loxP recombination system in efficient production of loxP-containing minicircles in vivo

Plasmid 53 (2005) 148–163 www.elsevier.com/locate/yplas

Limited use of the Cre/loxP recombination system in efficient production of loxP-containing minicircles in vivo Marian Sektas*, Maciej Specht Department of Microbiology, University of Gdansk, 80-822 Gdansk, ul.Kladki 24, Poland Received 4 September 2003, revised 8 April 2004 Available online 2 December 2004 Communicated by Grzegorz Wegrzyn

Abstract The Cre/loxP recombination system of bacteriophage P1 is one of the most powerful tools in genome engineering. We report, however, that the activity of the Cre/loxP system interferes with the stability of the multicopy loxP-bearing plasmids in Escherichia coli recA bacteria. Due to the predominantly unidirectional Cre-mediated high-order multimer formation of these plasmids, the number of their copies (overall yield) gradually decreases. Intermolecular recombination reduces the copy number of plasmids and eventually increases their segregational instability. We have found that in the presence of even the slightest amount of Cre activity, loxP-bearing plasmids continuously undergo multimerization, which very rapidly leads to loxP-plasmid free cells. Our results are compatible with the hypothesis of the multimer catastrophe [Cell, 1984 (36), 1097]. Ó 2004 Elsevier Inc. All rights reserved. Keywords: Cre/loxP recombination; Plasmid multimerization and demultimerization; Plasmid maintenance

1. Introduction The Cre/loxP recombination system of bacteriophage P1 is the most popular tool for genetic manipulation, both in vitro and in vivo (Buchholz et al., 1996; Kilby et al., 1993). Site-specific recombination allows for conditional gene targeting, *

Corresponding author. Fax: +48 58 320 2031. E-mail address: [email protected] (M. Sektas).

both, in knock-in reaction, when Cre mediates an integrative recombination (Buchholz and Bishop, 2001), or in knock-out reaction, when Cre catalyzes excision of the DNA fragment flanked by two parallel oriented loxP sites (Balbas and Gosset, 2001), including the switch of gene expression by translational frameshift. Broad utilization of the Cre/loxP recombination system provides an opportunity for understanding the genetic control of cell development enabling the study of those molecular path-

0147-619X/$ - see front matter Ó 2004 Elsevier Inc. All rights reserved. doi:10.1016/j.plasmid.2004.04.006

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ways which control development of cancer cells. Usually, it is achieved by means of gene knockout on the embryonic stem cells level or tissue-specific manner, followed by further analysis of knock-out phenotypes. All of the above examples demonstrate great potential for in vivo applications and direct manipulation of mammalian cells. However, it was shown that the efficiency of Cre excision level does not exceeded 75% and integration reaction is highly reversible (Abremski and Hoess, 1984; Ringrose et al., 1998). Low efficiency of excision is probably caused by high stability of the synaptic complex followed by re-integration reaction, however, in titration experiments intermolecular recombination products were not detected (Ringrose et al., 1998). Recently, some authors have reported formation of high-molecular multimers by lox-bearing plasmids, assuming cellular RecA activity (Bigger et al., 2001), and others have noticed some connections between Cre activity and inhibition of replication of the loxP-plasmids (Aranda et al., 2001; Sektas and Szybalski, 1998). In this paper, we investigate this phenomenon. We address the question as to what is the mechanism involved in the decrease of copy number of loxP-plasmids in the presence of Cre function. In addition, we present data on Escherichia coli Xer/ cer-independent demultimerization of pUC-based higher multimers, and discuss its consequences in RecA+ and RecA hosts.

2. Materials and methods 2.1. Bacteria and growth conditions Unless otherwise stated, Escherichia coli (E. coli) strains MM294 (Backman et al., 1976) or DH5a (Hanahan, 1983) and its derivative used in this study were grown at 37 °C in LB-broth or on LBagar plates (Sambrook et al., 1989). LB-media were supplemented with appropriate antibiotics at the following concentrations: ampicillin (Ap) 100 lg/ ml, chloramphenicol (Cm) 30 lg/ml, kanamycin (Km) 30 lg/ml, trimetoprim (Tp) 50 lg/ml, and tetracycline (Tc) 15 lg/ml. A set of the following isogenic E. coli strains containing different Xer/ cer deficient mutations was generously supplied

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by Dr. David Sherratt (University of Oxford, UK): DS856 (argRA9::fol), DS857 (pepA::Tn5), DS981 (xerC2), and DS9008 (xerD2::Tn10-9) (Table 1). 2.2. Plasmid constructions In most cases plasmid DNA was prepared from DH5a host or its derivative DH5 att::cre and MM294 by alkaline lysis procedure (Sambrook et al., 1989). All plasmids used in this work are listed in Table 1. To supply the Cre recombinase in a controllable fashion, we tested various, in respect to number of copy, replicons as a backbone and cre gene under control of E. coli arabinose operon—AraC/ParaBAD(PBAD) as a regulatory unit. This cassette was constructed by insertion downstream of the PBAD promoter of a 1.2-kb EcoRI–XbaI DNA fragment, containing cre gene without its own promoter and RBS region (Wild et al., 1998), into pBAD24 plasmid (Guzman et al., 1995). This construct, named pBAD24cre (Apr, pBR322 ori, Bolivar et al., 1997), was used as a source of araC-PBAD-cre cassette and it was subcloned into other replicons, after excision by Eco47III/HincII restriction enzymes. Two different plasmids in respect to the copy number level were obtained in this way, low-copy pSCaraCre (Tcr, pSC101 ori; Cohen et al., 1973), and medium-copy p15AraCre (Cmr, pACYC184 ori, Chang and Cohen, 1978), both compatible with recombination substrate loxPplasmids. Plasmid pJW165 with cre gene under lactose promoter PlacUV5 control was obtained from (Wild et al., 1998). To construct high copy test plasmids containing single or double loxP sequences we have used pSP-72 plasmid (Promega, Madison, WI, USA). Synthesized 42-bp SalI–BamHI DNA fragment containing single wtloxP sequence was inserted into SalI–BamHI sites of pSP-72, and resulted in pSP-loxP1 (2.6-kb). Plasmid pSP-loxP2 (2.78-kb) containing double loxP sites in parallel orientation was obtained after insertion of 611-bp ScaI–SmaI fragment of pBR322 (Bolivar et al., 1977) derivative—pBRloxP1, bearing loxP site, located between HindIII and EcoRV that came from pSP-loxP1.

