Enhanced DNA uptake and transfection in Escherichia coli cells grown in low-phosphate medium

384

BIOCHIMICAET BIOPHYSICAACTA

BBA 96948

E N H A N C E D DNA U P T A K E AND T R A N S F E C T I O N IN E S C H E R I C H I A CELLS GROWN IN L O W - P H O S P H A T E MEDIUM

COLI

MANUEL RIEBER AND NANCY MILLAN Center for Microbiology and Cell Biology, Instituto Venezolano de Investigacidnes Cientificas, Apartado i827, Caracas (Venezuela)

(Received March i8th, 1971)

SUMMARY I. The very limited ability of Escherichia coli wild type cells to take up DNA has been found to persist in endonuclease I-deficient cells, from which DNA can be recovered undegraded after co-incubation. 2. Addition of protamine sulphate promotes essentially reversible adsorption when incubation of DNA with the cells is calried out at 32 ° or 37 °. However, a significant enhancement of irreversible incorporation takes place at 4 °, both in protamine-treated cells and for partially starved cultures kept at the low temperature. 3. The greatest incorporation of DNA correlating with transfecting ability is found in osmotically treated EDTA-"permeable" cells grown in either Tiis-buffeled or phosphate-deficient media which incorporate much more DNA than similarly treated cells grown in phosphate-buffered medium. 4. On the basis of the greater DNA incorporation found both in nolmal and in osmotically shocked "permeable" cells grown in low-phosphate medium which correlates with a greater transfection efficiency for ~bXI74 DNA, it is postulated that a competent state of E. coli m a y be related to a state of phosphate limitation.

INTRODUCTION Even though DNA uptake is well established in various cellular systemsI,2, no such conditions have been well defined for Escherichia coli whole cells, otherwise ideally suited to the study of biochemical and genetic analysis. Although transfection of whole cells takes place in the presence of a helper3, othel systems for transfcction occur only in spheroplasts4,5. The problem of the cell wall of E. coli as a formidable permeability barrier has been extensively studied. Conditions of EDTA treatment and mild osmotic shock have been found to improve cell permeability significantlye,7. However, although DNA was shown to infect osmotically shocked E. colis, HEPPEL° further reported lack of success in demonstrating DNA uptake in such cells. Similar conclusions on the extremely low level of competence in E. coli whole cells have been reached by BENZINGERet a/.4. Neveltheless, the genetic transformation of various E. coli strains to a rather high frequency has been recently reported x°. Because of the prospects of genetic Biochim. Biophys. Acta, 246 (1971) 384-395

DNA TRANSFECTION IN LOW-PHOSPHATE MEDIUM

385

transformation in E. coli as a means of elucidating important questions on gene transfer mechanisms, we have tried to reproduce the procedure reported to yield transformable cells1°, but have been unsuccessful possibly due to the unavailability of the particular strains and perhaps because such a procedure is certainly not as efficient with many other E. coli strains. In order to have a clearer understanding of the rather general reluctance of E. coli to be transformed or even to take up DNA, the present studies were undertaken to investigate sequentially the various steps ultimately leading to a DNAmediated transformation and transfection. In this report we describe factors affecting DNA uptake and transfection, both of which are found to occur to a greater extent in cells grown in low-phosphate medium. A preliminary report on the inffial stages of DNA incorporation in E. coli has been presented 11.

MATERIALS AND METHODS

Bacterial strains The thymine-requiring X2I 7 (delivative of I5T-) and X5o3 (HfrOR2I-T68strs prototroph) were used for isolation of E. coli labeled DNA. HfrC 6 (met-), CIo (prototroph derivative of E. coli C) (both endonuclease 1-deficient) and X5Io (F- his- xylcyc r) were used as DNA recipients. For some experiments, E. coli B was used for T2 phage propagation or as a recipient for T2 DNA. The endonuclease 1-deficient cells were kindly sent by Dr. H. Hoffmann-Berling, and all the other strains described were generously supplied by Dr. Roy Curtiss III. Media Cells were propagated overnight in Penassay antibiotic medium 3 (Difco). The growth media used were: ( I ) M L phosphate-buffered medium 1~. Supplements consisted of 20 #g/ml of the required amino acid or 4/~g/ml of thymine for the thymine auxotroph. (2) Tris-buffered medium la modified to contain 3 % casamino acids (Difco) and 0.5 % glucose as carbon source. (3) Phosphate-deficient medium 1~containing NH4C1 to a final concentration of o.o12 M and K2HPO 4 to 0.006 M and 3 % casamino acids. Preparation o / D N A Bacterial DNA. For DNA labelling with thymidine, bacteria were grown in the ML phosphate containing either IO/zC/ml of IMe-~H~thymidine (18.3 C/mmole; Schwarz Bio Research) or I/~C/ml of E2-14Clthymidine (51.2 mC/mmole). DNA was extracted by the pronase-phenol method as follows: cells were harvested in mid-log phase from a Ioo-ml culture and resuspended in 2.5 ml of 0.05 M Tris-o.oo5 M EDTA-o.I M NaC1 (pH 8.5). Lysozyme (5 mg/ml) in 0.25 M Tris (pH 8.o) was added to a final concentration of 5oo#g/ml and the suspension maintained at 37 ° for io rain. Lysis was brought about by adding sodium dodecyl sulphate (20 °/o solution in I M Tris (pH 9.0)) to a concentration of 1 % , and pronase to I mg/ml (pre-heated at 80 ° for IO min). After incubation of the mixture at 5 °0 for 45 min, sodium p-aminosalicylate was added to 6 °/o and an equal volume of neutralized phenol and a half volume of chloroform were added as deproteinizing agents. FollowBiochim. Biophys. Acta, 246 (1971) 384-395

