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