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• W2028279314 abstract "The interaction of the two transcriptional regulators RcsA and RcsB with a specific operator is a common mechanism in the activation of capsule biosynthesis in enteric bacteria. We describe RcsAB binding sites in the wza promoter of the operon for colanic acid biosynthesis in Escherichia coliK-12, in the galF promoter of the operon for K2 antigen biosynthesis in Klebsiella pneumoniae, and in thetviA (vipR) promoter of the operon for Vi antigen biosynthesis in Salmonella typhi. We further show the interaction of RcsAB with the rcsA promoters of various species, indicating that rcsA autoregulation also depends on the presence of both proteins. The compilation of all identified RcsAB binding sites revealed the conserved core sequence TaAGaatatTCctA, which we propose to be termed RcsAB box. The RcsAB box is also part ofBordetella pertussis BvgA binding sites and may represent a more distributed recognition motif within the LuxR superfamily of transcriptional regulators. The RcsAB box is essential for the induction of Rcs-regulated promoters. Site-specific mutations of conserved nucleotides in the RcsAB boxes of the E. coli wzaand rcsA promoters resulted in an exopolysaccharide-negative phenotype and in the reduction of reporter gene expression. The interaction of the two transcriptional regulators RcsA and RcsB with a specific operator is a common mechanism in the activation of capsule biosynthesis in enteric bacteria. We describe RcsAB binding sites in the wza promoter of the operon for colanic acid biosynthesis in Escherichia coliK-12, in the galF promoter of the operon for K2 antigen biosynthesis in Klebsiella pneumoniae, and in thetviA (vipR) promoter of the operon for Vi antigen biosynthesis in Salmonella typhi. We further show the interaction of RcsAB with the rcsA promoters of various species, indicating that rcsA autoregulation also depends on the presence of both proteins. The compilation of all identified RcsAB binding sites revealed the conserved core sequence TaAGaatatTCctA, which we propose to be termed RcsAB box. The RcsAB box is also part ofBordetella pertussis BvgA binding sites and may represent a more distributed recognition motif within the LuxR superfamily of transcriptional regulators. The RcsAB box is essential for the induction of Rcs-regulated promoters. Site-specific mutations of conserved nucleotides in the RcsAB boxes of the E. coli wzaand rcsA promoters resulted in an exopolysaccharide-negative phenotype and in the reduction of reporter gene expression. exopolysaccharide base pair(s) kilobase pair(s) open reading frame polymerase chain reaction electrophoretic mobility shift assay surface plasmon resonance Encapsulation by exopolysaccharides (EPS)1 protects bacteria against a variety of unfavorable environmental conditions. The production of EPS furthermore represents an essential factor in the virulence of bacterial pathogens (1.Leigh J.A. Coplin D.L. Annu. Rev. Microbiol. 1992; 46: 307-346Crossref PubMed Scopus (291) Google Scholar). The biosynthesis of high molecular weight type I EPS in several enteric bacteria likeEscherichia coli (2.Gottesman S. Trisler P. Torres-Cabassa A. J. Bacteriol. 1985; 162: 1111-1119Crossref PubMed Google Scholar, 3.Jayaratne P. Keenleyside W.J. MacLachlan P.R. Dodgson C. Whitfield C. J. Bacteriol. 1993; 175: 5384-5394Crossref PubMed Google Scholar), Salmonella typhi (4.Virlogeux I. Waxin H. Ecobichon C. Lee J.O. Popoff M.Y. J. Bacteriol. 1996; 178: 1691-1698Crossref PubMed Scopus (80) Google Scholar),Klebsiella pneumoniae (5.Allen P. Hart C.A. Saunders J.R. J. Gen. Microbiol. 1987; 133: 331-340PubMed Google Scholar, 6.Mc Callum K.L. Whitfield C. Infect. Immun. 