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• W2046167506 abstract "Alginates are industrially important, linear copolymers of β-d-mannuronic acid (M) and its C-5-epimer α-l-guluronic acid (G). The G residues originate from a postpolymerization reaction catalyzed by mannuronan C-5-epimerases (MEs), leading to extensive variability in M/G ratios and distribution patterns. Alginates containing long continuous stretches of G residues (G blocks) can form strong gels, a polymer type not found in alginate-producing bacteria belonging to the genus Pseudomonas. Here we show that the Pseudomonas syringae genome encodes a Ca2+-dependent ME (PsmE) that efficiently forms such G blocks in vitro. The deduced PsmE protein consists of 1610 amino acids and is a modular enzyme related to the previously characterized family of Azotobacter vinelandii ME (AlgE1–7). A- and R-like modules with sequence similarity to those in the AlgE enzymes are found in PsmE, and the A module of PsmE (PsmEA) was found to be sufficient for epimerization. Interestingly, an R module from AlgE4 stimulated Ps-mEA activity. PsmE contains two regions designated M and RTX, both presumably involved in the binding of Ca2+. Bacterial alginates are partly acetylated, and such modified residues cannot be epimerized. Based on a detailed computer-assisted analysis and experimental studies another PsmE region, designated N, was found to encode an acetylhydrolase. By the combined action of N and A PsmE was capable of redesigning an extensively acetylated alginate low in G from a non gel-forming to a gel-forming state. Such a property has to our knowledge not been previously reported for an enzyme acting on a polysaccharide. Alginates are industrially important, linear copolymers of β-d-mannuronic acid (M) and its C-5-epimer α-l-guluronic acid (G). The G residues originate from a postpolymerization reaction catalyzed by mannuronan C-5-epimerases (MEs), leading to extensive variability in M/G ratios and distribution patterns. Alginates containing long continuous stretches of G residues (G blocks) can form strong gels, a polymer type not found in alginate-producing bacteria belonging to the genus Pseudomonas. Here we show that the Pseudomonas syringae genome encodes a Ca2+-dependent ME (PsmE) that efficiently forms such G blocks in vitro. The deduced PsmE protein consists of 1610 amino acids and is a modular enzyme related to the previously characterized family of Azotobacter vinelandii ME (AlgE1–7). A- and R-like modules with sequence similarity to those in the AlgE enzymes are found in PsmE, and the A module of PsmE (PsmEA) was found to be sufficient for epimerization. Interestingly, an R module from AlgE4 stimulated Ps-mEA activity. PsmE contains two regions designated M and RTX, both presumably involved in the binding of Ca2+. Bacterial alginates are partly acetylated, and such modified residues cannot be epimerized. Based on a detailed computer-assisted analysis and experimental studies another PsmE region, designated N, was found to encode an acetylhydrolase. By the combined action of N and A PsmE was capable of redesigning an extensively acetylated alginate low in G from a non gel-forming to a gel-forming state. Such a property has to our knowledge not been previously reported for an enzyme acting on a polysaccharide. Alginate is a family of biopolymers produced by brown algae and by some bacteria belonging to the genera Azotobacter and Pseudomonas (1.Stanford, E. C. C. (1881) British patent 142, LondonGoogle Scholar, 2Cote G.L. Krull L.H. Carbohydr. Res. 1988; 181: 143-152Crossref Scopus (59) Google Scholar, 3Gorin P.A.J. Spencer J.F.T. Can. J. Chem. 1966; 44: 993-998Crossref Google Scholar, 4Govan J.R.W. Fyfe J.A.M. Jarman T.R. J. Gen. Microbiol. 