386

M. RIEBER, N. MILLAN

ing separation of the upper phase b y centrifugation, the nucleic acids were collected on a glass rod upon addition of ethanol to 67 %. RNA was removed from the nucleic acid solution in citrate saline (O.Ol5 M trisodium citrate-o.I5 M NaC1) b y digestion with heat-treated ribonuclease, and a second phenol-chloroform extraction was performed. DNA was finally isolated either by addition of 2 vol. of cold ethanol or 2.5 vol. of methoxyethanol. Phage DNA. This was prepared essentially as described by BENZINGER et al.4.

Methylated albumin kieselguhr chromatography This was performed in i cm × IO cm columns prepared and run by stepwise elution 15. Procedure/or osmotic shock This was essentially a modification of the method followed by NOSSAL AND HEPPEL8 as follows: an overnight culture in Penassay medium was inoculated into the medium indicated in each case to an Ass onm = 0-05, and grown at 37 ° to an A 5 5 0 n m - = 0.25. After centrifugation at IO ooo × g for IO min the cell pellets were resuspended in 0.5 M sucrose-o.o5 M Tris-o.I mM E D T A (pH 7.5) (4° ml/g of cells). The suspension was shaken at 18o rev./min during 15 rain at 22 °, and then centrifuged at low speed. The sediment was then dispersed in the same volume of water and kept at o ° foi IO min, iecentrifuged and resuspended in the same volume of the original culture medium containing bovine serum albumin (Fraction V) to 1.5 % unless otherwise indicated. Cells were then either exposed directly to radioactive DNA, or preincubated alone prior to incubation with DNA, as described in the corresponding experiments. Radioactive measurements DNA uptake into whole cells was measured by extraction of washed cells with cold I M HC104, followed by hot I M HCIO 4 extraction for 15 min at 7 o°. HCIO 4 samples (0.5 ml) were blended with 5 ml of toluene-Triton X - i o o scintillator (30 % Triton X-ioo, 7 ° % toluene containing 4 g PPO and o.I g P O P O P per 1). In some other experiments, the cell pellets were treated with 0.3 M N a O H overnight, then neutralized with HC1 and made 5 % in trichloroacetic acid before filtration onto GF/A discs, with essentially similar results.

RESULTS

On the lack o/DNA uptake Preliminary assays to demonstrate significant uptake of radioactive DNA b y various strains, derivatives of E. coli B, K-I2 or C, from logarithmic or stationary cells grown in either rich or chemically defined media (see MATERIALSA N D M E T H O D S ) gave no encouraging results. The possibility that the failure to demonstrate radioactivity in the hot-acid fraction of the iecipient cells could be due to rapid enzyme degradation by a nuclease situated in the cell wall periplasm was taken into account by studying uptake in HfrCe, a m u t a n t 18 deficient in the membrane-bound endonuclease I, enzyme shown to be the predominant deoxyribonuclease in E. colilL Biochim. Biophys. Acta, 246 (I97I) 384-395

DNA TRANSFECTIONIN LOW-PHOSPHATEMEDIUM

387

Studies of DNA uptake using this HfrC8 mutant gave no enhanced incorporation as compared to wild-type cells. To determine whether lack of uptake was due to degradation during penetration caused by another nuclease, the HfrC e cells were incubated with radioactive DNA for 45 min. At 5-rain intelvals, radioactivity of the unadsorbed DNA was measured after low-speed centrifugation of the culture. Aliquots of the supernatant were loaded onto a methylated albumin column (see MATERIALS A N D M E T H O D S ) . Washing of the column with o.I and 0. 4 M NaC1 did not elute any significant radioactivity. However, elution with 0. 7 M NaC1 gave about 9° % recovery of the total radioactivity, eluting in every case at the same position as unincubated control DNA. This, together with the fact that no enhanced DNA incorporation was obselved in recipient cultures of endonuclease I-deficient cells as compared to wild-type cells, supports the assumption that lack of DNA uptake by E. coli cells does not fail primarily because of macromoleculai degradation during penetration.

