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Dipartimento di Scienze Biomolecolari e Biotecnologie, Università degli Studi di Milano, Via Celoria 26, 20133 Milano, Italy
Correspondence
D. Ghisotti
Daniela.Ghisotti{at}unimi.it
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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Present address: Max F. Perutz Laboratories at the Vienna Biocenter, Department of Microbiology and Genetics, University of Vienna, 1030 Vienna, Austria. 
Present address: Keryos, Via della Filanda 5, 20060 Gessate (MI), Italy.
Present address: Molmed SpA, Via Olgettina 58, 20132 Milano, Italy.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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, P2 and P4 integrate their genome into the host chromosome by site-specific recombination between the phage attP and the bacterial attB sites (Nash, 1981
; Pierson & Kahn, 1984, 1987; Richet et al., 1988; Yu et al., 1989; Saha et al., 1990; Cho et al., 2002; Frumerie et al., 2005). In the case of , the recombination event is mediated by a site-specific recombinase, the Int phage integrase. Recombination initiates with the pairing of two specific DNA sequences by a tetramer of recombinase molecules. A Holliday junction is formed by cleavage, exchange and ligation of one pair of strands, and is resolved by the exchange of the second pair of strands by isomerization of the recombinase tetramer (Biswas et al., 2005; Radman-Livaja et al., 2006). A DNA–protein complex named intasome is responsible for site-specific recombination, in which attP and attB sites, phage integrase, as well as bacterial factors, such as integration host factor (IHF) and Fis, are involved (de Moitoso & Landy, 1991; Patsey & Bruist, 1995; Swalla et al., 2003). The reverse excision event requires, in addition, a phage encoded excisionase that is part of the intasome complex (Numrych et al., 1991; Wu et al., 1998; Cho et al., 2002).
Bacteriophage P4 Int recombinase belongs to the large family of tyrosine recombinases which includes over 100 members that show very little sequence identity; however, a C-terminal region of P4 integrase that can be aligned with other tyrosine integrases presents typical motifs (Esposito & Scocca, 1997; Nunes-Düby et al., 1998). In particular, in P4 integrase two different DNA-recognition motifs for binding to the core and arm sites that are present in the attP site were identified (Fig. 1a; Argos et al., 1986; Pierson & Kahn, 1987; Nunes-Düby et al., 1998; Grainge & Jayaram, 1999). Upon integration, the phage genome is linearly integrated into the bacterial chromosome and the int gene is proximal to the attL site in the prophage as conventionally defined (Nash, 1981). Contrary to what occurs in and P2, P4 int is transcribed starting from attL towards the centre of the P4 genome (Pierson & Kahn, 1984, 1987; Ghisotti et al., 1990).
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; Kita et al., 1999; Bishop et al., 2005). Excision of the prophage not only requires the integrase protein, but also depends on the P4 Vis protein, which binds and bends DNA at the attP site. Vis is thought to favour the formation of the DNA–protein complex necessary for prophage excision (Calì et al., 2004).
In all temperate phages, expression of integrase is regulated according to the phage developmental pathways (Grainge & Jayaram, 1999; Radman-Livaja et al., 2003). In this work expression of P4 int gene has been analysed and its negative regulation by both Int and Vis is suggested.
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METHODS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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. The oligonucleotides used are listed below, with the upper-case letters corresponding to P4 sequence (coordinates are reported), the lower-case letters are sequences not corresponding to P4 (casual), R indicates complementary strand, the underlined sequences indicate the EcoRI or BamHI restriction sites: no. 524 (5'-ccaaggatccCGAAGTTAAGATGCCCC-3'; P4 3935–3952); no. 525 (5'-ggctgaattcTCGTCCTGCGTCTTCCG-3'; P4 4010–3994 R); no. 526 (5'-gcgcgaattcCTTACTTCCAAGACCATCCG-3'; P4 4076–4057 R); no. 527 (5'-ccgcgaattcTTCCGATTTATTGATGGGCT-3'; P4 4219–4200 R); no. 546 (5'-cacaggatccCCATCGGCCATTTTGTAGG-3'; P4 3848–3866); no. 547 (5'-cacaggatccGCGTTGAGCTTCATTTGG-3'; P4 3902–3919); no. 573 (5'-ccccggatcCATTTCTAATCGAAGTTAAGATGC-3'; P4 3925–3948); no. 663 (5'-cgcggaattcATCTTAACTTCGATTAGAAATGTGC-3'; P4 3922–3947 R). All the fragments obtained by PCR amplification were sequenced. LD broth was described by Ghisotti et al. (1992); ampicillin (100 µg ml–1) and chloramphenicol (30 µg ml–1) were added when necessary.
