Vol. 91, No. 4, 1979 December
BIOCHEMICAL
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
28, 1979
Pages 1556-1564
CLONINGOF A FOREIGNGENECODINGFOR a-AMYLASEIN BACILLUSSUBTILIS Yuko Yoneda, Scott Grahamand Frank E. Young Department of Microbiology, School of Medicine and Dentistry, University of Rochester, Rochester, New York 14620 Received
November
21,1979
SUMMARY Foreign DNAhas been introduced into the genomeof bacteriophage 03T, producing a specialized transducing bacteriophage containing the genetic information encoding cc-amylase from Bacillus amyloliquefaciensH. Genetic and physical studies demonstrated that the gene(s) is inserted into the bacteriophage genome. These bacteriophage carrying the gene(s) encoding cl-amylase lysogenized and replicated in Bacillus subtilis with normal efficiency. In these lysogens, the gene(s) encoding u-amylase appears to map near the bacteriophage attachment site rather than the chromosomal%E locus. This method of construction of specialized bacteriophage should be applicable to the cloning of other genes for which no primary selection exists. The Bacilli industrial
elaborate a wide variety
importance (1).
of extracellular
enzymes of
Although a number of different
o-amylases
are produced by various Bacilli,
the genetics and regulation
enzymes can only be examined in -B. subtilis model system. other Bacilli
which is a well characterized
Someof the genes encoding or regulating can be introduced into -B. subtilis
their
however, are not sufficiently DNAs to be integrated
related
o-amylase in
if there is extensive
genetic homology between the donor and recipient Bacilli,
strains
(2).
to -B. subtilis
during interspecific
DNA technology provides a method for inserting
multiple.types
an attractive
X/79/241
foreign genes into -B.
of strains
of
that synthesize
1976 NIlI Guidelines Appendix A (4), bacteriophage
cloning strategy
bacteriophage 03T is an intriguing
Copyright All rights
Recombinant
of u-amylase.
As discussed in.the
0006-291
(3).
Such procedures permit the analysis of genetic regulation
the various cr-amylases and construction
offer
Most to permit
transformation
Presumably, this is due to a lack of chromosomalhomology.
subtilis.
of these
in -B. subtilis.
carididate for such cloning procedures
556-09$01.00/0
@ 1979 by Academic Press, Inc. of re~r~ffction in anyform reserved.
In particular,
1556
BlOCHEMlCAL
Vol. 91, No. 4, 1979
(5) * This that
bacteriophage
can be used different
-B. subtilis
based
described
under
into
a recipient
with
the
for
that
ligated
for
of foreign
in the
an extension
DNA in -B. subtilis of bacteriophages
selection
is
laboratories for
(10,
are:
11).
in which
for
SP8 (6),
Recently,
bacteriophage
formation
of specialized
of auxotrophic
traits.
and the development carrying
genes
for
0105
fragments
(7), --et al.
of DNA
pll
and introduced
pll
(11).
Recombination
bacteriophage
genome
transducing The data
to permit
to be the cloning
of procedures which
in
system.
Kawamura
chromosomal
procedure
vectors
X model
and the homologous
of this
are now
cloning
of DNA from bacteriophage
a variety
isolation
MATERIALS The are listed maintenance (12,13), and phage
group
synthetase
- bacteriophage study
DNA of the vector
bacteriophages
describe
coli
was lysogenic
resulted
thymidylate
Several
intensive
cloning
fragments
of the lysogen
presented
most
RESEARCH COMMUNICATIONS
as candidates
on the Escherichia
a strategy
ligated
between
trait.
and the 03T-pll
were
a gene encoding
bacteriophages
The bacteriophages SP02 (5,8,9),
carries
as a selective
developing
AND BIOPHYSICAL
a method
for of primary
unavailable.
AND METHODS bacterial strains and the bacteriophages used in this study in Table I. Standard procedures were employed for the of bacterial cultures (12,13), propagation of bacteriophage isolation of DNA (12,14), transformation (13), transduction infection (13).