Temperature and ionic e]]ects on permeability In view of the finding that DNA is not degraded upon incubation with HfrC, cells, an alternative explanation for the failure of DNA to penetrate whole cells is that repulsion occurs between the negative charges can ied by the DNA at neutral pH and the negatively charged cell wall matrix. Therefore, we investigated the effect of neutlalization of the negative charges in the DNA, by complex formation with basic proteins. As shown in Table I, addition of protamine to the DNA to a concentration of 30/,g/ml before incubation with the cells gave a considerable enhancement of bound radioactivity. However, it was found that the criteria of irreversible binding z2 under which deoxyribonuclease-resistant DNA radioactivity becomes associated with the cell, were not applicable in assays in the presence of protamine. This was based on our observations that deoxyribonuclease addition to the DNA-protamine complex even prior to incubation with the cells only lowered incorporation by 40 % as compared with assays of deoxyribonuclease-untreated complex with E. coli cells. TABLE

I

EFFECT OF TEMPERATURE ON THE PROTAMINE-MEDIATED D N A UPTAKE Cells o f E. coli I-IfrC s w e r e g r o w n i n i o o c u l t u r e s o f M L p h o s p h a t e - b u f f e r e d m e d i u m of a c o n c e n t r a t i o n of 5 " lOS t o 2 • lO 8 cells p e r m l . A f t e r c e n t r i f u g a t i o n , t h e cell p e l l e t w a s r e s u s p e n d e d i n IO m l o f t h e s a m e m e d i u m . P r o t a m i n e s u l p h a t e ( 3 o / ~ g / m l ) a n d 3 H - l a b e l l e d D N A ( i 2 . 5 / , g ; 2 . lO 6 counts/min) were added and incubations carried out as indicated in each experiment. Prior to p r o c e s s i n g , cells w e r e t r e a t e d w i t h d e o x y r i b o n u c l e a s e ( i o / , g / m l ) a n d w a s h e d t w i c e i n c u l t u r e medium.

Expt. No.

Time at 4 ° (rain)

Time at 320 (rain)

Wash prior to deoxyribonuclease treatment water

i

--

60

2 3 4 5 6

-3° 3° 6o 6o

60 3° 3°

Radioctivity (counts/rain)

