Transfection with Mu-DNA

VIROLOGY

81, 152-159 (1977)

Transfection GERTH Department

PIETER C. WESTMAAS,

of Biochemistry,

University

with Mu-DNA VAN

DE AND

of Leiden,

PU’ITE, CAREL WIJFFELMAN

Wassenaarseweg

Accepted April

64, Leiden,

The Netherlands

1,1977

The frequency of transfection with Mu-DNA in a RecBC, SbcB strain treated with CaClz is extremely low. The frequency of transfection is 100- to 1000-fold higher when, prior to the CaCl, treatment, early Mu functions are expressed in the recipient cells. It was found that the products of the early Mu genes A and B, which are necessary for integration and replication, do not play a part in the observed stimulation of transfection. The gene which is responsible for the transfection stimulation could be mapped using XpMu and AdMu phages, which contain different portions of the early region of Mu, as helper phages in transfection experiments. This gene is located between genes B and C of Mu, very near the gene whose product causes the death of the host after thermoinduction of a defective Mu prophage. INTRODUCTION

In the past it has been shown that it is possible to transfect Escherichia coli spheroplasts with DNA extracted from different phages such as A and T4. However our attempts to transfect E. coli spheroplasts with Mu-DNA were not successful. The absence of a measurable transfection might be due to the fact that Mu has a linear genome, which cannot circularize by cohesive ends or terminal redundancy, leaving the double-stranded ends of the Mu-DNA susceptible to exonucleolytic breakdown in the spheroplasts. Successful transformation with linear DNA has been described using Ca2+-treated cells which are deficient in the ATP-dependent exonuclease, the product of the recBC genes (Mandel and Higa, 1970; Cosloy and Oishi, 1973; Wackernagel, 1972). With this system Mu transfection could be found, but the frequency was extremely low in comparison with transformation with linear E. coli DNA or transfection with A DNA. When the transfection experiments were repeated under conditions where early Mu proteins were present in the recipient cells at the time of transfection, a large stimulation of transfection was found.

In this paper we describe the tion experiments with Mu-DNA localization of an early Mu gene responsible for the stimulation of tion. MATERIALS

AND

transfecand the which is transfec-

METHODS

1. Bacterial strains, bacteriophages, and media. The bacterial strains used are listed in Table 1. Most of the transfection experiments were done in strain JC7620 or derivatives of this strain. Thermoinducible defective or amber Mu prophages integrated in the trp operon were introduced into this strain by Pl transduction of a CysB derivative (PP242 or PP243) to Cys+, followed by scoring for Trp-. The Mu bacteriophages used are: Mucts62, Mucts62Aam1093, Mucts62Bam1066, (Howe, 1973), Muc2000 (Wijffelman and Van de Putte, 1974), Mucts4Cam2005 (Wijffelman et al., 1973), and Mu-Aam (Toussaint and Faelen, 1973). The media and plating procedures for phage Mu have been described previously (Wijffelman et al., 1972). 2. Characterization of AdpglMu and XpMu phages. The AdMu phages were isolated in principle as described by Boram and Abelson (1973). We used strain PP136,

152 Copyright All rights

8 1977 by Academic Press, Inc. of reproduction in any form reserved.

ISSN

0042-6822

TRANSFECTION

WITH

TABLE

153

Mu-DNA

1

BACTERIAL STRAINS Chromosomal

Strain number

Origin

markers

PP 241

thr leu proA his thi argE sbcB recB recC tsz phx strA lacy galK xyl mtl ara sup-37 Markers JC 7620 AtonB-trp

PP 242

Markers

JC 7620

PP 243

Markers

JC 7620 except sup-37, cysB Su-

PP 244 PP 245

Markers JC 7620 trp::(Mucts62AD-P) Markers JC 7620 except trp:: ( + Mucts62AamlO93) SuMarkers JC 7620 except trp::(-Mucts62BamlO66) SuMarkers JC 7620, (-Mucts62BamlO66 Markers JC 7620, cysB (lam) thi galK thi galK trp::(Mucts62 AD-p) trp::(Mucts62 AD-p) thi thyA bio serA recA pgi pgl rel ttw tonA (lam) pgi pgl rel ttw tonA

