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University of Groningen Characterization of the lytic-lysogenic switch of the lactococcal bacteriophage Tuc2009 Kenny, JG; Leach, S; de la Hoz, AB; Venema, G; Kok, Jan; Fitzgerald, GF; Nauta, A; Alonso, JC; van Sinderen, D; Kenny, John G. Published in: Virology DOI: 10.1016/j.virol.2005.11.041 IMPORTANT NOTE: You are advised to consult the publisher's version (publisher's PDF) if you wish to cite from it. Please check the document version below. Document Version Publisher's PDF, also known as Version of record Publication date: 2006 Link to publication in University of Groningen/UMCG research database Citation for published version (APA): Kenny, J. G., Leach, S., de la Hoz, A. B., Venema, G., Kok, J., Fitzgerald, G. F., ... Alonso, J. C. (2006). Characterization of the lytic-lysogenic switch of the lactococcal bacteriophage Tuc2009. Virology, 347(2), 434-446. DOI: 10.1016/j.virol.2005.11.041 Copyright Other than for strictly personal use, it is not permitted to download or to forward/distribute the text or part of it without the consent of the author(s) and/or copyright holder(s), unless the work is under an open content license (like Creative Commons). Take-down policy If you believe that this document breaches copyright please contact us providing details, and we will remove access to the work immediately and investigate your claim. Downloaded from the University of Groningen/UMCG research database (Pure): http://www.rug.nl/research/portal. For technical reasons the number of authors shown on this cover page is limited to 10 maximum. Download date: 17-06-2017 Virology 347 (2006) 434 – 446 www.elsevier.com/locate/yviro Characterization of the lytic–lysogenic switch of the lactococcal bacteriophage Tuc2009 John G. Kenny a,*,1, Stephen Leach a,1, Ana B. de la Hoz b, Gerard Venema d, Jan Kok d, Gerald F. Fitzgerald a,c, Arjen Nauta d, Juan C. Alonso b, Douwe van Sinderen a,c a Department of Microbiology, National University of Ireland, Cork, Ireland Centro Nacional de Biotecnologia, Campus Universidad Autonoma de Madrid, Spain Alimentary Pharmabiotic Centre, Biosciences Institute, National University of Ireland, Cork, Ireland d Department of Molecular Genetics, University of Groningen, Haren, The Netherlands b c Received 30 September 2005; returned to author for revision 21 November 2005; accepted 28 November 2005 Available online 10 January 2006 Abstract Tuc2009 is a temperate bacteriophage of Lactococcus lactis subsp. cremoris UC509 which encodes a CI- and Cro-type lysogenic – lytic switch region. A helix-swap of the a3 helices of the closely related CI-type proteins from the lactococcal phages r1t and Tuc2009 revealed the crucial elements involved in DNA recognition while also pointing to conserved functional properties of phage CI proteins infecting different hosts. CItype proteins have been shown to bind to specific sequences located in the intergenic switch region, but to date, no detailed binding studies have been performed on lactococcal Cro analogues. Experiments shown here demonstrate alternative binding sites for these two proteins of Tuc2009. CI2009 binds to three inverted repeats, two within the intergenic region and one within the cro 2009 gene. This DNA-binding pattern appears to be conserved among repressors of lactococcal and streptococcal phages. The Cro2009 protein appears to bind to three direct repeats within the intergenic region causing distortion of the bound DNA. D 2005 Elsevier Inc. All rights reserved. Keywords: Phage; CI; Cro; DNA binding; DNA looping Introduction Lactic acid bacteria (LAB) are used as starter cultures in the production of fermented foods such as cheeses, yogurts, and sausages (McGrath et al., 2004). This has catalyzed research aimed at deciphering the processes involved in the multiplication of phages infecting LAB, including the genetic switch that governs the ‘‘decision’’ between the lytic and lysogenic lifecycles of a temperate bacteriophage. k Phage serves as the paradigm for this regulatory control of the lytic – lysogenic switch, yet even in such a well-studied system, theories concerning the mechanism of life-cycle control regularly undergo refinement (Gottesman and Weisberg, 2004; Kobiler et al., 2005; Svenningsen et al., 2005). The k repressor (CI) binds operator sites located within the * Corresponding author. Fax: +353 21 490 3101. E-mail address: [email protected] (J.G. Kenny). 1 Denotes that both authors contributed equally to this work. 0042-6822/$ - see front matter D 2005 Elsevier Inc. All rights reserved. doi:10.1016/j.virol.2005.11.041 intergenic switch region thereby excluding the RNA polymerase from binding to the lytic promoters and thus preventing the establishment of the lytic life-cycle (Revet et al., 1999). CI also performs an autoregulatory function as higher concentrations of the protein repress the activity of the PRM promoter responsible for expression of the cI gene. Cro protein exerts an opposite effect to that caused by CI by attaching to the same operators as CI but with an alternative order of occupancy allowing for the advancement of the lytic life-cycle (Friedman and Court, 2001; Ptashne, 1992). During induction of the phage from the lysogenic host, the cellular SOS response causes RecAmediated autocleavage of CI, thereby depleting levels of the intact protein and allowing the development of the lytic lifecycle (Ptashne, 1992). However, additional regulatory elements including CII, CIII, and Q are involved in the establishment of either life-cycle (Kobiler et al., 2005). The lysogenic phage Tuc2009 is a typical member of the P335 species of the Siphoviridae family of non-contractile tailed bacteriophages that was originally identified as a J.G. Kenny et al. / Virology 347 (2006) 434 – 446 prophage on the genome of Lactococcus lactis subsp. cremoris UC509, a strain used in Cheddar cheese production (Arendt et al., 1994; Proux et al., 2002; Seegers et al., 2004). Across the gamut of phages infecting LAB, various switch mechanisms