I M NH~C1 +

+ + + + +

138 1875 135o 4631 ii 915 1412

Biochim. Biophys. Acta, 2 4 6 ( 1 9 7 1 ) 3 8 4 - 3 9 5

3 88

M. RIEBER, N. MILLAN

Nevertheless, addition of I M NaC1 or NH4C1 prior to deoxyribonuclease treatment to the DNA-protamine complex did permit the assay for irreversibly binding because of the high salt-mediated reversible dissociation of the unincorporated complex. Table I shows that although a very significant amount of DNA is bound to the cells following incubation at 32 °, most of this binding is removed by washing the cells in culture medium containing high salt (I M) prior to deoxyribonuclease treatment. However, despite this disencouraging 1esult of plain reversible binding at 32 ° it was found that temperature played a most important role in the nature of the protamine-mediated DNA binding. Incubation of the cells with the protamine-DNA complex at 4 ° not only gave a considerably enhanced leversible binding (3-5-fold) but ploduced about a Io-fold increase in irreversible binding (Table I). The measured uptake at 4 ° obtained after NH4C1 washings and deoxyfibonuclease treatment was found to occur to a significant extent at that temperature and within 3-5 rain following DNA addition, provided that the cells were kept with protamine (30 #g/ml) at 4 ° prior to DNA addition for not less than 4 ° rain. Also, over 9 ° % of the accountable radioactivity was resistant to ethanol or cold HC1Q extraction but was extractable b y hot HC104 treatment, indicating that under such experimental conditions with protamine, there is no evident immediate degradation of the DNA to an acid-soluble form. It was interesting to notice that the stimulatory effect on DNA uptake given b y protamine at low temperature was not restricted to whole cells but also became evident, although to a lesser extent, at the spheroplast level. Assays of DNA uptake using lysozyme-EDTA spheroplast also showed a 2.6-fold gTeater macromolecular incorporation with protamine at 4 ° as compared with controls assayed at 37 °. This was consistent with our observation 39 that addition of 30 #g/ml of protamine to a ~-spheroplast assay 5, gave a Ioo-fold increase in phage DNA infectivity, in agreement with findings for phage Tt DNA infectivity in spheroplasts 18. In order to define whether protamine also causes some modification in the recipient cells, we added protamine (5°/zg/ml) to the cells prior to DNA addition. It was found that when cell preincubation with protamine is carried out during the first 3 ° min at 320 and then the following 30 min at 4 °, incorporation is lowered 4 ° % TABLE II EFFECT OF SODIUM ACETATE ON THE INCORPORATION OF D N A Cells of t h e h i s t i d i n e - r e q u i r i n g E. coli X5IO were g r o w n as i n d i c a t e d i n T a b l e I. Cells were r e s u s p e n d ed in i o ml of 2vIL p h o s p h a t e - b u f f e r e d m e d i u m c o n t a i n i n g o. 3 IV[ s o d i u m a c e t a t e (pH 6.0) w h e r e i n d i c a t e d . I n e x p e r i m e n t s w i t h p r e i n c u b a t i o n , t h i s w a s done a t 320 for 3 ° m i n a n d t h e n 3Hl a b e l l e d D N A (3/~g/ml; 2. 5 • lO 4 c o u n t s / m i n p e r / z g ) w a s a d d e d for 3 ° m i n a t t h e t e m p e r a t u r e i n d i c a t e d . F o l l o w i n g d e o x y r i b o n u c l e a s e t r e a t m e n t a n d w a s h i n g , cells were p r e p a r e d for c o u n t i n g as i n d i c a t e d in MATERIALS AND METHODS. Control a s s a y s in w h i c h t h e r e a c t i o n w a s s t o p p e d b y d e o x y r i b o n u c l e a s e a t zero t i m e a n d w a s h e d i m m e d i a t e l y , i n c o r p o r a t e d 57 c o u n t s / m i n u n d e r s i m i l a r e x p e r i m e n t a l conditions.

Expt. No.

Sodium acetate

I

--

2 3 4

+ + +

Histidine (l~g/ml)

_Preincubation

Counts/rain

Incubation temp.

5

+

5° 5 5

+ + +

13° 127 276 489

32° 320 32o 4°

Biochim. Biophys, Acta, 246 (1971) 384-395

DNA

38 9

TRANSFECTION IN LOW-PHOSPHATE MEDIUM

as compared with that found (978 counts/rain) when the first preincubation is done at 4 °. Nevertheless, it was found that washing of the cells ple-exposed to protamine prior to DNA addition cancelled the favourable action of the protamine, suggesting that this is just an effect on the DNA and not to a modification of the cells. Because of reports on the role of 0.3 M acetate (pH 6.0) in stimulating spheroplast formation in E. col# 9 we studied the possibility of increasing DNA uptake in cells exposed to acetate ions. It was found (Table II) that 0.3 M acetate did favour DNA uptake to some extent. This effect seemed more obvious following limited amino acid starvation resembling conditions used in the Bacillus subtilis 2° and E. col# ° transforming systems. However, as in the case of the protamine effect, incorporations were always greater with incubations at 4 °. Nevertheless, the significantly lower ability of acetate buffer in mediating DNA incorporation as compared with protamine may be due to its strictly autolytic stimulation, causing an effect different to that of charge neutralization and pIotection afforded by protamine. DATA uptake by cells with modi/ied permeability Subjection of E. coli cells grown in proteose peptone medium 4 to osmotic shock has been used to increase competence for phage nucleic acid infection to a level rather comparable to that of penicillin 21 or lysozyme-EDTA 5. However, such treatment was reported a to form spheroplast-like cells consisting of an heterogeneous mixture or largely abnormal bacteiia which had a considerable reduced viability. Nevertheless, it has been found that E. coli can be made to increase its permeability to actinomycin D (ref. 6) and to other metabolites 9 without a significant loss of viability. Modifications of such treatments that would permit DNA uptake in a chemically defined medium were investigated in view of reports 22 of a direct correlation between transformability and peImeability to actinomycin D in the B. subtilis system. Preliminary studies confirmed that EDTA treatment did improve cell permeability to actinomycin D (ref. 6), and to deoxyribonucleoside triphosphates 23 although in our hands such treatments gave no significant DNA uptake. However, considerable DNA incorporation was obtained by a modification of the osmotic treatment of NOSSAL AND HEPPEL8. Table III shows the effect of different shock media on the