1991; 59: 494-502Crossref PubMed Google Scholar), and the plant pathogenic bacteria Erwinia amylovora (7.Bernhard F. Poetter K. Geider K. Coplin D.L. Mol. Plant Microbe Interact. 1990; 3: 429-437Crossref PubMed Scopus (50) Google Scholar, 8.Chatterjee A. Chun W. Chatterjee A.K. Mol. Plant Microbe Interact. 1990; 3: 144-148Crossref Google Scholar, 9.Coleman M. Pearce R. Hitchin E. Busfield F. Mansfield J.W. Roberts I.S. J. Gen. Microbiol. 1990; 136: 1799-1806Crossref PubMed Scopus (27) Google Scholar, 10.Bereswill S. Geider K. J. Bacteriol. 1997; 179: 1354-1361Crossref PubMed Scopus (72) Google Scholar) and Pantoea stewartii (11.Poetter K. Coplin D.L. Mol. Gen. Genet. 1991; 229: 155-160Crossref PubMed Scopus (23) Google Scholar) is controlled by the Rcs (regulation ofcapsule synthesis) regulatory network. Two transcriptional regulators, RcsA and RcsB, are supposed to induce EPS biosynthesis cooperatively. The RcsB protein is highly conserved between different species with about 90% identity (10.Bereswill S. Geider K. J. Bacteriol. 1997; 179: 1354-1361Crossref PubMed Scopus (72) Google Scholar), and it represents the cytoplasmic activator of a classical bacterial two-component system. RcsB might be activated by the membrane-bound receptor RcsC (3.Jayaratne P. Keenleyside W.J. MacLachlan P.R. Dodgson C. Whitfield C. J. Bacteriol. 1993; 175: 5384-5394Crossref PubMed Google Scholar, 12.Stout V. Gottesman S. J. Bacteriol. 1990; 172: 659-669Crossref PubMed Google Scholar) via phosphotransfer to highly conserved aspartic acid residues in the N-terminal domain of RcsB. RcsA and RcsB are both characterized by a LuxR-type C-terminal DNA binding motif, but RcsA does not contain an N-terminal phosphorylation motif. The RcsA protein is limiting for the induction of EPS biosynthesis and is virtually not detectable in the uninduced cell due to its rapid degradation by the Lon protease (13.Torres-Cabassa A.S. Gottesman S. J. Bacteriol. 1987; 169: 981-989Crossref PubMed Scopus (184) Google Scholar, 14.Stout V. Torres-Cabassa A. Maurizi M.R. Gutnick D. Gottesman S. J. Bacteriol. 1991; 173: 1738-1747Crossref PubMed Google Scholar). The presence of RcsB is absolutely required for capsule biosynthesis, whereas an rcsA minus phenotype can be suppressed by multicopy rcsB (15.Brill J.A. Quinlan-Walshe C. Gottesman S. J. Bacteriol. 1988; 170: 2599-2611Crossref PubMed Google Scholar). RcsA might therefore act as a coinducer of EPS biosynthesis by enhancing the DNA binding activity of RcsB. Recently, genetic evidence for an autoregulation ofrcsA expression in E. coli has been reported and a DNA binding activity of RcsA at the rcsA promoter has been discussed (16.Ebel W. Trempy J.E. J. Bacteriol. 1999; 181: 577-584Crossref PubMed Google Scholar). We have previously shown that a heterodimer formed by one copy of RcsA and RcsB binds at corresponding regions approximately 500 bp upstream of the translational start sites of amsG andcpsA, the first open reading frames (ORF) in the E. amylovora ams operon for amylovoran biosynthesis, and in theP. stewartii cps operon for stewartan biosynthesis, respectively (17.Kelm O. Kiecker C. Geider K. Bernhard F. Mol. Gen. Genet. 1997; 256: 72-83Crossref PubMed Scopus (48) Google Scholar, 18.Wehland M. Kiecker C. Coplin D.L. Kelm O. Saenger W. Bernhard F. J. Biol. Chem. 1999; 274: 3300-3307Abstract Full Text Full Text PDF PubMed Scopus (50) Google Scholar). The two operons are highly homologous, and the activation of Rcs-dependent promoters by a RcsAB heterodimer as a general mechanism remained unclear. In addition, some evidence for modified Rcs-dependent regulation mechanisms in E. coli and S. typhi has been proposed (4.Virlogeux I. Waxin H. Ecobichon C. Lee J.O. Popoff M.Y. J. Bacteriol. 