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). In brief, total RNA (20 µg) extracted from E. coli C-1a and from P4-infected cells was fractionated on either 1.5 % formamide/formaldehyde agarose or 10 % polyacrylamide/urea denaturing gels, transferred to Hybond-N filter membranes (Amersham) and hybridized to the 32P-labelled RNA probe Int1, which covers the P4 nt 4035–3690 region. Hybridization and primer-extension analysis were performed as described by Briani et al. (1996) and by Boorstein & Craig (1989), respectively.
RNA electrophoretic mobility shift assay.
Glutathione S-transferase (GST)-Vis and GST purification were performed as described in Polo et al. (1996). 32P-labelled int RNA was synthesized by in vitro transcription with T7 RNA polymerase and pGM823 DNA as template, as described in Regonesi et al. (2004). The probe corresponds to the 3947–3848 P4 int transcript. Then, 1 pg (equivalent to 0.03 fmol) of the purified int RNA was incubated in the presence of either GST-Vis or GST in binding buffer [100 mM KCl, 10 mM Tris/HCl, 1 mM EDTA, 0.1 mM DTT, 5 % glycerol (v/v), 50 µg BSA ml–1] in the presence of RNasin (0.4 U) in a final volume of 8 µl at room temperature for 20 min. The samples were fractionated by native 5 % polyacrylamide gel electrophoresis at 4 °C.
-Galactosidase assay.
-Galactosidase activity was assayed in DH10B strains harbouring the reporter plasmids. Bacteria were grown in LD up to OD600=0.2–0.4 at 37 °C. -Galactosidase activity was measured using the method of Miller (1972). Each assay was repeated at least three times with independent extracts.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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; GenBank accession no. X51522
[GenBank]
). The presence of the insertion was confirmed by sequencing P4 int genes of different origin, such as P4 DNA extracted from E. coli C-1a/pP4, C-1a(P4) and three P4 DNA preparations from our phage collection. The presence of the insertion causes a frameshift in the terminal part of the Int polypeptide, creating a nonsense codon at 2649. The last 13 aa of the protein thus differ from the published sequence. The overall similarity with tyrosine-integrases of other phages was not modified, since the C-terminal end of P4 integrase is not significantly conserved.
Transcriptional profile of the int gene
Northern analysis of int transcripts after infection of E. coli C-1a with P4, using the Int1 probe (4035–3690), proximal to the Pint promoter, is reported in Fig. 2. In the first 40 min after infection, two transcripts covering the whole int gene (2.2 and 1.3 kb) were observed. A major signal was visible at low molecular masses (<0.6 kb). Upon hybridization of the same filter with either Int2 (3690–3216) or Int3 (3216–2622) probes, the 2.2 and 1.3 kb transcripts were still present, whereas the shorter RNAs disappeared (data not shown). Thus, the short transcripts mapped to the 5'-end of the int gene and may be due to premature transcription termination and/or transcript degradation. In the lysogenic state, the only transcripts identified were short transcripts; 2.2 and 1.3 kb RNAs were not visible.
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upstream and cII downstream of int, followed by a Rho-independent transcription termination site about 2.2 kb from Pint (Ghisotti et al., 1990), favours the latter hypothesis, suggesting that the 2.2 kb transcript encompasses int and cII.
P4 integrase negatively regulates Pint activity
Calì et al. (2004) identified the 5'-end of P4 int mRNA upon phage infection at 3947 (3946 in Calì et al., 2004) by primer extension. Canonical –10 (cATAAT) and –35 (TTGAaA) promoter consensus sequences located immediately upstream were proposed as the Pint promoter (see Fig. 1a).
