* Your assessment is very important for improving the workof artificial intelligence, which forms the content of this project
Download Purification and characterization of LasR as a DNA
Site-specific recombinase technology wikipedia , lookup
DNA supercoil wikipedia , lookup
Epigenetics in learning and memory wikipedia , lookup
Primary transcript wikipedia , lookup
No-SCAR (Scarless Cas9 Assisted Recombineering) Genome Editing wikipedia , lookup
Molecular cloning wikipedia , lookup
Microevolution wikipedia , lookup
Epigenetics of neurodegenerative diseases wikipedia , lookup
Deoxyribozyme wikipedia , lookup
Cancer epigenetics wikipedia , lookup
Gel electrophoresis of nucleic acids wikipedia , lookup
Extrachromosomal DNA wikipedia , lookup
Cre-Lox recombination wikipedia , lookup
Epigenetics of human development wikipedia , lookup
Epigenomics wikipedia , lookup
Vectors in gene therapy wikipedia , lookup
Non-coding DNA wikipedia , lookup
History of genetic engineering wikipedia , lookup
Protein moonlighting wikipedia , lookup
Nutriepigenomics wikipedia , lookup
DNA vaccination wikipedia , lookup
Helitron (biology) wikipedia , lookup
Point mutation wikipedia , lookup
ELSEVIER FEMS Microbiology Letters 142 (1996) 301-307 Purification and characterization of LasR as a DNA-binding protein Zhiying You a, Jun Fukushima ‘, Tetsuyoshi Ishiwata a, Baotong Chang a, Minoru Kurata ‘, Susumu Kawamoto a, Paul Williams b, Kenji Okuda aj* a Department of Bacteriology, Yokohama City University School b Department of Pharmaceutical Sciences, of Medicine, 3-9 Fukuura, University of Nottingham, Kanazawa-ku. University Park, Nottingham Yokohama 236, Japan NG7 ZRD, UK Received 1 July 1996; revised 11 July 1996; accepted 15 July 1996 Abstract In Pseudomonas aeruginosa, the activator protein LasR and a cognate autoinducer (AI) are required for expression of the elastase gene (lasB). In the present study, we investigated the binding properties of the P. aeruginosa IasR gene product. The LasR protein was overexpressed and purified as a glutathione S-transferase (GST) fusion protein. Using gel retardation and UV cross-linking analysis, we demonstrated that the GST-LasR could bind to a separate site in the ZasB upstream operator regions 1 and 3 in the presence of the autoinducer. Regions 1 and 3 are located at 105 and 42 base pairs upstream, respectively, from the IasB transcriptional start site. Our present results clearly demonstrate that LasR is a specific DNA-binding protein that regulates the transcription of the IasB gene in the presence of an autoinducer. Keywords: LasR; Autoinducer; DNA-binding protein; Transcriptional activator; 1. Introduction In some homologous regulatory systems such as the control of bioluminescence in Vibrio [l], the transcriptional activator protein might be activated by binding to a small diffusible molecule referred to as an autoinducer or N-acylhomoserine lactone to induce expression of different target genes [2]. The elastase gene (lasB), encoding extracellular pathogenic factors [3] has been cloned and sequenced [46], and its expression was shown to require the intact * Corresponding author. Tel.: +81 (45) 787 2511; Fax: +81 (45) 787 2509; E-mail: [email protected] 0378-1097/96/$12.00 IasB gene; Pseudomonas aeruginosa IasR gene and the P. aeruginosa autoinducer identified as N-(3-oxododecanoyl)homoserine lactone (OdDHL) [7,8]. According to current models of LasR, as well as of LuxR [9], the LasR polypeptide is presumed to consist of two domains [lO,ll]. For activation of transcription, it has been suggested that the conjugation of OdDHL to LasR protein is important [2,7]. However, there is no report which directly demonstrates binding of LasR and OdDHL complexes to the 1asB DNA fragment. This represents an obstacle hindering a better understanding of the mechanism by which LasR activates transcription of the 1asB gene in vitro. Thus, it