1996; 178: 1691-1698Crossref PubMed Scopus (80) Google Scholar,16.Ebel W. Trempy J.E. J. Bacteriol. 1999; 181: 577-584Crossref PubMed Google Scholar). In this work we demonstrate that the binding of RcsAB to regulatory DNA regions is a general principle in the Rcs-mediated activation of gene expression. We present RcsAB binding sites identified in the presumed main promoters of EPS biosynthetic operons of E. coli,K. pneumoniae, and S. typhi. RcsAB further modulates the rcsA autoregulation in those species by binding to the rcsA promoters. The compilation of all identified RcsAB binding sites allows us to define the RcsAB box as a new conserved bacterial operator. The RcsAB box was analyzed in vitro and in vivo, and it was found to be essential for full promoter activity in E. coli. The E. coli strains Xl1-Blue (19.Bullock W.O. Fernandez J.M. Stuart J.M. BioTechniques. 1987; 5: 376-379Google Scholar), BL21, C600, DH5α (Stratagene), and JB3034 (15.Brill J.A. Quinlan-Walshe C. Gottesman S. J. Bacteriol. 1988; 170: 2599-2611Crossref PubMed Google Scholar) and the plasmids pQE30 (Qiagen), pMalc2 (New England Biolabs), and pfdA8 (20.Geider K. Hohmeyer C. Haas R. Meyer T.F. Gene (Amst.). 1985; 33: 341-349Crossref PubMed Scopus (43) Google Scholar) were used for cloning and expression studies. Standard DNA techniques were done as described (21.Sambrook J. Fritsch E.F. Maniatis T. Molecular Cloning: A Laboratory Manual. 2nd Ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY1989Google Scholar). DNA fragments were amplified from chromosomal DNA of strain Xl1-Blue with Vent polymerase and suitable primers. The sequences of the oligonucleotides are available upon request. RcsA proteins were produced with the plasmids pM-RcsAEA, pM-RcsAEC, and pM-RcsAPS (17.Kelm O. Kiecker C. Geider K. Bernhard F. Mol. Gen. Genet. 1997; 256: 72-83Crossref PubMed Scopus (48) Google Scholar, 18.Wehland M. Kiecker C. Coplin D.L. Kelm O. Saenger W. Bernhard F. J. Biol. Chem. 1999; 274: 3300-3307Abstract Full Text Full Text PDF PubMed Scopus (50) Google Scholar) in strain BL21 as C-terminal fusions to the maltose-binding protein. RcsB proteins were produced with the plasmids pQ-RcsBEC and pQ-RcsBEA (17.Kelm O. Kiecker C. Geider K. Bernhard F. Mol. Gen. Genet. 1997; 256: 72-83Crossref PubMed Scopus (48) Google Scholar) with an N-terminal poly(His)6tag in the strain JB3034. The proteins were purified as described (17.Kelm O. Kiecker C. Geider K. Bernhard F. Mol. Gen. Genet. 1997; 256: 72-83Crossref PubMed Scopus (48) Google Scholar,18.Wehland M. Kiecker C. Coplin D.L. Kelm O. Saenger W. Bernhard F. J. Biol. Chem. 1999; 274: 3300-3307Abstract Full Text Full Text PDF PubMed Scopus (50) Google Scholar). If appropriate, the purified RcsA and RcsB proteins of E. coli, E. amylovora, and P. stewartii were named according to their origin RcsAEC, RcsAEA, RcsAPS, RcsBEC, and RcsBEA, respectively. A BamHI/HindIII restriction fragment from plasmid pQHB (22.Boucher P.E. Menozzi F.D. Locht C. J. Mol. Biol. 1994; 241: 363-377Crossref PubMed Scopus (69) Google Scholar) containing the coding region ofBordetella pertussis bvgA was cloned into the expression vector pMalc2, resulting in plasmid pM-bvgA. The BvgA protein was produced from strain DH5α × pM-bvgA and purified by affinity chromatography of the crude extract with a dextrin column. Radioactive DNA labeling with [α-32P]dATP and the EMSA technique were done as described previously (17.Kelm O. Kiecker C. Geider K. Bernhard F. Mol. Gen. Genet. 