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Table 1 Strains and plasmids used in this work Name

Relevant properties

Source

Bacteria DH5a DH5 attB::cre MM294 DS956 DS957 DS981 DS9008

supE44DlacU169 hsdR17 recA1 endA1 gyr96 thi1 relA1 deoR (/80dlacD(lacZM15) DH5a kattB::araC-PBAD-cre-Tcr supE44 hsdR2 endA1 pro thi-1 AB1157 recF lacIq lacZDM15 argRA9::fol (Tpr) AB1157 recF lacIq lacZDM15 pepA::Tn5 (Kmr) AB1157 recF lacIq lacZDM15 xerC (Kmr) AB1157 recF lacIq lacZDM15 xerD2::Tn10-9 (Kmr)

Hanahan (1983) This work Backman et al. (1976) Stirling et al. (1989) Flinn et al. (1989) Blakely et al. (1991) Blakely et al. (1993)

Plasmids pBR322 pBAD24 pBAD24cre pSC101 pSCaraCre pACYC184 p15AraCre pJW165 pSP-72 pSP-loxP1 pSP-loxP2 pMS107 pMSloxP1 pMS103 pTANTS pTANTS-cre.1 pINTtsCm

pMB1 ori, Apr, Tcr pMB1 ori of pBR322, Apr, Tcs, araC-ParaBAD pBAD24 with promoterless cre gene inserted between EcoRI/XbaI sites pSC101 ori, Tcr pSC101 with 2.5-kb araC-PBAD-cre inserted between NdeI–EcoRI sites p15A ori, Cmr, Tcr pACYC184 with 3.5-kb araC-PBAD-cre inserted between EcoRV and HincII, Cmr, Tcs p15A ori with cre under PlacUV5 promoter control pUC ori, Apr pSP-72 with single loxP site pSP-loxP1 derivative with two parallel oriented loxP sites separated by 520-bp pUC ori, Apr, loxP site 4.03-kb of pMS107 derivative with single loxP site pUC ori, Apr, loxP site Integrative plasmid, pBR322 ori, Tcr, k attP pTANTS plasmid with 2.5-kb araC-PBAD-cre inserted into SmaI site k-Int delivery plasmid, pSC ori Ts, Cmr

Bolivar et al. (1977) Guzman et al. (1995) This work Cohen et al. (1973) This work Chang and Cohen (1978) This work Wild et al. (1998) Promega Corporation This work This work Snaith et al. (1995) This work Snaith et al. (1995) Posfai et al. (1994) This work Hasan et al. (1994)

Plasmid pMSloxP1 was created by insertion of the second copy of loxP sequence from pMS107 plasmid (Snaith et al., 1995) into BglII site of the same plasmid, resulting in two parallel oriented loxP elements with FRT sequence in between. Then, by using in vivo Cre excision activity, final plasmid of about 4.03-kb containing one copy of loxP was obtained. 2.3. Construction of the cre+ inducible host DNA fragment from pBAD24cre, containing araC-PBAD-cre genes, was cut by HincII and Eco47III and cloned in SmaI sites of pTANTS tetracycline resistant integrative plasmid (Posfai et al., 1994) resulting in pTANTScre.1 plasmid. DH5a competent cells bearing pINTtsCm plasmid which supplies the Int integrase (Hasan et al., 1994), were transformed with derivative of pTANTScre.1 plasmid that contains araC-PBADcre cassette and k attP site, but has removed region

containing origin of replication (Sektas et al., 2001). This resulted in recombinant strain DH5 attB::cre (araC-PBAD-cre-Tcr) with conditionally expressed cre gene. 2.4. Generation times determination Generation times for a set of DH5a bacteria bearing various pSP-loxP1 multimeric forms were estimated by measuring their optical density (OD600 nm) at several stages of exponential phase of growth. Individual strains were cultivated in LB-broth (20 ml) with Ap at 37 °C. Obtained values are the mean of five experiments performed under similar growth conditions in respect to temperature, aeration, and initial inoculation ratio (1:200). 2.5. Growth competition assay The experiment was done as described by Summers et al. (1993). Initially, tested DH5a strains

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bearing monomeric or tetrameric form of pSPloxP1 plasmid were derived from a single colony grown on selective LB-agar plate. Strains were incubated overnight at 37 °C, as a 1.5 ml of LBbroth (containing Ap) cultures. Next day, each culture was diluted 106-fold and spread on LBplates containing Ap, to quantify the initial number of bacteria (cfu/ml) to be used for inoculation of a mixed culture. Further, the two tested strains were mixed in approximately 10:1 and 1:10 ratio, and diluted 104-fold into fresh 10 ml LB-broth containing Ap and grown overnight at 37 °C. The cycle of consecutive dilutions and growth of premixed cultures was repeated three times, and each time plasmid DNAs were isolated from 1 ml of a given culture. Control strains containing monomer or tetramer plasmid exclusively were grown independently under the same conditions. DNA was electrophoresed on a 0.8% agarose gel. 2.6. Determination of plasmid copy number of some multimeric forms of pSP-loxP1 plasmid Individual cultures of DH5a with cells carrying either pSP-loxP1 plasmid monomer or its higher multimers (from dimer to hexamer) were grown in 20 ml of LB-broth with Ap and sampled at different stages of growth. The number of bacteria was determined both by measuring the optical density at OD600 and by serial dilutions. Further, the amounts of cellular DNA content at a given stage of growth were determined by linearization of total isolated plasmid DNA with EcoRI and performing agarose gel electrophoresis with aliquots of each sample, followed by computer densitometric analysis of the gelÕs photography (ONE-Dscan 1.33 Program, Scanalytics, USA). Values of DNA amounts obtained were adjusted to the actual number of bacteria and to a size of particular multimer. The copy numbers were calculated in the monomeric equivalents. 2.7. Electron microscopy Electron microscopy analysis of DNA preparations of all multimeric forms of pSP-loxP1 plasmid was performed as described previously (Srutkowska et al., 1998).

