Amplification of λ plasmids in Escherichia coli relA mutants

Biotechnol ,ou!a\At

ELSEVIER

Journal of Biotechnology

OF

43 (1995) 139-143

Amplification of h plasmids in Escherichia coli relA mutants Grzegorz Wegrzyn Laboratory

of Molecular

Genetics,

Department

Received

of Molecular

Biology,

UniL’ersity of Gdarisk, Kiadki 24, 80-822

Gdarisk, Poland

18 April 1995; revised 3 1 July 1995; accepted 2 August 1995

Abstract

It was previously demonstrated that, contrary to wild-type stringent (reZ+) strains of Escherichin coli, in amino acid-starved relaxed (reZA) mutants the replication of A plasmid proceeds for several hours. The replication leads to amplification of A plasmid DNA. Here, the conditions for this amplification have been optimized. The amplification efficiency depends on the temperature as well as on the nature of amino acid starvation, but it is only little or totally not dependent on the pH value of the medium in a range from 6.0 to 8.0. It seems that the most efficient amplification can be achieved by overnight cultivation of E. coli relA arg strain harbouring A plasmid at 36-39°C in minimal medium containing Casamino acids. Under these conditions, the copy number of A plasmid increases from about 40 to about 300 per cell giving greater than 7-fold amplification. Keywords:

DNA amplification in vivo; A Plasmid; Amino acid starvation;

Relaxed mutant;

Escherichia

coli; Stringent

response;

Relaxed

response

There are many versatile and sophisticated phage vectors derived from bacteriophage A (for reviews

amounts. pCLIP vectors, like other A plasmids, are of medium copy number (20-50 copies per cell) in

see Sambrook et al., 1989; Chauthaiwale et al., 1992). Although plasmids derived from bacteriophage A (so-called A plasmids) have been known for a long time (Matsubara, 198 l), they have served as excellent models for studies on mechanisms of DNA replication, but (with very few exceptions, see Mukai et al., 1976) were not used as tools in molecular cloning. Recently, Boyd and Sherratt (1995) constructed a series of general-purpose plasmid vectors based on phage A origin of replication. These A plasmid vectors (called pCLIP) are compatible with most other vectors in common use, and are useful for routine plasmid cloning as well as other applications (for details see Boyd and Sherratt, 1995). One of the properties k>fa cloning vector should be a possibility of its effective isolation and purification in large

Escherichia

0168-1656/95/$09.50 0 1995 Elsevier Science B.V. All rights reserved SSDI 0168-1656(95)00132-8

coli

(Boyd

and

Sherratt,

1995;

Sza-

lewska et al., 1994). Therefore, an amplification of these plasmids would be very useful. The most

used vectors, as derivatives of pBR322 (Bolivar et al., 1977), pUC (Vieira and Messing, 1982, Vieira and Messing, 1991; Yanisch-Perron et al., 1985), and pACYC177 (Chang and Cohen, 1978), can be amplified by chloramphenicol method (Clewell, 1972). However, addition of chloramphenico1 to a culture of bacteria harbouring A plasmid results in only about 2-fold amplification of this plasmid (Wqgrzyn et al., 1991a), thus the method is inefficient in this case. Moreover, the pCLIP plasmids bear a chloramphenicol-resistance gene, thus chloramphenicol treatment cannot be used for amplification of these plasmids. Hecker et al. (1983) widely

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ofBiotechnology

demonstrated that replication of plasmid pBR322 is inhibited during the stringent control, whereas this plasmid replicates efficiently in amino acid-starved relaxed mutant (i.e., during the relaxed response). Then, amino acid starvation of E. cofi relA strains was proposed as an alternative method for amplification of plasmid pBR322 as well as other ColEl plasmids (Hecker et al., 1985; Schroeter et al., 1988; Riethdorf et al., 1989; Hofmann et al., 1990). Guzman et al. (1988) demonstrated that this method produces amplification of pBR322 with a better yield than chloramphenicol treatment. Recently, a mecha-