have been observed. Binding sites for the repressor within the intergenic region have been shown to vary greatly in number, from two in the case of r1t, øLC3, and TP901-1 (Blatny et al., 2001; Johansen et al., 2003; Nauta et al., 1996;), to three for the Lactobacillus casei phage A2 (Garcia et al., 1999), to as many as five for the Lactobacillus plantarum phage øg1e (Kakikawa et al., 2000). A repressor-bound operator positioned several hundred bases away from the intergenic region has been reported for a number of lactococcal and streptococcal phages including r1t, øLC3, ø31, TP901-1, and Sfi21 (Blatny et al., 2001; Bruttin et al., 2002; Durmaz et al., 2002; Johansen et al., 2003; Nauta et al., 1996), while a total of seven repressorbound operators have been detected for øg1e (Kakikawa et al., 2000). In most temperate LAB phages, a CI vs. Cro type system seems to be in operation where these two proteins vie for occupation of overlapping DNA-binding sites. However, exceptions to this system do occur. A protein – protein interaction between the regulatory elements of Sfi21 takes place (Bruttin et al., 2002), while the switch region of the Lactobacillus gasseri temperate phage øadh deviates from the analogous region of other temperate LAB phages due to the presence of convergent promoters (Engel et al., 1998). The lactococcal and streptococcal repressor proteins can be separated into two groups on the basis of their size and homologies (Johansen et al., 2003; Madsen and Hammer, 1998). The larger repressors including those from Tuc2009, øLC3, r1t, and BK5-T show high levels of identity over their C-termini which are believed to be involved in oligomerization of the proteins and possess RecA cleavage sites. These proteins differ over their N-termini which are believed to contact the specifically recognized DNA sequences bound by the repressors (Blatny et al., 2001; Madsen and Hammer, 1998; Boyce et al., 1995; Nauta et al., 1996). The repressors from ø31, a virulent phage which possesses a switch region but lacks a phage attachment site and an integrase, and Sfi21 are comparatively shorter and do not have a RecA cleavage site (Bruttin et al., 2002; Madsen et al., 2001). Interestingly, CI from TP901-1 does undergo oligomerization and despite lacking the RecA proteolysis site TP901-1 is inducible by mitomycin C (Johansen et al., 2003; Madsen et al., 1999). Studies on the homologue of the Tuc2009 repressor (cI 2009 ) in øLC3 (orf286) had identified two repressor-bound imperfect inverted repeats within the intergenic switch region of øLC3. This situation was confirmed here for CI2009 when the repressor was shown to bind two operators within the Tuc2009 intergenic region. In addition, we determined the binding site for CI2009 within the cro 2009 gene. For the first time, we ascertain the binding sites for a lactococcal Cro-type protein and describe the alternative, overlapping binding sites for CI2009 and Cro2009. The amino acids responsible for the DNA sequence recognition by two lactococcal repressors were also identified using a helix-swap approach. These studies suggest that many lactococcal and streptococcal 435 phages contain common elements in the life-cycle regulatory mechanism. Results The sequence binding specificities of CI2009 Comparative sequence analysis had previously identified the protein products of orf4 and orf5 of Tuc2009 (named here as cI 2009 and cro 2009 ) as the CI- and Cro-type regulators of the lysogenic –lytic life-cycles of this temperate phage, respectively (Fig. 1A) (Seegers et al., 2004; van de Guchte et al., 1994). DNase I footprinting experiments for the øLC3 homologue of CI2009, ORF286 (98% identical at the amino acid level), had shown that two DNA regions of the intergenic region from øLC3 were protected by this protein from enzymatic degradation (Blatny et al., 2001). Transcriptional analysis of Tuc2009 (Seegers et al., 2004) and øLC3 (Blatny et al., 2003) found that the repressors of both phages share a transcriptional start site. As expected, our experiments showed an essentially identical pattern of protection for CI2009 (Fig. 2A). We designated the CI2009 bound operators OL over the leftward, lysogenic, promoter and OR over the rightward, lytic, promoter. In contrast to what was reported for ORF286, we failed to show signs of hypersensitivity to DNase I upon binding of CI2009. CI2009 shows a preferential occupancy of OR at lower concentrations of the repressor as indicated by complete protection of this operator at 2.5 pmol of protein when compared to OL which becomes fully protected at 5 pmol of protein. Within the central protected region of OR, an inverted repeat CCACGAAAAGTGG is present. A second copy of this repeat, though imperfect, is also located over the 35 box of the lysogenic promoter, PL, at OL (CCACGTTTTGCAA) (see Fig. 1B). Binding of CI2009 to the inverted repeat at OR is also shown later in Fig. 5A. ORF286 of øLC3 was reported to bind a third operator approximately 500 bp upstream of orf286, although no detailed analysis of this binding region was performed (Blatny et al., 2001). CI2009 was also found by EMSA to bind to a third operator situated several hundred base pairs away from the intergenic switch region within the gene cro 2009 (data not shown). The precise CI2009 recognition sequence within cro 2009 was determined by DNase I footprinting (Fig. 2C). The sequence protected by the CI2009, AAAAGTCCACTAAAAGTGGTTTAGA, contains the same consensus CCACN5GTGG (Fig. 1D) inverted repeat found at OR. This operator was assigned the name OD given its distant location from the intergenic region (Fig. 1C). Within orf76, the cro-like gene of øLC3, the corresponding sequence reads AAAAGTCCACgAAAAGTGGTTTAGA which, with the exception of the lower case g, is identical to OD in Tuc2009. The sequence binding specificities of Cro2009 ORF76 of øLC3 was previously shown to bind the intergenic switch region of øLC3 (Blatny et al., 2003), while EMSA confirmed that Cro2009, which is 100% identical to 436 J.G. Kenny et al. / Virology 347 (2006) 434 – 446 Fig. 1. (A) Schematic representation of the area encoding the genetic switch region, including cI 2009 and cro 2009 , of Tuc2009. (B) Sequence of intergenic switch region from Tuc2009. The mRNA transcriptional start site for the cI 2009 gene is denoted by a right angle arrow. (C) Sequence of the CI2009-bound region from within cro 2009 . For both panels B and C, areas of protection against DNase I activity afforded by CI2009 and Cro2009 binding are denoted by black or open bars, respectively. The inverted repeats recognized by CI2009 are indicated by convergent arrows, while vertical arrows signify sites hypersensitive to DNase I activity upon Cro2009 binding. 