1997; 256: 72-83Crossref PubMed Scopus (48) Google Scholar, 18.Wehland M. Kiecker C. Coplin D.L. Kelm O. Saenger W. Bernhard F. J. Biol. Chem. 1999; 274: 3300-3307Abstract Full Text Full Text PDF PubMed Scopus (50) Google Scholar). The RcsAB heterodimer was obtained by mixing equimolar concentrations of the two proteins. For the reconstitution of double-stranded DNA fragments, about 1 μg of each of two complementary oligonucleotides were mixed in 200 mm Tris/HCl, pH 7.5, 100 mm NaCl and incubated for 5 min at 95 °C. The mixture was cooled slowly to room temperature and subsequently labeled. Phosphorylation of BvgA was obtained by incubating the protein in 50 mm Tris/HCl, pH 7.0, 20 mm MgCl2, 0.1 mmdithiothreitol, and 20 mm acetylphosphate for 20 min at 28 °C. SPR measurements were performed with a BIAcore X instrument (BIAcore, Uppsala, Sweden). Biotinylated DNA (about 60 resonance units) were coupled to the streptavidin-coated sensor chip SA as recommended by the manufacturer. The experiments were carried out at a flow rate of 50 μl/min. The DNA fragments and proteins were diluted in running buffer (50 mm Tris-HCl, pH 7.5, 100 mm NaCl, 10 mm dithiothreitol, 0.1 mm EDTA). Bovine serum albumin and λ-DNA were added to the protein solutions to a final concentration of 200 and 8 ng/μl, respectively. RcsA/RcsB mixtures of various concentrations ranging from about 47 nm to about 7.5 μm were injected allowing an association time of 120 s and a dissociation time of 300 s. A reference flow cell loaded with a random DNA target of same size as the probe DNA target was used to subtract unspecific DNA/protein interactions. Regeneration of the chip surface was achieved by removing all bound proteins with a pulse of 5 μl of 0.05% SDS in running buffer. Kinetic analyses were done using the BIAevaluation 3.0 program. To determine the binding properties of the proteins, 1:1 Langmuir kinetics provided by the software were used. Plasmid pMW31 was constructed by cloning a 3-kb DNA fragment starting seven nucleotides upstream of the RcsAB box of the wza gene into the BglII andPstI sites of the suicide vector pfdA8 (20.Geider K. Hohmeyer C. Haas R. Meyer T.F. Gene (Amst.). 1985; 33: 341-349Crossref PubMed Scopus (43) Google Scholar). In plasmid pMW29, four essential nucleotide positions in the RcsAB box TAAAGAAACTCCTA of the 3-kb fragment were modified by PCR, resulting in the sequence GAACTCAACTCCTA, where the mutated bases were underlined. The plasmids were transformed into E. coli C600 by electroporation and selected for kanamycin resistance. The correct insertion of the plasmids by homologous recombination was verified by PCR analysis of the isolated chromosomal DNA. An approximately 1-kb DNA fragment generated by PCR and containing the complete E. coli rcsA gene starting 38 bp upstream of the RcsAB box, was cloned into the vector pBluescript KS+resulting in plasmid prcsA-WT. Four essential positions of the RcsAB box TAAGGATTATCCGA in plasmid prcsA-WT were mutated by PCR upon introduction of an EcoRI restriction site and resulting in the sequence GAATTCTTATCCGA with the mutated bases underlined. EPS were quantified from cells grown on cellophane-covered LB-agar for 24 h at 37 °C. After harvesting, the cell number was determined and the capsules were washed off from the cells by vigorous shaking for 3 min. The cells were pelleted by ultracentrifugation, and the supernatant was dialyzed against water. The polysaccharides were determined as described (23.Dubois M. Giles K.A. Hamilton J.K. Rebers P.A. Smith F. Anal. Chem. 1956; 28: 350-356Crossref Scopus (39449) Google Scholar). The enzymatic activity of the β-galactosidase was determined with theo-nitrophenyl β-d-galactopyranoside assay after Miller (24.Miller J.H. Experiments in Molecular Genetics. