Laboratoire de Biologie et Génétique Moléculaire, Institut de Génétique et Microbiologie, UMR CNRS 8621, Bât. 400, Université Paris-Sud, F-91405 Orsay Cedex, France1
Author for correspondence: Alain Raynal. Tel: +33 1 69 15 62 10. Fax: +33 1 69 15 45 44. e-mail: alain.raynal{at}igmors.u-psud.fr
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ABSTRACT |
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Keywords: actinomycetes, integrase, site-specific recombination, attachment site
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INTRODUCTION |
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pSAM2 is an 11 kb element, originally isolated from Streptomyces ambofaciens, which produces the macrolide antibiotic spiramycin (Pernodet et al., 1984 ). pSAM2 can replicate, is self-transmissible, elicits the lethal zygosis reaction (pock formation) and mobilizes chromosomal markers (Smokvina et al., 1991
). Furthermore, pSAM2 has a recombination system that is very similar to that of temperate phages. pSAM2 is integrated into the chromosome through site-specific recombination between the element (attP) and the chromosomal (attB) sites. These regions share a 58 bp segment extending from the anticodon loop to the 3' end of a tRNAPro gene (Mazodier et al., 1990
; Boccard et al., 1989
).
In a previous study (Raynal et al., 1998 ), the pSAM2 site-specific recombination system was reconstructed in Escherichia coli and the chromosomal attB site was characterized by use of two compatible plasmids, one carrying the attP site and expressing the pSAM2 integrase and the other carrying various fragments of the chromosmal attB region. With this system, it was shown that Int is the only Streptomyces protein required for site-specific integration and that Int and Xis are both required for site-specific excision. The attB region required for efficient site-specific recombination was shown to be 26 bp long and centred around the anticodon loop. Comparison of this 26 bp region with attP suggested, according to the lambda model (Campbell, 1992
), that B and B', and C and C', core-type Int binding sites consist of 9 bp imperfect inverted repeats separated by a 5 bp overlap region.
This study aimed to characterize the attP site with respect to its minimal size and to the integrase binding sites.
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METHODS |
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Construction of attP-containing plasmids.
The complete attP region was isolated from pTS33 (Smokvina et al., 1990 ) as an AvrIIBglII fragment whose AvrII extremity was blunt-ended by Klenow treatment. The resulting fragment was cloned into EcoRV/BamHI-digested pMCL200 vector generating pOSattPpSAM2. The minimal attP site and parts of this site (a, b, c) (Fig. 2
) were cloned either by subcloning or by PCR amplification. pOSattPbc was derived from pOSattPpSAM2 after XbaIDraIII deletion in the 5' region followed by a SalISalI deletion in the 3' region. Deletion was performed by generating blunt ends with Klenow treatment, followed by ligation.
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Construction of the integrase expression plasmid.
To overproduce and to purify the pSAM2 integrase, the int gene was placed under the control of the T7 promoter of the E. coli plasmid pET-21a(+) (Novagen) in which the basal expression is very low in the absence of IPTG induction. The int gene was PCR amplified from pOSint1 (Raynal et al., 1998 ) with the Int5B/IntTag primer pair. A BamHI site was introduced immediately upstream of the int ATG start codon by use of the Int5B (5'-TTTGGATCCATGGCCAAGCGACGTAGC-3') synthetic sequence.
The IntTag sequence (5'-GTGCTCGAGTCGCGCCGGTCCCCGCTTG-3') hybridizes to the 3' end of the integrase coding sequence. The cloning of a BamHIXhoI amplified fragment in pET-21a(+) led to an in-frame fusion, providing a His-Tag coding sequence at the C-terminus of the integrase. pET-21int was obtained in DH5 and then introduced into BL21(DE3) to overproduce integrase after IPTG induction.
Purification of integrase.