In order to characterize the function of Pint, the RNA expressed from a set of plasmids harbouring different portions of the P4 int region was analysed by primer extension using an oligonucleotide complementary to the 3823–3840 P4 region, proximal to Pint (Fig. 3a). In pGM603, which carries the P4 4250–3690 region, a strong band was visible at 3947 (Fig. 3b). The same signal was less intense in pGM604 (4250–3594), and progressively fainter bands were observed in pGM605 (4250–3044) and pGM606 (4250–2475). Densitometric quantification indicated that the signal decreased to 42 % in pGM604, 16 % in pGM605 and 6 % in pGM606. Thus, when the P4 DNA fragment contained the whole int gene (pGM606) or at least 60 % of it (pGM605), the int mRNA abundance was greatly reduced, suggesting that Int negatively regulates transcription initiation at Pint. No band at 3947 was present with pGM597, which carries the 3970–3690 P4 DNA, lacking the –35 Pint sequence.
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), we reported that P4 Vis binds to the attP region and is able to repress Pint activity. To characterize further the effect of Vis on Pint, we constructed several plasmids carrying Pint fragments upstream of the reporter lacZ. The plasmids differed in the 5'-extension of the P4 region that included either BoxII (from coordinate 4010; pGM791 in Fig. 4), BoxI (from coordinate 4076; pGM792) and the core region (from coordinate 4219; pGM793). E. coli DH10B strains carrying pGZ119EH (control plasmid) or pGM677 (expressing Vis) were transformed with the plasmids and -galactosidase activity was measured. The results are reported in Fig. 4.
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-galactosidase in the absence of Vis. The highest activity was seen with the shortest fragment, carried by pGM791, suggesting that the upstream regions have a negative effect on Pint activity. This negative regulation might be caused by DNA conformation of the upstream region and/or binding of some bacterial factor.
In the presence of Vis, -galactosidase activity was reduced. In all three plasmids the amount of reduction was around 23–29 %, without a clear effect due to the presence of BoxI and the core region. Thus, BoxII appears to be sufficient for full repression by Vis.
Identification of P4 integrase start codon
The translation initiation codon of P4 integrase has not been experimentally determined. The int coding sequence presents three ATG codons in the first 90 nt downstream of the 5'-end of the int mRNA, none preceded by a potential ribosome-binding site (Fig. 1a). In order to determine which ATG serves as the int start codon, we cloned Pint and fragments extending to the first, second and third ATG, creating translational fusions with lacZ (Fig. 4). Strains DH10B/pGZ119EH and DH10B/pGM677 were transformed with the plasmids and -galactosidase activity was measured. Fusion with the first ATG (pGM805) did not express -galactosidase (6 Miller units), indicating that this codon is not used for translation initiation. On the other hand, fusions with the second ATG (pGM796) expressed -galactosidase weakly (802 Miller units), whereas the highest level of -galactosidase expression was obtained in fusions with the third ATG (pGM798; 3355 Miller units). These results suggest that both the second and the third ATG may contribute to integrase translation in P4. It should be noted that the fusions with the second and third ATG also include the first three codons downstream. It has been shown that not only ATG, but also codons in +2 to +5 positions may alter translation efficiency (Gonzalez de Valdivia & Isaksson, 2004, 2005). Therefore, the constructs pGM796 and pGM798 reflect the translation initiation efficiency of a larger initiation region, and suggest that the third ATG substantially contributes to int translation.
All these plasmids were regulated negatively by the presence of Vis: the level of -galactosidase expression was reduced to 2 %. This effect is stronger than that observed with plasmids carrying a transcriptional fusion with lacZ, in which about 25 % of the activity was still present. Thus, it appears that Vis has a dual negative effect on both transcription and translation.
Vis appears to favour int mRNA processing
The effect of Vis on Pint was analysed further by performing primer-extension experiments in the presence and absence of Vis on RNA extracted from E. coli DH10B/pGZ119EH and DH10B/pGM677 transformed with pGM798, which carries a translational int–lacZ fusion. A strong band at 3947 and weaker downstream signals were present in the absence of Vis (Fig. 5). In the presence of Vis, the signal at 3947 was reduced to about 30 %, but a downstream band at 3890 was intensified. This band was barely visible in the absence of Vis. Thus, Vis appears to intensify a specific 5'-end downstream of the transcription start site. This new 5'-end could be due to Vis-induced mRNA processing or, less likely, to a new transcription initiation point.