JC 7620

PP 246 PP 562 PP 296 KA 52 KMBL 1699 KMBL 3260 KMBL 1368 DF 1311 PP 132 PP133 KMBL PP 135 PP 136 KMBL KMBL KMBL KMBL KMBL

1164

1001 1014 2302 2304 2332

pgi thi thi thi

suSu thi thi thi

lam galK trp:: (Mucts62AamlO93) galK trp:: (Mucts62Bam1066) galK trp:: (Mucts4Cam200.5)

a A lysogen in which Muc+Aam3011 is integrated in the chlD gene, so that the pgl gene is located between the A and the Mu prophage. After uv induction of the A prophage, XdpglMu phages can be found among the A+, Pgl+ transductants by testing them for Mu immunity. However we selected directly for AdpglMuc+ hybrids by using a recipient strain lysogenic for a Mu&s62 in the thyA gene (PP133) and selecting for Pgl+ transductants at 43”, after expression at 32” overnight. Because c+ is dominant over cts and the c+ gene is carried only by those Adpgl phages which have picked up part of the Mu prophage, every Pgl+ transductant is due to a AdpglMuc+phage. From these transductams the Mutts prophage was removed by

et al., (1971)

sup-37,

JC 7620, selection for spontaneous TonB mutants, followed by replica-plating for TrPPP 241, cysB introduced by Pl transduction, with selection for Trp+ CysPP 242, made by conjugation with an HfrH, Su strain PP 242, see text PP 243, see text

sup-37,

PP 243. see text

cysB

pgl rel ttw tonA thyA::(Mucts62) Alac-pro supE Alac-pro supE (lam) Alac-pro supE (lam)chlD::(MuAam

Kushner

AJ-6)

3011)

PP 242, see text PP 242 Buttin Westmaas et al. (1976) Westmaas et al. 1976) Wijffelman et al. (1973) Kupor and Fraenkel (1969) DF 1311, cured for lambda prophage Ab2i434 PP 132 Collection of Van de Putte KMBL 1164 PP 134 W 1485 KMBL 1001 Wijffelman et al. (1974) Wijffelman et al. (1974) KA 52

with

Pl transduction with selection on Thy+. HFT lysates were prepared by uv induction. Eighty independently isolated AdMu phages were tested for the presence of Mu genes by complementation and markerrescue experiment. For that purpose sustrains lysogenic for MuctsAam (KMBL (KMBL 2304) or 2302), MuctsBam MuctsCan (KMBL 2332) were grown at 28”. At a concentration of lo8 cells/ml the bacteria were infected at 43” with the HFT lysates (m.o.i. of A+ = 10). After 90 min of growth at 42” the production of Mu amber phages (complementation) was measured on PP135 and that of wild-type Mu phage (marker-rescue) on KMBL 1014. The data of the AdMu phages used in this paper are shown in Table 2. The ApMu phages used

