A plasmid cloning system utilizing replication and packaging functions of the filamentous bacteriophage fd

341

Gene, 33 (1985) 341-349 Elsevier GENE

1210

A plasmid cloning system utilizing replication and packaging functions of the fdamentous hacteriophage fd (Recombinant DNA; vectors; multicopy plasmids; fd gene 2; E. coli host; phage I lysogens; on’; antibiotic resistance markers; transposons)

Klaus Geider, Christine Hohmeyer,

Rainer Haas and Thomas F. Meyer

Max-Planck-Institut ftir Medizinbche Forschung, Abteilung Molekulare Biologie. Jahnstrasse 29, 06900 Heidelberg (F. R. G.) Tel. 06221-486 340 (Received

May 18th, 1984)

(Revision

received

(Accepted

October

October

5th, 1984)

8th, 1984)

SUMMARY

DNA cloning vectors were developed which utilize the replication origin (ori) of bacteriophage fd for their propagation. These vectors depend on the expression of viral gene 2 that was inserted into phage I, which in turn was integrated into the host genome. The constitutive expression of gene 2 in the host cells is sufficient for the propagation of at least 100 pfd plasmids per cell. In addition to the fd ori, the pfd vectors carry various antibiotic-resistance genes and unique restriction sites. Some of these vectors have no homologies to commonly used pBR plasmids or to il DNA. The nucleotide sequence of the vectors can be deduced from published sequences. Large DNA inserts can be stably propagated in pfd vectors; these are more stable than similar DNA fragments cloned in intact genomes of tilamentous bacteriophage. Inclusion of phage sequences required for efficient phage packaging and infection with a helper phage resulted in formation of phage particles containing single-stranded plasmid genomes. Growth at 42’ C without selective pressure results in loss of pfd plasmids.

INTRODUCTION

Commonly used prokaryotic cloning vectors have been mostly derived from ColEl-type plasmids or phage I (Vosberg, 1977; see also Maniatis et al., 1982). Other vectors have been taken from plasmids propagated in low copy numbers (RP4, F episome, Abbreviations: phenicol;

Ap, ampicillin;

frg., fragment(s);

multiplicity

of infection;

bp, base

pairs;

Cm, chloram-

kb, 1000 bp; Km, kanamycin; oti, origin

of replication;

pos., position(s)

on the map (in bp); R(superscript),

Tc, tetracycline;

Tn, transposon;

plasmid

carrier

state; (), indicates

0378-l 119/85/$03.30

0

UV, ultraviolet;

resistance; [I, indicates

prophage.

1985 Elsevier

Science

moi,

p, plasmid;

Publishers

pSC101). Single-stranded DNA of derivatives from the filamentous bacteriophage Ml3 (Messing et al., 1977) is a valuable tool for nucleotide sequencing (Messing, 1983). These and other filamentous bacteriophages (fd, fl) can also be used as vectors for subcloning of restriction fragments, because they are not homologous to many of those vectors mentioned above. We have recently constructed a novel vector system based on replication functions of bacteriophage fd. A plasmid with the fd on’ (pfdA1) depends on the coexistence of fd gene 2 in the same cell, which was provided by cloning the gene into pBR325

342

(Meyer and Geider, 1981). Here we present data which extend this cloning system to a series of vectors which stably propagate also insertions of large DNA fragments as double-stranded DNA. One of the pfd vectors constructed can be packaged into a phage particle. Since the DNA fragment coding for fd gene 2 was inserted into phage 1 which was used to lysogenize the host cell, the pfd plasmids do not longer depend on the presence of a helper plasmid.

