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RESEARCH LETTER Two DNA sites for MelR in the same orientation are sufficient for optimal MelR-dependent repression at the Escherichia coli melR promoter Mohamed S. Elrobh1,2, Christine L. Webster3, Shivanthi Samarasinghe3, Danielle Durose3 & Stephen J.W. Busby3 1 Biochemistry Department, College of Science, King Saud University, Riyadh, Saudi Arabia; 2Biochemistry Department, Faculty of Science, Ain Shams University, Cairo, Egypt; and 3School of Biosciences, University of Birmingham, Birmingham, UK Correspondence: Stephen Busby, School of Biosciences, University of Birmingham, Edgbaston, Birmingham B15 2TT, UK. Tel.: 44 121 414 5439; fax: 44 121 414 5925; e-mail: [email protected] Received 31 July 2012; revised 3 October 2012; accepted 4 October 2012. Final version published online 8 November 2012. DOI: 10.1111/1574-6968.12027 Abstract The Escherichia coli melR gene encodes the MelR transcription factor that controls melibiose utilization. Expression of melR is autoregulated by MelR, which represses the melR promoter by binding to a target that overlaps the transcript start. Here, we show that MelR-dependent repression of the melR promoter can be enhanced by the presence of a second single DNA site for MelR located up to 250 base pairs upstream. Parallels with AraC-dependent repression at the araC–araBAD regulatory region and the possibility of the MelR-dependent repression loop formation are discussed. The results show that MelR bound at two distal loci can cooperate together in transcriptional repression. MICROBIOLOGY LETTERS Editor: Olga Ozoline Keywords Escherichia coli; melibiose operon regulatory region; promoters; DNA sites for MelR; repression; loop formation. Introduction The activity of many bacterial promoters is controlled by transcription repressors, and many cases have now been described where efficient repression requires interaction between repressors bound at two separated DNA targets, resulting in looping of the intervening DNA (Browning & Busby, 2004). One of the first cases to be described was repression by the Escherichia coli AraC protein at the araC-araBAD intergenic regulatory region, which requires AraC binding to two target sites, I1 and O2, separated by 210 base pairs (reviewed by Schleif, 2010). In previous work, we have studied the interactions of MelR at the E. coli melibiose operon regulatory region (Wade et al., 2000, 2001). MelR is a member of the AraC family of transcription factors and is essential for melibiose-dependent triggering of the melAB operon that encodes products needed for melibiose catabolism and transport. The melR gene is located upstream of the ª 2012 Federation of European Microbiological Societies Published by Blackwell Publishing Ltd. All rights reserved melAB operon, and the melR and melAB promoters are divergent, with the transcript start sites separated by 256 base pairs (Webster et al., 1987). We showed that transcription activation at the melAB promoter requires binding of MelR to four target sites (denoted 1′, 1, 2 and 2′) and binding of the global regulator, cyclic AMP receptor protein (CRP) to a target site located between MelR sites 1 and 2 (Fig. 1a). Transcription initiation at the melR promoter is dependent on activation by CRP and is repressed by MelR binding to a single target site (denoted R) overlapping the melR transcript start. Wade et al. (2000) reported that efficient MelR-dependent repression of the melR promoter requires upstream sequences that covered the melAB promoter and that the most important element in repression is MelR binding at target site 2. Further detailed analysis by Samarasinghe et al. (2008) showed that MelR bound at sites 1 and 1′ plays a role in repression, and images from atomic force microscopy suggested that repression is due to a nucleoprotein FEMS Microbiol Lett 338 (2013) 62–67 Optimal repression by E. coli MelR. 63 melR promoter (a) melAB 2’ 2 1 1’ R melAB promoter –174.5 2 (b) 1 melR +2.5 R 1’ TB22 TB23 TB31 TB28 TB33 –174.5 +2.5 % Activity (c) 150 –MelR +MelR 100 50 0 TB22 TB23 TB31 TB28 TB33 Fig. 1. The Escherichia coli melibiose operon regulatory region and MelR-dependent repression of the melR promoter. (a) Schematic diagram of the intergenic region between the divergent melAB and melR transcription units. The bent arrows indicate the transcription start sites for the melAB and melR promoters, and triangles denote the corresponding -10 hexamer elements. The locations of the different 18-bp DNA sites for MelR (sites 2′, 2, 1, 1′ and R) are indicated by shaded rectangles, and the DNA sites for CRP are denoted by pairs of ovals. (b) Diagram