The int gene was cloned in the expression vector, pET21a+, under the control of the IPTG-regulated T7 promoter, yielding pET21-int. A His-tag was added to the C-terminal end of the integrase. Five millilitres of a 37 °C overnight preculture of E. coli BL21(DE3) containing pET-21int were inoculated into 500 ml LB medium containing 100 µg ampicillin ml-1. The culture was grown at 22 °C with shaking to an OD650 of 0·45. IPTG was then added (1·0 mM) and the cells were grown for a further 5 h at the same temperature and harvested by centrifugation. After washing once in 50 mM Tris, 100 mM NaCl, pH 8, the pellet was resuspended in 5 ml of the same buffer and frozen at -20 °C before lysis. Lysozyme (1 mg ml-1) was added and the sample was lysed for 30 min at room temperature. After centrifugation the pellet was resuspended in the same buffer containing 0·1% sodium deoxycholate. The resulting suspension was denatured by sonication and centrifuged at 13000 g for 10 min. The resulting supernatant was further purified, as described by Novagen, and the soluble fraction was found to contain only a small amount of integrase. Therefore, integrase was purified from inclusion bodies present in the pellet following the last centrifugation step. The pellet was resuspended in binding buffer (5 mM imidazole, 0·5 M NaCl, 20 mM Tris/HCl pH 7·9) containing 6 M urea (final concentration). All further purification steps (loading, washing and elution) were performed with buffer containing 6 M urea. Proteins were eluted in 300 mM imidazole, 0·5 M NaCl, 20 mM Tris/HCl pH 7·9, 6 M urea. Fractions (1 ml) were eluted from the His-bind resin column, and the A595 revealed that integrase was in fractions 2, 3 and 4. Urea was gradually removed by successive dialysis to a final urea concentration of 1·5 M to allow protein refolding. A 20% (v/v) glycerol solution containing 0·6 mg integrase ml-1 was split into aliquots and stored at -20 °C. SDS-PAGE was carried out in 10% acrylamide gels according to Sambrook et al. (1989) . Proteins were detected by Coomassie blue staining.
DNase I footprinting.
DNase I footprinting experiments were performed by use of the Promega Core Footprinting System. The probes consisted of fragments carrying a 3' and a 5' protruding end isolated from the cloned attP minimal site. The 5' end was radio-labelled by filling in with the Klenow fragment of DNA polymerase I and [-32P]CTP. The labelled fragment was loaded onto an agarose gel and purified by use of the GFX Gel Band Purification Kit (Amersham Pharmacia Biotech). Binding reactions were carried out in 25 mM Tris/HCl pH 8·0, 6·25 mM MgCl2, 0·5 mM EDTA, 0·5 mM DTT, 200 mM KCl and 10% (v/v) glycerol buffer. Purified integrase (10 µg) was incubated with 50000 c.p.m. of labelled probe at 25 °C for 1 h. Reactions were treated with 1·5 U DNase I (RQ1 DNase Promega) for 1 min at 25 °C and the resulting product was analysed on a 6% sequencing gel.
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RESULTS |
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An E. coli DH5 strain harbouring pOSint
P for the production of pSAM2 integrase was first transformed by pOSattP. The resulting clones were selected as ApR/CmR colonies at 37 °C. The plasmids used are stably maintained in E. coli, at least in the presence of a selection pressure, and before transformation with the third plasmid, recipient cells retained the two other plasmids. These strains were further transformed by pOSattB. After 4 h expression in the presence of IPTG (5x10-4 M) to allow expression of the SmR phenotype, incubation was continued overnight at 30 °C in the presence of both Sm (20 µg ml-1) and IPTG. Finally, SmR clones were selected at 30 °C and 42 °C, on plates containing Sm (40 µg ml-1) and IPTG. As pOSattB, conferring SmR, cannot replicate at 42 °C, the growth of SmR clones at 42 °C should indicate the formation of pOSattB-pOSattP co-integrates by site-specific recombination. SmR colonies obtained at 42 °C were replica plated to test for CmR at 42 °C. All the tested SmR colonies isolated at 42 °C were CmR. In all cases, restriction analysis on plasmids isolated from these strains confirmed that they were co-integrates resulting from recombination between attB and attP, even when the recombination efficiency was very low. In the absence of the int gene, no SmR colonies were obtained at 42 °C. The co-integrates were therefore due to site-specific recombination promoted by Int.