Table 1 Amplification

of A plasmid DNA a in amino acid-starved

Culture medium ’ and the nature of starvation d

Plasmid content

(“0

plasmid amount (ng OD-unitbefore amplification

f

nism of regulation of pBR322 replication in amino acid-starved bacteria has been partially elucidated (Herman et al., 1994a). Other studies revealed that replication of h plasmids is also under the stringent control (Wggrzyn et al., 1991b), i.e., the replication proceeds in amino acid-starved relA mutants but not in rel+ bacteria. Then it was demonstrated that inhibition of h plasmid replication in amino acidstarved wild-type bacteria results from ppGpp (guanosine 5’-diphosphate-3’-diphosphate)-mediated inhibition of transcriptional activation of orih (Szalewska-Palasz et al., 1994; Szalewska-Palasz and

Escherichia

Temperature

43 (1995) 139-143

coli refA cells b

’

’)

after amplification

Amplification factor h

Plasmid copy number per cell

s

before amplification

f

after amplification

s

MM+aa: isoleucine starvation

30 33 36 39 42

55 64 126 120 33

119 160 411 474 76

8.2 9.5 18.8 17.9 4.9

17.7 23.7 61.3 70.7 11.2

2.2 2.5 3.3 3.9 2.3

MM + Ca: arginine starvation

30 33 36 39 42

160 195 300 276 72

726 885 1998 1987 300

23.8 29.1 44.7 41.1 10.7

108.0 132.1 297.7 295.9 44.5

4.5 4.5 6.7 7.2 4.2

a h plasmid pKB2 has been already described (Kur et al., 1987; Szalewska et al., 1994). b E. coli K-12 strain CW9 (leu arg thr his thi reL4 rem; Fiil and Friesen, 1968) was used. ’ Minimal media 1 and 2 (Wegrzyn et al., 1991 b) were used. Apart from salts, thiamine and glucose the medium contains 1% Casamino acids (medium 1 = MM + Cal, or instead of Casamino acids, 50 pg ml- ’ each required amino acids (L-leucine, L-arginine, t_-threonine and t_-histidine; medium 2 = MM + aa). Unless otherwise indicated, the pH of the medium was adjusted to 7.0. It is worth mentioning that very similar results were obtained when other commonly used minimal media were employed (data not shown). d Jsoleucine starvation of bacteria growing in medium 2 (MM + aa, i.e., minimal medium supplemented with appropriate amino acids) was induced by addition of r_-valine to final concentration 1 mg ml- ’ Arginine starvation of arg- bacteria was induced by exhaustion of this amino acid from medium 1 (MM + Ca, i.e., minimal medium supplemented with Casamino acids), when the stationary phase of growth started, according to Hofmann et al. (1990). ’ Measurement of h plasmid DNA content was performed as described previously (Wegrzyn et al., 199lb; Herman et al., 1994a). Briefly, samples (0.8 OD,,, units) of cell culture were centrifuged, the pellets were washed with 0.9% NaCl and then frozen at -20°C. After thawing, the crude lysis and agarose gel electrophoresis were performed (for details see Wegrzyn et al., 199lb). h plasmid bands were $uantitated by densitometry fusing WP E.A.S.Y Enhanced Analysis System, Cambridge, UK). Plasmid content was estimated in exponentially growing (nonstarved) bacteria. s Plasmid content was estimated three fin the case of isoleucine starvation) or six (in the case of arginine starvation) h after the onset of the starvation. Jsoleucine starvation longer than 3 h, as well as arginine starvation longer than 6 h, did not influence significantly the value of y amplification factor. The amplification factor was calculated as the ratio of plasmid content per bacterial mass 3 (in the case of isoleucine starvation) or 6 (in the case of arginine starvation) h after the onset of starvation, to the plasmid content per bacterial mass in the exponentially growing (nonstarved) bacteria. Thus, the amplification factor = 1 represents the value obtained for nonstarved bacterial culture.