MATERIALSANDMETHODS

(a) Bacterial strains, bacteriophages

and plasmids

Escherichia coli BHB2600 supE, supF was obtained

from B. Hohn; 1101 has been described (Meyer and Geider, 1981). Phage pfd103 was obtained from R. Herrmann (Herrmann et al., 1980) and phage jZNM616 was kindly provided by N. Murray (Mileham et al., 1980). The sources of other plasmids are mentioned in RESULTS, sections a-c. (b) Plasmid constructions

Cleavage and in vitro recombination of DNA was performed as described (Meyer and Geider, 1981). Transformation of the cells was as described by Hanahan (1983). Those and other procedures have been summarized by Maniatis et al. (1982). For growth of cells carrying plasmids agar and media contained the following concentrations of antibiotics: 100 pg Ap/ml, 50 pg Cm/ml, 20 pg Km/ml, or 20 pg Tc/ml. The basic vector consists of a 262-bp DNA fragment, containing the fd on’ and the KmR gene. Propagation of this replicon requires fd gene 2 protein, which was originally supplied by the helper plasmid pTM12 (Meyer and Geider 1981). To circumvent the two-plasmid system, which results in contamination of pfdA1 with pTM12, we inserted fd gene 2 into the host genome by means of the 1 prophage.

RESULTS

(a) Construction

of E. coli

strains

lysogenic

for

phage I carrying fd gene 2

A central fragment of phage INM6 16, a derivative of 1 plac5 with the wild type repressor gene, was replaced by DNA with fd gene 2 as outlined in Fig. 1. The phage requires suppressor functions of the host for lytic propagation. One of the clones obtained (Fig. 1) was further analyzed. The prophage 1CH6 16 was induced by UV irradiation and propagated lytically on plates. Combined phages from several plates were purified by CsCl banding and their DNA was phenol-extracted. Cleavage with EcoRI produced a fragment which was similar in size to the corresponding EcoRI-HaeII fragment from pTM12. This fragment contains the information for fd gene 2 protein but in addition to phage fd DNA a 200-bp fragment from the gene for TcR of pBR325, where the fragment was originally inserted. Since the fragment is replicated as part of the host genome, the extent of homology and the copy number are not sufficient to detect homology with pBR-plasmid DNA by standard colony hybridization techniques. To lysogenize E. coli sup strains with phage XH616, we infected cells with this phage at an moi of 5. The cells were further grown overnight and then treated with transformation buffer for DNA uptake (Harmhan, 1983). The competent cells were transformed with DNA from plasmid pfdA1 and screened on agar plates containing Km. Only cells lysogenic for phage ACH616 are KmR. The recipient strain was further identified by determination of genetic markers. Curing of the cells from the pfdA1 plasmid can be achieved by growth at 42 ‘C as described in RESULTS, section d. (b) Construction of plasmids derived from the synthetic plasmid pfdA1

In addition to the phage fd replication origin, plasmid pfdA1 bears fragments conferring KmR and a part of CmR (Meyer and Geider, 1981). The CmR fragment was removed, and the new vector with the fd ori and KmR was named pfdA2 (Fig. 2). This vector contains unique restriction sites for EcoRI and HaeII outside the resistance gene which can be used for insertions of foreign DNA.

343

1 NM616

Eco RI

Eco RI

\1

>

insert

HpaI

EcoRI

EcoRI

ICH616

c<

_> fd gene 2

1 Fig. 1. Insertion

of a DNA fragment

A DNA fragment

by cleavage

After terminal

degradation

with DNA polymerase

was ligated

into EcoRI-cleaved

in vitro (Collins and Hohn,

and the phage phage I-resistant About

hybridized

of phage 1NM616.

Prophage

1CH616

pTM12 (Meyer and Geider,

of strain BHB2600

phage

was constructed

1981) with restriction

into the

as follows.

enzyme HaeII.

and the DNA was cleaved with EcoRI.

a DNA insertion.

produced

labeled

for the corresponding

phage plaques

After ligation

the phage

which were blotted

fd DNA. Positive

plaques

phage. These cells were screened

DNA was

on nitrocellulose

(5%) were used to pick for propagation

of pfdA1.

the fd plasmid.