is important to clarify the binding of LasR and the ZasB gene. During Copyright 0 1996 Federation of European Microbiological Societies. Published by Elsevier Science B.V. PZlSO378-1097(96)00286-8 Z. You et ul. I FEMS 302 h4icrohiolog.r Letters 142 (1996) 301-307 the course of LasR overproduction experiments in coli, an insoluble inclusion body appeared in the cells. In this study, we have overcome this difficulty by constructing a plasmid that overproduces the 1asR gene product using the prokaryotic expression vector pGEX in E. coli and by developing a procedure for purifying this fusion protein. Using the purified LasR protein, we established that the LasR protein exhibits DNA-binding activity in the presence of OdDHL. We also identified the two 1asB operators that are involved in transcriptional activation of 1asB. Escherichia 2. Materials and methods 2. I. Bacterial strains, plasmids, and culture conditions P. aeruginosa IF03455 [3] was obtained from the for Fermentation, Osaka, Japan. The P. aeruginosa non-elastase-producing strain, PA103 [3], was donated by P.V. Homma (University of Louisville School of Medicine, Louisville, KY). E. coli JMlOl was used as the host strain for the plasmid that overproduced LasR protein. E. coli cultures were grown in 2XYT or agar at 37°C with ampicillin (100 ltg ml-‘) as needed. Isopropyl-P-p-thiogalactoside (IPTG, 100 uM), synthetic OdDHL (10 pM) and protease inhibitors (aprotinin 2 ug ml-‘. leupeptin 1 pg ml-‘, PMSF 50 lrg ml-‘) were added as indicated. The procedure used for synthesis of OdDHL has been previously described [12]. 40) containing protease inhibitors and placed in an ice bath followed by two 30-s sonication steps. For fusion protein recovery using glutathione-Sepharose 4B (Pharmacia Biotech), GST-LasR extracts were resuspended in 2 ~01s. of EBC-DTT buffer (EBC buffer containing 5 mM DTT). Aliquots (15 ml) were rocked for 30 min at 4°C with 2.5 ml of equilibrated glutathione-Sepharose 4B beads which had been previously washed three times and resuspended (final concentration 1: 1, v/v) in EDC-DTT buffer containing 0.075% SDS. The beads were then mixed gently with 500 pl of glutathione elution buffer (10 mM reduced glutathione, 50 mM Tris-HCl, pH 8.0) and incubated at 4°C for 30 min to elute the fusion protein from the matrix. Purified LasR fusion proteins were collected by centrifugation for 10 s at 12000 rpm and stored at -70°C. 2.3. DNA-binding experiments Institute 2.2. Expression and purification of glutathione S-transferase (GSTJ-LasR fusion proteins 1asR genes derived from P. and PA103 was amplified by PCR. PCR products were ligated in frame into the BamHI site present within the pGEX-3X (Pharmacia Biotech) polylinker. Plasmids encoding pGEXLasR3455 and pGEX-LasR103, respectively, were introduced into E. coli JMlOl containing the tat promoter for overproduction of LasR protein. Extracts were prepared from cells harvested at Asa = 1.5 after IPTG induction. The cell pellets were resuspended in 8 ml of sonication EBC buffer (50 mM Tris-HCl, pH 8.0, 120 mM NaCl, 0.5% NP- The DNA encoding aeruginosa IF03455 Interaction of LasR with the regulatory regions of and UV crosslinking tests. The probes were generated by PCR and consisted of sequences from - 118 to - 12 bp containing regions 1 and 3 upstream of 1asB and forming the region 1 probe sequence from - 118 to -92 (+ 1 designates the transcription start site[l3]). The region 3 probe has a 20 base pair oligonucleotide from the -52 to -33 bp region of the 1asB promoter. The DNA-binding assay was performed according to established procedures [14]. The 20 l.rl reaction mixture, containing approx. 