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3. Results 3.1. Plasmid yield analysis The main function of Cre/lox recombination acting during vegetative P1 bacteriophage life-cycle is to prevent accumulation of chromosome dimers by their conversion to monomers (Austin et al., 1981). This mechanism partly ensures stable maintenance of virus plasmid in the population of lysogenic bacteria. Surprisingly, in the course of constructing some vectors utilizing the Cre/loxP system in E. coli strains, we have found that the lower the level of repression of cre expression, the faster decrease of the copy number of the tested loxP-plasmids. To investigate whether the Cre function is involved in the maintenance of the loxP-bearing plasmids we examined their stability and overall yield in the presence of Cre, which was expressed from a gene located in trans. We chose E. coli recA bacteria as a host, to avoid an overlapping effect of the cellular homologous recombination on the structure and maintenance of tested plasmids. We used various delivery plasmids as a source of Cre activity, mostly under arabinose AraC/PBAD repressor/promoter control, or recombinant DH5a strain (named DH5 attB::cre), with cre gene under PBAD promoter control, integrated into k attB site by the means of Int/att recombination. An example of the phenomenon of decrease in the copy number of loxP-plasmids in the presence of Cre is shown in Fig. 1. Effect of the influence of Cre recombination on maintenance and stability of loxP-bearing plasmids was investigated in a relatively tight (araC-PBAD arabinose repressor/promoter, Fig. 1A) and weak (lacIq-PlacUV5 lactose repressor/promoter, Fig. 1B) control systems of cre expression. High copy single loxP-bearing plasmid pMSloxP1 was maintained together with conditionally Cre-supplying plasmid pSCaraCre. It was shown (Fig. 1A), that the amount of loxPplasmid harvested one day after introduction into bacteria already bearing resident low-copy Cresupplying plasmid pSCaraCre is comparable to that obtained from the control Cre bacteria (Fig. 1A, lanes 1 and 5, respectively). In this experiment bacteria were cultivated overnight at 37 °C

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Fig. 1. Example of the influence of Cre recombination on maintenance of loxP-bearing plasmids. Comparative plasmid yields analysis displayed by a 0.8% agarose gel electrophoresis. (A) Study in relatively tight cre expression system. E. coli DH5a bacteria containing Cre-delivery plasmid pSCaraCre under araC-PBAD arabinose repressor/promoter control and single loxP-bearing plasmid-pMSloxP1 were cultivated overnight in LB-broth at 37 °C in the presence of antibiotics. All preparations of plasmids were obtained roughly from the same amount of bacteria, and followed by EcoRI digestion. Lane 1, plasmid DNA extracted from bacteria cultivated overnight, after inoculation with freshly transformed bacterial colony immediately from LB-agar plate; lane 2, plasmids isolated from bacteria grown overnight under Cre inducing conditions (0.02% arabinose); lane 3, plasmids isolated from culture inoculated with deep freezer stock bacteria; lane 4, plasmids isolated from bacteria inoculated with three days old colonies from an agar plate kept in refrigerator; lane 5, pMSloxP1 alone; lane 6, pSCaraCre alone; and lane 7, 1 kb DNA Ladder (Promega, USA). (B) Study in relatively weak cre expression system. E. coli DH5a bacteria containing Cre-delivery plasmid pJW165 under lacIq-PlacUV5 lactose repressor/promoter control and single loxP-bearing plasmid—pMS103 or pMS107 (Snaith et al., 1995) were cultivated overnight in LB-broth supplemented with Ap and Cm. Isolated plasmids were EcoRI digested. Lane 1, pJW165 alone; lane 2, pMS103 alone; lane 3, plasmids isolated from freshly transformed bacteria grown overnight; lane 4, pMS107 alone; lane 5, plasmids from freshly transformed bacteria; and lane 6, HindIII digested k DNA.

under repressed conditions, in LB-broth with selective pressure of all appropriate antibiotics. However, after overnight induction of cre gene

recombinase expression (at final concentration of 0.02% L -arabinose) a strong influence on copy number of pMSloxP1 can be seen (Fig. 1A, lane 2). Similarly, gradual tendency to yield decline in pMSloxP1 plasmid is observed, when DNA was isolated from cultures inoculated by frozen stock bacteria (Fig. 1A, lane 3), or from the cultures of bacteria inoculated by three days old colonies from agar plates stored in a refrigerator (lane 4). Reducing amount of pMSloxP1 plasmid harvested from bacteria cultivated under repressed conditions can be only explained as a cumulative effect of the leakage of cre gene expression. A much stronger effect of the reduction of copy number of loxP-plasmids can be observed in the weaker control system of cre expression, provided by plasmid pJW165 (Fig. 1B). The amounts of loxP-plasmids (pMS103 and pMS107) isolated just one day after transformation of bacteria with resident, conditionally Cre-supplying plasmid pJW165 are very low (Fig. 1B, lanes 3 and 5, respectively). Similar effect of decreasing of plasmid copy number was observed in the case of plasmid pSP-loxP2 (2.78-kb; Fig. 2A) containing two parallel oriented loxP sites. In this experiment DH5 attB::cre host or DH5a bearing p15AraCre plasmid were transformed with pSP-loxP2. After Cre excisive recombination one should expect two smaller plasmids, one of about 2.25-kb (named ploxA), containing the origin of replication and the ampicillin resistance gene, and a second, origin-less circle of about 0.53-kb (named ploxB). In fact, we could see these two excision products, but in addition, also products of reverse reaction resulting from plasmids integration. By using XhoI restriction enzyme, we were able to uniquely cleave shorter loxPÕs interregion, and observed linear forms of ploxB ori plasmid and integrants made from pSP-loxP2 and ploxA with the following sizes: 5.03 , 7.28 , 9.53 , 11.78 , and 14.03 kb (Fig. 2A, lanes 2–4; see also Fig. 2B). Under these digestion conditions, regions of plasmids built from ploxB units arrayed tandemly are cut-out and are visible at the bottom of the gel as a single band (L-ploxB). Using ScaI restriction enzyme, whose recognition site is located in bla gene, we were able to linearize all of the possible fusion plasmids created in vivo, except the smallest one,