154

VAN

DE PUTTE,

WESTMAAS,

AND

WIJFFELMAN

are XpMuctsA (96-491, hpMuctsAB (98-961, same volume of ice-cold 100 mikf CaCl,, XpMuctsABFEiZ (102), and XpMuctsABkiZand kept in ice for 20 min. Finally the cells Clys (504) (Magazin, Allet, and Howe, in were centrifuged and resuspended in 100 preparation). The presence of the kil gene mM CaCl, in l/10 of the original vol. Muwas determined by M. Gassler (personal DNA (5 ~1) was added to 100 ~1 of cells and communication). the mixture was incubated at 40” for 10 3. Isolation of Mu-DNA. The Mu-DNA min. After a lo-fold dilution in LC broth used in transfection experiments was iso- and an incubation of 30 min at 37”, infective centers were measured on a suitable lated from Muc2000, a clear plaque mutant which gave high titers when grown by con- indicator strain. Control experiments fluent lysis (Wijffelman et al., 1974). showed that under these conditions the first free phages were produced 45 min Phage stocks were purified in CsCl equiafter the start of the incubation at 37”. librium density gradients. The phages In the case where A-Mu phages were were dialysed against 2x SSC (1 x SSC: 0.15 M NaCl, 0.015 M Na-citrate). To the used as helper phages, the induction at 43” was omitted and replaced by an adsorption dialyzed sample (3 * 1012 phageslml), 1 vol of phenol, saturated with 2x SSC was period for 20 min at 37”. added. The mixture was gently shaken at RESULTS room temperature for 5 min, and then centrifuged at 5000 rpm for 10 min. The upper 1. Transfection with Mu-DNA aqueous phase was taken and extracted with phenol for a second time. Finally the The transfection frequency of Mu-DNA DNA solution was extensively dialyzed in a RecBC, SbcB strain was extremely against 0.1 x SSC. low (Table 3, lines 1 and 2). However when 4. Transfection produced. The transfectransfection was performed in cells in tion procedure was slightly modified from which early Mu proteins were present, the that described by Coslow and Oishi (1973). frequency of transfection was strongly Recipient strains were grown overnight in stimulated. For this purpose PP244 was LC broth at 28”, diluted into fresh me- used, in which a thermoinducible defective dium, and grown to 2 * lo8 cells/ml. Strains Mu prophage is present, containing the c, containing defective or amber Mu pro- A, B, C, and Zys genes of Mu. On induction ocphages were then induced for 20 min at of this strain no Mu-DNA replication 43”. After centrifugation the cells were re- curs (Goosen, personal communication) and only early RNA synthesis takes place suspended in 0.5 vol of ice-cold 10 mM CaCl,, pelleted, resuspended again in the (Wijffelman and Van de Putte, 1974). By TABLE COMPLEMENTATION Isolate numher

Phages (No./ml) Aam KMBL

(Ksyre: 1014 110

1

38 16 3 10 A (control)

AND MARKER-RESCUE

3.8 4.6 3.8 3.7

x lo3 x lo3 x lo3 x 103
n The A gene in the hdMu

iF2)

mea-

PP 135”

2.0 2.0 1.8 1.7 1.9 2.0

x 104

x x x x x

104 lo4 104 104 104

after infection

2

DATA

OBTAINED

Barn (KsMFeIj 203no4)meaKMBL

1014

WITH

of a su- host, lysogenic

PP 135

5.0 5.5 2.0 3.0 2.7 5.0

AdpglMu

Cam (Ksffe: KMBL

PHAGES

for

1014

Mu genes present iF23

mea-

PP 135

x 103

10

x 10’

10 20 106

1.2 2.0 1.5 1.2 1.1

10

1.4 x 1056

x 103

x 10’

x 10’ x 103


x x x x x

105 105 105 105 10’

C

CA CAB CAB cABC -

phages can be shown to be present only by marker-rescue, because the hdMu a MuAam3011 insertion in the chZD gene which does not complement

phage was derived from MuctsAamlO93. b This value is rather high for a control. The reason for this is that it has recently been shown that A can complement partially for the C gene function of Mu (M. Gassler, personal communication).

TRANSFECTION

WITH

Mu-DNA

155

that the recipient cells are RecBC, SbcB, because when the early Mu genes are exTHE INFLUENCE OF BACTERIAL AND MU PROPHAGE pressed in Ret+ cells the transfection freMUTATIONS ON THE FREQUENCY OF TRANSFECTION” quency is found to be extremely low (Table TransfecRelevant characteristics Strain tion fre3, line 8). So, even in the presence of the quency” transfection-stimulating factor, the Mu<10-T PP 242 recBC sbcB DNA can still be broken down by exonu
3