1 ng of a (5’-32P)-endlabeled DNA fragment (10 000 cpm) and the protein extracts in 10 mM Tris-HCl, pH 7.8, 50 mM KCl, 1 mM DTT, 1 mM EDTA, 1 mM MgCls, 5% glycerol, 50 pg ml-’ BSA, 5 yg l,rl’ salmon sperm DNA, was incubated for 30 min at 25°C. The sample was loaded onto a 5% polyacrylamide gel and electrophoresed in 0.5 X TBE buffer (45 mM Tris-borate, 1 mM EDTA) at 8 V/cm for the gel retardation assay. Alternatively, an equal volume of 2X SDSPAGE gel loading buffer was added, and the same reaction products were resolved on a 420% SDSPAGE gel by UV cross-linking. 1asB was studied using gel retardation Z. You et al. IFEMS Microbiology Letters 142 (1996) 301-307 2.4. SDS-polyacrylamide gel electrophoresis 303 and Western blotting A 4-20% resolving SDS-polyacrylamide gel was prepared and stained with Coomassie blue R-250. Western blotting was performed following published procedures [ 151 with anti-GST antibody (Pharmacia Biotech) and anti-LasR antibody. Anti-LasR serum was obtained from rabbits immunized with synthesized multi antigenic peptide methods [16]. Antibody titers could still be observed in the 211 diluted samples by ELISA using LasR peptides as coating antigen. 3. Results and discussion 3.1. Overproduction, purcfkation and identt&ation of the fusion protein as GST-LasR To establish the existence of a LasR protein capable of regulating 1usB regions, we first carried out purification of LasR fusion protein. Due to the insolubility of the inclusion bodies in the bacteria, initial efforts to express LasR protein in these cells proved difficult. However, when LasR protein was fused to the carboxyl-terminal of GST, it became soluble. We generated a series of GST-LasR fusion proteins (Fig. 1) which were purified using a glutathione-Sepharose 4B column. Fig. 1. Glutathione S-transferase-LasR fusion proteins. Diagram of the pGEX encoded LasR protein described in this report. LasR is shown with the proposed C-terminal domain responsible for DNA binding and activation of the IasB gene. The N-terminal domain binds to the autoinducer (AI) which then binds directly to its transcriptional activator protein, and DNA binding and gene activation then occur. IF03455 is a positive elastaseproducing strain and PA103 is a non-elastase-producing strain. The arrow indicates an amino acid substitution of R179 to W of PA103. 1 123456 2 3 kDa Fig. 2. Expression and purification of glutathione S-transferase fusion proteins. (A) SDS-PAGE. Whole-cell lysates of bacterial clones expressing the pGEX-3X-encoded glutathione S-transferase leader sequence (lane 4) GST-LasR3455 (lane 5). and GSTLasR103 (lane 6) were prepared as described in Section 2. Each bacterial sonicate was also prepared and incubated with glutathione-Sepharose 4B (lanes 1-3, respectively). Proteins were resolved by electrophoresis in a 420% SDS-polyacrylamide gel and visualized by Coomassie blue staining. The arrow indicates the 53 kDa position of the purified GST-LasR fusion protein. (B) Western immunoblot using anti-LasR antibody. Lane 1, purified GST protein, Lanes 2,3, purified GST-LasR3455 and GSTLasR103. Expression and purification of the LasR fusion proteins are shown in Fig. 2. Total protein fractions extracted from E. coli JMlOl carrying the IasR gene transformed with parental pGEX-3X (lane 4) or with various pGEX-3X recombinants (lanes $6) were resolved by SDS-polyacrylamide gel electrophoresis and visualized by Coomassie blue staining. Sonicated samples of these bacterial clones were also prepared and passed through a glutathione-Sepharose 4B column (lanes l-3). The fusion protein that specifically bound to glutathione-Sepharose had a molecular mass of 53 kDa, consistent with the size of the LasR, i.e. 27 kDa plus the molecular mass of GST, 26 kDa. We next examined whether these purified proteins were GST-LasR fusion proteins by using anti-GST antibody (data not shown) and anti-LasR antibody. Each of the 53 kDa GST-LasR fusion proteins was detected (Fig. 2B). From these results it is clear that the LasR fusion protein was nearly homogeneous. 