10 and 35 consensus sequences are boxed. The location of the G to T mutation in Tuc2009-mut is signified by an asterisk above the wild-type base. A shaded block arrow indicates the 3Vend of the PCR product used in lanes 1 and 2 of Fig. 3A. (D) Alignment of the half sites of the inverted repeats bound by CI2009 and the resultant consensus sequence. ORF76 at the amino acid level, binds the switch region from Tuc2009 (data not shown). In order to determine the sequences recognized by Cro2009 DNase I protection assays were performed on the DNA encoding the intergenic region in the presence of Cro2009 (Fig. 2D). Two protected regions were observed, which are overlapping but not identical to those exhibited upon repressor binding, indicating that Cro2009 recognizes a sequence that differs from that bound by CI2009 (Fig. 1B). In addition, a number of hypersensitivity sites were apparent upon Cro2009 binding, an indicator of DNA bending (Schleif, 1992). We encountered the same Cro instability to freezing and thawing as reported by Blatny et al. (2003) for ORF76 of øLC3. Blatny et al. (2003) were unable to show specific binding sites for ORF76 by DNase I footprinting, possibly due to this protein instability, which may also account for our inability to discern an order of occupancy for the Cro2009-bound direct repeats. The rightward region protected by Cro2009 (designated ORCro) was noticeably 17 bp longer than the Cro2009-protected leftward region (named OLCro), and this area of extended protection spread between the divergent promoters (Fig. 1B). Careful inspection of the ORCro region revealed two 13 bp direct repeats. Interestingly, within OLCro, a 13-bp sequence was observed that is similar to the two direct repeats located within ORCro (Fig. 3A). The binding by Cro2009 to the two direct repeats within ORCro and the single repeat within OLCro would explain the 17-bp difference in length between the two protected regions observed during footprinting experiments. Another compelling finding which substantiates the notion that CI2009 and Cro2009 do have alternative binding specificities was obtained through an EMSA in which two 100-bp fragments of DNA encompassing OD or containing OL and OLCro were incubated in the presence or absence of 100 pmol of the Cro2009 protein. Cro2009 was shown to bind the DNA containing OLCro but not the DNA which encompasses OD (Fig. 3B). In contrast, CI2009 was found to bind both the OD and the OL containing DNA fragments (data not shown). DNA-binding specificities of the mutated repressor proteins CI2009 and its functional analogue of the lactococcal phage r1t, Rro, show over 95% similarity over their C-terminal regions but much less so over their 80 N-termini amino acids which in the case of Rro were predicted to encompass the DNA-binding helix– turn – helix domains (Nauta et al., 1996). The predicted a3 helices of such phage repressors are believed to contact the DNA and recognize the specific operator regions, thereby allowing control of transcription from the lytic and lysogenic promoters (Ptashne, 1992). In an effort to establish whether this a3 helix had been correctly allocated for these proteins a helix-swap experiment was undertaken of the type performed previously for DNA-binding proteins of phages infecting Gram-negative bacteria (Wharton et al., 1984; Wharton and Ptashne, 1985). A mutated version of CI2009 J.G. Kenny et al. / Virology 347 (2006) 434 – 446 437 Fig. 2. DNase I footprinting assays using 0, 2.5 and 5 pmol of CI2009 and CIN:Rro, or 0, 20, 40, and 60 pmol of Cro2009. The experiments shown in panels A, B, and D were performed using a 227 bp g32P-labeled upper strand of the DNA encoding the intergenic region (generated by PCR using the primer pair FP-F and FP-R) bound to CI2009, CIN:Rro, or Cro2009, respectively. Panel C was performed using a g32P-labeled 100 bp PCR fragment (generated using the primer pair EMSA/FPODF and EMSA/FP-ODR) from within the cro 2009 gene and incubated with CI2009. Regions of protection are indicated by black bars, while hypersensitivity sites are assigned black arrows. A band due to double-stranded DNA of the intergenic region is indicated by an open arrow. called CIMutH3 contains six amino acid substitutions in the assumed turn and a3 helix of CI2009, corresponding to six amino acids in the predicted turn and a3 helix of Rro (see Fig. 4A). In EMSA experiments involving lysates of Escherichia coli cells overproducing these proteins, the introduced amino acid substitutions were shown to confer the DNA recognition specificities of Rro to CIMutH3 (Fig. 4B). Interestingly, when the extreme 5V portion of cI 2009 , encoding the 80 N-terminal amino acids of CI2009, was translationally fused to the entire rro gene (cIN:Rro), it was shown that the resultant protein was capable of binding to the lysogenic –lytic intergenic regions of both Tuc2009 and r1t (Fig. 2B). Furthermore, CI2009 and CIN:Rro generated an identical protection pattern in a DNase I footprinting assay, indicating that the presence of the extra amino acids specifying the r1t repressor does not appear to affect the binding of the protein to the Tuc2009 intergenic switch region (Figs. 2A and B). CI2009 and Rro were only able to bind DNA derived from the switch region of their respective phages (Fig. 4B). To ensure the functional relevance of the EMSAs, in vivo