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1972Google Scholar). The 485-bp PCR fragment Pwza485 containing the putative promoter and the first nine codons ofwza, the first ORF in the operon for colanic acid biosynthesis in E. coli, interacted with RcsABECin EMSAs. Terminal deletions revealed the 55-bp fragment Pwza 55 spanning nucleotide positions −119 to −65 relative to the transcriptional start site of wza (Fig.1) as sufficient for a retardation by the RcsABEC heterodimer (Fig. 2). The extent of retardation was diminished with the 41-bp fragment Pwza 41 from positions −119 to −79, and no retardation was observed with the 27-bp fragment Pwza 27 spanning nucleotide positions −106 to −80. This was also observed with the 38-bp fragment Pwza 38 spanning nucleotide positions −119 to −82. This indicated that the 28-bp region spanning nucleotide positions −106 to −79 relative to the transcriptional start site ofwza was essential but not sufficient for the binding of RcsABEC (Figs. 1 and 2). An extension of the 3′-end of the 55-bp fragment did not further contribute to a better binding of RcsABEC, and the extent of retardation of the 72-bp fragment Pwza 72 spanning positions −119 to −48 was comparable to that of Pwza 55 (data not shown). Incubation at 28 °C compared with 37 °C prior to electrophoresis increased the percent of retardation of the RcsABEC/DNA complex about 3-fold.Figure 2Retardation of the E. coli wzapromoter by RcsAB. Fragments Pwza 27, Pwza 38, Pwza 41, and Pwza 48 were analyzed in EMSAs with RcsABEC at standard conditions. The retardation of fragment Pwza 55 was furthermore analyzed with heterologous RcsAB proteins: RcsAEC/RcsBEC(lane 1), RcsAEC/RcsBEA(lane 2), RcsAEA/RcsBEA(lane 3), and RcsAPS/RcsBEA (lane 4). Proteins were used in concentrations of 1.7 μm for RcsBEC and RcsBAE, and of 5.7 μmfor RcsAEC, RcsAEA, and RcsAPS, respectively. I, retarded DNA fragments; II, free DNA.View Large Image Figure ViewerDownload Hi-res image Download (PPT) The alignment of the 55-bp fragment with the RcsAB binding site of theE. amylovora amsG promoter (18.Wehland M. Kiecker C. Coplin D.L. Kelm O. Saenger W. Bernhard F. J. Biol. Chem. 1999; 274: 3300-3307Abstract Full Text Full Text PDF PubMed Scopus (50) Google Scholar) implicated several nucleotides in the region from −115 to −96 relative to the transcriptional start site of wza as putative targets for RcsAB (Fig. 1). Suspected positions were further analyzed by the introduction of putative up and down mutations according to the RcsABamsG consensus (TableI). The replacement of the degenerated adenine at position −110 by a conserved guanine in the fragments Pwza 72 (G−109) and Pwza 72 (G−109A−108) increased the extent of retardation by RcsABEC (TableII). In contrast, the retardation was considerably reduced after replacing the highly conserved thymine at position −112 in fragment Pwza 72(G−112) by guanine. The retardation was completely abolished in fragment Pwza 72(C−110T−108C−106) carrying mutations in three conserved positions. A decreased percent of retardation in an EMSA was furthermore observed after the replacement of two less conserved adenines by cytosines in the fragment Pwza 72 (C−98C−96). Interestingly, two mutations adjacent to the putative RcsAB consensus also reduced the extent of retardation of fragment Pwza 72 (G−91C−90) by the two proteins (Table II). This gives evidence for additional DNA/protein interactions neighboring the consensus motif.Table IDefinition of the RcsAB box in ResAB binding sitesGeneSpeciesRcsAB boxaThe RcsAB box is in bold, palindromic nucleotide positions within the RcsAB box are underlined, and the colon represents the center of symmetry.LocationbNucleotide positions relative to the translational start site.Ref.cReference for the corresponding sequences.wzaE. coli K12aacc ta aagaa:actccta aaaa−452 /−439(25.Stout V. J. Bacteriol. 