1981; 125: 217-220PubMed Google Scholar, 5Linker A. Jones R.S. Nature. 1964; 204: 187-188Crossref PubMed Scopus (103) Google Scholar, 6Osman S.F. Fett W.F. Fishman M.L. J. Bacteriol. 1986; 166: 66-71Crossref PubMed Google Scholar). The polymer consists of 1–4 linked β-d-mannuronic acid (M) 1The abbreviations used are: M, β-d-mannuronic acid; G, α-l-guluronic acid; G-blocks, stretches of contiguous G residues; M-blocks, stretches of contiguous M residues; ME, mannuronan C-5-epimerase; MG-blocks, stretches of contiguous alternating structure ((MG)n); MOPS, 3-(N-morpholino)propanesulfonic acid; ORF, open reading frame; PAF-AH, platelet-activating factor acetylhydrolase; RGAE, rhamnogalacturonan acetylesterase.1The abbreviations used are: M, β-d-mannuronic acid; G, α-l-guluronic acid; G-blocks, stretches of contiguous G residues; M-blocks, stretches of contiguous M residues; ME, mannuronan C-5-epimerase; MG-blocks, stretches of contiguous alternating structure ((MG)n); MOPS, 3-(N-morpholino)propanesulfonic acid; ORF, open reading frame; PAF-AH, platelet-activating factor acetylhydrolase; RGAE, rhamnogalacturonan acetylesterase. and its C-5-epimer α-l-guluronic acid (G) (7Smidsrød O. Draget K.I. Carbohydrates Eur. 1996; 14: 6-13Google Scholar), and the M and G residues are organized in blocks of consecutive M or G residues (M or G blocks) or alternating M and G (MG blocks) (8Haug A. Larsen B. Smidsrød O. Carbohydr. Res. 1974; 32: 217-225Crossref Scopus (396) Google Scholar, 9Haug A. Myklestad S. Larsen B. Smidsrød O. Acta Chem. Scand. 1967; 21: 758-778Google Scholar). The main difference between bacterial and algal alginates is the acetylation of the former polymers at the O2 and/or O3 position of some M residues (10Davidson I.W. Sutherland I.W. Lawson C.J. J. Gen. Microbiol. 1977; 98: 603-606Crossref Scopus (73) Google Scholar, 11Skjåk-Bræk G. Larsen B. Grasdalen H. Carbohydr. Res. 1985; 145: 169-174Crossref Scopus (43) Google Scholar, 12Skjåk-Bræk G. Grasdalen H. Larsen B. Carbohydr. Res. 1986; 154: 239-250Crossref PubMed Scopus (241) Google Scholar). Probably in all species, alginate is first synthesized as mannuronan, and, in a post-polymerization step, M residues are converted to G by mannuronan C-5-epimerases (ME) (13Valla S. Li J. Ertesvåg H. Barbeyron T. Lindahl U. Biochimie (Paris). 2001; 83: 819-830Crossref PubMed Scopus (42) Google Scholar). Acetyl groups protect the residue from epimerization or depolymerization. In Azotobacter vinelandii, which expresses extracellular epimerases, this mechanism controls the degree of epimerization (11Skjåk-Bræk G. Larsen B. Grasdalen H. Carbohydr. Res. 1985; 145: 169-174Crossref Scopus (43) Google Scholar, 14Linker A. Evans L.R. J. Bacteriol. 1984; 159: 958-964Crossref PubMed Google Scholar). In bacteria a periplasmic ME is encoded by algG, which is found in the alginate biosynthetic gene cluster (15Franklin M.J. Chitnis C.E. Gacesa P. Sonesson A. White D.C. Ohman D.E. J. Bacteriol. 1994; 176: 1821-1830Crossref PubMed Google Scholar, 16Peñaloza-Vázquez A. Kidambi S.P. Chakrabarty A.M. Bender C.L. J. Bacteriol. 1997; 179: 4464-4472Crossref PubMed Google Scholar, 17Rehm B.H. Ertesvåg H. Valla S. J. Bacteriol. 