156

VAN

DE PUTTE,

WESTMAAS,

ment in which a MuctsBam defective prophage, deleted from the 0 end, is induced in a permissive RecBC, SbcB host before transfection (PP562). As expected, a strong stimulation of transfection was found (Table 3, line 6). If the stimulation of transfection was due to recombination, some of the infective centers would consist of B amber phages. For this reason the infective centers were allowed to grow for one cycle (90 min at 37”) before analysis. Less than 0.5% of the progeny consisted of amber phages, indicating that recombination does not play a significant part in the stimulation of transfection. 2. Localization of the Transfection-Stimulating Factor on the Mu Genome To test whether the A or B gene products of Mu are responsible for the observed stimulation of transfection a nonpermissive RecBC, SbcB strain lysogenic for MuctsAam or MuctsBam was used. It has been shown previously that on induction of an A or B amber lysogen only early RNA synthesis takes place (Wijffelman et al., 1974). Therefore the only known difference between induction of a defective prophage and induction of the amber lysogens is the absence of the A or B gene products. As shown in Table 3 (lines 4 and 5) the stimulation of transfection is also found with these amber mutants. So the products of the genes A and B are not responsible for the observed stimulation of transfection. This stimulation is probably due to the activity of another early gene of Mu. We know that besides the genes A and B at least two other genes are present in the early region of Mu. There is a gene called ner, which is responsible for the negative regulation of early RNA synthesis (Wijffelman et al., 1974) and a gene called kil which is responsible for the killing of the host under conditions where only early Mu functions are expressed (Westmaas et al., 1976). We decided to map the transfectionstimulating factor using h-Mu phages which contain varying amounts of the early region of Mu. Instead of inducing a

AND

WIJFFELMAN

thermoinducible defective Mu prophage in the recipient cells, these cells without a defective Mu prophage were now infected with the A-Mu phages before the transfection procedure. The recipient cells were lysogenic for A so that after infection with the phages only the Mu genes are expressed. Two clases of h-Mu phages were used, defective and plaque-forming ones. The isolation of the defective phages is described in Materials and Methods. These XdpglMuc+ phages were classified by testing them by marker-rescue and complementation for the presence of the early Mu genes A and B and gene C. The presence of the kil gene of Mu was determined by using the phages for specialized transduction of the pgl gene. When transduction is performed with a h-Mu phage which carries the kil gene, the number of Pgl+ transductants in a Mu nonimmune recipient is low, due to the expression of the kil gene in the nonimmune cells. The 80 independently isolated AdpgZMu phages tested in this way could be divided in two classes: In one class the transduction frequency is approximately %O-fold lower in the Mu-sensitive host. These A-Mu phages are considered to be kil+ and contain in all cases at least the A and B genes of Mu. In this way the kil gene of Mu was mapped between genes B and C. The results of the transduction experiments of a number of AdpgZMu phages falling in different classes are shown in Table 4. These A-Mu phages were tested for the ability to stimulate the transfection with Mu-DNA. The results are shown in Table 5. All A-Mu phages which contain the kil gene give a stimulation of transfection comparable to the stimulation obtained after induction of a defective prophage containing the early region of Mu. With the A-Mu phages which lack the kil gene the transfection frequencies were lower by a factor of 10-20. Therefore the transfection-stimulating function maps very close to the kil gene. Plaque-forming A-Mu phages were tested for their ability to stimulate transfection. The advantage of these phages in that the multiplicity of infection can be more strictly controlled. The results are shown in Table 6 and are comparable with

TRANSFECTION TABLE

4

SPECIALIZED TRANSDUCTION OF THE pgl GENE WITH ApglMu PHAGES” Pgl+ transductants “Killing ISOPhage late (No&O4 recipients) property” number PP 133 PP 132 (Mu-sen- (Iv&i;sit&) 1 38

AdpglMuc AdpglMucAmb

200 153

200 168

-

16 3 10

AdpglMucAB AdpglMucAB AdDglMucABC

173 43 22

185 600 500

+ +

a Exponentially growing recipient cells (PP 132 and PP 133) were concentrated to 2 x lo9 cells/ml and mixed with an equal volume of an HFT lysate containing A+ and AdMu phages. Multiplicity of infection was approximately 10, based on the A titer. After adsorption of the phage, the cells were plated on minimal plates containing maltose (selection Pgl+) and thymine. Incubation of the plates was at 32”. b The index “m” at a gene indicates that the gene is detectable only by marker-rescue and not by complementation. TABLE STIMULATION Isolate

number 1 38 16

3 10

5

OF TRANSFECTION

Helper phage AdpglMuc ApglMucA ApglMucAB

ApglMwABkil ApglMucABkilC A+

BY A-Mu

PHAGES’

Tranfection frequency 4.0 x 10-1

4.0 x 10-1 l-2 2.0 2.5 2.0

x 10-6 x 10-S

x 1O-5 x 10-1

(L Cells of PP296 were infected with different helper phages (m.o.i. = 10, based on the AC titer) treated with 100 mM Ca*+ and Mu-DNA was taken up at 40” for 10 min. Infective centers were measured on PP 135.