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Fig. 2. Analysis of the Cre-recombination products of pSP-loxP2 containing double loxP sequences in direct repeat. (A) Agarose gel analysis. E. coli DH5 att::cre bacteria (lanes 1, 2, 6, and 7) or DH5a bearing Cre-delivery plasmid p15AraCre (lanes 3, 4, 8, and 9) (cre gene is under araC-PBAD arabinose repressor/promoter control in both cases), containing test plasmid pSP-loxP2 with two loxP sites parallel oriented, were cultivated up to late stationary phase. Isolated plasmids were either XhoI (lanes 1–5) or ScaI digested (lanes 6– 10) and run on a 0.8% agarose gel. To see more details, the bottom part of the gel was overexposed. Lanes 1 and 6, pSP-loxP2 replicated under repressed conditions for Cre; lanes 2 and 7, pSP-loxP2 replicated under Cre induction (4.5-h, 0.02% arabinose); lanes 3 and 8, p15AraCre and pSP-loxP2 isolated from bacteria grown under repressed conditions; lanes 4 and 9, preparations of plasmids isolated from bacteria grown under 4.5-h arabinose (0.02%) induction; lanes 5 and 10, p15AraCre alone; lane 11, 1 kb DNA Ladder (Promega, USA). Plasmids ploxA and ploxB are Cre excision products. Note that plasmid p15AraCre is not cut by ScaI. L, linear form of plasmid. Arrows within lanes 9 and 10 show multimers of supercoiled ploxB. For clarity, only positions of multimers detected by XhoI digestion are indicated by arrows on the left side of the picture. (B) Schematic representation of the structure of recombination products of pSP-loxP2 deduced from XhoI and ScaI restriction analysis. Graphical symbols: triangle, loxP site; filled circle, origin of replication; heavy black line, 530 bp ori region; and light gray line, DNA region encoding resistance to ampicillin.

ploxB. Therefore, we could see linear form of pSP-loxP2 (2.78-kb) and its shorter derivative ploxA (2.25-kb) and moreover, integration products of pSP-loxP2 (or ploxA) with ploxB, i.e., bands with sizes of about 3.31-, 3.84-, 4.37-, and 4.9-kb, respectively (Fig. 2A, lanes 7–9; see also Fig. 2B). We are aware of some limitations in restriction analysis employed as a tool to detect all of the variety of pSP-loxP2 recombination products. We are not able to see fusion plasmids built from tandem arrays of combined poly-ploxA

and poly-ploxB (Fig. 2B). Moreover, molecular concentrations of some simple plasmid forms like monomer of pSP-loxP2, ploxA, and ploxB might be easily overestimated based on the gel picture, because they are accumulated after excision from more complexed plasmid structures. Significant loss in the amount of pSP-loxP2 plasmid and its derivatives can be observed after 4.5-h arabinose induction (at final concentration of 0.02%), when Cre activity is supplied by p15AraCre plasmid (Fig. 2A, lanes 4 and 9). It seems that non-multi-

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merized forms of plasmids like monomer of pSPloxP2 and ploxA are more stable under these conditions (lane 4). Only a little effect on plasmid loss can be seen after 4.5-h arabinose induction of cre gene expression, when it was located on chromosome (lanes 2 and 7), as well as in bacteria with plasmid p15AraCre cultivated under repressed conditions (lanes 3 and 8). In the last case leakage of cre transcription produces both types of recombination, excision or integration. Apparently, in case of high copy number loxP-plasmids used here, integrative recombination is random and more efficient than multimer resolution. We confirmed that quantitatively by densitometric measurement of the amounts of native forms of the formed multimers in comparison to the amount of intact pSP-loxP1 and pSP-loxP2 monomers of the DNA preparations analyzed above (not shown). We proved in the control experiments that cellular Cre activity has not affected the yield of any no-loxP-plasmids (data not shown). 3.2. Cre mediated multimerization of loxP-bearing plasmids To mimic cellular conditions with permanent low level of Cre activity, we have gently induced (0.02% arabinose) DH5 attB::cre bacteria bearing pSP-loxP1 plasmid with single loxP site. Then, the plasmid was isolated from bacteria after overnight cultivation, and its native forms were studied by separation on an agarose gel (Fig. 3A). We could observe a ladder of plasmids bigger than monomers raised by Cre integrative recombination, which made up about 75% of the overall amount (Fig. 3A, lane 2). Electron micrograph of this plasmid preparation (Fig. 3C) confirmed our predictions about formation of the covalently closed multimers. Many types of multimers ranging from monomer to octamer can be seen on the micrograph. To examine a preferential type of Cre mediated recombination in our experimental approach, the trimer of pSP-loxP1 plasmid arisen from Cre recombination was isolated from the agarose gel, and then was transformed into either DH5a cells (Fig. 3B, lane 1) or DH5 attB::cre host (lanes 2 and 3). Further, both types of trans-