(13)
Construction of specialized transducing bacteriophage containing foreign DNA was modified from the method described by Kawamura --et al. Chromosomal DNA isolated from B. amyloliquefaciensH was first (11). digested with B&II, and the enzyme inactivated by heat at 68OC for 15 min. Bacteriophage 03T DNA was similarly treated with B&II and the reaction terminated by heat inactivation. The samples were combined and ligated by the procedure of Tanaka and Weisblum (15). This ligated mixture of DNA was added to a preparation of chromosomal DNA from -B. subtilis strain RUB200 that was prototrophic for threonine and defective in a-amylase biosynthesis. The entire mixture was incubated with a recipient strain of B. subtilis (RUB2Ol) that is lysogenic for bacteriophage 03T and auxotrophic gr threonine. The transformants that could grow on minimal medium containing glucose, 1% soluble starch and the appropriate amino acid supplements but not threonine were analyzed for the production of cr-amylase by the appearance of halos when the agar was flooded with I2' The analysis of bacteriophage DNA by site-specific was performed as described previously (8). The activity a-amylase was analyzed by electrophoresis using standard
1557
endonucleases and type of methodology
(16).
Vol. 91, No. 4, 1979
BIOCHEMICAL
TABLE I.
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
Bacterial
strains
and bacteriophages
Genotype
Strain
NA64
metB5 purB6 %E' -
mR2
NA64L
-metB5 purB6 j.&iR 3E+
NA64-1
-aro1906 %E+
RUB200
-aro1906
RUB201
=R2
xR2
-aro1906
3R2 . -thr xEO7 xR2
(03T)
RUB202
-arc1906
*A
=R2
RUB203
-aro1906
-linR %EO7
RUB204
-aro1906
*A
=B
xEO7
xR2
(03TAmy+>
RUB205
-aro1906
*A
*B
sEO7
sR2
(03T-)
RUB206a
-aro1906
sEO7
RUB207
-aro1906 J&R 9EO7
RUB208
-linR sEO7
sR2
RUB209
-linR zEO7
9R2
RUB500
Bacillus
03T
Thyp3+
83T-
Thyp3-
03TAmy+
ThyP3+Amy+
a One of the original
5EO7
*B
sEO7 xR2
-RR2
(03TAmy') sR2
(03TAmy+)
(03TAmy+)
amyloliquefaciensH
03TAmy+ transformants
of RUB201.
The site-specific endonuclease B&II was prepared and used as T4 DNA ligase was obtained from New described by Duncan --et al. (17). Amino acid supplements were reagent grade. England Biolabs. RESULTS Because can take
up more
population for
the
another
transformation
competent than
one fragment
of transformants trait
cell
will
when homologous (introduction
in
the -B. subtilis
transformation
of DNA, approximately be multiply DNA is
of foreign
1558
transformed used.
DNA), &
To enrich subtilis
system
4 to 6% of any (congression) for
heterologous
RUB201 was
Vol. 91, No. 4, 1979
Figure
1.
BIOCHEMICAL
DNA Fragment profiles
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS
of 03T and 03TAmy+.
03T DNA and @LITAmy' DNA (that of clone 5 is shown) were digested with the site-specific endonucleases EcoRI or I&II and the resulting fragments displayed on an agarose gel= described in Maserials and Methods. Samples 1 and 3, DNA from bacteriophage 03TAmy digested with EcoRI and B&II, respectively; Samples 2 and 4, DNA from bacteriophage 63T digested with &RI and g&II, respectively. The photograph is a composite taken from two gels in order to simultaneously compare both high molecular weight fragments and low molecular weight fragments.
15.59
Vol. 91, No. 4, 1979
transformed threonine
BIOCHEMICAL
with
a mixture
and the
ligated
DNA as described transformants from
five
generated trait
of these
(Fig
1).
stocks the
mixture
acquisition
03T was also
demonstrated
03T-specific
gene,
transformants
bacteriophage
containing
Table Donor DNA
A B
C
the Amy+
five
Genetic Selected
bacteriophage
All were
linked
the bacteriophage
21 of the Amy+ Thy+. to
Thus,
analysis Non-selected Marker
Non-selected Ja Selected Traits
RUB204
RUB205
Thy+
AmY+
RUB208
RUB200
Are+ LinR
AmY+
o/120 O/120
AmY+
21/120
RUB203
RUB200
Aro+ LinR
AmY+ AmY+
O/120 O/120
NA64L
RUB200
Are+ LillR
AmY+ AmY+
581120 241120
RUB208
NA64-I
Are+ LinR
AmY-
471120
AmY-
18J120
Are+ LinR
AmY-
49J120
Each of the transformants The non-selected trait Numerator: non-selected selected by transformation.
AmY-
231120
was selected for the indicated trait. was scored on the appropriate medium. denominator: total number of clones trait;
1560
strain
the bacteriophage.
Marker
NA64-I
and
of Amy+ to bacteriophage
A).