TABLE III EFFECT OF DIFFERENT SHOCK MEDIA. ON THE DIN'A-INCORPORATING ABILITY Cells of HfrC e were g r o w n in I o o - m l c u l t u r e s of ML m e d i u m from a n A 5s0nm O. I t o a n A 55ohm 0.45. A f t e r c e n t r i f u g a t i o n a t IO ooo × g for IO m i n t h e cell pe l l e t s w e re w a s h e d in o.oi IV[ T r i s - o . o 3 M N a C I ( p H 8.o), a n d t r e a t e d for o s m o t i c s h o c k in t h e m e d i a a b o v e d e s c r i b e d as i n d i c a t e d in MATERIALS AND METHODS. The cells were f u r t h e r c e n t r i f u g e d a n d r e s u s p e n d e d in t h e s a m e v o l u m e of N I L + o . 3 % b o v i n e s e r u m a l b u m i n . A f t e r 3 ° rain p r e i n c u b a t i o n , [3H]DNA (3 p g / m l ; 2.5 • io4 c o u n t s / r a i n p e r #g) w a s a d d e d d u r i n g 45 rain p r i o r t o d e o x y r i b o n u c l e a s e a d d i t i o n ( i o / ~ g / m l ) . =

Resuspension medium

DNA incorporation (counts/rain)

0. 4 mM MgC12 o.I mM MgC12 C o m p l e t e ML m e d i u m o. oi M T r i s - o . o 3 M NaC1 Water

224 325 75 129 16o2

=

Biochim. Biophys. Acta, 246 (1971) 384-395

39 °

M. RIEBER, N. MILLfi,N

relative incorporation of DNA. I t can be seen t h a t a Io-fold greater incorporation was observed in cells shocked into water, as compared to that found for cells shocked into 0. 4 mM MgC12 (ref. 8). Although freshly shocked cells showed considerable DNA uptake this result gave some variability. I t was found that preincubation of shocked cells for 15 rain, stationary at 32°, gave a more reproducible incorporation. This time interval m a y be related to a partial requirement fol membrane 1egeneration similar to that observed in B. subtilis 23. Consistent with this explanation was the fact that bovine serum albumin, known to favour the stabilization of spheroplast-like cells 24 did increase DNA uptake (Table IV) in our system. Incorporation under such conditions reached a plateau after a 4 ° min incubation with about 50 % maximal incorporation taking place 15 min after DNA addition. TABLE IV OF BOVINE SERUM ALBUMIN ON D N A UPTAKE Ceils of HfrC, were g r o w n in Tris m e d i u m , a n d o s m o t i c a l l y s h o c k e d as d e s c r i b e d i n MATERIALS AND METHODS. Cells were f i n a l l y r e s u s p e n d e d in Tris m e d i u m w i t h t h e a d d i t i o n of b o v i n e s e r u m a l b u m i n i n d i c a t e d , p r e i n c u b a t e d s t a t i o n a r y a t 3 2o for 15 rain a n d 3H-labelled D N A (3/~g/ml; 2. 5 • Io 4 c o u n t s / m i n p e r / ~ g ) a d d e d for 6o rain a t 3 2°. D e o x y r i b o n u c l e a s e ( i o / ~ g / m l ) w a s t h e n a d d e d a n d s a m p l e s were processed as d e s c r i b e d in MATERIALS AND METHODS. S i m i l a r q u a l i t a t i v e r e s u l t s were o b t a i n e d in e x p e r i m e n t s w h e r e i n c u b a t i o n w i t h D N A t o o k p l a c e for o n l y 5 rain. EFFECT

Bovine serum albumin (%, w/v)

Radioactivity (counts/rain)

-o.15 o.3o 1.5 ° 3.oo

347 666 822 32Ol 1277

In order to test whether some lipid components normally associated with bovine serum albumin were responsible for the enhanced DNA incorporation (Table IV), experiments were carlied out using also albumin treated to remove adhered f a t t y acids ~5. In such experiments, no significant differences were found between treated and untreated bovine serum albumin samples regarding their ability to promote DNA incorporation. The possibility that the same cells propagated in different media would respond differently to osmotic shock was tested b y comparing their response following propagation in either a phosphate-buffered ML medium, or a Tris-buffered medium (see MATERIALSAND METHODS). A significant and reproducible effect was observed for cells grown in Tris-buffered medium (Table V) which usually exhibited a 3-5-fold greater DNA incorporation, as compared to the same cells grown in ML-phosphate medium. That low phosphate in the medium and not the effect of Tris is responsible for this effect was shown b y the similar incorporation obtained in Tris-grown cells x3 containing either casamino acids or the required amino acids at a concentration of 50 #g/ml. Also, cells grown in minimal medium low in phosphate (6 raM) 14 behaved essentially very similar to the Tris-grown cells, whereas no such behaviour was observed when phosphate ions were added to those media, in a proportion similar to that occurring in the normal phosphate medium. Biochim. Biophys. Acta, 246 (1971) 384-395