Fig. 3. Cre mediated multimerization of loxP-bearing plasmids. (A) pSP-loxP1 plasmid was isolated from DH5 attB::cre host after cultivation up to stationary phase in the absence (lane 1) or presence of 0.02% arabinose for Cre induction (4.5 h, lane 2). Native forms of plasmids were run on a 0.7% agarose gel with ethidium bromide. 1SC, 2SC, 3SC, and 4SC: supercoiled forms of monomer, dimer, trimer, and tetramer, respectively; Ch, chromosome. (B). Examination of the preference to the either Cre recombination type in vivo. Trimer of Cre recombined pSPloxP1 plasmid was isolated from agarose gel, and was then transformed into DH5a cells (lane 1) or DH5 attB::cre host (lanes 2 and 3). Further, both types of transformants were cultivated overnight in LB-medium with ampicillin, and then plasmid DNA was isolated and electrophoresed on agarose gel. Moreover, DH5 attB::cre bacteria containing trimers of pSPloxP1 were induced by 0.02% arabinose overnight (lane 3). Lane 4, HindIII digested k DNA. (C) Representative electron micrograph of pSP-loxP1 plasmid DNA isolated from E. coli DH5 attB::cre strain, after 4.5-h induction by 0.02% arabinose. Arrows show multimeric forms of the plasmid: monomer (M), dimer (D), tetramer (D), and (O) octamer. Bar represents 1.25 kb.

formants were cultivated overnight in LB-broth with ampicillin, and then plasmid DNA was isolated and electrophoresed on agarose gel (Fig. 3B, lanes 1 and 2, respectively). Moreover, DH5 attB::cre bacteria containing trimers of pSP-

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loxP1 were induced by 0.02% arabinose overnight to produce Cre recombinase (lane 3). It is shown that Cre is capable to perform both types of reactions, but integrative recombination dominates over the resolution one. 3.3. Study of the influence of Cre activity on loxP-plasmids maintenance At this point of our study we address the question what is the molecular explanation of decreasing of the overall yield of the loxP-plasmids. We designed an experiment to detect stability and maintenance of loxP-plasmid—pSP-loxP1, in the presence of Cre activity under nonselective conditions for pSP-loxP1 and pSP-72, used here as a loxP control (Fig. 4A). Initially, the overnight cultures with all appropriate antibiotics were diluted 1:500 in fresh LB-broth medium without ampicillin, and then were cultivated to exponential phase of growth. We examined set of the following hosts with appropriate plasmids: E. coli DH5 attB::cre carrying the pSP-loxP1, E. coli DH5a carrying p15AraCre resident plasmid with pSP-72 control plasmid or pSP-loxP1 plasmid. Moreover, control DH5 attB::cre host containing pSP-loxP1 test plasmid was induced with 0.2% arabinose. In addition, the media were supplemented with tetracycline for DH5 attB::cre strain selection or with chloramphenicol for p15AraCre plasmid. Appropriate dilutions of refreshed cultures were performed after 0, 3, 6, and 9 h of incubation at 37 °C, and then bacteria were plated on plain LB-agar and incubated overnight. Next day, individual colonies were transferred to LBagar supplemented with ampicillin. At least 150 colonies were tested for each culture at every time point. The results are shown in Fig. 4A. Maintenance of pSP-loxP1 plasmid is stable in DH5 attB::cre strain under non-inducing conditions even without ampicillin pressure (row 1). In striking contrast, the same plasmid is lost very rapidly in the presence of low but permanent level of Cre activity in DH5a host bearing p15AraCre plasmid (row 4). We could see the same effect under inducing conditions in the case of DH5 attB::cre carrying pSP-loxP1 (row 3). After 3 h of cultivation, 33% of LB-agar grown colonies were plasmid free,

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and after the next 3 h number of bacteria that lost plasmid increased up to 85%. We confirmed the fact of plasmid loss by examining the plasmid content in bacteria containing pSP-loxP1 and p15AraCre, which were grown on plain LB-agar, after spreading them from the diluted culture at time 0 of incubation in LB-broth. Twenty colonies taken from agar were suspended in 100-ll of lysis buffer and plasmid isolation was carried out (Fig. 4B). After agarose gel electrophoresis we could see fraction of pSP-72 control plasmid, isolated from DH5a host bearing p15AraCre plasmid (lane 2), but could not see pSP-loxP1 plasmid in DH5a with resident p15AraCre (lane 3). In addition, we have tested multimeric state of pSP-loxP1 plasmid in DH5a att::cre host grown on LB-broth with Ap, after 6 h of cre induction (0.2% arabinose). Then, bacteria were spread on LB-agar plate supplemented with Ap. We picked 143 individual colonies directly from plate and isolated the total plasmid DNA by alkaline lysis method (Sambrook et al., 1989). Whole DNA preparations from the independent colonies were run on a 0.8% agarose gel and compared to the mixture of pSP-loxP1 multimers used as a marker (Fig. 4C). Each colony tested contained diverse pool of multimers with dominant tetrameric and pentameric forms of pSP-loxP1 plasmid. Among the colonies investigated, only 22 contained traces of monomeric form of plasmid visible on the gel. Furthermore, we asked what is the fate of particular multimeric forms in single bacterial clone, generation by generation. We spread 20 generation old individual bacterial colonies on fresh LB-agar supplemented with Ap, and after overnight growth (40 generations since Cre recombination was abolished) we analyzed the multimer structure of plasmid in several individual colonies derived from the same original clone. Surprisingly, majority of colonies tested contained only one particular dominant multimer out of several that existed in the starting pool (Fig. 4D, lanes 2–14). Assuming random distribution of plasmids during cell division, it looks like many copies (if not all) of the same type of multimers might be grouped (or not yet diffused after replication cycle) in the same cluster, hence, probability of segregation of plasmids in such complexes into one daughter cell would be