To use pfdA2 for cloning of restriction fragments derived by cleavage with other restriction enzymes we inserted a polylinker into the EcoRI site of pfdA2 with unique cleavage sites for the enzymes BglII, PstI, Sal1 (HincII), andXba1. In the plasmid, named pfdA8 (Fig. 3), the EcoRI site next to the fd origin fragment was lost and an insertion of about 20 bp occurred around position 1400. A second drug resistance marker was introduced by replacement of the CmR fragment in pfdA1 with the ApR from pBR322, resulting in plasmid pfdA3 (Fig. 2). Three adjacent Hue11 sites from pfdA1 were still present after the cleavage and ligation procedure. Another marker, CmR from transposon Tn9, was inserted into a pfdAZderivative (without the EcoRI site) and gave plasmid pfdA4 (Fig. 2). (c) Introduction

1

which replaces

to radioactively

integration

E. coli chromosome

I, EcoRI linkers were ligated to the fragment

which were lysogenic

l/4 of the clones could propagate

of plasmid

phage 1NM616,

1978). Infection

DNA was subsequently minicolonies

PBR 0.2 kb

with fd gene 2 into the genome

with fd gene 2 was obtained

The fragment packaged

I

of the fd packaging

signal into

pfdA2

As shown in Table I, superinfection of cells carrying plasmid pfdA2 with an fd helper phage produces

phages with the pfdA2 genome at a low level. It has been demonstrated (Schaller 1979; Dotto et al., 1981) that DNA sequences adjacent to the replication origin for complementary strand and viral strand synthesis of filamentous phages (Meyer and Geider, 1980) in the direcion of gene 4 on the phage genome carry signals for phage morphogenesis. Such a DNA sequence from nucleotide 5417 to nucleotide 5830 on the fd genome (Beck et al., 1978) has been cloned into pBR322 as plasmid PIG-414RB (R. Sommer, R. Herrmann and H. Schaller, unpublished). The fd on’ in pfdA2 was replaced by this fragment to give pfdB2 (Fig. 4). Attempts to remove an 85bp duplication arising from the cloning procedure were not successful. The F episome was transferred into strain BHB2600(ICH616)[pfdB2] to allow infection by a helper phage required for packaging experiments. Selection for an antibiotic resistance marker of the helper (Table I) eliminates uninfected cells. Since packaged viral strands of pfd-plasmids cannot form plaques, they have to be determined by colony

344 EcoRI

Hind111

/

/

B*mH’

Ap-frgm. from pBR322

EcoRI 3.30/o

PVUI - PSI1

Hat=11

Fig. 2. Construction DNA polymerase was cleaved

of pfdA vectors.

Plasmid

pfdA2: Plasmid

I, EcoRI linkers were ligated to the fragment.

pfdA1 was partially

again with Hue11 to remove two small Hue11 fragments

with Hue11 and EcoRI. The ApR was recovered

from pSV2-CAT,

and EcoRI

Plasmid

cleaved

and ligated into the pfdA fragment.

plasmid

unpublished)

<(EcoRI)

with DNA polymerase

were cleaved

from:

with HueII,

pBA

Hind111 BglII

I and blunt end joining

fN-JR9 &I

BumHI

which were left from pfdA1. Plasmid

a pBR322 derivative

pfdA4: The EcoRI

and the 1590-bp fragment

-

EcoRI

90

cleaved with HueII. After terminal

degradation

with

The DNA was then cleaved with EcoRI and ligated. The plasmid obtained

(pfdA2E).

(Gorman

site in pfdA2 was removed This plasmid

pfdA3: pfdA1 was cleaved

et al., 1982), after cleavage

and pHM2,

with CmR was ligated into pfdA2E.

with Hue11

by filling the ends of the EcoRI pBR322

with Tn9 (H. Matzura,

( ), indicates

site abolished.

formation of infected cells. For the helper phage pfd103 we also measured the plaque titer which was found to be larger than the titer determined by drug-resistant colonies by a factor of 20 to 100. As listed in Table I, packaging of the defective phage pfdB2 was usually one order of magnitude lower than that of the helper phage pfd103, and packaging of pfdA2 was lower by three orders of magnitude relative to pfd103. DNA of purified phages from a packaging experiment was also visualized on gels. No band was visible in the position expected for single-stranded pfdA2 DNA. The DNA band corresponding to single-stranded pfdB2 DNA

Fig. 3. Insertion

of a polylinker

tion of pfdA8: Plasmids

fragment

pfdA2, pUR222

into pfdA2. Construcand pBA were cleaved

with EcoRI and PsrI and ligated. The linker insert was derived from two EcoRI-PstI 23 bp from pUR222

fragments:

67 bp from pBA (2.8 kb) and

(2.7 kb). ( ), indicates

site abolished.