of the starting TB22 EcoRI–HindIII melR promoter fragment carrying the segment of the melibiose operon regulatory region indicated by the dotted lines joining panels A and B, drawn the same symbols as in panel A. The figure also illustrates the TB23, TB31, TB28 and TB33 derivatives, with substituted sequences denoted by the thickened black line. The orientation of each of the DNA sites for MelR is denoted by a horizontal arrow, and the locations of centre of some of these sites are indicated. (c) The figure illustrates measured levels of b-galactosidase in E. coli WAM1321 cells carrying melR promoter::lac fusions, encoded by pRW50 with the indicated promoter fragments. b-Galactosidase expression was measured in cells containing plasmid pJW15 that encodes MelR (+MelR: black filled bars), or the control pJW15DmelR plasmid with no melR insert ( MelR: open bars) as outlined in Materials and methods. For each promoter, the activity +MelR is expressed as a% of the activity MelR. The results are averages from at least three independent determinations. complex consisting of four MelR subunits and ~170 base pairs of DNA between MelR-binding target site 2 and target site R. FEMS Microbiol Lett 338 (2013) 62–67 Most members of the AraC family of transcription regulators function as homodimers of two subunits with the N-terminal domain of each subunit involved in ligand binding and dimerization, and the C-terminal domain responsible for DNA binding (Gallegos et al., 1997). C-terminal domains of AraC family members are highly conserved, carry two helix-turn-helix motifs and bind to asymmetric ~18 base pair target operator sequences. As it is well established that effective transcriptional repression can result from the two subunits of a single AraC dimer binding to two separated target sites (Schleif, 2010), and as MelR has been shown to dimerise (Bourgerie et al., 1997; Kahramanoglou et al., 2006), we revisited the E. coli melibiose operon regulatory region to investigate whether two DNA sites for MelR could be manipulated to produce efficient MelR-dependent repression of the melR promoter. Materials and methods In this work, we exploited the low-copy-number lac expression vector plasmid, pRW50, encoding resistance to tetracycline (Lodge et al., 1992). The starting points of the work were pRW50 derivatives carrying the TB22 and TB23 EcoRI-HindIII fragments (Fig. 1b) containing the E. coli melR promoter, as described by Samarasinghe et al. (2008). These recombinant pRW50 derivatives each carry a melR promoter::lacZ fusion, and they were propagated in the WAM1321 E. coli K-12 Dlac Dmel strain to measure melR promoter activity. Cells were grown in minimal medium with fructose, as a carbon source, and 35 lg mL 1 tetracycline, as in the study by Samarasinghe et al. (2008), and the Miller (1972) method was used to quantify b-galactosidase expression. For the different melR promoter fusions studied here in our conditions in the absence of MelR, b-galactosidase activity levels range from 360 to 400 standard Miller units. To quantify repression by MelR, cells also carried pJW15, encoding melR or empty vector, pJW15DmelR, and 80 lg mL 1 ampicillin was included in the media, as described by Kahramanoglou et al. (2006). In experiments to measure effects due to MalI, cells also carried pACYC–malI, encoding malI or empty vector, pACYC-DHN (Lloyd et al., 2010), and 10 lg mL 1 chloramphenicol was included in the media. Derivatives of the TB22 and TB23 EcoRI-HindIII fragments, illustrated in Figs 1–4, were constructed by standard recombinant DNA technology using synthetic oligos purchased from Alta Bioscience (http://www.altabioscience.com/) and cloned into pRW50. The complete annotated base sequence of each fragment is listed in the Data S1 (Supporting information), and the DNA sequences were checked by the functional genomics facility ª 2012 Federation of European Microbiological Societies Published by Blackwell Publishing Ltd. All rights reserved 64 of the University of Birmingham College of Life and Environmental Sciences (http://www.genomics.bham.ac.uk/). Results and discussion Optimal MelR-dependent repression due to two DNA sites for MelR in the same orientation To investigate MelR-dependent repression at the melR promoter, we exploited different melR promoter::lac fusions carried by derivatives of the pRW50 lowcopy-number lac expression plasmid, and b-galactosidase expression was measured in the WAM1321 E. coli K-12 Dlac Dmel host strain, containing either plasmid pJW15, encoding melR or empty vector. The starting experiment compared MelR-dependent repression of the melR promoter carried on the TB22 and the TB23 