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Fig. 4. Maintenance stability of the loxP-bearing plasmids in the presence of Cre recombinase. (A) Study under growth conditions without antibiotic selective pressure. Overnight cultures with all appropriate antibiotics were diluted 1:500 in fresh LB-medium and exponentially growing cells of either E. coli DH5 attB::cre carrying a pSP-loxP1 (row 1) or E. coli DH5a carrying p15AraCre resident plasmid with pSP-72 (row 2) or pSP-loxP1 (row 4), were examined for stability of pSP-loxP1 in the presence of tetracycline (DH5 attB::cre) or chloramphenicol (p15AraCre), respectively, but without ampicillin (nonselective conditions for pSP-72 and pSP-loxP1 maintenance). In addition, DH5 attB::cre host containing pSP-loxP1 test plasmid was induced by 0.2% arabinose (row 3). Appropriate dilutions of each culture were performed after 0, 3, 6, and 9 h of cultivation at 37 °C, and then bacteria were plated on plain LB-agar and incubated overnight. Next day, individual colonies were restreaked on the Ap containing LB-agar plate. At least 150 colonies were tested for each culture at indicated times. (B) Analysis of plasmid pSP-loxP1 inheritance in bacteria with resident Cre-delivery plasmid. A 0.7% agarose gel electrophoresis of plasmids isolated immediately from 20 colonies grown on plain LB-agar that were spread at time 0 of incubation in LB-broth. Lane 1, HindIII digested k DNA; lane 2, EcoRI digested plasmids p15AraCre and pSP-72 isolated from DH5a host; lane 3, EcoRI digested plasmids p15AraCre and pSP-loxP1 isolated from DH5a host. (C) Segregation profile of the various forms of pSP-loxP1 in 18 individual DH5 att::cre colonies (lanes 2–19) previously grown under 0.2% arabinose inducing conditions. Lane 1, plasmid monomer; lane 20, ladder of multimers. (D) Segregation of pSP-loxP1multimers in 13 individual colonies (lanes 2–14) after 20 growth generations in the absence of Cre induction, originated from one particular clone of bacteria, previously cre expressed. Lane 1, multimer pattern from original pool; lane 15, ladder of pSP-loxP1 multimers. (E) Segregation profile of native forms of pSP-loxP1 in 18 individual Cre colonies after 40 generations (lanes 2–19). Lane 1, plasmid monomer; lane 20, ladder of plasmid multimers. M, D, T, and Ttr—indicate monomeric, dimeric, trimeric, and tetrameric forms of plasmid, respectively.

very high. Otherwise, all tested colonies would have had similar multimer profile. In the control experiment, we checked distribution of pSP-loxP1 forms in Cre deficient DH5a culture. Out of 117 individual colonies tested grown on LB-agar supplemented with ampicillin, only four colonies carried plasmid dimers, and the rest of them contained plasmid monomers or mixture consisting of dominant monomers with traces of dimeric

forms (Fig. 4E). The established ratio in distribution of monomers and dimers in the studied bacterial populations are in good agreement with the earlier obtained data for Rec+ bacteria (Summers et al., 1993). It is also noteworthy that most colonies of bacteria bearing mixture of higher multimers produce much less amount of plasmid DNA in comparison to those which carry predominantly monomers (Fig. 4E).

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3.4. Bacteria with monomeric form of plasmid overgrow bacteria with higher multimers To compare some vital features of bacteria carrying monomers to those which contain higher multimers of pSP-loxP1 plasmid, DH5a cells were selectively transformed with dimeric, trimeric, and tetrameric forms of it. In the light of the fact of appearance of a few subpopulations of clones containing only one particular multimer in a relatively short-period of time (Fig. 4D), it seems that such a comparative study has a reasonable basis. We determined generation times of particular exponentially growing strains, cultivated in LB-broth with Ap (Table 2). The fastest growth (41.1 ± 3.1 min) was obtained for a culture of bacteria with monomers, the slowest for bacteria with tetramers (49.8 ± 6.8 min). In fact, overnight cultures of bacteria with tetramers usually reached lower cell density than those with monomers (not shown). Intermediate values of generation times were obtained for bacteria carrying dimers or trimers, 44.5 ± 3.5 and 47.5 ± 3.5, respectively. In addition, we estimated the approximate copy number of various forms of pSP-loxP1 plasmid with respect to the copy number of the monomer. Samples were taken at different stages of culture growth and the total amount of plasmid DNA per bacterial mass was estimated (Table 2). Multimeric plasmids (up to tetramers) are replicated more efficiently than monomeric in the time range starting from early exponential phase to early stationary. Then, the relative copy number of multimeric forms drops below that of monomeric and

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is maintained stably overnight (Table 2). We revealed that greater than tetramer multimers were highly unstable in respect to their structure, maintenance and copy number (not shown). We observed Cre/loxP as well as Xer/cer-independent conversion of higher-order multimers to simpler, more stable forms (Fig. 5). We tested the structural changes of pUC-based loxP -multimer plasmids in Cre background and in a set of Xer/cer deficient strains (argR, pepA, xerC, and xerD, Table 1) starting with single transformant colony. The observed multimersÕ decay was more efficient in E. coli recA+ bacteria (MM294) than in recA (DH5a) or recF (either Xer/cer deficient strain). In case of MM294 host, pSP-loxP1 tetramers, pentamers, and hexamers are partially resolved to monomers (or dimers) within the first 20 generations after transformation (agar plate colonies, Fig. 5). In DH5a cells as well as in any recF Xer/cer deficient strains similar demultimerization events took place, but they were shifted in time by about 20–40 generations in comparison to recA+ background. Plasmid decay was general and common for many tested pUC-based replicons (data not shown). Similar observation was reported earlier for plasmids in RecA+ bacteria as well as in some combinations of different rec mutations (James et al., 1982). One of apparent mechanisms responsible for this conversion was proposed to be an intramolecular conservative homologous recombination, which includes one exchange or any odd number of exchanges. This would result in the formation of circular plasmids with reduced size in respect to the original multimer. In fact, we

Table 2 Generation times (min) and plasmid copy number in DH5a cells carrying various multimeric forms of pSP-loxP1 plasmid Plasmid multimer form

Monomer Dimer Trimer Tetramer

Generation time (min)a

41.1 ± 3.1 44.5 ± 3.5 47.5 ± 3.5 49.8 ± 6.8

Copy number ratiob OD600 0.17–0.22

OD600 0.55–0.65

OD600 1.3–1.5

OD600 overnight

1 4.36 ± 0.53 3.35 ± 0.35 2.80 ± 0.30

1 2.47 ± 0.4 2.20 ± 0.28 1.80 ± 0.24

1 0.94 ± 0.26 0.80 ± 0.21 0.51 ± 0.17

1 0.89 ± 0.14 0.65 ± 0.12 0.45 ± 0.12

a Estimated for bacterial cultures at exponential phase of growth (OD600 0.08–0.4). Values are the mean of at least five independent experiments. b Copy number of particular multimer was normalized to the copy number of pSP-loxP1 monomers. Results obtained at a given stage of culture growth represent the average of at least three independent experiments.