1996; 178: 4273-4280Crossref PubMed Google Scholar)galF (orf1)K. pneumoniae K2aaaa t aagatt:attctca cttc−181 /−168(26.Arakawa Y. Wacharotayankun R. Ngatsuka T. Ito H. Kato N. Ohta M. J. Bacteriol. 1995; 177: 1788-1796Crossref PubMed Google Scholar)tviA (vipR)S. typhicgat ta ggaat:attctta tttt−322 /−309(27.Hashimoto Y. Li N. Yokoyama H. Ezaki T. J. Bacteriol. 1993; 175: 4456-4465Crossref PubMed Google Scholar)amsGE. amylovoraatat t gagaat:aatctta attt−550 /−537(28.Bugert P. Geider K. Mol. Microbiol. 1995; 15: 917-935Crossref PubMed Scopus (211) Google Scholar)cpsAP. stewartiiaaca t ggaata:aatctga tttt−537 /−524(18.Wehland M. Kiecker C. Coplin D.L. Kelm O. Saenger W. Bernhard F. J. Biol. Chem. 1999; 274: 3300-3307Abstract Full Text Full Text PDF PubMed Scopus (50) Google Scholar)rcsAE. coli K12atcc t aaggat:tatccga aaaa−264 /−251(29.Blattner F.R. Plunkett III, G. Bloch C.A. Perna N.T. Burlan V. Riley M. Collado-Vides J. Glasner J.D. Rode C.K. Mayhew G.F. Gregor J. Davis N.W. Kirkpatrick H.A. Goeden M.A. Rose D.J. Mau B. Shao Y. Science. 1997; 277: 1453-1474Crossref PubMed Scopus (5935) Google Scholar)rcsAS. typhittac t aaggtt:tatccga aaat−265 /−252(4.Virlogeux I. Waxin H. Ecobichon C. Lee J.O. Popoff M.Y. J. Bacteriol. 1996; 178: 1691-1698Crossref PubMed Scopus (80) Google Scholar)rcsAK. pneumoniaegaag t aaggaa:attctga aagt−257 /−244(5.Allen P. Hart C.A. Saunders J.R. J. Gen. Microbiol. 1987; 133: 331-340PubMed Google Scholar)rcsAE. amylovoraaatt ta agaat:agtccta tcat−318 /−305(7.Bernhard F. Poetter K. Geider K. Coplin D.L. Mol. Plant Microbe Interact. 1990; 3: 429-437Crossref PubMed Scopus (50) Google Scholar–9.Coleman M. Pearce R. Hitchin E. Busfield F. Mansfield J.W. Roberts I.S. J. Gen. Microbiol. 1990; 136: 1799-1806Crossref PubMed Scopus (27) Google Scholar)bvgAB. parapertussisgaat t cagaat:tttccta tttt−175 /−162(30.Scarlato V. Prugnola A. Aricó B. Rappuoli R. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 6753-6757Crossref PubMed Scopus (64) Google Scholar)bvgAB. pertussisgaat t cagact:tttccta tttt−176 /−163(30.Scarlato V. Prugnola A. Aricó B. Rappuoli R. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 6753-6757Crossref PubMed Scopus (64) Google Scholar)fhaB. pertussistgac ta agaaa:tttccta caag−165 /−152(30.Scarlato V. Prugnola A. Aricó B. Rappuoli R. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 6753-6757Crossref PubMed Scopus (64) Google Scholar)ConsensusdConsensus of the 12 presented sequences showing the degree of conservation: uppercase letter ≥ 70%, lower case letter ≥ 50%, dot < 50%.aaa. Ta AGaat:atTCctA .tttThis workRcsABamsGeConsensus of the in vitro selected RcsAB box in the context of the E. amylovora amsG promoter; R = A + G, V = A + C + G, W = A + T, S = G + C, Y = C + T.TRVGAAW:AWTSYGR(18.Wehland M. Kiecker C. Coplin D.L. Kelm O. Saenger W. Bernhard F. J. Biol. Chem. 1999; 274: 3300-3307Abstract Full Text Full Text PDF PubMed Scopus (50) Google Scholar)luxIbox acc tgtagga:tcgtaca ggt(31)a The RcsAB box is in bold, palindromic nucleotide positions within the RcsAB box are underlined, and the colon represents the center of symmetry.b Nucleotide positions relative to the translational start site.c Reference for the corresponding sequences.d Consensus of the 12 presented sequences showing the degree of conservation: uppercase letter ≥ 70%, lower case letter ≥ 50%, dot < 50%.e Consensus of the in vitro selected RcsAB box in the context of the E. amylovora amsG promoter; R = A + G, V = A + C + G, W = A + T, S = G + C, Y = C + T. Open table in a new tab Table IIIn vitro analysis of the RcsAB binding site in the E. coli wza promoterFragmentaDNA fragments of 72 bp were used for the EMSA.wza promoter sequencebRelative to the wza transcriptional start site from position −112 to −96 and further 3′ located nucleotides if applicable. Nucleotide positions analysed by mutagenesis are underlined.RetardationcProtein concentrations used for EMSA analysis were 2 μm RcsABEC.