A Bacillus subtilis plasmid that can be packaged as single-stranded DNA in Escherichia coli: use for oligodeoxynucleotide-directed mutagenesis

Gmr. 41 (1986) 331-335

331

Elsevier GENE

1521

A Bacillus subtilis plasmid that can he packaged as single-stranded for oligodeoxynucleotide-directed mutagenesis (Recombinant

DNA;

shuttle vectors;

phages fl and IRl;

nucleotide

DNA in Escherichia

coli: use

sequencing)

Brian J. Schmidt, Jeanne Strasser and Charles W. Saunders * Gerlev Corporntion. Gaithersburg, MD 20877 (U.S.A.) Tel. (301)258-0552 (Received

August

(Revision

received

(Accepted

12th. 1985) October

November

29th,

1985)

7th. 1985)

SUMMARY

A Bacillus subtilis/Escherichia coli shuttle vector was modified to contain the origin of DNA replication of the E. coli tilamentous phage fl, in both orientations. Upon superinfection with an fl-related phage of an E. coli strain containing either of the modified vectors, the single-stranded (ss) form of the plasmid was packaged in virions and released to the culture medium. Each of these ss DNAs has been purified from the virions and used as a template for oligodeoxynucleotide-directed mutagenesis. The resulting mutations were demonstrated by DNA sequencing. The capacity of these vectors to be isolated as phage ss DNA from E. coli and to replicate as plasmids in B. subtilis makes them convenient substrates for the production of oligodeoxynucleotide-directed mutations for studies in B. subtilis.

INTRODUCTION

Oligodeoxynucleotide-directed (o-d) mutagenesis is most readily performed on ss DNA templates (Zoller and Smith, 1982). To study the effects in

* To whom correspondence

and

reprint

requests

should

be

bp, base pair(s); dd, dideoxy;

ds,

addressed. Abbreviations: double

Ap, ampicillin;

stranded:

EtdBr,

ethidium

bromide;

nucleotide(s);

o-d, oligodeoxynucleotide-directed;

forming

PolIk,

units;

Klenow

polymerase

I; n, resistant,

transposon;

[ 1,designates

0378-l 119186/$03.50

0

(large)

resistance;

1000 bp; nt, pfu, plaque-

of E. co/i DNA

fragment

ss, single stranded;

plasmid-carrier

1986 Elsevier

kb,

state.

Science

Publishers

Tn,

B. subtilis of mutations constructed in this way, the procedure, as originally described, would involve (a) subcloning a restriction fragment into a vector based on coliphage M13, (b) performing o-d mutagenesis and nt sequencing on the ss DNA isolated from virions produced from E. coli, and (c) subcloning the restriction fragment carrying the mutation of interest from the replicative form of the phage DNA into a vector capable of replication in B. subtilis (Dubnau, 1985; Vasantha et al., 1984). If this procedure were to be used to introduce a large number of o-d mutations into a particular gene, considerable time would be spent subcloning fragments from the mutagenized Ml3 derivatives to the B. subtilis vector. To avoid such subcloning steps, we have devel-

B.V. (Biomedical

Division)

332

oped a shuttle E. coli to

vector

able to generate ss DNA as plasmid in B.