repressor-mediated super-infection immunity assays against Tuc2009 and r1t were performed in their respective hosts. The 438 J.G. Kenny et al. / Virology 347 (2006) 434 – 446 Fig. 3. (A) Alignment of the three direct repeats located within OLCro and ORCro. The positions of the sequences within the Tuc2009 genome are indicated beside each repeat. Residues shared by at least two of the direct repeats are highlighted by black boxes. (B) EMSA using radio-labeled probes encoding OL and OLCro (lanes 1 and 2) or OD (lanes 3 and 4) (generated by PCR using the primer pairs EMSA-OLF and EMSA-OLR, and EMSA/FP-ODF and EMSA/FP-ODR, respectively). Lanes 1 and 3 are negative controls without protein. Lanes 2 and 4 contain 100 pmol of purified Cro2009. different repressor genes were cloned into the hosts in the expression vector pNZ44 to allow constitutive protein expression in L. lactis (McGrath et al., 2001). The results obtained concurred with the binding assays, since cells expressing CI2009 conferred resistance against Tuc2009 only. Cells which expressed Rro or CIMutH3 were only resistant to r1t infection, while those carrying the cIN:Rro gene were resistant to both phages (Fig. 4C). The repressor- and Cro-type proteins of Tuc2009 and r1t show conserved DNA-binding patterns To confirm the binding sites for CI2009 shown by footprinting assays, additional gel-shift experiments were performed using short synthetic pieces of DNA produced by hybridizing complementary oligonucleotides encoding the repressor-bound operator sites OR of Tuc2009 and O1 of r1t (Fig. 5A). As Fig. 4. (A) Relevant protein sequences of the N-terminal sections of CI2009, Rro, and CIMutH3 with the changed amino acid residues boxed and their location within the secondary structure of the DNA-binding domains indicated. (B) EMSA using g32P-labeled 571-bp (generated by using the primer pair EMSA-TucF and EMSA-TucR) and 617-bp DNA fragments (primer pair EMSA-r1Tf and EMSA-r1tR) encoding the lysogenic – lytic switch regions of Tuc2009 or r1t, respectively, and incubated with 1 Ag of protein from the E. coli lysates containing the corresponding overexpressed protein. The control lysate was from M15 cells carrying the pQE60 plasmid without any insert and induced with IPTG. (C) Table showing the results of super-infection immunity assays of repressor against Tuc2009 and r1t. + Indicates that the host containing the corresponding plasmid showed an EOP of 10 8 when challenged with the infecting phage. Similarly, indicates an EOP of 1. J.G. Kenny et al. / Virology 347 (2006) 434 – 446 439 designated Tuc2009-mut, that showed an EOP of ¨10 5. This phage is a thousand-fold less sensitive to the presence of CI2009 than its wild-type antecedent which showed an EOP of ¨10 8. Such lower sensitivity to super-infection immunity has previously been shown to be the phenotypic manifestation of altered DNA sequence in the operators recognized by the phage repressor proteins (Durmaz et al., 2002). Subsequent sequencing of the switch region of Tuc2009 mapped this mutation to a G to T substitution within OL (see Fig. 1B). In order to determine if this mutation did indeed affect repressor binding properties, DNA fragments encoding the switch region of wildtype and Tuc2009-mut were amplified, and their binding characteristics to CI2009 and Cro2009 were determined by EMSA (Fig. 5B). The G to T substitution destroyed the only consensus base pair in that half of the repressor-bound inverted repeat (CCACN5GCAA to CCACN5TCAA) and reduced the affinity of CI2009 for OL. As predicted by the footprinting and the EMSA with synthetic DNA, binding to the switch region by Cro2009 did not seem to be adversely affected by this mutation, as it was outside the direct repeat recognized by that protein. Examination of the PL and PR promoter activities Fig. 5. (A) EMSA using 24 bp- or 21-bp segments of synthetically produced DNA encoding the repressor recognition sequences for Tuc2009 and r1t, respectively. These DNA segments were created by annealing the complementary oligonucleotide pairs Tuc-F-OR and Tuc-R-OR, or r1t-Oa and r1t-Ob. Proteins included in incubations are as indicated. All binding reactions, with the exception of the negative control, involved 50 pmol of purified protein. (B) EMSA experiments to determine the effect of the G – T change in Tuc2009-mut on CI2009 and Cro2009 binding. To the left in each gel is the DNA fragment derived from Tuc2009, to the right, the fragment from Tuc2009-mut. The upper gel shows binding by CI2009, the lower binding by Cro2009. Purified protein was added in increments of 1, 2.5, 5, and 10 pmol for CI2009 and 5, 10, 20, 30, and 40 pmol for Cro2009. The negative control lanes (Con) do not contain any protein. expected, the pattern of repressor binding to Tuc2009 and/or r1t DNA was the same as that shown in Fig. 4B, confirming that the amino acid substitutions and additions introduced into CIMutH3 and CIN:Rro specifically change their DNA recognition abilities. However, neither Cro2009 nor the topological equivalent of k Cro in r1t known as Tec was capable of binding those regions of DNA recognized by their corresponding repressors. This is despite the fact that the OR of Tuc2009 used contains half of the direct repeat depicted in Fig. 3A. These findings agree with those of the EMSA and DNase I footprinting experiments described above which had indicated that CI2009 and Cro2009 bind to different DNA sequences within the intergenic region of Tuc2009. A single base pair substitution within the CI2009 and Cro2009 protected sites inhibits binding of CI2009 During the course of the analysis of the CI2009-mediated super-infection immunity, we isolated a variant of Tuc2009, To examine the roles of CI2009 and Cro2009 in the control of PL and PR activity, four different promoter probe constructs were made. The general design of the constructs is schematically displayed in Fig. 6A. These were transformed into L. lactis NZ9000 in the presence of pNZ8048 with or without the cI 2009 gene. The nisin-inducible promoter present