from which the insert had been
removed was subsequently used to clone EcoRI fragments
of ~105 DNA to determine whether the excision process
had altered the ability of the chloramphenicol acutely-
transfers gene to produce high-level resistance.
There appeared to be no difference between this plasm id
and pow in terms of the ability of inserts to promote
Char. Relevant data for fragments cloned in ply
from EcoRI* digests of ply and SPY and EcoRI digests
of s. licheniformis DNA are shown in Table I. In each
case, removal of the cloned insert regenerated a plasm id
comparable to ply.
EcoRI fragments of B. subtilis DNA were cloned into
ply by as procedure differing from the above only in
that the transformation recipient was a Russ derivative
of BRIE, BRIAR, and the protoplasm transformation
system was used [Clang, S. and Cohen, SUN. Mol. con.
Junta 160~ 115 (1979)]. By this procedure, the
frequency of Char transform ants was on the order of
1,000 per go of input DNA. Each of more than I trays-
formats examined contained ply harboring an insert.
Three classes of DNA fragments were identified on the
basis of ability to promote expression of Char when
inserted into ply. The EcoRI-generated complex
minting fragment previously cloned from B pummels
[Keg gins, KIM., et at., J. Bacterial. 134:514-520 (1978);
and Keg gins, KIM., et at., Pro. Neil. Aged. Sat,
Use. 75:1423-1427 (1978)] did not promote Char when
inserted into ply in either orientation, although
tropic complementing activity was expressed in both often-
stations. EcoRI fragment F of ~105 DNA [Curly, DO and
Garbo, A J. Viral. 3~:789-791 (1980)] promoted Char
in one orientation (ply) but not in the reverse
orientation (ply). However, the ~105 immunity property
of fragment F was expressed in both orientations. Lastly,
the EcoRI* fragment cloned from ply into ply [chimera,
designated ply (shown in Table IT promoted Char in
either orientation. Orientations of all inserts were
judged by Hind III cleavage patterns.
Sequences that promote Char expression have been cloned
from a variety of DNA sources, and it therefore is
likely that these contain regulatory signals common to
-12-
many genes. Whether these regulatory signals exert
their control at -the transcriptional or translational
level remains to be determined.
The recombinant vector plasm id, ply, contains an
unexpressed CAT gene preceded by a unique EcoRI site
and permits the direct cloning of DNA fragments that
have promoter activity in B. subtilis. Promoter
fragments cloned into the unique EcoRI site result in
high level expression of the CAT gene. The cloned
promoter in plasm id ply described more fully herein-
after is capable of permitting expression of both foreign
procaryotic and eucaryotic genes in B. subtilis. ply
is therefore the first vector plasm id for B. subtilis
capable of expressing cloned procaryotic and eucaryotlc
genes.
CLONING E. COLT TRY GENES IN B. SUBTILIS
In order to determine whether splicing a gene not normally
expressed in _ subtilis into the cloned CAT gene in
ply would allow expression of the foreign DNA, the
Hind III site was chosen for cloning because inserts at
this location were found to inactivate all CAT activity.
The Hind III generated DNA fragment spanning the F.
golf tropic gene was cloned into ply from HB101 chromosome
DNA and pVH5 (See Materials and Methods which hollow
for details). Composite plasrnids containing a try
insert cloned from chromosome digest (ply) or a
digest of pVH5 (ply) were distinguishable by several
criteria. Therefore, relevant properties for only
ply are described. ply (5.0 My, shown in
Fig. 2) complemented mutations in trod, tropic and trpF
genes of B. subtilis, but not mutations in type, trpB,
or trap. Removal of the 1.7 My insert from ply by
Hind III cleavage and ligation, regenerated the Char
phenotype of the vector and deleted the try _ _ F
-13-
complementing activity. The 1.7 My cloned insert in
ply and ply comigrated with a Hind III fragment
of pVH5. Nick translated pVH5 hybridized to the 1.7 My
fragment in Hind III digests of ply and 608-3,
demonstrating identity among the 1.7 My fragments in
the plasmids. In this experiment, approximately 1 go
of digested plasm id was subjected to electrophoresis as
described previously, [Moorer, R. and Levitt, P.S., J.
Bacterial. 1~3:879-886 (1980)]. Hind III digested DNA
was employed as reference. The gel was blotted to
nitrocellulose paper to which was hybridized nick trays-
fated pVH5.