To enable we

of ss approach

Zagursky

Berman

virtues

of

vectors

which

as plasmids

DNA in E. coli,

of Dente et

(1983) and

(1984). They

lilamentous

phages

the

pBR322 and the lack most of

in

and plasmids

in

of replication

of

As these

fl genome

they replicate

the host

is

2/ Y ‘H2

am;@ *ffzJ

~~~~

+(,,

of

Fig. I. Construction pEMBL9(

fl

origin-containing

+ ) (Dente et al., 1983) was modified

plasmids. by introduction

ofEcoR1 linkers at the unique 0~11 site ofthe plasmid, pGX2469. EXPERIMENTAL

AND

DISCUSSION

We then inserted

fl origin of replication subtilis shuttle

(a) To as an

phages

and fl), and the fl origin

replication pEMBL9( +

fragment, et 1983; 1). Phage 1 (Dotto and Horiuchi, 198 l), a derivative of fl that shows resistance to negative interference of miniphages, was used to superinfect derivativesofE. cofiGX1210 [F’truD36proA+B+] Ia0 lucZAM15 A(lucpro) supE thi zig::TnlO hsdR2 (J.J. Anderson, unpublished) which contain plasmids with the fl origin of replication (pGX3802, pGX3804, pGX3805). In each case, two species of DNA were isolated from phage in the culture supernatant (Fig. 2). One DNA was the size of IRl. From the work of Dente et al. (1983) the more slowly migrating species would be expected to be the ss version of the host plasmid. This is apparently true, as the DNA was insensitive to restriction enzymes that cut the ds form of the plasmid (Fig. 3, and data not shown) and served as a template for dd sequencing and o-d mutagenesis (see below). In contrast, only IRl DNA was isolated from virions

which

the EcoRl

plasmid,

generating

pGX3802,

structed

by cloning

Pv, Pvull;

the

fl

and

pGX3805, A control

EcoRl

fragment

sites are shown. indicated

R, EcoRl; of pEl94

gcnc, and ori

H2, Hincll. sequences.

the

an E. co/i/B.

of the fl sequences.

et al., 1984). Only the relevant

clease recognition boundary

pGX3804

generating

containing

which lacks a B. subtilis replicon

plasmid, (Saunders

fragment

(heavy line) into pGX2464,

differ in the orientation

(II 194

was con-

into

pGX315

restriction

endonu-

as follows: C, Clal;

The wavy lines represent

cmlp refers to the pBR322

the ApK

to the ColEI origin of replication.

resulting from superinfection of GX1210 or derivatives carrying either pGX3 15 or pGX2464, which lack the fl origin of replication (Fig. 2). Further, no DNA was isolated from virion preparations from the culture supernatant of GX 12 10 [pGX3804] treated similarly except that IRl was not added. The presence of the fl ori did not affect the ability of the shuttle vectors to be maintained in B. subtilis. The ds plasmids pGX2464, pGX3804, and pGX3805 each transformed B. subtilis BR151 (trpC2 metB 10 lys-3) (Lovett et al., 1976) efficiently. After three days of daily passage of BR151[pGX2464], BR151[pGX3804], and BR151[pGX3805] on a drug-free medium, about 60% of the isolates from

333

ABCDEFGH

ABCDE ss pGX3805

-

ds pGX3805

-

ss IRl DNA -

ss pGX3805 ss IRl DNA. ss pGX3802

Fig. 3. Virion ss DNA from superinfected insensitive ments

to the restriction

were

Samples

performed

scribed

(Saunders

graphed

upon ss

GX1210[pGX3805]; isolated Fig. 2. Elcctrophoretic IRl

plasmids.

analysis

superinfection

of vrrion-packaged

of E. co/i GX1210

One ml of an overnight

into YT broth

(Zoller

37’ C. IRl (approx.

culture

and Smith,

superinfected

was diluted

100.fold

pGX3805;

1982) and aerated

for 2 h at

was added,

supcrnatant

was filtered through

to give final concentrations sample was incubated

Tris

GSA

rotor

The

a 0.45 pm filter (Nal-

glycol M, 8000 (Sigma) were added

either overnight and

The

at 4” C or at room temper-

for IO min at 10000 rev.,‘min

resuspended

in

2 ml

of

HCI-1 mM EDTA, pH 8. Debris was removed

10 mM

by pelleting

for 10 min at 15000 x g. The supernatant

was extracted

with an equal volume of phenol-chloroform

(1 :l) and precipitat-

ed with 0.1 vol. of 3 M Na’acetate, ethanol. water

The ethanol containing

precipitate

0.01 pgjml

(IO ~1) were electrophoresed Tris-borate-EDTA EtdBr,

and photographed Phage

(A) GXl2lO[pGX315], [pGX3804], treated

upon

illumination

from

Lane G contains

of I DNA.

species is indicated. apparently

agarose

Samples (Sigma)

et al., 1984), stained

of virion

ss DNA (C) EcoRl

and virion ss DNA from

(D) EcoRl not treated.

treatment

of ds

The identity

of

is indicated.

cultures

of

(D) GX1210-

(F) GXl210[pGX2464], an extract

ofGXl210[pGX3804] The identity

from a similarly but no superLane C contains

a

of some of the DNA

Note that, as expected,

ss pGX3804

have the same gel electrophoretic

pGX3805 were purified from the corresponding BR 15 1 derivatives and used to transform GX 12 10 to Ap resistance. Six ApR transformants generated by each plasmid were then superinfected with IRl, and phage DNA was purified. The electrophoretic profile of the phage DNA from all 12 transformants was indistinguishable from that produced from the original GX1210[pGX3804] and GX1210[pGX3805] strains (not shown). (b) Oligodeoxynucleotide-directed

mutagenesis

in

with

by a Fotodyne

superinfected

IRl had been added to the culture.

Hind111 digest pGX3805

0.83;

(B) GX1210[pGX3802],

culture supernatant

infecting

through

(E) GXl210[pGX3805],