As CmRSmR colonies growing at 42 °C were only obtained if co-integrates were formed, the ratio of SmR colonies isolated in non-permissive (42 °C) and permissive (30 °C) conditions for pOSattB replication allowed us to measure the relative frequency of recombination. With pOSattPpSAM2, harbouring a 829 bp fragment containing the attP region, this ratio was 94%, confirming that pSAM2 site-specific recombination is very efficient in E. coli (Table 1) even when Int acts in trans on attP sites. Intermolecular recombination between attP and attB was assayed in our experiments and the conclusions reached about the sequences required do not necessarily apply for the excision reaction.
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Purification of integrase
Before purifying integrase, the functionality of the His-tagged integrase was studied in vivo. As for the study of the attP site, we used pOSattPmin and pOSattB to provide recombinant sites and pET21-int to produce the His-tagged integrase in recombination experiments performed in the expression strain BL21(DE3). The frequency of recombination was 85% with pET21-int, compared to 92% with pOSint1P in DH5
. These results clearly show that the His-tagged integrase encoded by pET21-int was functional and bound efficiently to its target sequences.
Integrase was purified from inclusion bodies using His-bind resin. After the purification of these inclusion bodies, integrase was recovered under denaturing conditions (6 M urea) followed by partial renaturation. Approximately 2 mg purified integrase was obtained from 500 ml culture. Analysis of the final product by 10% SDS-PAGE showed a unique band at 43 kDa, corresponding to the purified integrase (Fig. 3).
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If we represent the pSAM2 minimal attP site with a coordinate 0 at the centre of our previously described core site, the 17 bp repeat sequences lie between positions -100/-116 in the 5' region and +102/+118 in the 3' region, represented as P1 and P2 in Fig. 5. Thus, the two defined arm sites, which strongly bind integrase, appear to be symmetrical with regard to the core site at which the recombination event occurs.
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DISCUSSION |
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As pSAM2 site-specific recombination is efficient in E. coli (Raynal et al., 1998 ), no Streptomyces-specific protein, besides Int, and Int together with Xis for excision, seems to be required for this process. However, the involvement of some E. coli proteins, e.g. histone-like proteins, cannot be excluded.
pSAM2 integrase resembles other integrases in its C-terminal part (Nunes-Düby et al., 1998 ) and this part is most probably involved in the catalytic activity. For the N-terminal part of pSAM2 Int, possibly involved in arm-binding activity, no obvious conserved region was detected after comparison with other integrases. pSAM2 integrase appears to have a strong affinity for arm-type sites, but no integrase binding could be detected on the core-type site, even in low KCl concentrations, which decrease binding specificity. It seems that the binding of Int to arm-type sites is a prerequisite for proper recognition of the core binding site during integration.
The overall organization of the pSAM2 attP site can be compared to those of other phages such as P22, P2, lambda and L5 (Pena et al., 1997 ) (Fig. 6
). In all these cases, the arm-type sites are located between -60/-130 and +60/+120 nt on both sides of the core site. For pSAM2, only two arm-type sites, consisting of 17 nt direct repeat sequences with a single mismatch, are required. In lambda (Ross & Landy, 1982
), P2 (Yu & Haggard-Ljungquist, 1993
), P22 (Smith-Mungo et al., 1994
) and mycobacteriophage L5 (Pena et al., 1997
), the sequences of the arm-type sites are shorter (about 10 nt). For lambda and L5 phages, arm-type binding sites consist of four or five repeats of shorter sequences, but some of them do not appear to be essential, for example P'1/P2 for lambda (Bauer et al., 1986
), P3/P67 for L5 (Pena et al., 1997
). There is little or no sequence similarity between the arm-type sites of these four phages. Nevertheless, the site we described for pSAM2 shares five nucleotides with the lambda consensus (GTCAC). In the five arm-type binding sites described for lambda, the nucleotides TCA are perfectly conserved in the consensus sequence. In addition, arm-type mutants that contain a C-to-T transition, located in the lambda P1, P'2 or P'3 arm-type site, exhibit a 10- to 100-fold reduction in integrative recombination (Bauer et al., 1986
). In contrast to phage lambda, for L5, P2 and P22, arm-type sites are arranged in pairs and it was suggested for L5 that Int might bind co-operatively to pairs of sites. However, recent results (Pena et al., 1999
, 2000
) clearly indicate that the formation of site-specific recombination complexes in L5 is quite different to that in lambda.
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Received 9 July 2001;
revised 26 September 2001;
accepted 27 September 2001.