G. W~grzyn/Journal

ofBiotechnology

Wegrzyn, 1994; Szalewska-Palasz and Wegrzyn, 1995; Herman and Wggrzyn, 1995). ppGpp is not accumulated in amino acid-starved reZA mutants, and during the relaxed response the replication of A plasmid is carried out by the A0 initiator-containing replication complex (Wegrzyn et al., 1992, 1995a) that was assembled prior to the onset of amino acid starvation and is inherited by one of two daughter plasmid copies in each replication cycle (Wegrzyn and Taylor, 1992; Wegrzyn et al., 1995b). This replication leads to amplification of h plasmid DNA (Wggrzyn et al., 1991b). The aim of this work was to optimize the conditions for A plasmid amplification in amino acid-starved relaxed mutants of E. coli. The addition of L-valine to a bacterial culture growing in minimal medium is the most convenient and quickest way for induction of isoleucine starvation of E. coli K-12 strains (see for example Hecker et al., 1983; Wegrzyn et al., 1991b; Herman et al., 1994b). It was previously demonstrated that in the case of pBR322 plasmid amplification during the relaxed response, the yield of amplification depends on growth temperature (Riethdorf et al., 1989). Therefore, the efficiency of A plasmid amplification in isoleucine-starved relA strain was estimated at different temperatures and the optimal temperature was found to be between 36 and 39°C (Table 1). Isoleucine starvation was usually induced at OD,,, of bacterial culture close to 0.2. When the starvation for isoleucine was provoked at considerably higher OD (0.5 or higher), the amplification factor was significantly lower (data not shown). It was shown previously that replication (and thus eventual amplification) of different plasmids during the stringent and relaxed response is dependent on the kind of deprived amino acid (Riethdorf et al., 1989; Herman et al., 1994b). The most spectacular differences were observed between starvation for isoleucine and arginine (Herman et al., 1994b). Therefore, the estimation of the amplification factor at different temperatures during arginine starvation of relA arg bacteria growing in a minimal medium supplemented with Casamino acids pas repeated. Starvation for arginine was induced at the beginning of the stationary phase of growth, as described by Hofmann et al. (1990). The same optimum temperature was found as in the case of isoleucine starvation; however, the yield of amplification was signifi-

43 (1995) 139-143

141

cantly higher in arginine-starved relA strain (Table 1). Similar results were obtained when starvation for arginine was induced by removal of arginine from a minimal medium which was previously supplemented with appropriate amino acids (data not shown). Nevertheless, it is clear that the former means of achieving arginine starvation is much more convenient in a laboratory practice. It is interesting that A plasmid content in unstarved relA cells was also the highest at temperature of 36-39°C (Table 1). Therefore, the differences in an actual yield of plasmid DNA after its amplification at different temperatures were even bigger than those assumed only on the basis of the amplification factor. Reinikainen et al. (1989) found that the efficiency of ColEl plasmid amplification by chloramphenicol treatment depends on the pH value of the growth medium in a range from 6.0 to 8.0. That is not the case during A plasmid amplification in isoleucine- as well as arginine-starved relA mutants. I found that the amplification factor was very similar at different pH values (in a range from 6.0 to 8.0) of the medium (data not shown). It seems that arginine starvation of relA at-g cells, induced by exhaustion of this amino acid from a minimal medium supplemented with Casamino acids when the stationary phase of growth starts, at a temperature of about 37°C is the most effective way to amplification of A plasmids. Therefore, the kinetics of this amplification were investigated. Apart from the estimation of plasmid content, A plasmid DNA synthesis was monitored by pulse-labelling with [ 3Hlthymidine (Fig. 1). The relative amount of plasmid DNA per bacterial mass remained constant during the exponential growth. The onset of the stationary phase, which was also the onset of arginine starvation (Hofmann et al., 1990), resulted in increase of the plasmid content per bacterial mass. This increase proceeded for about 6 h. Under these conditions the synthesis of plasmid DNA occurred at the same level. This is in accordance with previous reports which demonstrated that the number of the active A replication complexes remains at a constant level in amino acid-starved relA cells (Wegrzyn and Taylor, 1992; Szalewska-Palasz et al., 1994). After about 6-h amplification, the synthesis of plasmid DNA dropped and the plasmid content achieved