In order to test whether sequences in ply were
essential to the expression of the cloned E. golf try
fragment, the orientation of the fragment was
reversed by Hind III cleavage and ligation. Each of
three plasmids which were Curs and did not complement
_r~2 in BD224 contained the 1.7 My try insert in the
reverse orientation as judged by digestions with Hint
II and EcoRI endonucleases. Secondly, removal of the
0.2 My promoting fragment from ply by EcoRI
cleavage and ligation resulted in loss of tropic come
plementing activity in BD224. Lastly, the 1.7 My try
fragment was transferred from ply to the single
Hind III site in a joint vector constructed by ligating
pBR322 and pueblo at their EcoRI sites. This plasm id
complemented the mutation in E. golf strain ISSUE,
but did not complement the tropic mutation in BD224
within 36 hour incubation at 37C. Louvre, prolonged
30 incubation (72 hours) of BD224 harboring such composite
plasmids did result in sparse growth on tryptophan-free
media. Accordingly, sequences present in ply appear
essential to expression of the cloned E. golf tropic
gene.
CHLOR~lPHENICOL INDUCTION OF E. COLT
tropic EXPRESSION IN PLY
P
The CAT activity specified by ply was 10-fold higher
in host cells grown in the presence of Cur than in the
same cells grown in drug free broth [Williams, DIM.,
et at., J. Bacterial. 146:1162-1165 (1981)]. This
increase is believed to result from Cur induction of
CAT. It was thought therefore that the expression of
foreign genes, such as the E. golf tropic fragment,
inserted into the CAT gene might also be inducible by
Cur. BD224 (ply) was grown to the middle of the
exponential growth phase in Mix OH containing 100 gel
of tryptophan. The culture was split and a concentration
of Cur which is sub inhibitory to BD224, 0.1 gel was
added to one. Within 100 mix after addition of Cur to
BD224 (ply, a 7-fold increase in the level of the
tropic gene product InGPS was detected (as shown in Fig. 3).
BD224 (ply) not exposed to Cur showed no increase
in the specific activity of InGPS (Fig. 3). In contrast
to the apparent induction of the cloned pi expression
by Cur, the expression of the cloned appeared
unaffected by the level of tryptophan added to the
growth medium. BD224 (ply) grown in Mix OH or Mix
OH containing 0.5 gel of tryptophan or 200 gel of
tryptophan contained approximately the same level of
InGPS.
EXPRESSION OF MOUSE DHFR CLONED IN B. SUBTILIS
In order to test whether ply would allow expression
of a cloned eucaryotic gene, the mouse gene specifying
DHFR previously cloned in E. golf on a Sty fragment
was chosen [Clang, AWOKE., et at., Nature 275:617-624
(1978)].
-15-
Mammalian DHFR is resistant to the drug trimethoprim whereas
the bacterial enzyme is sensitive. Selection of cells that
were capable of expressing the mouse DHFR gene was -therefore
based on their resistance to 25 gel of trimethoprim.
pPL608-TRl was a derivative of ply containing to 1.0 My
mouse DHFR gene inserted at the P I site (as shown in Fig.
4) and confirmed by agrees gel electrophoresis of Sty and
BglII digested ply and pDHFRll which showed the 1.0 My
Sty mouse DNA fragment present in both ply and
pDHFRll. Each of more than 200 neomycin-resistant
transform ants of B. subtilis strain BGSCIS53 generated with
pPL608-TRl were resistant to 25 gel of trimethoprim.
Removal of either the promoting fragment from pPL608-TRl (by
EcoRI cleavage) on the mouse DHFR fragment (by Sly
cleavage) resulted in deleted plasm id forms that no longer
specified trimethoprim resistance in strain BGSCIS53.
ply Try confers Char indicating insertion of the fragment
of mouse DNA into the Sty site did not inactivate the CAT
gene on ply.
DHFR activity in extracts of ASB298 ~pPL608-TRl) was
insensitive to 10 6 M trimethoprim, but was reduced by 93
prevent when 10 6 M methotrexate was present in the reaction
mixture (shown in Table II). The sensitivity to
methotrexate, and resistance to trimethoprim, are
characteristics typical of mammalian DHFR [Clang, AWOKE., et
at., Nature 275:617-624 (1978)]. The level of DHFR was
similar regardless of where BGSCIS53 (pPL608-TRl) cells were
grown in no Cur or 0.1 ug/ml of Cur. These data suggest
that expression of a gene inserted at the Sty site is not
Cur inducible.
- 16 -
ABLE II
DHFR ACTIVITY IN EXTRACTS OF B. SUBTILIS HARBORING
pPL608-TRlC
Inhibitors Added % Activity Remaining CAM
none 100% 3614
Trimethoprim (10 EM) 100% 3626
Trimethoprim (10 EM)
+ 7% 247
Methotrexate (10 EM)
c. BGSCIS53 (pPL608-TRl) cells were grown in puns broth
and harvested during mid exponential growth. Cells were
washed and resuspended in 0.1 M KHP04, pi 5.8, and extracts
prepared as described for CAT [Skew, WOVE., Methods Enamel.