on pNZ8048 allows an increase in promoter activity with increasing nisin concentrations, thus allowing nisin-controlled expression of CI2009 (de Ruyter et al., 1997; Kuipers et al., 1995). To exclude the possibility of clonal variation of the type seen for TP901-1 (Madsen et al., 1999), five randomly chosen clones of each of the pPTPL constructs were grown overnight with antibiotic but without nisin and assayed for h-galactosidase activity. These clones did not show any significant differences in Miller units (data not shown). Background levels for pPTPL without any insert are 0.5 T 0.4 Miller Units. Control experiments involving strains containing the pPTPL constructs with pNZ8048 lacking the cI 2009 gene with and without nisin did not show any significant difference in h-galactosidase activity to those containing pNZ804-CI2009 in the absence of nisin (data not shown). For strains containing the pNZ804-CI2009 plasmid, a stepwise reduction in the activity of both PL and PR promoters was observed upon the addition of increasing concentrations of nisin. Levels of h-galactosidase activity were the same at 1 or 10 ng/ml of nisin and only slightly higher at 0.5 ng/ml (data not shown). For all four pPTPL constructs, CI2009 downregulates the activity of the promoters (Fig. 6B). This is consistent with the binding results of the footprinting experiments which showed that the repressor binds to operators located over each promoter. Interestingly, in the absence of nisin, there is over a 30-fold difference in activity between the pMut-croPL and pWTPL constructs, indicating that Cro2009 strongly represses transcrip- 440 J.G. Kenny et al. / Virology 347 (2006) 434 – 446 Fig. 6. (A) Schematic representation of the pPTPL constructs used. The arrows indicate the orfs corresponding to cI 2009 and cro 2009 . The black bars below these arrows represent the DNA fragments which were cloned into the pPTPL vector, in either orientation, to create the four constructs studied. A vertical line in the Mut cro line indicates the position of two stop codons in the cro 2009 gene. (B) Results of the h-galactosidase assays employing the four constructs depicted in panel A. For each construct, the figures shown correspond to the Miller units recorded without nisin added and with nisin added to a final concentration of 1 ng/ml. The fold reduction for each construct upon the addition of nisin is also displayed as are the standard deviations for each reading. tion from the lysogenic promoter. This is a much stronger repression than that achieved by overexpressed CI2009 against either PL or PR. The same is not true of the lytic promoter, PR, where a slight upregulation of the promoter was observed in the presence of a functional cro 2009 gene. Interestingly, two of the Cro2009-bound direct repeats are located on or beside the PR promoter. Also, results shown here indicate that pWTPR shows a greater than 40- or 80-fold higher activity than pWTPL in the absence or presence of overexpressed CI2009, respectively. This is indicative of a regulatory system favoring a high level of downregulation of the lysogenic life-cycle promoter, PL, by Cro2009 and a corresponding progression of the lytic life-cycle. Discussion This study analyzed the DNA-binding properties of CI2009 and Cro2009 from Tuc2009 and Rro from r1t. To our knowledge, no structure – function analyses had previously been reported in phages infecting Gram-positive hosts regarding the repressor N-terminal helix –turn – helix structure. The six amino acids exchanged between Rro and CI2009 were shown to be responsible for DNA recognition since CIMutH3 only binds DNA encompassing the intergenic switch region of r1t and not that of Tuc2009. Similarly, the N-terminus of CI2009 was shown to dictate DNA binding as the fusion protein, CIN:Rro, bound sequences from both r1t and Tuc2009. This pattern of binding by these repressors was confirmed in vivo by super-infection immunity assays. DNase I footprinting allowed the identification of two binding sites for CI2009 and Cro2009 within the intergenic region at the lysogenic – lytic switch. The CI2009-bound operators at PL and PR (OL and OR) cover the predicted 35, and 35 and 10 boxes of these promoters, respectively. The Cro2009-bound operators (OLCro and ORCro) overlap the 10, and 35 boxes of PL and PR, respectively. The OL and OR both contain an homologous inverted repeat which we propose to be the recognition sequence for CI2009. EMSA and DNase I footprinting experiments denoted that a third operator (OD) is also bound by CI2009 and is located within the gene encoding cro 2009 . OD also contains our proposed CI2009-specific inverted repeat (CCACN5GTGG) which is closely related to the CGTGGTT sequence reported to be recognized by the highly homologous ORF286 of øLC3 (Blatny et al., 2001). This work showed different binding affinities by CI2009 for OL and OR (OR > OL). This order of affinity is in accordance with the level of conservation of the CI2009-recognized inverted repeat (CCACN5GTGG) as described here, and differences in the sequences between operators have been assigned as an important factor in affinity discrimination (Brennan et al., 1990). Given the agreement of OD with the consensus sequence, one would expect CI2009 to occupy both OR and OD in preference to occupying OL. This pattern of repressor – operator affinities coincides with that seen for r1t, ø31, and Sfi21 (Bruttin et al., 2002; Durmaz et al., 2002; Nauta et al., 1996). Interestingly, for the repressors of ø31 and Sfi21, no binding was reported to the predicted operator sites which were located at the lysogenic promoter (Bruttin et al., 2002; Durmaz et al., 2002). This is the first time that binding by a lactococcal Cro protein has been investigated by footprinting assays. The length of the protected DNA segment afforded by Cro2009 binding to the ORCro region as compared to OLCro is striking and, in conjunction with the direct repeats shown in Fig. 3A, promotes the idea of two Cro2009-bound direct repeats within ORCro and one