345

TABLE

I

Formation

of infectious

The cells (column 1 h at 37°C supernatant

particles

upon superinfection

titered for plaque-forming

a dilution of the phage supernatant Then 0.2 ml were spread

cells infected

for the infective

and the culture particles

was further

on strain

overnight

cells of BHB2600

on Km plates indicated

infectious

carrying

F + (ICH616).

were centrifuged

resistance

particles

with pfd DNA (column

D). In some experiments

markers,

and the 0.1 ml of

They were left at 37°C for 30 min.

The Km plates were incubated packaging

at 37°C for I day and

C), colonies

on plates with

of pfdA2 or pfdB2 was tenfold

listed in column C.

Cells

Plaque

Km

Cm

(A)

(B)

(C)

(D)

titer of fd103

Cells resistant

BHB2600

F+ (ICH616)[pfdA2]

5 x 10”

2x

BHB2600

F+(,lCH616)[pfdB2]

7 x 10”

6 x lo*

to

106

6x

IO9

6 x 10’

was about 50-fold lower than that for single-stranded DNA of phage pfd103 reflecting the titer and the size of the two phages.

EcoRI BamHI

with phage fd103 (moi = 5). After

at 30°C. The cultures

B). To titer the particles

of plates with Km or Cm, respectively.

with the helper phage fd103 (column

centers

of 3 x lO’/ml and infected

aerated

1101 (column

were mixed with 1 ml of log-phase

on the surface

the Cm plates up to 2 days. Colonies increased

pfd plasmids

A) were grown at 37°C with 20 ng Km/ml to a density

50 ng Cm/ml were added

Cm represented

of cells carrying

&

(d) General properties of pfd plasmids

HaeII

Hind111

’ T SmaI

PVUI

insertion of fd packaging-signal from pIG414-RR EcoRI

The nucleotide sequence of all plasmids constructed can be essentially deduced from published sequences (Table II). The DNA fragment conferring KmR (NPTI) from plasmid R6-5, formerly inserted into pACYC177 (Chang and Cohen, 1978) and then into phage fd106 (Herrmann et al., 1980) was found to be about 60 nucleotides shorter than expected from Hue11 cleavage of Tn903 (Oka et al., 1981). The BamHI site in the fd replication origin can be used for insertion of DNA (Zahm et al., 1984), but replacement of the BamHI-HaeII fragment of pfdA2 by foreign DNA (Simon, 1983) or its deletion causes an extreme reduction of the plasmid copy number, since these procedures remove part of the replication origin for complementary strand initiation (Geider et al., 1978). Other parts on the genome may then substitute for initiation functions (Cleary and Ray, 1981; Kim et al., 1981). In cells without fd gene 2,

Fig. 4. Construction signal. Plasmids were cleaved pfdA2”

XhoI

of pfdAl.

ofvector

pfdA2”

pfdB2 with the phage fd packaging

and PIG-414RB

with EcoRI

and BamHl

(RESULTS,

section c)

and the DNA

is a version of pfdA2 with the three adjacent

ligated.

HaeII sites

346

TABLE

II

Referenced

list of sequenced

DNA fragments

used in pfd vector

constructions

The maps in the Figs. and the fragments

in this table are presented

such that the DNA fragment

has 5’-to-3’

All other DNA fragments

are aligned according

Additions

direction

in the viral strand.

or deletions

can be found in RESULTS,

(1)

Fragments

(A)

fd origin pos. 5568 (HueII)

(B)

fd

with the origin of phage fd replication

to their ligation to the phage DNA (panel I).

a-c.

sections

used in construction

origin

including

References

to 5829 (HueHI-/&RI)

packaging

sequence

Beck et al., 1978 pos. 5415

(HueIII-BamHI)b

to

pos. 5829

Beck et al., 1978

and pBR322 residue (pos.

Beck et al., 1978

(HueHI-/&RI) (C)

fd gene 2 fragment, approx.

pos. 5829 (EcoRI of fdll)”

to pos. 1014 (EcoRII)

Sutcliffe,

30 to 232)

(E)

KmR (NPTI)

of Tn903 (601,55) approx.