DNA

391

TRANSFECTION IN LOW-PHOSPHATE MEDIUM

TABLE V DNA Cells of HfrC 8 from a 20- h culture in P e n a s s a y m e d i u m were added to give an initial inoculum of A ~ 5 0 n m = 0.05 in the media indicated above and then aliquots were osmotically shocked as described in MATERIALS AND METHODS. Controls were similarly centrifuged and resuspended as the t r e a t e d cells in the same original medium. Bovine s e r u m a l b u m i n (1. 5 %) was added to the shocked cells and following a 3o-min preincubation at 32°, ~H-labelled D N A was added for a i5-min period. Results of incorporation are expressed as percentage of the radioactivity incorporated b y Tris-shocked cells (lOl 7 counts/min).

E F F E C T OF C U L T U R E M E D I U M ON T H E U P T A K E OF

Culture medium

Osmotic schock

Percent o~ radioactivity incorporated

ML ML Tris Tris

+ -+

27 16 ioo 4°

--

Experiments using E. colt B to compare the relative uptake of native, renatured and alkali-denatured 32P-labelled T2 DNA gave comparable results for native and renatured DNA and a 1.5-4.5-fold greater incorporation of denatured DNA. This resembles the results with both osmotic shock spheroplasts 4 and penicillin-induced spheroplasts ~1which have also been shown to have a greater affinity for single-stranded DNA. That treated cells take up single-stranded DNA was confirmed by the infectivity obtained in om system using # X I 7 4 DNA against cells of the endonuclease I-deficient E. colt C io. It can be seen that the results shown previously, demonstrating a greater DNA incorporation in either Tris-grown cells or in osmotically treated grown Triscells, correlate directly with the transfection experiments using # X I 7 4 DNA now shown in Table VI. It should be emphasized that in a number of experiments using osmotically treated Tris-grown cells, we obtained transfection frequencies IO-IOO times higher than those indicated in Table VI, which are comparable to those ohT A B L E

EFFECT

VI OF C U L T U R E M E D I U M ON T H E R E L A T I V E T R A N S F E C T I N G

ABILITY

OF ~X

174 DNA

E. coli Cio was grown and processed for osmotic shock as indicated in MATERIALS AND METHODS. Controls were taken t h r o u g h a similar procedure except t h a t resuspension took place always in the same original medium. Following a 2o-min s t a t i o n a r y preincubation at 32 °, i ml of ~ X 174 D N A (i g/ml in o.o5M Tris-MgCl~, o.ooi M - o . o o i 1V[ CaClz) was added to I ml of each culture and the m i x t u r e incubated s t a t i o n a r y at 320 for 15 rain. Then 8 ml of P e n a s s a y m e d i u m (Difco) were added and incubation continued for 15 min at 37 ° at IOO rev./min. Following addition of chloroform the culture was kept at 37 ° for 20 rain, and a p p r o p r i a t e dilutions were made on 2.5 ml of soft agar seeded with o.1 ml of Cio indicator cells.

Experiment

Plaque-/orming units per ml

Control cells grown in "Iris m e d i u m Control cells g r o w n in p h o s p h a t e -buffered m e d i u m Osmotically treated cells g r o w n in Tris-buffered m e d i u m Osmotically treated cells g r o w n in phosphate-buffered medium Deoxyribonuclease -treated cells grown in Tris-buffered m e d i u m with or w i t h o u t osmotic t r e a t m e n t

2 " IO 2 O

8 • IC

s

IO 3

Biochim. Biophys. Acta, 246 (1971) 384-395

392

M. RIEBER, N. MILL.~N

tained by YOUNG AND SINSHEIMER~n using lysozyme-EDTA spheroplasts at similar saturating concentrations of q~XI74 DNA. Nevertheless, although this absolute variability in transfection frequency did occur between one experiment and another, no such variability was found for the lelative transfection values described. It should also be mentioned that although higher relative frequencies were obtained for Tlisfree, low-phosphate grown cells than for phosphate-grown cells, such results were nevertheless lower than those obtained with cells grown in Tris-buffered medium. Under conditions in which DNA concentration is not limiting, up to 5 % of the labelled DNA is adsorbed in a deoxyribonuclease-insensitive manner by the treated cells about lO-15 rain after incubation. However, even then, following deoxyribonuclease addition there is a requirement for further incubation for at least 5 rain at 37 ° in oIder to recover most of the label in the purified DNA fraction. This resembles findings in the B. subtilis system 27 in which DNA which attains deoxyribonuclease insensitivity is not necessarily in the cell interior but stored in a region peripheral to the membrane.