Cro2009-bound repeat within OLCro. In addition, the observed hypersensitivity pattern is indicative of distortion of the DNA by Cro2009. The overlapping but non-identical binding sites for CI2009 and Cro2009 were confirmed by various EMSAs. These results agree with DNA-binding studies for ORF286 and ORF76 of øLC3 where these two proteins were shown by EMSA to compete for the same PCR product (Blatny et al., 2003). J.G. Kenny et al. / Virology 347 (2006) 434 – 446 Transcriptional studies described here show that CI2009 downregulates both the PL and the PR promoters of Tuc2009 and agree with similar studies performed in other lactococcal phages TP901-1 and ø31 (Johansen et al., 2003; Madsen et al., 2001). This is largely similar to the findings of Blatny et al. (2003) for studies performed on the highly homologous øLC3 switch region. They did find, however, that in constructs possessing the orf76 gene, the repressor actually caused a 10-fold increase in the activity of the lysogenic promoter. The same studies also found that while ORF76 downregulated the lysogenic promoter by almost 14-fold, it also downregulated the lytic promoter by 5-fold, which conflicts with the results found here where Cro2009 slightly upregulated the lytic promoter. The ability of a Cro-like protein to upregulate a promoter is not without precedent since Mor of TP901-1 was shown to upregulate the activity the lysogenic promoter of TP901-1 (Madsen et al., 1999). However, for ø31, in the absence of the repressor, the lytic and lysogenic promoters were downregulated by the Cro protein by more than 55- and 400-fold, respectively (Madsen et al., 2001). It should be noted that the constructs used in previous studies that did not contain the relative cro genes also lacked the repressor-bound operators located at the 3Vend of those cro genes. In this study, we introduced two stop codons into the cro 2009 gene allowing us to monitor the promoter activities in the presence of all three repressorbound operators. Under the conditions used here, the balance of promoter activity for Tuc2009 favors the progression of the majority of the infecting phages into a lytic life-cycle. The different results from promoter studies described above from different investigations highlight the overall complexity of the problem facing us in deciphering the switch mechanism of phages infecting Gram-positive bacteria. Promoter studies on the switch region of TP901-1 have demonstrated a clonal variability, thereby indicating a role of other phage factors in life-cycle control (Madsen et al., 1999). Although no clonal variability was observed here, we did not have both the repressor and cro-like genes encoded on a single promoter 441 probe construct, and both of these factors were shown to be required for clonal variability in TP901-1 (Madsen et al., 1999). The orfs downstream of the cro-like genes in Tuc2009, TP-J34, and ø31 have been identified as putative antirepressors which may have a role in the regulation of the switch region (Madsen et al., 2001; Neve et al., 1998; Seegers et al., 2004). Analysis of other lactococcal and streptococcal phages shows a conserved distribution of three operator sites though not a conserved regulatory mechanism (Fig. 7) (Blatny et al., 2001; Bruttin et al., 2002; Durmaz et al., 2002; Johansen et al., 2003; Nauta et al., 1996). In the case of BK5-T, three operator sites were originally assigned to within the switch intergenic region by analogy with k (Boyce et al., 1995). A slight alteration of the inverted repeat for putative repressor binding to ACCGAN6TCGGT (where N6 is composed of A and T residues) changes the distribution of three operators to one occupying each promoter region and one at the 3Vend of the crolike gene. Precisely what function the OD operator serves has yet to be elucidated. It may act as a road-block to transcription of the cro gene or, alternatively, it may allow looping of the DNA through interactions between the bound repressors. While the idea of DNA looping as a regulatory mechanism in Tuc2009 remains a hypothetical model, it is known to occur in phages infecting Gram-negative bacteria (Revet et al., 1999; Dodd et al., 2001; Dodd and Egan, 2002). Studies are ongoing to show the role of OD in the regulation of the lysogenic –lytic switch of Tuc2009 and how this fits into the process of control over the lysogenic –lytic switch in Tuc2009. Materials and methods Bacterial strains, plasmids, bacteriophages, and growth media Bacteriophages, bacterial strains, and plasmids used in this study are listed in Table 1. L. lactis strains were grown in GM17 (Oxoid) broth or agar (1.4%) supplemented with 0.5% glucose at 30 -C, while E. coli strains were cultivated in LuriaBertani broth or agar (1.4%) at 37 -C (Sambrook et al., 1989). Fig. 7. Diagram showing the lysogenic – lytic switch region of a number of lactococcal and streptococcal phages where the orfs are depicted as block arrows, the operators that agree with the consensus for repressor binding as dark boxes, and the promoters as right angled arrows. Details on these operator consensus sequences (with the exception of BK5-T) can be found in Blatny et al. (2001), Bruttin et al. (2002), Durmaz et al. (2002), Johansen et al. (2003), Nauta et al. (1996). 