03~ which
of DNA
of lysogeny
of Amy+ with
the Amy+ trait
II.
endonuclease
Recipient
RUB209 a
Linkage
l'hr+
or deletion
any of these
2, experiment
for
be induced
the establishment
production.
(Table
contained
Experiment
insertion
by co-transformation
mP3
RUB204 is a lysogen
03T could
03T carrying
with
between
of a-amylase
lo5
of the site-specific
RUB200 with
gave a 100% correlation
Seven of the
bacteriophage
consistent
of strain
was prototrophic
DNA and heterologous
Bacteriophage
Comparison
alterations
RESEARCH COMMUNICATIONS
of bacteriophage
cl-amylase.
of DNA from
Infection
DNA that
and Methods.
clones.
fragments
revealed
of homologous
in Materials produced
AND BIOPHYSICAL
Vol. 91, No. 4, 1979
To determine
whether
integrates %E
BIOCHEMICAL
the bacteriophage
at the normal
locus,
mapping
experiment
B.
AND BIOPHYSICAL
03~ carrying
bacteriophage
experiments
performed
The amyE locus
in -B. subtilis
Therefore,
loci.
lack
of linkage
strains
RUB203 and RUB208 to either
of these
control
strain
site
than
whether
subtilis
=E
we performed donor
number
the
DNA from
still
strains
integrated
present
in the chromosome
confirm
the
at the =E
contention
transduction
the Amy+ trait strain
corresponding thyP3
linked
three-factor
genome.
mapping
The a-amylase distinct
from
the 2B
produced
an a-amylase
type
a-amylase
with
the
gene. structural
attachment
gene for
observations Preliminary indicate
loci;
the original
an inactive
gene linked that
one
to the
the Amy+ trait
bacteriophage
03T,
definitive
must be performed.
amyloliquefaciens
H is
a-amylase.
2).
the bacteriophage
still
Therefore,
likely
for
Because
observations)
locus.
All
electrophoretically
evidence
is
maps elsewhere.
is
C.
the Amy+ trait gene
and an active
site
gEO7
same relative
These
There
electrophoretically
seven
identical
that
those
strains
03T carrying
B amyloliquefaciens --L
1561
Amy+ transformants to that
was no evidence
in any of the Amy+ transformants.
genetic
Thus
=EO7
two a-amylase
it
to the
To explore
2, part
that
unpublished
Although
subtilis
-B. amyloliquefaciensH(Fig.
subtilis
RUB208.
%E
analyses of &
B).
in Table
to the aP3
to the -B. subtilis
at the normal
the &
and that
and
with
as compared
2, experiment
the Amy+ trait
RUB206 contained
bacteriophage
integrates
locus
the -aro1
2,
is at a different
must be concluded
(S. Phillips,
is
markers
described
of -B. subtilis
that
experiments
recombinant
it
in Table
between
RUB208 and RUB209 gave the
of Amy- transformants
or at the
as described
the Amy' trait (Table
(13)
of the Amy+ trait
contains
experiments
has not
that
gene
the Amy+ recombinant
gene, the
the &
that
site
lies
the lin
NA64L establishes
the Amy+ trait
integration
were
the
RESEARCH COMMUNICATIONS
of a 2B
This contain
subtilis-
result
is
an inactive
the Amy+ trait H a-amylase
of
contain
and not
consistent =E the
an activator
of
Vol. 91, No. 4, 1979
BIOCHEMICAL
2
I
Figure
2.
Polyacrylamide
AND BIOPHYSICAL
3
5
4
gel profile
6
RESEARCH COMMUNICATIONS
7
of a-amylase
8
9
activity.
The production of a-amylase was assayed using polyacrylamide gel Samples are as electrophoresis as described in Materials and Methods. 1, J!. amyloliquefaciensH (RUB500); 2, g. subtilis (NA64); and follows: 3-9 tha seven transductants of RUB201 obtained with bacteriophage 03TAmy . Upper arrow: a-amylase. -B. subtilis
2. subtilis
%E
Furthermore,
locus.
the Amy+ trait
do not
In preliminary
experiments
inhibit
the
were
lysogenic
for
which
also
contained
an active
activity
bacteriophage
and -B. subtilis
arrow:
g. amyloliquefaciensH
the bacteriophage
that
strains
03T carrying
=E
locus
cr-amylase
03T that
of the -B. subtilis
we established
which
a-amylase
lower
a-amylase;
produce
(Yoneda
=E
gene.
of -B. subtilis the Amy+ trait
both
et al.,
carry
&
and
amyloliquefaciens
unpublished
observations).