43:737-755 (1975)~. Assays -For DHFR [Little field, JAW.,
Pro. Neil. Aged. Sat., USA, 62:88-95 (1969)] were done in
duplicate and varied by no more than I percent. DHFR
activity assayed without inhibitors equaled 1.5 units where
1.0 unit will convert lnmole of foliate to tetrahydroFolate
per minute at 37C.
_ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _
ply appears to be a valuable vector for achieving foreign
gene expression in B. subtilis. The present study
demonstrates Cur inducible expression of a foreign
procaryotic gene inserted at the Hind III site and
expression of the mouse DHFR gene inserted at the Sty site.
Moreover, the promoter fragment within ply is readily
removed From the plasm id allowing for the insertion of other
promoters that may enhance or reduce the expression of
cloned Foreign genes.
., I,
-17-
The 1.7 My Hind III E. golf try fragment cloned in
ply from chromosome digest or from pVH5 was identified
by complementation of a genetic defect in the recipient.
The cloned fragment complements mutations in the B.
subtilis try genes D, C, and F. It has been previously
shown that two Hind III sites occur within the E. golf
try operon flanking the E. golf tropic gene [Crawford,
IMP., et at., J. Mol. Blot. 142:489_502 (1980); Nichols,
BY et at., J. Mol. Blot. 142:503-517 (1980), and
Christie, GEE. and Plait, T., J. Mol. Blot. 142:519-530
(1980)]. One site is located in the beginning of trpB
and the other is approximately in the middle of trod.
The E. golf tropic gene specifies a protein that has
two activities, each of which is specified by a separate
gene in B. isubtilis, tropic and [Crawford, IMP.,
Bacterial. Rev. 39:87-120 (1975)]. The E. golf trod
citron specifies a protein that also exhibits two
activities, each of which is specified by a separate
gene in B. subtilis, trig and trod [Crawford, IMP.,
Bacterial. Rev. 39:87-120 (1975)]. Since the cloned E.
golf fragment complements a mutation in the B. subtilis
trod gene, that portion of the _ icoli trod gene
corresponding to the B. subtilis trod gene is being
expressed. The to_ activity is associated with the
car boxy terminal portion of -the trod product, and
the corresponding portion of the gene is present on the
cloned fragment. The translation initiation cordon
necessary for trod expression may reside in the plasm id
vector, and may result in the fusion of the amino terminal
portion of a vector specified protein (probably CAT) to
the car boxy terminal peptize specified by the cloned
trod gene.
Expression of the CAT gene in pledge, the tropic gene in
ply or the DHFR gene in pPL608-TRl was dependent
on the presence of the promoter fragment. Therefore,
the promoter fragment likely provides sequences necessary
-18-
for transcription initiation and that transcription
occurs from the promoting fragment through the CAT
structural gene. Insertion of genes between the Sty
and Hind III sites on ply presumably places them
under control of a strong promoter that is efficiently
recognized my B. subtilis RNA polymers.
Production of palpated upon expression of the cloned
mouse DHFR gene has been confirmed. Initial molecular
weight characterization established that the enzyme
DHFR was in fact produced.
In summary, the mouse dihydrofolate reeducates gene and
a segment of the Escherichia golf try operon genetically
express in Bacillus subtilis when cloned in the novel
plasm id ply. The cloned mouse gene confers in-
methoprim resistance on B. subtilis and the cloned try
fragment complements mutations in the B. subtilis try D
C and F genes. Expression of both cloned fragments is
dependent on a promoter present in the vector plasm id.
The E. golf try fragment is cloned in a Eland III site
within a chloramphenicol acetyltransferase gene present
on ply, and as a result, expression of the E. golf
tropic gene product is inducible by chloramphenicol. The
mouse gene is inserted at a Sty site preceding the
chloramphenicol acetyltransferase gene and its expression
is not chloramphenicol inducible. The replication
functions and neomycin-resistance of pluck are derived
from pub. accordingly, ply is stably maintained
at high copy number in B. subtilis.
MATERIALS AND METHODS
Bacteria, plasmids and media
Strains of B. subtilis and E. golf used are listed in
Table III. Plasmids pub, pCM194, pVH5 and pBR322
have been described [Believer, F., et at., Gene 2:95-113
I
(1977); Gryczan, TO et at., J. Bacterial. 134:318-
323 (1978); and Hershfield, V., et at., Pro. ayatollah.