442 J.G. Kenny et al. / Virology 347 (2006) 434 – 446 Table 1 Bacteriophages, bacterial strains, and plasmids used in this study Phage, strain, or plasmid Phage Tuc2009 Tuc2009-mut r1t E. coli M15 EC101 EC1000 L. lactis UC509.9 R1K10 NZ9000 Plasmids pUK21 Relevant feature Isolated from L. lactis subsp. cremoris UC509 Derivative of Tuc2009 with a G to T change at 2813 Isolated from L. lactis subsp. cremoris R1 Costello (1988) Host for pQE30 and pQE60, contains pREP4, KanR JM101 with chromosomally encoded repA Kmr; MC1000 derivative, carrying a single copy of pWV01 repA in glgB Qiagen Prophage cured derivative UC509, host for Tuc2009 Prophage cured derivative of R1, host for r1t MG1363 pepN::nisRK; wild-type strain Cloning vector, KmR pQE30 pQE60 pNZ44 E. coli expression vector, AmpR E. coli expression vector, AmpR L. lactis expression vector, CmR pNZ8048 L. lactis expression vector, CmR pPTPL Promoter-screening vector containing promoterless lacZ gene, TcR, pPTP derivative pUK21 derivative containing cIMutH3 pQE30 derivative containing cI 2009 pQE60 derivative containing cro 2009 pQE30 derivative containing rro pQE30 derivative containing tec pQE30 derivative containing cIMutH3 pQE30 derivative containing cIN:rro pNZ44 derivative containing cI 2009 pNZ44 derivative containing rro pNZ44 derivative containing cIMutH3 pNZ44 derivative containing cIN:rro pNZ8048 derivative containing cI 2009 pPTPL containing the Tuc2009 switch region 2733 – 3303 bp including cro 2009 PR-lacZ pUK21-CIMutH3 pQE-CI2009 pQE-Cro2009 pQE-Rro pQE-Tec pQE-CIMutH3 pQE-CIN:Rro pNZ44-CI2009 pNZ44-Rro pNZ44-CIMutH3 pNZ44-CIN:Rro pNZ8048-CI2009 pWTPL Source or reference This study Table 1 (continued ) Phage, strain, or plasmid Plasmids pMut-croPL pWTPR Lowrie (1974) pMut-croPR Relevant feature Source or reference pPTPL containing the Tuc2009 switch region 2733 – 3303 bp with stop codons in cro 2009 PL-lacZ pPTPL containing the Tuc2009 switch region 2733 – 3303 bp including cro 2009 PR-lacZ pPTPL containing the Tuc2009 switch region 2733 – 3303 bp with stop codons in cro 2009 PR-lacZ This study This study This study Law et al. (1995) Leenhouts et al. (1996) Costello (1988) Lowrie (1974) Kuipers et al. (1998) Vieira and Messing (1991) Qiagen Qiagen McGrath et al. (2001) de Ruyter et al. (1997) O’Driscoll et al. (2004) This study This study This study Bacteriophage Tuc2009 was propagated on L. lactis subsp. cremoris UC509.9. E. coli M15 cells containing pQE60, pQE30, or derivatives thereof were grown in the presence of 100 Ag ml 1 ampicillin and 25 Ag ml 1 kanamycin. pNZ44 and pNZ8048, or their derivatives, were maintained in cells of L. lactis using chloramphenicol at a concentration of 10 Ag ml 1, while pPTPL-based constructs were similarly maintained using tetracycline at a concentration of 5 Ag ml 1. Phages were purified using CsCl density gradient centrifugation (Sambrook et al., 1989). Plaque assays were performed as described by Lillehaug with the inclusion of chloramphenicol in the media at a final concentration of 5 Ag ml 1 (Lillehaug, 1997). Sequence analysis Database searches and pfam allocations were performed using BLASTN and BLASTP (Altschul et al., 1997) and conserved domain search programs, respectively, located at the following URL (http://www.ncbi.nlm.nih.gov/). Sequence alignments were performed using the clustal alignment method of MEGALIGN 3.16 software from the DNASTAR 2002 Version 5 software package. DNA manipulations and sequencing This study This study This study This study This study This study This study This study This study This study PCR amplifications were carried out using EXPAND long template PCR system (Roche) according to the manufacturer’s instructions with a Gene Amp PCR system 2400 thermal cycler (Perkin-Elmer). Oligonucleotides were manufactured by MWG (Ebersberg, Germany) with L. lactis subsp. cremoris UC509 and L. lactis subsp. cremoris R1 DNA as templates for PCR. Restriction enzymes, shrimp alkaline phosphatase, T4 DNA ligase, and DNase I were supplied by Roche and employed as recommended by the manufacturer. Electrotransformation of plasmid DNA into E. coli was performed as described by Sambrook et al. (1989), while that of L. lactis was performed as described by Wells et al. (1993). All DNA cloning steps were performed using E. coli as the host. The integrity of clones was checked by restriction profiling and DNA sequencing. Plasmid purifications from E. coli were performed using the Wizard Plus SV miniprep kit (Promega). Plasmid DNA preparations from L. lactis were completed using the protocol of O’Sullivan and Klaenhammer J.G. Kenny et al. / Virology 347 (2006) 434 – 446 (1993). Sequence analysis was performed by MWG (Ebersberg, Germany). 443 Table 2 Oligonucleotides used in the construction of plasmids and to generate fragments of DNA by PCR for use in GEMSA or footprinting studies Plasmid constructions DNA encoding the cI 2009 , cro 2009 , rro, and tec genes were amplified by PCR using appropriate primers (Table 2). The cI 2009 , rro, and tec genes were cloned into the expression vector pQE30 using BamHI and HindIII sites, while cro 2009 was cloned into pQE60 using the restriction enzymes NcoI and BglII. pQE30 and pQE60 permit the overexpression of proteins fused to a 6 His tag at the N- or C-termini, respectively. To produce pQE-CIMutH3, the primers Hs1 and Hs2 were designed to introduce changes into the coding region of cI 2009 to effect substitution of 6 amino acids in the N-terminus of the resultant protein as compared to the wild type (Fig. 3A). Primers Hs1 and QE-cIF were used to amplify a 140-bp region encompassing the extreme 5V end of the cI 2009 gene prior to digestion with EcoRV and BamHI and subsequent cloning into similarly restricted pUK21 (Vieira and Messing, 1991). The resultant plasmid was digested with HindIII and EcoRV, in order to insert the PCR product that encodes the 3Vportion of the cI 2009 gene, obtained by PCR using the hs2 and QE-cI/rroR oligonucleotides. The resultant plasmid, pUK21-CIMutH3, was restricted with BamHI and HindIII, and the fragment corresponding to the redesigned version of cI 2009 , designated cIMutH3, was inserted into the expression vector pQE30, creating pQE30-CIMutH3. To produce a fusion protein containing the N-terminus of CI2009 and the intact Rro, the 5Vportion of cI 2009 was amplified using appropriately designed primers containing BamHI sites. The resulting PCR product was cloned into the unique BamHI site of pQE-Rro generating the plasmid pQE-CIN:Rro in an orientation that causes the first 80 codons of the cI 2009 gene to be translationally fused to the first codon of the rro gene. The resulting protein was named CIN:Rro. To generate pNZ44 derivatives, oligonucleotides, one containing a PstI site, the other an XbaI site, were used to amplify the cI 2009 , rro, cIN:Rro, and