1996; 178: 5884-5889Crossref PubMed Google Scholar). Previous studies demonstrated that epimerase-defective algG mutants of Pseudomonas aeruginosa or Pseudomonas fluorescens produce pure polymannuronic acid, which suggests that algG is the sole ME in these bacteria (15Franklin M.J. Chitnis C.E. Gacesa P. Sonesson A. White D.C. Ohman D.E. J. Bacteriol. 1994; 176: 1821-1830Crossref PubMed Google Scholar, 18Gimmestad M. Sletta H. Ertesvåg H. Bakkevig K. Jain S. Suh S.J. Skjåk-Bræk G. Ellingsen T.E. Ohman D.E. Valla S. J. Bacteriol. 2003; 185: 3515-3523Crossref PubMed Scopus (117) Google Scholar). A. vinelandii encodes a family of seven members (AlgE1–7) of Ca2+-dependent ME that are secreted to the surface and extracellular environment. The genes encoding these isoenzymes have been sequenced, cloned, and expressed in our laboratory (19Ertesvåg H. Doseth B. Larsen B. Skjåk-Bræk G. Valla S. J. Bacteriol. 1994; 176: 2846-2853Crossref PubMed Google Scholar, 20Ertesvåg H. Høidal H.K. Hals I.K. Rian A. Doseth B. Valla S. Mol. Microbiol. 1995; 16: 719-731Crossref PubMed Scopus (118) Google Scholar, 21Svanem B.I. Skjåk-Bræk G. Ertesvåg H. Valla S. J. Bacteriol. 1999; 181: 68-77Crossref PubMed Google Scholar). These enzymes can be divided into G block-producing (e.g. AlgE2), and MG block-forming (e.g. AlgE4) enzymes (22Ertesvåg H. Høidal H.K. Schjerven H. Svanem B.I. Valla S. Metab. Eng. 1999; 1: 262-269Crossref PubMed Scopus (55) Google Scholar, 23Høidal H.K. Ertesvåg H. Skjåk-Bræk G. Stokke B.T. Valla S. J. Biol. Chem. 1999; 274: 12316-12322Abstract Full Text Full Text PDF PubMed Scopus (68) Google Scholar, 24Ramstad M.V. Ellingsen T.E. Josefsen K.D. Høidal H.K. Valla S. Skjåk-Bræk G. Levine D. Enzyme Microb. Technol. 1999; 24: 636-646Crossref Scopus (20) Google Scholar), which are composed of varying numbers of two modules, A (about 385 amino acids) and R (about 150 amino acids). By using AlgE1 as a model, the A module was shown to determine the epimerization pattern and to be sufficient for epimerization, whereas the reaction rate is influenced by the R modules (25Ertesvåg H. Valla S. J. Bacteriol. 1999; 181: 3033-3038Crossref PubMed Google Scholar). Plant pathogenic bacteria are able to sense changes in their environment and can adapt accordingly by altering the expression of genes specifically required during pathogenesis or epiphytic growth. For example, P. syringae pv. glycinea PG4180 causes typical bacterial blight symptoms on soybean plants when the bacteria are grown at 18 °C prior to inoculation, but not from bacteria grown at 28 °C (26Budde I.P. Ullrich M.S. Mol. Plant Microbe Interact. 2000; 13: 951-961Crossref PubMed Scopus (47) Google Scholar). Consistent with this, PG4180 produced optimal levels of the virulence factor coronatine at 18 °C and negligible amounts at 28 °C (27Palmer D.A. Bender C.L. Appl. Environ. Microbiol. 1993; 59: 1619-1626Crossref PubMed Google Scholar). In addition to coronatine, alginate is produced as a loosely attached capsule by many P. syringae strains, and the production seems to be correlated with virulence (28Keith R.C. Keith L.M. Hernandez-Guzman G. Uppalapati S.R. Bender C.L. Microbiology. 2003; 149: 1127-1138Crossref PubMed Scopus (50) Google Scholar, 29Yu J. Peñaloza-Vázquez A. Chakrabarty A.M. Bender C.L. Mol. Microbiol. 1999; 33: 712-720Crossref PubMed Scopus (143) Google Scholar). Although PG4180 also produces alginate (30.Keith, R. C. (2002) Expression of Alginate in Response to Environmental Stress and Plant Signals. M.S. thesis, 91 pp., Oklahoma State UniversityGoogle Scholar), temperature-dependent production of alginate has not been reported for this strain. Unlike alginates from Azotobacter sp., those produced by Pseudomonas sp. are not known to contain homopolymeric G blocks (31Skjåk-Bræk G. Espevik T. Carbohydrates Eur. 1996; 14: 19-25Google Scholar). Ullrich et al. (32Ullrich M.S. Schergaut M. Boch J. Ullrich B. Microbiology. 