WITH

157

Mu-DNA

the time of transfection. It has also been shown recently by others that for a successful Mu transfection a helper function is needed (Kahmann, et al., 1976). The early gene which is mainly responsible for this has been mapped and is located between genes B and C. This gene maps very close to the kil gene of Mu, which also maps between genes B and C. This is deduced from the observation that all the hMu hybrids containing an intact kil gene also stimulate Mu transfection, when they are used as helper phages in transfection. The stimulation of transfection appears to be specific for Mu. No effect was found on E. coli transformation. That an early gene product is responsible for the stimulation of transfection has recently become even more probable because we have been able to isolate polar mutants in the early region of Mu mutants (Westmaas and Van de Putte, in preparation). These mutants show among other things a strongly reduced ability to help in Mu transfection. Besides the obvious stimulation of transfection by phages which contain the kil gene we also found a significant stimulation with the helper phage hMuctsAB (Table 6, line 2, and Table 5, line 31, especially when used with high m.o.i. (results not shown). There are several nossible explanations for this phenomenon: First, the transfecting DNA could be rescued by recombination with the helper phage. This must then be a consequence of the high m.o.i. of the helper phage, because, when a defective B amber prophage was used to stimulate the transfection, recombination TABLE

those obtained with the defective A-MU phages. Again the phages which carry the kil gene strongly stimulate transfection. By increasing the m.o.i., there is also an increase in stimulation of transfection but in all cases the difference between a kil+ and a kill phage remains approximately a factor of 10-20. DISCUSSION

We have shown that for an optimal transfection with Mu-DNA, early Mu gene products have to be present in the cell at

6

THE EFFECT OF INFECTION OF ApMu PHAGES ON THE FREQUENCY OF TRANSFECTIONO

Helper

phage

ApMuctsA ApMuctsAB ApMuctsABkil ApMuctsABkilClys,b AC1857

Transfection quency (96-49) (98-96)

(102) (504)

2.0 5.0 6.0 7.0 6.0

fre-

x 10-G x 10-6 x 1O-5 x 10-s

x lo-’

a For experimental conditions see footnote to Table 5. The m.o.i. was 5. b The index “m” at a gene indicates that the presence of the gene can be shown only by markerrescue and not by complementation.

158

VAN

DE PUTTE,

WESTMAAS,

was found to be extremely low (see Results, Section 1). Second, there could be an unknown gene, located between A and kil, whose product plays a minor part in transfection stimulation. The third and most plausible explanation is that at a high m.o.i. with ApMuctsAB the recipient cells are supplied with such high levels ofA and B gene products that the transfecting DNA is protected long enough to express its own early gene which is needed for the transfection. The extremely low transfection frequency in the absence of helper functions proves that the early gene product is needed in a very early stage of phage development, even before early RNA synthesis takes place, because for all the transfection experiments the DNA was isolated from Mu phages which carry the complete early region. Therefore we propose that on normal infection with Mu, this product is injected together with the DNA into the host cell. However, this hypothesis needs further confirmation. That a Mu protein is needed for successful transfection with Mu-DNA is probably due to the particular features of the MuDNA itself. In the first place the Mu-DNA enters the cell as a double-stranded linear DNA molecule, which can not circularize by sticky ends or by terminal redundancy. However it is plausible that the Mu-DNA has to circularize before it can integrate. This circularization could be performed by a protein, as for instance that found with the Bacillus subtilis phage 429. In this case the same protein also seems to be essential for transfection (Ortin et al., 1971; Hirokawa, 1972; Yanofsky et al., 1976). In the second place the vegetative Mu-DNA contains bacterial DNA at both ends (Daniel1 et al., 1975; Bukhari and Allet, 1975). This bacterial DNA is lost during integration (Hsu and Davidson, 1974; Bukhari and Allet, 1975). So the MuDNA has not only to circularize but also to get rid of its bacterial DNA during the integration process. The early gene product which is essential for Mu transfection could play a role in one or both of these processes. We thank