%Pwza 72TAAAGAAACTCCTAAAA25.2 ± 3.4Pwza 72(G−112)GAAAGAAACTCCTAAAA3.6 ± 0.1Pwza 72(C−110T−108C−106)TACATACACTCCTAAAA0Pwza 72(G−109)TAAGGAAACTCCTAAAA33.6 ± 1.2Pwza 72(G−109A−108)TAAGAAAACTCCTAAAA30.3 ± 2.2Pwza 72(C−98C−96)TAAAGAAACTCCTACAC8.6 ± 1.4Pwza 72(G−112C−109T−108C−107)GAACTCAACTCCTAAAA0Pwza 72(G−91C−90)TAAAGAAACTCCTAAAA-N4-GC14.3 ± 2.1Pwza 72(C−70G−69G−68A−67)TAAAGAAACTCCTAAAA-N25-CGGA22.2 ± 1.8a DNA fragments of 72 bp were used for the EMSA.b Relative to the wza transcriptional start site from position −112 to −96 and further 3′ located nucleotides if applicable. Nucleotide positions analysed by mutagenesis are underlined.c Protein concentrations used for EMSA analysis were 2 μm RcsABEC. Open table in a new tab To analyze whether the wza promoter exhibits some preference for the recognition by the homologous RcsABEC proteins, we used the 55-bp fragment of the wza promoter as a target in EMSAs with various combinations of the RcsAEA, RcsAEC, RcsAPS, RcsBEA, and RcsBEC proteins (Fig. 2). The wza promoter was recognized by the heterologous Rcs proteins in all combinations tested, and we could not detect significant differences in the extent of retardation of the DNA fragment. We observed no binding of RcsAEC or RcsBEC alone to the 55-bp fragment, or to the 485-bp fragment containing the complete wzapromoter in concentrations up to 4.5 μm. In contrast, approximately 0.2 μm of the two proteins together already retarded these DNA fragments in EMSAs. The DNA fragment Pwza 48 spanning the nucleotide positions −95 to −48, including an inverted repeat sequence, was retarded neither by RcsAEC nor by RcsABEC (Fig. 2). In addition, the mutation of four bases in the inverted repeat sequence of the fragment Pwza 72(C−68G−67G−66A−65) did not show any effect on the retardation by RcsABEC(Table II). The binding kinetics of the RcsABEC heterodimer at Pwza 72 was analyzed by the surface plasmon resonance technique (Fig. 3). With protein concentrations in a range of 47 nm and 7.5 μm, the ka was calculated to 5.4 ± 3.3 × 104m−1s−1 and the kd to 1.4 ± 0.4 × 10−3 s−1, resulting in aKD of 77 ± 28 nm. TheKD of RcsABEC at the E. coli wza promoter corresponds to the KD of RcsABEA at the E. amylovora amsG promoter previously determined by the EMSA technique (18.Wehland M. Kiecker C. Coplin D.L. Kelm O. Saenger W. Bernhard F. J. Biol. Chem. 1999; 274: 3300-3307Abstract Full Text Full Text PDF PubMed Scopus (50) Google Scholar). We furthermore analyzed the DNA fragment Pwza 72(G−112C−109T−108C−107), containing four point mutations in highly conserved positions (Fig. 1). This fragment was not retarded by RcsAB in an EMSA (Table II). Theka was calculated to 560 ± 140m−1 s−1 and thekd to 2.4 ± 1.0 × 10−2s−1, resulting in an approximately 103-fold increased KD of 50 ± 30 μm. The chromosomal merodiploids MW29 and MW31 of theE. coli K12 strain C600 were constructed after integration of the two plasmids pMW29 and pMW31 by homologous recombination. Thewza promoter of strain MW31 was truncated just upstream of the RcsAB box, and strain MW29 contained additionally four point mutations in essential bases within the identified RcsAB binding site. The phenotypes of the mutants were assayed after introduction of plasmid pEA101 containing the E. amylovora rcsA gene, which resulted in the wild type strain C600 in the induction of colanic acid biosynthesis by activation of the wza promoter. The EPS production and the phenotype of