(F)

ApR (blu) from pBR322,

(G)

Cm-resistance

pos. 2721 (HueII)

Sutcliffe,

to pos. 4362/O (EcoRI)

(cut) of Tn9, pos. 1 to 1102 with part of flanking

IS Z-DNA, pos. 138 to 1 (HueII)

Vectors

pfdA2:

constructed

fragments

using the above fragments

F + E + A

pfdA4:

fragments

E’ + G + A’

pfdA8:

linkerf + fragments

source

site of pfdll

b Addition

1978

or after modification

E + A

E + repeatg

of gene 2 protein:

a EcoRI

1979

and Ohtsubo,

E + A

pfdA3: fragments

pfdB2: fragment

directly

1978; 1979

Alton and Vapnek, Ohtsubo

and 768 to 416 (HueII)d

(2)

1979

Oka et al., 1981

pos. 2200 to pos. 830 (HueII)

+ fragment

prophage

(Herrmann

of BumHI

linker

B

ICH616;

the central

EcoRI

fragment

in INM616

was replaced

by fragment

C.

et al., 1981). to blunt

end

after

Hue111

cleavage

of fdll-DNA

(R. Sommer,

R. Herrmann,

and

H. Schaller,

unpublished). ’ Meyer and Geider

(1981).

d Tn9 was transposed

into pBR322

e Filling of EcoRI-site

in pfdA2 and removal

’ See linker sequence

of plink322

a BumHI

(pHM2) (Maniatis

(H. Matzura,

unpublished).

of second Hue11 site of the fd-fragment. et al., 1982: p. 7) and pUR222

(Rtlther

et al., 1980).

site at pos. 5646 to Hue11 site at pos. 5560 (Beck et al., 1978).

pfd plasmids can give rise to a few antibioticresistant colonies (less than 10e3 compared to cells lysogenic for phage KH616), which contain an autonomously replicating plasmid carrying fd gene 2. The mechanism of this rare recombination event is unknown. The pfd plasmids were estimated to replicate at about 100 copies per cell. This number does not only depend on the inserted DNA, but also on the growth temperature. The latter property is due to the thermolability of fd gene 2 protein (Lin and Pratt, 1974; Meyer and Geider, 1979). Cell growth at 42°C strongly reduces the cellular copies of the pfd plasmids, which can thus be eliminated in the absence of selective pressure (Table III).

The pfd vectors constructed were used in cloning parts of the Ti-plasmid of A. tumefuciens, the pilus gene of Neisseria gonorrhoeae and to insert fragments with an antibiotic resistance. Fragments up to 15 kb could be stably propagated (Table IV).

DISCUSSION

We have developed novel vectors for DNA cloning which consist of the fd otiand resistance markers. The replication of these pfd plasmids depends on the synthesis of fd gene 2 protein in the same cell. To circumvent the use of a helper plasmid, a fragment with fd gene 2 from pTM12 (Meyer and Geider,

347 TABLE III Loss of pfd piasmids and of phage fd by propagation of carrier cells at 42°C The cells (column A) were diluted 1: 100 from a stationary culture into rich medium or spread on plates and then grown at 42°C overnight. They were then plated on rich agar (column B) or spread on plates containing 20 pg Km/ml (pfd plasmids) or 50 pg Cm/ml (fdiO3) (column C). From the culture alter the first passage, cells were diluted 1: 100 into fresh medium and grown again at 42°C overnight. The titer of cells still carrying pfd plasmids is listed in column D. Control experiments at 37°C with strain BHB26~(~CH616)[pfdA2] showed no loss of the pfd plasmid alter two passages in medium without Km. Liquid culture

Cell titer of culture (colonies/ml)

Titer on selective plates (colonies/ml) First passage

Second passage

(A)

(B)

(C)

(D)

BHB2600(nCH616)[pfdA2] BHB26~(~CH616~[pfdB2] 1lOl[fd103]

5 x 108 f x IO9 5 x 10s

2x 10’ 3 x 10’ 2x 10’