and (H) GX1210.

in 150 PI of

of RNase A (Sigma).

DNA

twice

pH 6, and 2.5 vols of 95”; was resuspended

buffer (Saunders

UV light source.

and (E) ds pGX3805,

source.

superinfected

and the

of 5.8% and 2.8yj0, respectively.

ature for 1 h. Phage were pelleted a

treatment

of ds pGX3805

some of the DNA species

UV light

from

GXl210[pGX3805];

GXl2lO[pGX3805];

1982). as de-

for 5 h at 37°C. The culture was then centri-

gene). NaCl and polyethylene

in

(B)EtoRl

et al., agarose

with EtdBr, and photo-

isolated

superinfected

of a mixture

various

fuged for 10 min at 10000 rev.imin in a GSA rotor (Sorvall). culture

treatment

carrying

lOI* pfujml of culture)

culture was aerated

ss DNA fol-

from

0.8”,,

by a Fotodync

DNA

is

The EcoRl treat-

(Maniatis

through

et al., 1984) stained

illumination

GX1210[pGX3805]

EcoRl.

as described

were electrophoresed

(A) Virion

lowing

enzyme

and ss mobility.

each strain had retained the erythromycin-resistance phenotype. In addition, the fl origin was apparently unaffected by passage through B. subtilis. pGX3804 and

As an initial example, we used a 5 1-nt oligodeoxynucleotide primer to insert 14 bp and delete 8 bp from plasmid pGX3805 (Fig. 4). As this replacement will delete a unique XhoI site (and a Hind111 site as well), we enriched for the desired mutation by digestion with XhoI before transformation. The XhoIdigested DNA produced 149 ApR transformants of E. coli HB 101, whereas, without XhoI digestion, 334 ApR transformants were recovered. Analysis of plasmids from 12 transformants arising from the XhoI-digested DNA suggested that three contained the desired mutation. Each of these had a restriction pattern similar to that of the parental pGX3805 except that they were not cut with Hind111 or XhoI, the restriction enzymes whose recognition sites

334

afzJi -- a;fzzJ Ollgonucleotlde. DNA Polymerase, hgase; transform E. co11to ApR )

pE194

pE194

cCAA

pGX3805: Oligonucleotlde:

5’CCG GGC

ATT TAA

ACA TGT

AAA TTT

ACA TGT

TCA AGT

Fig. 4. Oligodeoxynucleotide-directed to a mismatched

(Zoller and Smith, 1982). Equivalent et al., 1982) in the presence reeA 13 atwlilproA2 a strain carrying pictured

mutagenesis

51.nt oligodeoxynucleotide,

to create

TCC

pGX3812.

‘T ,A

CCA

CAC

reaction

pGX3812.

Restriction

heteroduplex

CGA GCT

GAT CTA

CAC GTG

in restriction

endonuclease

recognition

sites: R, EcoRI;

enzyme reaction

buffer (Maniatis HBlOl

1979) to ApR. Among the transformants Pv, PvuII;

was

of PolIk and T4 ligase

of (15 units) XhoI for 30 nun at 37°C. The DNAs were then used to transform

between

AAG3’ TTC

from GX1210[pGX3805]

for 90 min in the presence

were incubated

GCA CTG

i GC

A phage ss DNA preparation

lacy1 gulK2 rpsL20 qi-5 mtl-I supE44) (Bolivar and Backman,

the presumed

AAA

and the primer was elongated

volumes of the elongation

or absence

TC,

GCd CGG

i

annealed

GCT

X. Xhol; H, HindIll;

(h.sd-20

was found

C, C/t/I. Below is

the 51-mer and ss pGX3805.

should have been removed by the mutation. We used one of these altered plasmids, pGX3812, to transform GX1210 to purify the altered plasmid from any parental plasmid that might have also arisen from the heteroduplex donor DNA. The resulting transformant was superinfected with phage IRl, and ss DNA was isolated from virions in the culture supernatant. Sequence analysis (Sanger et al., 1977) of pGX38 12 confirmed the presence of the predicted nt sequence. As a second example of o-d mutagenesis with such vectors, we have used a 43-nt oligodeoxynucleotide to insert a 9-bp sequence containing a BarnHI site into pGX3804. In this case, there was no loss of a restriction site to allow enrichment for mutants. Ten randomly picked transformants were screened, and one contained a plasmid with a BumHI site. DNA sequencing confirmed the mutation to be the one designed.

In summary, we have demonstrated the feasibility of o-d mutagenesis using vectors that can replicate to produce ss phage DNA in E. coli and replicate as plasmids in B. subtilis. Strausberg et al. (1984) have similarly performed o-d mutagenesis on shuttle vectors comprised of the E. cofi filamentous phage M 13 DNA and a yeast expression vector, an approach that has similar virtues. For studies of the effects of o-d mutations in hosts other than E. coli, it should be generally useful to construct vectors that can replicate in the appropriate host organism and can also replicate to produce ss phage DNA in E. coli.

ACKNOWLEDGEMENTS

We thank the Genex DNA Chemistry oligonucleotide synthesis, Mark Guyer,

Group for Ethel Jak-

335

son, and Steve Fahnestock manuscript, paration

for critical review of the

Gene Krauss

and Robin Lynch for pre-

of the figures, and Sally Young for expert

secretarial

Sanger.

F., Nicklen, S. and Coulson,

chain terminating

A.R.: DNA sequencing

with

Proc. Natl. Acad. Sci. USA 74

(1977) 5463-5467. Saunders,

assistance.

inhibitors.

C.W., Schmidt,

and Guyer,

B.J., Mirot,

M.S., Thompson,

M.S.: Use of chromosomal

L.D.

integration

in the

establishment and expression of hlaZ, a Smphylococcus aweus blactamase gene, in Bacillus suhrili.~.J. Bacterial. 157 (1984) 718-726. Strausberg,

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