fragments, illustrated in Fig. 1b. The 251 base pair TB22 EcoRI-HindIII fragment carries DNA sequence from 192 base pairs upstream of the melR promoter transcript start (position 192) to 59 base pairs downstream (+59) and includes MelR target site 2, whilst in the 227 base pair TB23 fragment, MelR target site 2 is deleted. Results illustrated in Fig. 1c show that, as expected, the deletion of site 2 in the TB23 fragment causes a clear reduction in MelRdependent repression of the melR promoter and confirms previous observations (Wade et al., 2000). Previously, we identified the DNA target site for MelR subunits as an 18 base pair asymmetric sequence (Webster et al., 1987; Wade et al., 2001). By convention, we denote the location of each site by its centre with respect to the target promoter. Hence, at the melR promoter, MelR-binding site R is located at position +2.5 (i.e. between base pairs 2 and 3 downstream from the melR promoter transcript start) and MelR-binding site 2 is located at position 174.5 (i.e. between base pairs 174 and 175 upstream from the melR promoter transcript start). To investigate whether the binding of two MelR subunits could be sufficient to repress the melR promoter efficiently, we constructed the TB31, TB28 and TB33 fragments, illustrated in Fig. 1b. TB31 carries the core melR promoter sequences exactly as in TB22 and TB23, but DNA sequence upstream of position 80 is replaced by unrelated sequence. TB28 and TB33 are derivatives of TB31 carrying a single consensus 18 base pair site for MelR at position 174.5. In the TB28 fragment, this site has the same orientation as site 2 in the starting TB22 fragment, whilst, in TB33, this site has the opposite orientation, which is the same as for site R. Results illustrated in Fig. 1c show that MelR-dependent repression of the melR promoter in the TB31 and TB28 fragments is weak, but is increased to ~90% with the TB33 fragment. ª 2012 Federation of European Microbiological Societies Published by Blackwell Publishing Ltd. All rights reserved M.S. Elrobh et al. The weak repression of the melR promoter carried on the TB31 fragment must be due to MelR binding to the target site R alone, and this result is consistent with the study by Wade et al. (2000). The ~90% repression found with the TB33 fragment must be due to MelR binding to the targets at both positions 174.5 and +2.5 and interaction between MelR bound at the two loci. Strikingly, repression is greatly reduced with the TB28 fragment (Fig. 1b and c), and this was expected from our previous work in which we replaced MelR target sites 1 and 1′ and the adjacent DNA site for CRP (Samarasinghe et al., 2008). Hence, as for AraC-dependent repression at the araC-araBAD intergenic region, efficient repression with just two bound regulator molecules depends on both target sequences being in the same orientation (Carra & Schleif, 1993). MelR-dependent repression at the TB33 melR promoter is insensitive to spacing but reduced by a decoy DNA site for MelR The centre-to-centre distance between the two DNA sites for MelR in the TB33 fragment is 176 base pairs. To investigate the relation between spacing and repression, we constructed a series of derivative fragments with the upstream MelR target at different locations, ranging from position 254.5 to position – 83.5. This is illustrated in Fig. 2, which also lists the percentage MelRdependent repression for each case. The data show that repression is largely unaffected as the upstream DNA site for MelR is moved through ~170 base pairs, including translocation by five base pairs to the opposite face of the DNA helix (compare repression with TB33, TB332 and TB333). A simple explanation for our observations is that repression of the melR promoter in the TB33 fragment and its derivatives is due to a bridging interaction between MelR bound at the upstream and downstream DNA sites and subsequent loop formation, and this interaction must be sufficiently flexible to accommodate different distances and different face of the DNA helix juxtapositions between the sites. We suppose that the lack of efficient repression with the TB23 fragment (Fig. 1c) must be due to interactions between MelR bound at site 1 and site 1′ that preclude interaction with site R (Fig. 1b). To investigate this, we constructed the TB33P and TB33R derivatives illustrated in Fig. 3a. These fragments are derivatives of TB33 that contain a supplementary upstream DNA site for MelR organised in either the same orientation (TB33P) or opposite orientation (TB33R). Results illustrated in Fig. 3b show that the presence of the supplementary DNA site for MelR significantly reduces MelR-dependent