1 x 105 4x 106

Plate culture

Single colonies tested

Growth on selective plates

BHB26OO(XH616)[pfdA2] second passage

14 5

5 0

TABLE IV Examples of DNA cloning in pfd vectors Basic vector (cloning site used)

Insert

Screening procedure

pfdA2 (EcoRI)

EcoRI-frgm. 1 from pTi-C58 (14.25 kb) HindHI-frg. 23 from pTi-C58 (3.2 kb) HindHI-frg. 23 from pTi-C58 (3.2 kb) structural gene for pilus protein from N. gonorrhoeae (0.53 kb)b intact transposon Tn3 from RSF1050/67-24 (5.2 kb)

colony hybridization with fragment in pBR325

pfdA3 (HindIIIf pfdA4 (HindHI) pfdA8 (EcoRI)

pfdB2 (EcoRI)

subclone from above fragment, gel elution, inactivation of KmR, size determination of insert recloning of fragment from pfdA3 insertion; inactivation of KmR

colony hybridization with larger fragment in pBR322

resistance to Ap and Km

a Schweitzer et al. (1980); b Meyer et al. (1982); c Heffron et al. (1978).

348

1981) was inserted into the host by means of a I prophage. Because the pfd vectors strictly require fd gene 2 protein for replication, these plasmids are biologically safe cloning vectors with the limitation that some of the pfd plasmids occasionally pick up the fragment with fd gene 2 from the lysogenic phage 1CH616 to form an autonomously growing plasmid. The fd cloning vectors pfdA2 and pfdB2 are not homologous to other cloning vectors commonly used. They are therefore a convenient tool for subcloning of restriction fragments. Cells with a pfd vector can be cured from the plasmid by growing the cells at 42°C in the absence of antibiotic. The plasmids can therefore be used as a removable marker for cells to do genetic manipulations. The pfd plasmids are also useful as donors for transposons. Cells may be left with the donor plasmid carrying a transposon insertion for an appropriate time, and then they can be either cured, or transposition to another plasmid can be analyzed with isolated plasmid DNA. Plasmid pfdB2 can be packaged as single-stranded DNA into phage particles after infection with a helper phage. This plasmid contains the packaging signal, which comprises an fd sequence located between fd gene 4 and the replication origin (Schaller, 1979; Dotto et al., 1981; 1984). Plasmids carrying a DNA segment comprising the intergenic region of filamentous phage fl have been shown to be efficiently packaged (Dotto and Horiuchi, 1981; Dente et al., 1983). The relatively low packaging rate of pfdB2 compared to the helper phage can be explained with a less efficient mode of DNA replication. Because the fragment from the intergenic region used in the construction of pfd vectors does not cover the whole recognition sequence for viral strand replication, the viral gene 2 protein might be more readily available for the infecting helper phage than for propagation of plasmid pfdB2. This interpretation agrees with the observed phenotypic suppression of mutations in the viral strand replication origin by an increased level of gene 2 protein (Dotto and Zinder, 1984) and the finding that cloning of the whole intergenic region shows a reduction of the titer of an infecting helper phage, whereas deletion of sequences within the initiation signal for viral strand synthesis abolishes interference with the helper phage (Zagursky and Berman, 1984).

The insertion of a 15-kb fragment into the EcoRI site of pfdA2 resulted in a stable hybrid DNA, which was not reduced in size even after long time passaging. This is often not the case for cloning of large DNA fragments into the whole fd or Ml3 genome (Herrmann et al., 1980; Dente et al., 1983), although for certain Ml3 vectors stable propagation of large inserts has been reported (Barnes et al., 1983). We assume that packaging and infection of cells with filamentous phages but not replication of the phage DNA (Meyer and Geider, 1982) cause the instability of large DNA inserts. Since the nucleotide sequences of filamentous bacteriophage differ only in a few positions (Beck and Zink, 1981) without a known functional impact on phage complementation, many data on pfd cloning vectors presented here can also be applied to cloning vectors derived from replication functions of bacteriophage M 13 and fl.

ACKNOWLEDGEMENTS

We thank E. Piaskowski for help in the phage packaging experiments and U. Albrecht and H. Scherer for assistance in the analysis of pfd plasmids.

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