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could observe such molecule species on agarose gels in case of each bacterial clone bearing a particular multimer. Eventually, subpopulation of cells with monomers (or dimers) was established, which was the most competitive and successful in such a culture. In case of our RecA Cre+ phenotype investigated, alternative recA-independent kind of plasmid demultimerization mechanism, as suggested by Dianov et al. (1991), was not so effective to restore and keep stable balance between various plasmid forms in a single cell, to provide for heritable stability of plasmid.

To check whether the differences in rate of growth can really affect viability of particular recA (DH5a) bacteria bearing multimers we performed growth competition assay (LB medium with Ap), by mixing cultures of bacteria bearing monomer-only and tetramer-only plasmids (Fig. 6, Summers et al., 1993). Regardless of the initial ratio of the mixed strains of bacteria, after approximately 60 generations of growth bacteria with monomers overcame bacteria with tetramers. Interestingly, under these growth conditions (antibiotic pressure, absence of Cre protein) bacteria

Fig. 5. Cre/loxP and Xer/cer-independent conversion of higher-order multimers to monomers. (A) pSP-loxP1 tetramers (lanes 1–6) or hexamers (lanes 8–13) decays in DH5a (recA1 cre ) host, and tetramers (lanes 15–17) and pentamers (lanes 18–20) decays in MM294 (recA+ cre ) host. Plasmid DNA were isolated from 1 ml of consecutive cultures, and DNA aliquots were run on a 0.8% agarose gel. Each lane corresponds to approximately 20 generations of bacterial growth. Lanes 7, 14, and 21 contain ladder of pSP-loxP1 multimers. (B) pSP72 (loxP ) tetramers decay in MM294 (lanes 1–4) and DH5a (lanes 6–9) hosts, respectively. Lane 5, ladder of pSP72 multimers. (C) Decay of pSP-loxP1pentamer form in E. coli Xer/cer recF deficient strains: pepA (lanes 1–3), xerC (lanes 5–7), argR (lanes 9–12) and xerD (lanes 14–16), respectively. Lanes 4, 8, and 13, contain ladder of pSP-loxP1 multimers. M, D, T, Ttr, and H— indicate monomeric, dimeric, trimeric, tetrameric, and hexameric forms of plasmid, respectively.

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Fig. 6. Growth competition assay of DH5a bacteria bearing monomeric or tetrameric forms of pSP-loxP1. (A) Competition under antibiotic selection. Bacteria were cultivated in LB-broth containing Ap at 37 °C and refreshed after 22–24-h by 104-fold dilution for three or four consecutive days. Plasmids were isolated from 1 ml of each culture and 1/10th of total DNA was run on a 0.8% agarose gel. Lanes 1–4, plasmids from bacteria with monomers; lanes 5–8, plasmids from bacteria with tetramers; lanes 10–12, plasmids extracted from mixed culture of monomers and tetramers (initial ratio 10:1, respectively; lane 9, starting DNA ratio of the premixed cultures (P)); lanes 14–16; plasmids extracted from mixed culture of monomers and tetramers (initial ratio 1:10, respectively; lane 13, starting DNA ratio of the premixed cultures (P)). 1SC, 4SC: supercoiled forms of monomers and tetramers, respectively; Ch, chromosome. It is assumed that one day growth corresponds to 20 generations of bacteria. (B) Growth competition between plasmidfree bacteria and plasmid containing bacteria. Stability of plasmids was investigated during growth in the absence of ampicillin for four days. Cultures were inoculated with a single colony formed by bacteria containing either monomers or tetramers. Consecutive overnight cultures were 104-fold diluted into fresh LB-medium, followed by a 22–24-h growth period. One milliliter sample of each culture was lysed and plasmid DNA was extracted and then aliquots were run on a 0.8% agarose gel. Lanes 1–4, plasmids from bacteria with monomers; lanes 5–8, plasmids from bacteria with tetramers.

bearing tetramers cultivated alone were relatively stable within the first 60 generations (Fig. 6, lanes 5–7), and then underwent conversion to monomers (lane 8). Dimers and trimers containing bacteria cultivated under similar conditions stably maintained them within 180 generations of growth (not shown). In this case, we did not observe any conversion to structurally simpler forms. Since molecular concentration of monomeric forms in

cellular pool in Cre-recombination proficient bacteria undergoes constant reduction (Fig. 4D), we investigated maintenance stability of tetrameric form of pSP-loxP1 in the culture grown without antibiotic pressure. We treat this as a growth competition test between bacteria containing tetramers and plasmid-free bacteria, appearing during prolonged growth period. In this experiment, two strains containing monomeric or tetrameric forms

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of pSP-loxP1 were cultivated independently trough 80 generations in the absence of ampicillin. Under these growth conditions monomers are relatively stably maintained (Fig. 6B, lanes 1–4), but in contrast, tetramers are gradually lost (Fig. 6B, lanes 5–8). After 60 generations of growth only 3% of the tested colonies retained tetramers in comparison to 84% of cells bearing monomers.