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of Biotechnology43

Fig. 1. The kinetics of A plasrnid (pKB2) amplification in E. coli reL4 arg mutant (CP79) growing in the minimal medium supplemented with Casamino acids (MM-t-Cal at 37°C. The OD,,, of bacterial culture (0). relative plasmid DNA content (0) and relative plasmid DNA synthesis ( n ) are presented. The plasmid pKB2, the strain CP79, the culture medium, and the method for estimation of plasmid DNA content are described in Table 1 (footnotes: a, b, c, and e, respectively). Synthesis of plasmid DNA was estimated by measurement of [‘Hlthymidine incorporation during 5-min pulses as described previously (Wegrzyn and Taylor, 1992), but deoxyadenosine was added to each sample to final concentration 100 pg ml - ’ in order to inhibit intracellular thymidine synthesis.

new, roughly constant value, several-fold higher than the basal level. The amount of A plasmid DNA did not decreased during next several hours. This is important in the light of the results obtained by Hofmann et al. (1990). They found that in very similar conditions, after the onset of arginine starvation the amount of plasmid pBR322 increased, reached maximum after 6-8 h and then started to decrease. Since in arginine-starved reL4 arg bacteria the amount of A plasmid DNA does not decrease for several hours after reaching a maximum, it is not necessary to monitor the plasmid content during cultivation or to look after the best time for the end of cultivation. Moreover, for A plasmid amplification, it is not necessary to do anything except inoculation of appropriate strain into appropriate medium and overnight cultivation, as arginine starvation starts spontaneously. Therefore, a recipe of how to obtain the highest A plasmid amplification seems to be extremely simple: inoculate an overnight bacterial culture into a fresh minimal medium supplemented

(I9951 139-143

with Casamino acids, cultivate at 37°C for at least 10 h (usually overnight), and isolate plasmid DNA. The experiments presented above, in which A plasmid pKB2 (Kur et al., 1987) was used, were repeated with >another A plasmid, pCBl04, which was one of plasmids used by Boyd and Sherratt (1995) during c onstruction of the pCLIP series, and very similar results were obtained (data not shown). Also, the results of other studies, in which the replication of different A plasmids was investigated during the stringent and relaxed response, indicate that all orih-based plasmids can replicate in amino acidstarved relA mutants on the condition that appropriate plasmid and host functions, necessary for A DNA replication, are present in the cell (Wegrzyn et al., 1991a, b; Wegrzyn and Taylor, 1992; Herman et al., 1994b; Szalewska-Palasz et al., 1994; SzalewskaPalasz and Wggrzyn, 1994; Wqgrzyn et al., 1995b). The method of amplification of plasmid DNA in amino acid-starved relA mutants was first proposed by Hecker et al. (1985) and concerned plasmid pBR322. Then it was demonstrated that this method is useful for amplification of other ColEl-derived plasmids and is more efficient than commonly used chloramphenicol method (Guzman et al., 1988; Hofmann et al., 1990). It was also suggested that starvation for arginine can be used as a method for DNA amplification in the case of ori-pSC101 based plasmids (Herman et al., 1994b). Here it was demonstrated that A plasmids may be effectively amplified in a similar way. Amplification of plasmid DNA in amino acid-starved bacteria provides also a mean of achieving very efficient overproduction of a protein when corresponding gene is cloned on the plasmid. It was shown that very efficient overexpression of a gene cloned on the plasmid occurs after simple addition of deprived amino acid following plasmid amplification (Hecker et al., 1988). Finally, the described method of amplification is extremely cheap, as neither addition of chemicals nor other treatments of bacterial culture are necessary.

Acknowledgements This work was supported by the University Gdarisk (grant BW-0010-5-0068-5).

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