the control mutant MW31 × pEA101 was not altered compared with the wild type strain C600 × pEA101 (Table III). Thus, the approximately 450-bp fragment upstream of wza is sufficient for full promoter activity. In contrast, the EPS production of the mutant MW29 × pEA101 was drastically reduced and the mutant showed a butyrous colony type (Table III). These results demonstrate the importance of the identified RcsAB binding site for the activation of colanic acid biosynthesis, and they indicate that RcsAB might also bindin vivo to that region.Table IIIPhenotype of mutated RcsAB boxes in E. coliStrainaRelevant genotype. (genotype)PlasmidColony typebColony type determined after 24 h of growth on LB-agar at 37 °C.EPS productioncAfter 24 h of growth on LB-agar at 37 °C and estimated with the anthron assay. Means of at least three determinations. —, not applicable.cpsBexpressiondβ-Galactosidase units estimated after Miller (24). Means of at least three determinations.μg glucose/10 8 cellsLacZ unitsC600 (wt)B——pEA101F5.8 ± 0.6—MW31B——pEA101F6.0 ± 0.4—MW29B——pEA101B0.3 ± 0.1—DH5α (wt)B——prcsA-WTF15.2 ± 0.3—prcsA-M4B0.3 ± 0.1—SG1087 (rcsA,lon)B——prcsA-M4F9.9 ± 0.6—JB3034 (rcsA, lon,cpsB∷lacZ)——2 ± 0.1prcsA-WT——382 ± 84prcsA-M4——67 ± 38a Relevant genotype.b Colony type determined after 24 h of growth on LB-agar at 37 °C.c After 24 h of growth on LB-agar at 37 °C and estimated with the anthron assay. Means of at least three determinations. —, not applicable.d β-Galactosidase units estimated after Miller (24.Miller J.H. Experiments in Molecular Genetics. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1972Google Scholar). Means of at least three determinations. Open table in a new tab The autoregulation of E. coli rcsA has been reported previously (16.Ebel W. Trempy J.E. J. Bacteriol. 1999; 181: 577-584Crossref PubMed Google Scholar), and we investigated whether the activation of rcsA promoters is also directed via DNA binding of RcsAB. The 277-bp PCR fragment PrcsA EC277 containing the intergenic region between the E. coli fliR and rcsA genes including the start codon of rcsA EC was clearly retarded by RcsABEC (Fig.4 A). A putative RcsAB binding site was detected at nucleotide positions −264 to −251 relative to the translational start site of rcsA EC (TableI). Accordingly, the reconstituted 34-bp DNA fragment PrcsA EC34 spanning nucleotide positions −274 to −241 was retarded by RcsABEC (Fig. 4 A). Among the most critical positions for RcsAB binding are three conserved purines most likely represented by the sequence GGA at positions −261 to −259 in the rcsA EC promoter. The mutation of the three purines to the sequence TTC in fragment PrcsA ECM completely abolished retardation of the 277-bp fragment by RcsABEC (Fig. 4 A). This demonstrated that the proposed sequence is essential for the in vitro binding of the Rcs proteins to thercsA EC promoter. The rcsAautoregulation appears to be dependent on the presence of both proteins, as neither RcsA nor RcsB alone in concentrations of up to 4.5 μm were able to shift the 277-bp fragment of thercsA EC promoter in EMSAs. If the autoregulation of rcsA expression is a conserved mechanism, an RcsAB binding site should also be present in thercsA promoters of other species. We detected putative RcsAB binding sites in the promoter regions of rcsA genes fromE. amylovora, K. pneumoniae and S. typhi at locations between −321 to − 244 relative to the translational start sites (Table I). We could demonstrate an interaction of RcsABEC with the 29-bp fragment PrcsA EA from −331 to −302 of E" @default.
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• W2028279314 title "The RcsAB Box" @default.
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