2. You ef al. I FEMS Microbiology Letters 142 (1996) 301-307 304 A. ORF Fig. 3. (A) Gel retardation assay using an IasB upstream probe and LasR protein. The end-labeled IrsB upstream probe used extends from -118 to -12 bp which includes regions 1 and 3. The LasR3455 protein used was the GST-LasR fusion protein. A mobility shift assay was performed by 5% polyacrylamide gel/TBE electrophoresis of a (5’.s’P)-labeled DNA fragment. Region 3 competitor DNA was used at 140-fold molar excess in lane 3. 1 ul anti-LasR antibody was added to lane 2. (B) Organization of the upstream region and probes of IasB. 3.2. LasR binds to the regulatory regions c$lusB LasR has an amino acid sequence similar to the DNA-binding domain of the helix-turn-helix (H-TH) type of DNA-binding proteins [17]. We have also determined the location of sequences required for efficient promoter activity that exists from -135 to -86 bp (containing region 1) and from -63 to -27 bp (containing region 3) in the 1asB promoter region using the CAT assay system with our deletion mutants (Fukushima et al., submitted). We next studied whether LasR protein binds to the regulatory region containing regions 1 and 3 upstream of 1usB (Fig. 3B). LasR protein was used for DNA-binding analyses to determine if DNA binding in vitro is correlated with in vivo transcriptional activation [13]. A gel retardation assay was performed to determine whether purified LasR binds to the DNA fragment containing regulatory regions 1 and 3 of lusB in the presence of OdDHL. The electrophoretic mobility of the DNA fragment containing these 1usB regulatory regions was significantly retarded by LasR (two bands were observed; Fig. 3A, lane 1). To ex- amine the sequence specificity of binding, a 140-fold excess of unlabeled oligo DNA containing regulatory region 3 was added to this reaction mixture. Retardation of a lower band was markedly inhibited by the unlabeled DNA fragment (Fig. 3A, lane 3). On the other hand, retardation of this band was inhibited by the addition of anti-LasR antibody, and the highest super-shift band appeared (Fig. 3A, lane 2). This retardation was not observed using unrelated control antibodies or unrelated DNA fragments (data not shown). These observations indicate that LasR protein binds to the regulatory regions of 1asB and that region 3 is one of the binding sites. Binding of LasR protein to the DNA fragment containing regions 1 and 3 was further examined by a UV cross-linking study. As shown in Fig. 4, two retarded bands were detected by the simultaneous addition of both LasR fusion protein and OdDHL (lane 6). Furthermore, a lOO-fold excess of unlabeled region 1 or a 140-fold excess of unlabeled region 3 DNA fragments were added as competitors, and retardation of the band was inhibited (lanes 3,4 in Fig. 4). Purified GST protein was not detected as Z. You et al. IFEMS Microbiology Letters 142 (1996) 301-307 a distinct band (lane 7). These data confirm the importance of these two DNA fragment regions when LasR protein binds to the upstream DNA sequence of the 1asB gene. Since a complex of LasR and OdDHL has been reported [7] to be important for the activation of LasR protein, we tested LasR binding with this purified protein. No shifted band was detected (Fig. 4, lane 5). On the other hand, the extent of retardation increased when the concentration of OdDHL was increased (lanes 1,2 in Fig. 4) indicated that an autoinducer is necessary for LasR protein to bind to regulatory regions of 1asB. We hypothesized that the IasR gene contains two domains, an autoinducer response domain and DNA binding domain. We assumed that the autoinducer 12345678 Fig. 4. W cross-linking analysis of the 1asE upstream region with the LasR fusion protein using regions 1 and 3 DNA as a probe. The end-labeled 1asB upstream probe used extends from -118 to - 12 bp. The reaction mixtures were fractionated on a 12% SDS-polyacrylamide gel. Lane I,2 contained 10 pM OdDHL and 100 pM OdDHL; lanes 3,4, addition of 140-fold excess of region 3 oligo DNA and loo-fold excess of region 1 fragment DNA as competitor; lane 5, no OdDHL; lane 6, no competitor; lane 7, only purified GST protein. 