DISCUSSION Genetic
exchange
limited
and has been
very
described subtilis. described cloning isolated
in
this
This
paper
demonstrated
provide
of heterologous
(11).
only
by modifying
The original
DNA because
the defective
for
and -B. subtilis a few genes.
a means to introduce
was made possible
by Kawamura
from
-B. amyloliquefaciens
between
2B
it
subtilis
1562
foreign
a technique
technique used bacterial bacteriophage
would
is
The techniques DNA into
-B.
originally not permit
chromosomal
fragments
PBSX as the
source
BIOCHEMICAL
Vol. 91, No. 4, 1979
of DNA for cloning
cloning
(11).
AND BIOPHYSICAL
The modification
of heterologous
chromosomal
the -B. amyloliquefaciens
cr-amylase
inserting
a variety
into
bacteriophage
this
technique
into
the -B. subtilis
of metabolic pll
will
permit
fragment
(congression),
it
no primary
selection
for
which
cells
4 a 10 -fold
enrichment
formidable
screening
should
allow
the effects
cl-amylase
in B. - subtilis
2B
liklihood,
to be inserted
by more than genes,
one DNA
of congression
one.
of Bacillus
of
such as a-amylase,
o-amylase,
managed
reducing
gave a
This
approach
genes
encoding
subtilis. in -B. subtilis
of extracellular of these
In all
of the ability
Utilization
containing
inserting
and -B. licheniformis
any source advantage
for
the
03T and for
data).
to clone
exists.
of loci
synthesis
determine
By taking
possible
both
from -B. pumilus
to be transformed is
true
does permit
bacteriophage
DNA from
to an easily
into
is
here
unpublished
of a variety
are a number
on the
et al.,
clones
task
enzymes
There effects
for
the insertion
extracellular
gene into
chromosome.
-B. subtilis
This
DNA.
purified
competent
reported
markers
(Yoneda
RESEARCH COMMUNICATIONS
loci
which
enzymes on the production
exert
(18).
synergistic Experiments
to
of -B. amyloliquefaciens
are in progress.
ACKNOWLEDGEMENTS We thank course their
Dr.
of this technical
Gary Wilson
study.
We also
assistance.
for
his
thank This
valuable
suggestions
Tim Evans
and Scott
study
was funded
during Sutton
by a grant
the for
from Miles
Laboratories. REFERENCES 1. 2. 3. 4. 5. 6. 7.
Priest, Yoneda, (1974) Wilson, Young, Young, Chemical Zahler, (1977) Perkins, 403-407.
F.G. (1977) Bacterial. Reviews 41, 711-753. Y., Yamane, K., Yamaguchi, K., Nagata, Y., and Maruo, B. J. Bacterial. 120, 1144-1150. G.A. and Young, F.E. (1972) 3. Bacterial. 111, 705-716. F.E. (1976) Federal Register 41, 27923. F.E. and Wilson, G.A. (1978) Genetic Engineering, pp. 145-157, Rubber Press, Inc. S.A., Korman, R.Z., Rosenthal, R., and Hemphill, H.E. J. Bacterial. 129, 556-558. J-B., Zarley, C.D., and Dean, D.H. (1978) J. Virol. 28,
1563
Vol. 91, No. 4, 1979
8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18.
BIOCHEMICAL
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
Yoneda, Y., Graham, S., and Young, F.E. (1979) Gene 7, 51-68. Graham, S., Yoneda, Y., and Young, F.E. (1979) Gene 7, 69-77. Cregg, J.M. and Ito, J. (1979) Gene 6, 199-219. Kawamura, F., Saito, H., and Ikeda, Y. (1979) Gene 5, 87-91. Yasbin, R.E., Wilson, G.A. and Young, F.E. (1975) J. Bacterial. 121, 296-304. Williams, M.T. and Young, F.E. (1977) J. Virol. 21, 522-529. G.A. (1977) Gene 1, 169-180. Graham, S., Young, F.E. and Wilson, Tanaka, T. and Weisblum, B. (1975) J. Bacterial. 121, 354-362. Sasaki, T., Yanasaki, M., Maruo, B., Yoneda, Y., Yamane, K., Takatsuki, A. and Tamura, G., (1976) Biochem. Biophys. Res. Commun. 70, 125-131. G.A. and Young, F.E. (1978) J. Bacterial. Duncan, C.H., Wilson, 134, 338-344. and Environ. Microbial., in press. Yoneda, Y., J. Applied
1564