4. Discussion In conclusion, we showed that Cre mediated loxP-plasmid multimerization process leads to plasmid free cells in recA genetic background. Our results are compatible with the hypothesis of the multimer catastrophe (Summers and Sherratt, 1984). According to this hypothesis high-order multimers should out-replicate lower order multimers, which leads rapidly to the appearance of multimer-only cells. Moreover, those authors demonstrated that ratio of multimers declined because cells of such a type grew more slowly (Summers et al., 1993). We confirmed that this is the case under our experimental conditions. Constitutive expression of Cre recombinase generates loxP-plasmid multimers very efficiently (Figs. 2A and 3A). Multimers become dominant in heterogeneous pool of cellular plasmids, especially after induction of cre expression, when monomerÕs depletion by their fusion takes part (Fig. 4C). Cells containing multimers grow significantly slower (Table 2) and are overgrown by plasmid-free bacteria. We showed this directly in growth competition assay performed under nonselective conditions in recombination-deficient strains. In consecutive cultures cultivated, tetramers were overgrown by plasmidfree bacteria and lost within 60 generations (3% bacteria retained plasmid, Fig. 6B). In contrast, monomers were quite stably maintained at least 80 generations under the same conditions. Similar results were obtained when we challenged the viability features of the mixed culture consisting of Cre RecA bacteria (to preserve their structure unchanged) with tetramers or monomers of pSPloxP1 in growth competition assay (Fig. 6A). This was performed under selective conditions and we chose monomers bearing bacteria as an example

of faster growing bacteria. Process of the loss of loxP-plasmids can be very fast, especially in a liquid medium in the presence of ampicillin as a selection factor which is regarded as short-lived because of extracellular presence of b-lactamase. We showed that in the presence of active Cre under nonselective conditions for maintenance of loxP-plasmids, the process of their loss is really very fast (Figs. 4A and B). With the exception of the relatively short-time during an exponential phase of growth, multimers are maintained at a lower copy number than monomers in the following way: the more complexed multimer, the lower the number of its copy per cell (Table 2). It is very likely that rapid loss of potentially high-copy ColE1-type multimers is possible thanks to their clustered character during their replication cycle and further segregation (Pogliano et al., 2001). We showed, that progeny of clones initially containing diverse pool of multimers very often inherit only one particular multimer. Moreover, our results show that all of the tested types of loxP-plasmids undergo Cre-mediated multimerization process, irrespective of number or orientation of loxP sites (not shown) and type of replication origin (e.g., oriS/mini-P1 copy-up mutants, pMB1, p15A, oriV/RK2, not shown). Since excision is an intramolecular event it was thought it should be therefore favored over integration reaction. It is not clear why integrative reaction is more efficient than excision but it sheds a new light on utilization of Cre/loxP system in manipulation techniques with high-copy loxP-plasmids. One explanation of this might be the different frequency of replication initiation observed among single-origin plasmids and multi-origin plasmids (Martin-Parras et al., 1992; Summers et al., 1993 and this study). Multi-origin plasmids are amplified faster and thus quickly become molecularly significant in substrate pool for further Cre recombination. In other words, replication process plays prominent role in elevation of the copy of high-order multimers formed by Cre, but obviously is not potent to create them by itself. It is worth to say that Cre activity leads to gradual loss of monomers. Conversely to earlier suggestion (Bigger et al., 2001), we showed that origin-less circular molecules formed during excisive recombination,

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which are slowly lost because of their replication deficiency, can be recruited as a substrate in alternating integration processes. It is reflected first, by the appearing of dimers and trimers of ori circles and second, by the presence of tandem arrays of poly-ori units flanked by loxP sites in recombinant plasmid structures. Interestingly, while studying stability of the particular type of multimer in the independent recA cultures, we revealed Cre/loxP and Xer/cer independent demutlimerization event, acting most effectively on tetramers and higher-order plasmids. Similar observations were reported earlier in literature. In the most comparative study James et al. (1982) proposed RecA/RecF dependent mechanism of intramolecular recombination of tetrameric plasmids. Our results show that intramolecular recombination is significantly reduced but not completely blocked in either recA or recF mutated bacteria, thus its mechanism must be a mixture of recA-dependent and independent events, which was suggested earlier by Dianov et al. (1991). Despite, the presence of such plasmid structure regulation occurring in vivo, apparently it is not so efficient in recA-deficient hosts to maintain stable balance between various plasmid forms in single cell, and to provide their stable inheritance. Paradoxically, RecA phenotype, believed to have deleterious effect on plasmid stability (reviewed by Summers, 1998), with its ability for effective highorder plasmid demultimerization, and thus restoration of more stable plasmid structure, seems to offer better phenotypic conditions for stable maintenance of plasmids, which are affected by the very active ‘‘multimerizing’’ factors, like for example Cre recombinase. The conditions where dual type of recombinations influence plasmid structure and its maintenance require more detailed investigation, but obviously RecA-dependent plasmid structure fluctuations can also bring some adaptive benefits like toxic gene deletions, primary gene structure restorations, accumulation of advantageous mutations, and more efficient plasmid establishment (reviewed by Yarmolinsky, 2000). It is important to emphasize that in such in vivo attempts where very efficient production of loxPminicircles wants to be achieved, the Cre/lox system should be used with caution or altered by

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alternative recombination system. Strengthened control of Cre activity can only attenuate or ‘‘slow-down’’ random multimerization process but not completely eliminate it. To bypass this problem a more intense search for new specific mutant sequences and/or mutant protein that would act in unidirectional fashion, favoring strictly only one type of recombination should be done (Albert et al., 1995; Araki et al., 1997; Buchholz et al., 1998; Langer et al., 2002; Lee and Saito, 1998; Senecoff et al., 1988). In addition, Flp/FRT recombination system behaves essentially as described here for Cre/loxP (not shown), but thankfully, activity of Flp protein can be regulated by temperature shift (Buchholz et al., 1996).

Acknowledgments We wish to express many thanks to Grazyna Konopa (University of Gdansk, Gdansk, Poland) for excellent work on the electron microscopy and to Waclaw Szybalski (University of Wisconsin, Madison, USA) who partially supported this work. Plasmids were kindly provided by J. Wild (pJW165, University of Wisconsin, Madison, USA), J.A. Murray (pMS103 and pMS107, Cambridge University, Cambridge, UK), L. M. Guzman (pBAD24; Harvard University, Boston, USA) and M. Koob (pTANTS and pINTtsCm, University of Minnesota, Minneapolis, USA). Xer/cer deficient strains were kindly provided by David Sherratt (University of Oxford, Oxford, UK). We are also grateful to Kasia Potrykus for her excellent help at the stage of the manuscript editing. This work was supported, in part, by KBN Grant BW 1170-5-0219-4 (Warsaw, Poland).

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