12 34 305 56 Fig. 5. Cross-linking assay of the LasR fusion protein with the IusB upstream region 1 using region 1 DNA as a probe. The end-labeled IasB upstream probe used was the fragment from -118 to -92 bp DNA. Lane 1, no competitor; lane 2, IO-fold excess of region 1 fragment DNA; lane 3, 150-fold excess of non-specific salmon sperm DNA; lane 4, 2 fl anti-LasR antibody; lane 5, 140-fold excess of region 3 oligo DNA as competitor. binds to the N-terminal domains of the regulatory proteins, thereby stimulating binding of the C-terminal domains to DNA target sites [18]. Therefore, the active form of LasR regulates expression of lu,sB. To further examine the binding position of the upstream 1asB promoter, the 1asB binding region was more precisely studied using the region 1 DNA fragment as a probe. Upon mixing the probe with LasR protein, a single band with retarded mobility could be clearly recognized (Fig. 5, lane 1). Furthermore, competitive experiments with a lo-fold of unlabeled DNA fragment of region 1 and a 140-fold unlabeled oligo DNA fragment of region 3 resulted in inhibition of the retardation band (Fig. 5, lanes 2,5), and retardation was not observed using a 150fold unrelated salmon sperm DNA (Fig. 5, lane 3) indicating that LasR specifically bound to the 1asB regulatory region. 306 Z. You et ul. I FEMS Microbiology Rust et al. [13] have recently shown that the regulatory region of the 1asB promoter contains two operator sequences that are involved in LasR- and OdDHL-mediated 1asB activation. The two operator sequences reported by these investigators are included in our binding regions (regions 1 and 3). The results of DNA-binding analysis of LasR protein to the upstream region of the 1asB promoter suggest that the combination of LasR and OdDHL is necessary for 1asB transcription, and control of its expression. In addition, regions 1 and 3 are shown to be involved in 1asB activation. The region 3 sequence is centered 42 bp upstream from the 1asB transcriptional start site which is located 141 bp upstream of the translation initiation codon [13], containing a lux box-like 20 bp palindrome. A similar sequence in region 1 upstream of 1asB contains a half lux boxlike region as well. Both binding regions are similar to the reported consensus sequence for other genes controlled by an autoinducer [l 1] and the two LasRbinding regions contain 14 bp repeats that are 70% homologous. LuxR studies have also revealed that the DNA-binding region specifically binds to sequences upstream from the lux operator [19,20]. Furthermore, we have shown that conserved amphipathic a-helix sequences in LasR protein precede the H-T-H DNA binding motif [17]. We predicted that these motifs including H-T-H contribute to DNA binding. This previous discovery accompanied by our experimental results confirms that the LasROdDHL complex interacts with the upstream region 3 (from -52 to -33 bp) and region 1 (from -118 to -92 bp) of 1asB and that is regulated to activate transcription of 1asB expression. This report on in vitro DNA binding is the first to use purified, full length transcriptional activator LasR complexes and to characterize the binding of LasR protein to 1asB DNA. Acknowledgments We are grateful to Ms. T. Kaneko, Niikura for technical assistance. I. Oiji, and K. Letters 142 (1996) 301-307 References [l] Meighen, E.A. (1993) Bacterial bioluminescence: organization, regulation, and application of the lux genes. FASEB. J. 7, 10161022. [2] Gambello, M.J. and Iglewski, B.H. (1991) Cloning and characterization of the Pseudomonas aeruginosa 1asR gene, a transcriptional activator of elastase expression, J. Bacterial. 