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). By increasing the amount of GST-Vis, the RNA fragment was shifted, indicating that Vis is able to bind the transcript. The binding ability essentially depended on Vis, since the effect of GST alone on RNA mobility was barely detectable. The addition of increasing amounts of poly(A) efficiently competed Vis binding. Moreover, we also tested Vis binding to a different RNA fragment (the lacZ RNA) and found a similar binding efficiency for Vis (data not shown).
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) of the Vis polypeptide identified a putative S4 RNA-binding motif in the protein, between residues 28 and 64, as shown in Fig. 6(c). |
DISCUSSION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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). The effect of the negative control was confirmed by monitoring Pint activity in different constructs, carrying either the whole int coding sequence (pGM606) or about 60 % of it (pGM605). In both plasmids, the Pint signal was severely decreased. P4 integrase protein and its N-terminal portion have not been identified, thus nothing is known about the stability of either protein. Nevertheless, our results indicate that the truncated N-terminal portion of P4 integrase is sufficient for repression of Pint. The main products of int transcription are short RNAs that cover the 5'-end of the gene. These transcripts may be produced by processing of longer transcripts and/or by premature transcription termination. In any case, the presence of these short RNAs in all phases of the P4 life-cycle, including the lysogenic condition, indicates that Pint basal level activity is always substantial and thus post-transcription initiation regulatory mechanisms may play a relevant role in control of Pint expression.
P4 Vis is involved in Pint regulation
Our data indicate that Vis, the P4 excisionase (Calì et al., 2004), modulates Int expression both at transcription initiation and post-transcriptional levels. Expression of the lacZ reporter gene in plasmids that carry either a transcriptional or a translational fusion downstream of Pint decreased in the presence of Vis. In transcriptional fusions, the -galactosidase activity was reduced to less than 30 % and the presence of BoxII was sufficient for observing the effect. The Vis-binding site at BoxII overlaps the –35 sequence of Pint and this may account for the negative effect on Pint activity. Further upstream regions do not seem to be implicated in this regulatory mechanism.
When the plasmids carry a translational int–lacZ fusion, Vis had a much greater effect on the level of -galactosidase activity, reducing it to about 2 %. This suggests that Vis is also involved in post-transcriptional regulation of int expression. We demonstrated that Vis binds an int RNA, and found an S4 RNA-binding motif within the protein. Moreover, in the presence of Vis, the abundance of a 5'-end at 3890, within the int coding sequence, is enhanced. It may be suggested that Vis binds int mRNA, preventing int translation and promoting an endonucleolytic cut of int RNA that functionally inactivates the int mRNA. Vis-dependent negative control on int synthesis is likely to be relevant for preventing int expression upon prophage excision and avoid reintegration of P4 in the chromosome.
On the other hand, P4 prophage genome excision requires the presence of both Int and Vis (Calì et al., 2004). A basal level of Int is likely to be continuously expressed, as suggested by repression of transcription from Pint. Expression of Vis from PLL is the first event that occurs upon P4 prophage derepression (Polo et al., 1996). This may provide the amount of Vis required for the formation of the excision complex and the production of a free circular P4 genome. Expression of vis will then be autoregulated by Vis repression of PLL.
In conclusion, we have shown a dual effect of Int and Vis on P4 int expression that might modulate the amount of integrase present in the cell and adapt it to the different conditions in P4 life-cycle.
P4 integrase: one or two proteins?
It appears that two different ATG codons at 3915 and 3858 (pGM796 and pGM798) are used for Int translation. The two open reading frames are 422 and 403 aa long and the two proteins differ by 19 aa at their N-terminal end. Our data suggest that P4 integrase may be expressed in two forms. Whether these proteins have a different functional role in P4 lysogenization was not established. In Tn5, the two proteins differing by 55 aa at the N-terminal end are the transposase and its inhibitor, respectively (de la Cruz et al., 1993; Davies et al., 1999; Reznikoff, 2003; Steiniger-White et al., 2004). Translation of the inhibitor utilizes a distinct initiation site relative to the transposase, and, in vivo, the inhibitor protein is a transdominant negative regulator of transposition and acts presumably by forming heteromultimers with transposase. A similar role could be performed by the two forms of P4 integrase, in which the longer protein may be functional in integration, whereas the shorter could be involved in inhibition of integrase activity.
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ACKNOWLEDGEMENTS |
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Received 27 January 2006;
accepted 3 April 2006.
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