ACKNOWLEDGMENTS Nice Guijt, Bep Lotterman,

Willy

Bos-

AND

WIJFFELMAN

man, and Yvonne Venema for technical assistance. Part of these investigations had been carried out under the auspices of the Netherlands Foundation for Chemical Research (S.O.N.) and with financial aid from the Netherlands Organization for the Advancement of Pure Scientific Research (Z.W.0.). REFERENCES BORAM, W., and ABELSON, J. (1973). Bacteriophage Mu integration: On the orientation of the prophage. Virology 54, 102-108. BUKHARI, A. I., and ALLET, B. (1975). Plaque-forming h-Mu hybrids. Virology 63, 30-39. COSLOY, S. D., and OISHI, M. (1973). Genetic transformation in Escherichia coli K12. Proc. Nut. Acad. Sci. USA 70, 84-87. DANIELL, E., KOHNE, D. E., and ABELSON, J. (1975). Characterization of the inhomogeneous DNA in virions of bacteriophage Mu by DNA reannealing kinetics. J. Viral. 15, 739-743. HIROKAWA, H. (1972). Transfecting deoxyribonucleic acid of Bacillus bacteriophage 429 that is protease sensitive. Proc. Nat. Acad. Sci. USA 69, 1555-1559. HOWE, M. M. (1973). Prophage deletion mapping of bacteriophage Mu-l. Virology 54, 93-101. Hsu, M., and DAVIDSON, M. (1972). Structure of inserted Mu-l DNA and physical mapping of bacterial genes by Mu-l insertion. Proc. Nat. Acad. Sci. USA 69, 2823-2827. KAHMANN, R., KAMP, D., and ZIPSER, D. (1976). Transfection ofEscherichia coli by Mu DNA. Mol. Gen. Genet. 149, 323-328. KUPOR, R. S., and FRAENKEL, D. G. (1969). 6-Phosphogluconolactonase mutants of Escherichia coli and a maltose blue gene. J. Bacterial. 100, 12961304. KUSHNER, S. R., NAGAISHI, H., TEMPLIN, A., and CLARK, A. J. (1971). Genetic recombination in Escherichia coEi: The role of exonuclease I. Proc. Nat. Acad. Sci. USA 68, 824-827. MANDEL, M., and HIGA, A. (1970). Calcium-dependent bacteriophage DNA infection. J. Mol. Biol. 53, 159-162. ORTIN, J., VIRUELA, E., and SALAS, M. (1971). DNA-protein complex in circular DNA from phage +29. Nature New Biol. 234, 275-277. TOUSSAINT, A., and FAELEN, M. (1973). Connecting two unrelated DNA sequences with a Mu dimer. Nature New Biol. 242, l-4. VAN DE PUTTE, P., WESTMAAS, G., GIPHART, M., and WIJFFELMAN, C. (1976). On the kil gene of bacteriophage Mu. In ‘DNA Insertions” (to be published). Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y. WACKERNAGEL, W. (1972). An improved spheroplast assay for A-DNA and the influence of the bacterial genotype on the transfection rate. Virology 48,94103.

TRANSFECTION WE~TMAAEI, G. C., VAN DER MAAS, W. L., and VAN DE PUTTE, P. (1976). Defective prophages of bacteriophage Mu. Mol. Gen. Genet. 145, 81-87. WIJFFELMAN, C. A., WESTMAAS, G. C., and VAN DE PUTTE, P. (1972). Vegetative recombination of bacteriophage Mu-l inEscherichia coli. Mol. Gen. Genet. 116, 40-46. WIJFFELMAN, C. A., WESTMAAS, G. C., and VAN DE PUTTE, P. (1973). Similarity of vegetative map and prophage map of bacteriophage Mu-l. Virology 54, 125-134.

WITH

Mu-DNA

159

WIJFFELMAN, C. A., GASSLER, M., STEVENS, F. W., and VAN DE PUTTE, P. (1974). On the control of transcription of bacteriophage Mu. Mol. Gen. Genet. 131, 85-96. WIJFFJZLMAN, C. A., and VAN DE PUTTE, P. (1974). Transcription of bacteriophage Mu. Mol. Gen. Genet. 135, 327-337. YANOWKY, S., KAWAMURA, F., and ITO, J. (1976). Thermolabile transfecting DNA from temperature-sensitive mutant of phage 429. Nature (London) 259, 60-63.