173, 3000-3009. [3] Morihara, K. and Homma, J.Y. (1985) Pseudomonas protease. In: Bacterial Enzymes and Virulence (Holder, I.A., Ed.), pp. 41-79, CRC Press, Boca Raton, FL. [4] Bever, R.A. and Iglewski. B.H. (1988) Molecular characterization and nucleotide sequence of the Pseudomonas neruginosa elastase structural gene. J. Bacterial. 170, 430994314. [5] Fukushima, J., Yamamoto, S., Morihala, K., Atsumi, Y., Takeuchi, H., Kawamoto, S. and Okuda, K. (1989) Structural gene and complete amino acid sequence of Pseudomonas aeruginosu IF03455 elastase. J. Bacterial. 171. 169881704. 161Yamamoto, S., Fukusima. J., Atsumi, Y., Takeuchi, H., Kawamoto, S., Okuda. K. and Morihara, K. (1988) Cloning and characterization of elastase structural gene from Pseudomonas aeruginosa IF03455. Biochem. Biophys. Res. Commun. 152. 1117-1122. [71 Passador, L., Cook, J.M., Gambello, M.J., Rust, L. and Iglewski., B.H. (1993) Expression of Pseudomonas aeruginosa virulence genes requires cell-to-cell communication. Science 260. 1127-l 130. PI Pearson, J.P., Gray, K.M., Passador, L., Tucker, K.D., Eberhard, A., Iglewski, B.H. and Greenberg, E.P. (1994) Structure of the autoinducer required for expression of Pseudomonas ueruginosa virulence genes. Proc. Natl. Acad. Sci. USA 91, 197-201. [91 Choi, S.H. and Greenberg, E.P. (1992) Genetic evidence for multimerization of LuxR, the transcriptional activator of Vihriojischeri luminescence. Mol. Mar. Biol. Biotechnol. 1, 4088 413. UOI Fuqua, W.C., Winans, S.C. and Greenberg, E.P. (1994) Quorum sensing in bacteria: the LuxR-LuxI family of cell densityresponsive transcriptional regulators. J. Bacterial. 176, 2699 275. 11II Gray, K.M., Passador, L., Iglewski, B.H. and Greenberg, E.P. (1994) Interchangeability and specificity of components from the quorum-sensing regulatory systems of Vibrio fischeri and Pesudomonas aeruginosu. J. Bacterial. 176, 30763080. [I21 Chhabra, S.R., Stead, P., Bainton, N.J., Salmond, G.P.C.. Stewart, G.S.A.B., Williams, P. and Bycroft, B.W. (1993) Autoregulation of carbapenem biosynthesis in Erwinia carotovora by analogues of N-(3-oxohexanoyl)-r-homoserine lactone. J. Antibiot. 46, 44-449. P31 Rust, L., Pesci, E.C. and Iglewski, B.H. (1996) Analysis of the Pseudomonas aeruginosa elastase (lasE) regulatory region. J. Bacterial. 178, 1134-l 140. u41 Fried, M. and Crothers, D.M. (1981) Equilibria and kinetics of lac repressor-operator interactions by polyacrylamide gel electrophoresis. Nucleic Acids Res. 9, 6505-6525. [15] Shinagawa, H., Iwasaki, H., Kato, T. and Nakata, .4. (1988) 2. You ef al. IFEMS Microbiology Letters 142 (1996) 301-307 RecA protein dependent cleavage of UmuD protein and SOS mutagensis. Proc. Natl. Acad. Sci. USA 85, 18061810. [16] Nardelly, B., Lu, Y.A., Shiu, D.R., Delpierre-Defoort, C., Proty, A.T. and Tam, J.P. (1992) A chemically defined synthetic vaccine model for HIV-l. J. Immunol. 148, 914-920. [17] Fukushima, J., Ishiwata, T., Kurata, M., You, Z.Y. and Okuda, K. (1994) Intracellular receptor-type transcription factor, LasR, contains a highly conserved amphipathic region which precedes the putative helix-turn-helix DNA binding motif. Nucleic Acids Res. 22, 37063707. [18] Bainton, N.J., Bycroft, B.W., Chhabra, S.R., Stead, P., Gledhill, L., Hill, P.J., Rees, C.E.D., Winson, M.K., Sahnond, 307 G.P.C., Stewart, G.S.A.B. and Williams, P. (1993) A general role for the lux autoinducer in bacterial cell signalling: control of antibiotic biosynthesis in Erwinia. Gene 116, 87-92. [19] Miyamoto, C.M., Smith, E.E., Swartzman, E., Cao, J.G., Graham, A.F. and Meighen, E.A. (1994) Proximal and distalsites bind LuxR independently and activate expression of the Vibrio harveyi lux operon. Mol. Microbial. 14, 255-262. [20] Stevens, A.M., Dolan, K.M. and Greenberg, E.P. (1994) Synergistic binding of the Vibrio fischeri LuxR transcriptional activator domain and RNA polymerase to the lux promoter region. Proc. Natl. Acad. Sci. USA 91, 12619-12623.