DISCUSSION It has been correctly pointed out 1° t h a t the amenability of donor cells to yield biologically active DNA is a determining parameter foi a successful DNA-mediated transformation in E. coli. Nevertheless, we have found t h a t the main difficulty for transformation in most E. coli strains resides in their general reluctance to take up DNA. Neither wild type nor endonuclease 1-deficient strains show significant incolpotation of homologous or heterologous DNA, nor of small DNA molecules extractable intact such as 2 or q~XI74 DNA, under normal conditions of growth. This inability of E. coli B to take up DNA has also been reported b y PIECHOWSKA AND SHUGAR2s confirming much earlier observations b y LERMAN AND TOLMACH29. Various modifications have been studied in order to enhance the rather low level of spontaneous competence for DNA uptake existing in most E. coli strains 4. It has been found that conditions that favour neutralization of the negative charge carried by the DNA at neutral pH, such as addition of protamine sulphate, enhance reversible adsorption. However, no actual uptake occurs unless incubation of the cells with protamine, either prior to or during DNA addition, is carried out at 4 °. This exposure to low temperature increases both reversible and irreversible adsorption, and does not appear to be due to a decreased nuclease activity at 4 ° because a similar behaviour is obselved for endonuclease I-deficient strains, resembling the observations described in animal systems a° for the protamine-enhanced infectivity of polio virus RNA. Also, some enhancing effect of low temperature for DNA uptake has been found under conditions of partial amino acid starvation and/or in the presence of sodium acetate as stimulator of autolysis 19. This observed increase of DNA uptake taking place by pre-exposing the cells to 4 ° under the experimental conditions described m a y be related to alterations in lipid composition reported to take place at o-4 ° in E. coli al. This low-temperature enhancing effect on polynucleotide uptake has also been observed in mouse ascites tumor cells 3z and leukemic cells33 which show a definitely greater polynucleotide incorporation at 4 ° than that measured at 37 °. Other very sigBiochim. Biophys. Acta, 246 (i97 I) 384-395

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nificant changes of permeability at 4 ° have been observed ~ in studies of the amino acid transport system in Streptomyces hydrogenans. Transfection experiments such as those described in Table VI using protamine at low temperature gave variable results regarding qgXI74 DNA infectivity although results were always reproducible in uptake experiments. This may be due to the fact that the recipient cell although becoming saturated with biologically active DNA is not necessarily competent to process the genetic information received35, or perhaps because there is an inefficient release of the DNA from its complex with the basic protein. Nevertheless, some manifestations of the biological effect of the protaminemediated uptake are better seen by the enhanced infectivity given by DNA-protamine complexes in spheroplast assays 89 also observed by others is. This protamine effect in spheroplasts may be related to observations ~6 that penicillin and lysozyme spheroplasts retain part of their original cell wall-outer membrane complex, as shown by theiI ability to adsorb coliphages. In contrast with the protamine effect which ceases essentially upon removal of the basic protein, permeability alterations can cause better defined modifications in the cells which remain alter removal ot the responsible agents. The EDTA-Tris tIeatment known to promote the permeability to actinomycin D (ref. 6) and to deoxyribonucleoside triphosphates 23 has been found to be insufficient for the demonstration of DNA uptake and transfection, although significant DNA incorporation and activity was observed by our modification of the EDTA-Tris osmotic shock procedure of NOSSAL AND HEPPEL 8.