repression of the melR FEMS Microbiol Lett 338 (2013) 62–67 Optimal repression by E. coli MelR. 65 % MelR-dependent Repression TB344 (–254.5) 89 TB342 (–214.5) 87 TB341 (–194.5) 84 TB33 (–174.5) 90 TB332 (–169.5) 92 TB333 (–164.5) 94 TB334 (–136.5) 88 TB331 (–123.5) 95 TB335 (–116.5) 90 TB336 (–103.5) 82 TB337 (–83.5) 92 TB31 28 +2.5 TB33 –194.5 TB33P –204.5 Fig. 2. MelR-dependent repression of the melR promoter and upstream-bound MelR. The figure illustrates a series of EcoRI-HindIII melR promoter fragments derived from TB33, with the upstream DNA site for MelR at different locations as listed in brackets. Conventions for the different symbols are as in Fig. 1. The numbers in the column on the right side of the figure denote the% repression of melR promoter activity by MelR, measured as outlined in the legend to Fig. 1. promoter, presumably because the supplementary site acts as a decoy for MelR–MelR interactions. Effects of MalI binding between MelR targets on MelR-dependent repression The flexibility in the spacing of the two DNA sites for MelR observed in the experiment illustrated in Fig. 2 suggested that it would be interesting to insert an intervening site for another DNA-binding protein. In recent work, Lloyd et al. (2010) identified the DNA site for the E. coli MalI repressor (that is a member of the LacI family) as a symmetric 16 base pair sequence element. Figure 4a illustrates an experiment where one or two of these elements were inserted between the two DNA sites for MelR in the TB334 fragment. Results in Fig. 4b show that in the absence of a plasmid encoding MalI, as expected, these insertions have but small effects on MelR-dependent repression of the melR promoter. However, with plasmid pACYC-malI, which encodes MalI, there is a clear small significant relief of repression with the TB334I-1 and TB334I-2 fragments carrying one or two MalI operator elements, but no relief with the control TB31, TB33 or TB334 fragments. FEMS Microbiol Lett 338 (2013) 62–67 –174.5 (a) TB33R TB31 (b) 120 –MelR +MelR % Activity 100 80 60 40 20 0 TB33 TB33P TB33R TB31 Fig. 3. Decoy MelR affects MelR-dependent repression of the melR promoter. (a) Schematic diagram of EcoRI-HindIII melR promoter fragments derived from TB33 with an upstream decoy DNA site for MelR. Conventions for the different symbols are as in Fig. 1. (b) The figure illustrates b-galactosidase expression in Escherichia coli WAM1321 cells carrying melR promoter::lac fusions, encoded by pRW50 with the indicated promoter fragments. Expression was measured in cells containing plasmid pJW15 (+MelR: black filled bars) or the control pJW15DmelR plasmid (-MelR: open bars) as outlined in Materials and methods. For each promoter, the activity +MelR is expressed as a% of the activity MelR, and each result is the average from at least three independent determinations. Conclusions The expression of many transcription repressors is autoregulated by repression (Browning & Busby, 2004). Kahramanoglou et al. (2006) proposed a two-state model for MelR in which, in the absence of its ligand, melibiose, MelR acts as an autorepressor of its own production by repressing the melR promoter. Samarasinghe et al. (2008) showed that this repression was due to the formation of a nucleoprotein complex involving four MelR subunits. Here, we report that it is possible to construct simpler derivatives of the melR promoter where only two MelR targets are needed for efficient repression (Fig. 1), and there are clear parallels between this and AraC-dependent repression at the araC–araBAD intergenic region, where repression is dependent on interaction between two AraC subunits bound to targets separated by 210 base pairs (Schleif, 2010). An explanation for the observed repression with the TB33 fragment is that MelR subunits bound ª 2012 Federation of European Microbiological Societies Published by Blackwell Publishing Ltd. All rights reserved M.S. Elrobh et al. 66 (a) TB31 –174.5 +2.5 TB33 –136.5 TB334 –156.5 TB334I-1 In the new constructs described here, efficient repression of the melR promoter by MelR requires interaction between MelR bound immediately adjacent to the transcript start and upstream-bound MelR, and this can be subverted by the insertion of a supplementary DNA site for MelR (Fig. 3). Hence, efficient repression results from two, but not from three, DNA sites for MelR. Our experiments underline the diversity of protein–DNA architectures that can be responsible for transcription repression. Acknowledgements –176.5 TB334I-2 This work was supported by the UK BBSRC with a project grant to S.J.W.B. and a summer studentship to D.D. (b) Promoter Fragment % MelR-dependent