Aged. Sat., U.S.A. 71:3455-3459 (1974)]. pDHFRll is a
pBR322 derivative containing a 1.0 My Sty fragment
specifying mouse dihydrofolate reeducates [Clang, AWOKE.,
et at., Nature 275:617-624 (1978)]. Tryouts blood
ajar base and puns broth were from Disco. Munich
medium has been described [Spizizen, J. Pro. Neil.
Aged. Sat., U.S.A. 44:1072-1078 (1958)]. Minimal ajar
for E. golf was My. All incubations were at 37C.
Enzyme assays
CAT was assayed as described by Skew [Skew, WOVE., Methods
Enzymol. 43:737-755 (1975)]. InGPS was assayed according
to the methods of Hook and Crawford [Hook, SO. and
Crawford, IMP., J. Bacterial. 116:685-693 (1973)], and
DHFR was measured by the procedure of Little field.
[Little field, JAW., Pro. Neil. Aged. Sat., U.S.A.
62:342-357 (1969)]. The Bradford method was used for
protein determinations. [Bradford, M., Anal. ~iochem.
72:248-254 (1976)].
press ion plasm id ply
The construction of ply is described hereinabove and
in Williams, DIM., et Allah J. Bacterial. 146:1162-1165
(1981)]. The plasm id has a mass of about 3.3 My and
consists of a major portion of pueblo joined to a 0.8 My
segment of B. pummels DNA containing a CAT gene plus a
0.2 My EcoRI* promoter fragment cloned from phase SPY
DNA. The cloned promoter permits expression of the CAT
gene, thereby allowing ply to confer Char on B.
subtilis. Unique restriction sites for Hind III and
Sty exist downstream from the promoter (see Fig. 2).
.
I
-20-
The specific activity of CAT in BRIE (ply) was 10- to
20-fold higher in cells grown in Cur (5 gel than in cells
grown in drug free broth. The molecular size and copy
number (approximately 50) were -the same regardless of
whether host cells were grown in the presence or absence of
Cur. Thus, the increased CAT activity was not due to
increased gene dosage. Exposure of BD224 (ply) to a
sub inhibitory concentration of Cur (0.1 gel caused a
10-fold increase in the specific activity of CAT within 100
minutes. It therefore appears that CAT is inducible by Cur.
TABLE III
BACTERIAL STRAINS
Organism Strain Relevant properties
B. subtilis 168 BRIE tropic Metro Lucy
BD224 tropic Thor Russ
To trap
T20 trpB
T12 trpF
To tropic
T22 trod
T24 type
BGSCIS53 spy
_ golf HB101 Troupe
ISSUE tropic
___ _ __ _ _ _ _ ___ _ _ ___ __ _ __ _
i
-21-
CLONING ESCHERICHIA COLT tropic IN B. SUBTILIS
Hind III digests of HB101 chromosome DNA (2 I or plasm id
pVH5 (lug) were combined with 0.5 us of Hind III cleaved
ply, annealed, ligated [Levitt, P.S. and Keg gins, KIM.,
Methods Enzymol. 68:342-357 (1979)] and transformed into
competent BD224 [Boil, OF and Wilson GUY., J. Bacterial.
94:562-570 (1967)] at 1 gel of DNA. Cells were plated on
Mix OH containing lO~ug/ml of neomycin sulfate. Fourteen
Try+ transform ants were recovered by cloning from HB101 DNA,
and 93 were obtained by cloning from pVH5. ply and
ply are Try derivatives of ply containing Try
inserts from HB101 and pVH5, respectively. The ability of
the cloned try fragment to complement mutations in each of
the six _ subtilis try genes was performed as previously
described. [Keg gins, KIM., et at., Pro. Natalie. Aged. Sat.,
U.S.A. 75:1423-1427 (1978)]. Southern transfers, nick
translation, hybridizations and agrees gel elec-trophoresis
were as previously described [Moorer, R. and Levitt, P.S.,
J. Bacterial. 143:879-886 (1980)i and Southern, EM., J.
Mol. Blot. 98:503-517 (1975)].
CLONING MOUSE DIHYDROFOLATE
REEDUCATES (DHFR) IN B. SUBTILIS
pDHFRll (l~ug/ml) and ply (lvug/ml) were digested with
Sty annealed, ligated and transformed into _ subtilis
strain BGSCIS53. Neomycin-resistant transform ants were
selected and each was picked to appropriately supplemented
Mix OH containing 25~ug/ml of trimethoprim. Approximately 2
percent of the transform ants were trimethoprim resistant.
The plasm id from one such clone, pPL608-TRl, was
characterized.