In contrast with the four deoxyribonucleoside triphosphate-dependent de novo incorporation into DNA which occurs in EDTA-Tris treated cells, and which is lowered either following osmotic shock or when conducted in endonuclease I-deficient cells~3, we found that DNA uptake following EDTA-Tris osmotic shock takes place in the absence of the triphosphate precursor and to a rather high degree both in endonuclease I-deficient and in wild type cells. As a result of the osmotic shock there is a subsequent temporary lag in DNA synthesis under conditions in which DNA uptake is maximal. Also, under conditions in which hydroxyurea (o.I M) strongly inhibits the incorporation of [SH]thymidine into DNA, there is no decrease in the measured amount of DNA incorporated by the osmotically treated cells. Additional evidence that DNA and not a degradation and reutilization product is found after incubation of DNA with osmotically treated cells is given by the transfecting activity of 2 DNA (ref. 8) and # X I 7 4 DNA (Table VI) by such procedures. This is supported by the corlelation between the greater transfecting ability of # X I 7 4 in such cells. It should be pointed out that although low temperature favoured DNA uptake under the experimental conditions described, it was found that uptake by osmotically treated cells with altered permeability occurred to a greatei (I.5-fold) extent in the range 32-37 ° than at 4 °, suggestive of a different mechanism in uptake. In the low-temperature procedures, there may be a reversible modification within the lipid layer ~ determining a widening of membrane pores or some other temporary alteration. However, in osmotically shocked cells pretieated with EDTA there is an actual release of cell wall membrane material ~7. Although the osmotic shock treatment for increased permeability has been found to give cells behaving as the best recipients for transfection experiments, optimal conditions ot this permeability treatment have not yet been found regarding the Biochim. Biophys. Acta, 246 (1971) 384-395

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expression of chromosomal DNA in transformation experiments. This could be attributed to alteration in membranous components of "permeable" cells which may affect recombinational events necessary for transformation. The present observations that cells grown in Tris or low-phosphate medium exhibit greater DNA uptake and transfection than similar cells grown in phosphatebuffered medium suggests the search of a competent state for E. coli in low-phosphate grown cells. The behaviour of such cells especially evident after EDTA-osmotic shock treatment may be related to findings of a significant decrease in the total phospholipid content described 14 for cells grown in low phosphate. This would be compatible with a lower content of hydrophobic lipid or charged phospholipid in the E. coli membrane, exerting a lower repulsion of the charged DNA. It may also be relevant that exposure of E. coli cells to EDTA-Tris has been found to give a significantly greater degree of permeability alterations than cells exposed to EDTA-phosphate ~3,2s.

ACKNOWLEDGEMENT

Some partial financial support from the Consejo Nacional de Investigaciones Cientlficas y Tecnol6gicas (CONICIT) Venezuela, is gratefully acknowledged.

ADDENDUM

After the completion of this manuscript, we learned that MANDEL AND HIGA4° demonstrated a calcium-dependent phage DNA infection in the absence of helper phage. However, although not emphasized by the authors, it stems from their experimental data that, in agreement with our observations on DNA uptake at low temperature by partially starved cells, they used partially starved cells at low temperature, conditions under which they did obtain significant DNA uptake and transfection. It seems also significant that as a medium for the competent cells they used the P medium of RADDING AND KAISER41 and not the normal growth H medium otherwise identical although with a 5-fold higher phosphate content than the P medium. REFERENCES I L. LEDOUX, in J. N. DAVlDSON AND W. COHN Progress in Nucleic Acid Research and Molecular Biology, A c a d e m i c Press, N e w York, i965, p. 231. 2 A. ToMAsz, Ann. Rev. Genet., 3 (I969) 2I 7. 3 A. D. KAISER AND D. S. HOGNESS, J. Mol. Biol., I (196o) 392. 4 !~. BENZlNGI~R, H. DELIUS, R. JAENISCH AND P. ~-I. HOFSCHNEIDER, Eur. J. Biochem., 2 (1967) 414 . 5 G. D. GUTHRIE AND R. L. SINSHEIMER, J. Mol. Biol., 2 (196o) 297. 6 L. LEIVE, Proc. Natl. Acad. Sci. U.S., 53 (1965) 745. 7 H. C. NEU AND L. A. HEPPEL, J. Biol. Chem., 240 (1965) 3685 . 8 1%. G. NOSSAL AND L. A. HEPPEL, J. Biol. Chem., 241 (1966) 3055 . 9 A. HEPPEL, in L. GROSSMAN AND K. MOLDAVE, Methods in Enzymology, A c a d e m i c Press, N e w York , 1968, p. 841. I o N. G. AVADHANI, B. M. MEHTA AND D. V. REGE, J. Mol. Biol., 42 (1969) 413 . i i IV[. I~IEBER, in L. L E n o u x , Uptake o[ In[ormative Macromolecules by Living Cells, N o r t h H o l l a n d , A m s t e r d a m , i97 o, in t h e press. i 2 R. CURTXSS I I I , J. Bacteriol., 89 (1965) 28. 13 D. t3. CLEWELL AND D. R. HELINSKI, Proc. Natl. Acad. Sci. U.S., 62 (1969) 1159.

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