cIMutH3 genes from their pQE30 derivatives (see description above). These PCRgenerated fragments were then digested with the two restriction enzymes and ligated into similarly restricted pNZ44. To construct a pNZ8048 derivative expressing cI 2009 under the control of the nisin-inducible promoter, an oligonucleotide pair, one containing an NcoI site, the other an XbaI site, was used to amplify cI 2009 which was then digested and inserted into pNZ8048, generating plasmid pNZ8048-CI2009. To produce transcriptional fusions to a promoterless lacZ, the following pPTPL derivatives were constructed. The primer pairs PL-switch-F and PL-switch-R, and PR-switch-F and PRswitch-R (see Table 2) were used to amplify the DNA from Tuc2009 encompassing the complete intergenic switch region and cro 2009 gene. Cloning of the cro 2009 gene lacking the first 15 bp was performed using DNA produced by PCR employing the primer pairs Mutcro-F and PL-switch-R, or Mutcro-F and PR-switch-R, respectively, between SalI and BglII sites or SalI and BamHI sites, depending on the desired orientation within BamHI and HindIII sites are underlined once and twice, respectively. BglII sites are in curly brackets { }, EcoRV sites in double-pointed brackets ? X, NcoI sites in square brackets [ ], PstI sites in quotation marks ‘‘ ’’, SalI sites in single pointed brackets < >, and XbaI sites in parentheses ( ). 444 J.G. Kenny et al. / Virology 347 (2006) 434 – 446 the plasmid. Subsequently, the intergenic region together with the first 15 bp from cro 2009 was amplified and cloned between the BamHI and SalI sites using the primers PL-switch-F and Mutcro-R, or the BglII and SalI sites using the primers PRswitch-F and Mutcro-R. Mutcro-R introduces two stop codons into the cro 2009 gene by changing GAGTTA to TAGTAA. This generates lacZ fusions with the Tuc2009 switch containing an intact OD without the presence of a functional cro 2009 . These constructs were transformed into E. coli EC1000 and subsequently transferred to L. lactis NZ9000 cells containing pNZ8048 or pNZ8048-CI2009. The pPTPL plasmid contains stop codons in all three reading frames after the cloning site to eliminate translational inhibition. Protein expression and purification Overexpression and purification of target proteins were achieved using the E. coli expression plasmids pQE30 and pQE60 as described in Kenny et al. (2004). The purified proteins, all of which were of the expected size, were determined to be >95% pure by SDS-PAGE (data not shown) (Laemmli, 1970). Following purification, the proteins were dialyzed against a buffer containing 50 mM Tris –HCl, pH 7.5, 50 mM NaCl, 10 mM MgCl2, 20% glycerol overnight at 4 -C before being aliquoted and stored at 80 -C for no longer than 3 months. Each aliquot of purified protein was thawed once and subsequently discarded. Protein concentrations were determined using the Bio-Rad protein assay in conjunction with a bovine serum albumin standard curve. Gel retardation assays The oligonucleotide pairs used to produce the PCR-derived DNA fragments for these assays are denoted in Table 2. Oligonucleotides were end labeled using g32P-ATP and T4 polynucleotide kinase (PNK) (New England Biolabs) and PCRs performed to produce DNA fragments incorporating the lysogenic – lytic switch regions of Tuc2009, Tuc2009-mut, and r1t. Alternatively, PCR fragments encompassing individual operator regions were generated, which were subsequently labeled using T4 PNK and g32P-ATP. The PCR fragments were purified using the Jet Quick PCR purification kit (Genomed) and the level of radioactive labeling measured using the Beckman LS6500 multi-purpose scintillation counter. Where synthetic DNA fragments were required, 10 pmol of labeled oligonucleotide was annealed to 20 pmol of its non-radioactive complementary strand by heating at 90 -C for 5 min in HIN buffer (6 mM Tris – HCl, pH 7.5, 6 mM MgCl, 50 mM NaCl, 1 mM DTT) followed by cooling to room temperature overnight. Binding reactions were performed in a binding buffer of 50 mM Tris – HCl, pH 7.5, 50 mM NaCl, 10 mM MgCl2 in final volumes of 20 Al containing the labeled probe and appropriate concentrations of protein in the presence of 1 Ag of poly (dIdC) or 1 Ag of calf thymus DNA. Following incubation at room temperature for 30 min, samples were run on non-denaturing 4% polyacrylamide gels in 0.5 TBE and then dried. Bands were visualized by autoradiography at 70 -C using Kodak Biomax MR Film and intensifying screens. DNase I footprinting assays Individually labeled DNA fragments covering the lysogenic – lytic region of Tuc2009 were amplified by PCR using combinations of a 5Vradioactively labeled oligonucleotide and a secondary unlabeled oligonucleotide. Binding reactions were performed as described above for the EMSA. The DNase I footprinting assay was performed essentially as described by Ladero et al. (1999). Following the binding reactions, 1 Al of a concentration of DNase I was added which cleaved on average one bond per DNA fragment. This digestion was stopped after 5 min at 37 -C by the addition of 0.5 Al of 0.5 M EDTA, pH 8. The DNA was precipitated, resuspended in loading buffer, and subjected to electrophoresis on a denaturing 6% polyacrylamide gel in TBE buffer, dried and autoradiographed as described above. G + A sequencing reactions were performed as described by the Suretrack Footprinting Kit (Pharmacia). b-Galactosidase assays L. lactis NZ9000 cells harboring pPTPL derivatives were inoculated into GM17 containing appropriate antibiotics with 2% of an overnight culture. Cells were grown to an OD600 of between 0.2 and 0.3, and nisin, when included, was then added at a final concentration of 0.1, 0.2, 0.5, 1, and 10 ng/ml to cells harboring the pNZ8048-CI2009 plasmid. Control experiments for each promoter construct were also performed where nisin, when included, was added to a final concentration of 10 ng/ml to strains containing the pPTPL constructs and the pNZ8048 plasmid lacking a cI 2009 gene. 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