Repression TB31 –MalI 33 (1.1) +MalI 32 (2.4) TB33 93 (0.7) 94 (0.7) TB334 94 (0.4) 93 (0.8) TB334I-1 92* (0.7) 84* (0.6) TB334I-2 83* (1.4) 64* (1.9) Fig. 4. Effects of MalI binding on MelR-dependent repression of the melR promoter. (a) Schematic diagram of EcoRI-HindIII melR promoter fragments derived from TB33 with inserted DNA sites for MalI. Conventions for the different symbols are as in Fig. 1, with open hexagons representing 16 base pair DNA sites for MalI. (b) The numbers in the Table denote the% repression of melR promoter activity in each fragment by MelR, measured as in the legend to Fig. 1. Measurements were taken in cells that carried either pACYC malI, encoding malI (+MalI) or empty vector, pACYC-DHN ( MalI). Each listed result is given as the mean of five independent determinations, with the standard deviation in brackets. Asterisks denote data pairs with P-values <0.05 according to the ANOVA calculator. at the upstream and downstream DNA targets interact and result in loop formation, as for AraC. However, there appears to be more flexibility in how the two DNA sites for MelR can be juxtaposed, compared to AraC. Hence, AraC-dependent repression is disrupted by +5 base pair insertions (Lee & Schleif, 1989), whilst MelR-dependent repression is not (Fig. 2). The simplest explanation for this would be that the linker joining the N- and C-terminal domains is more flexible in MelR than in AraC. This flexibility is underscored by the experiment in Fig. 4 where MalI binding failed to completely disrupt repression. This experiment also argues that the mechanism of MelR-dependent repression with TB33 is different to the mechanism operating at the more complex wild type melibiose operon regulatory region in TB22 (Fig. 1), where repression depends on the formation of a nucleoprotein complex. ª 2012 Federation of European Microbiological Societies Published by Blackwell Publishing Ltd. All rights reserved References Bourgerie SJ, Michan CM, Thomas MS, Busby SJ & Hyde EI (1997) DNA binding and DNA bending by the MelR transcription activator protein from Escherichia coli. Nucleic Acids Res 25: 1685–1693. Browning DF & Busby SJ (2004) The regulation of bacterial transcription initiation. Nat Rev Microbiol 2: 57–65. Carra JH & Schleif RF (1993) Variation of half-site organization and DNA looping by AraC protein. EMBO J 12: 35–44. Gallegos MT, Schleif R, Bairoch A, Hofmann K & Ramos JL (1997) Arac/XylS family of transcriptional regulators. Microbiol Mol Biol Rev 61: 393–410. Kahramanoglou C, Webster CL, El-Robh MS, Belyaeva TA & Busby SJ (2006) Mutational analysis of the Escherichia coli melR gene suggests a two-state concerted model to explain transcriptional activation and repression in the melibiose operon. J Bacteriol 188: 3199–3207. Lee DH & Schleif RF (1989) In vivo DNA loops in araCBAD: size limits and helical repeat. P Natl Acad Sci USA 86: 476– 480. Lloyd GS, Godfrey RE & Busby SJ (2010) Targets for the MalI repressor at the divergent Escherichia coli K-12 malX-malI promoters. FEMS Microbiol Lett 305: 28–34. Lodge J, Fear J, Busby S, Gunasekaran P & Kamini NR (1992) Broad host-range plasmids carrying the E. coli lactose and galactose operons. FEMS Microbiol Lett 95: 271–276. Miller J (1972) Experiments in Molecular Genetics. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY. Samarasinghe S, El-Robh MS, Grainger DC, Zhang W, Soultanas P & Busby SJ (2008) Autoregulation of the Escherichia coli melR promoter: repression involves four molecules of MelR. Nucleic Acids Res 36: 2667–2676. Schleif R (2010) AraC protein, regulation of the L-arabinose operon in Escherichia coli, and the light switch mechanism of AraC action. FEMS Microbiol Rev 34: 779–796. Wade JT, Belyaeva TA, Hyde EI & Busby SJ (2000) Repression of the Escherichia coli melR promoter by MelR: evidence FEMS Microbiol Lett 338 (2013) 62–67 Optimal repression by E. coli MelR. that efficient repression requires the formation of a repression loop. Mol Microbiol 36: 223–229. Wade JT, Belyaeva TA, Hyde EI & Busby SJ (2001) A simple mechanism for co-dependence on two activators at an Escherichia coli promoter. EMBO J 20: 7160–7167. Webster CL, Kempsell K, Booth I & Busby S (1987) Organization of the regulatory region of the Escherichia coli melibiose operon. Gene 59: 253–263. FEMS Microbiol Lett 338 (2013) 62–67 67 Supporting Information Additional Supporting Information may be found in the online version of this article: Data S1. Complete base sequence of each of the EcoRIHindIII fragments carrying the melR promoter. ª 2012 Federation of European Microbiological Societies Published by Blackwell Publishing Ltd. All rights reserved