2000; 146: 2457-2468Crossref PubMed Scopus (22) Google Scholar) used a promoter-trapping strategy to identify P. syringae PG4180 promoters with induced expression at 18 °C when compared with 28 °C. Sequencing of several hundred nucleotides of the transcriptional fusion contained in plasmid p561 revealed the presence of an open reading frame with 53% amino acid identity to the extracellular epimerase AlgE2 from A. vinelandii (32Ullrich M.S. Schergaut M. Boch J. Ullrich B. Microbiology. 2000; 146: 2457-2468Crossref PubMed Scopus (22) Google Scholar). In this report, we describe the molecular cloning and characterization of this gene (designated psmE) and show that it encodes a bifunctional enzyme possessing both G block-forming ME activity and mannuronan-O-acetylhydrolase activity. Growth of Bacteria—The bacterial strains and plasmids used in this study are listed in Table I. Bacteria were grown at 37 °C in L broth or on L agar supplemented with 200 μg/ml ampicillin or 12.5 μg/ml tetracycline when appropriate.Table IBacterial strains and plasmidsStrains or plasmidsRelevant characteristicsReferencesEscherichia coliDH5α(33Sambrook J. Russell D. Molecular Cloning: A Laboratory Manual. Third Ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY2001Google Scholar)ER2566Contains T7 RNA polymeraseNew England BiolabsP. syringae pv. glycineaPG4180Pathogenic on soybeans, isolated from soybeans cultivated in New Zealand, 1975(69Michell R.E. Physiol. Plant Pathol. 1982; 20: 83-89Crossref Scopus (81) Google Scholar, 70Young J.M. International Collection of Microorganisms from Plants.Catalogue to Accessions 1–9519. Plant Diseases Division, Department of Scientific and Industrial Research, Auckland, New Zealand1988Google Scholar)PlasmidspBluescript II SK+ColEI replicon, cloning vector, ApRStratagenepET-21aContains a T7 promoter and His tag peptide, ApRNovagenpTrc99AColE1-replicon, lacIq, ApR(71Amann E. Ochs B. Abel K.J. Gene (Amst.). 1988; 69: 301-315Crossref PubMed Scopus (877) Google Scholar)pRK7813Cosmid vector, TcR(72Jones J.D. Gutterson N. Gene (Amst.). 1987; 61: 299-306Crossref PubMed Scopus (118) Google Scholar)p561Contains 2.0 kb of PG4180 DNA in pBluescript II SK+, ApR(32Ullrich M.S. Schergaut M. Boch J. Ullrich B. Microbiology. 2000; 146: 2457-2468Crossref PubMed Scopus (22) Google Scholar)pBL5Derivative of pTrc99A encoding algE4 from A. vinelandii, Apr(73Bjerkan T.M. Lillehov B.E. Strand W.I. Skjåk-Bræk G. Valla S. Ertesvåg H. Biochem. J. 2004; (in press)Google Scholar)pMF9Cosmid clone from a P. syringae pv. glycinea PG4180 library containing a ∼40-kb insert in pRK7813, TcRThis studypMF9.1Derivative of pBluescript II SK+ containing a 4.4-kb AflIII fragment from pMF9 end-filled with Klenow and ligated into the SmaI site of the vector, ApRThis studypMF9.2Derivative of pET21a where a 4.4-kb EcoRI-SacI fragment of pMF9.1 was ligated into the corresponding sites of the vector, ApRThis studypMT9.2Derivative of pET21a where a 11-kb HindIII fragment of pMF9 was ligated into the corresponding site of the vector, ApRThis studypTB46Derivative of pMF9.2 in which a 1.7-kb EcoRI (T4 filled-in)-Acc65I fragment from pBL5 was ligated into the XhoI (T4 filled-in)-Acc65I sites of the vector, ApRThis studypTB47Derivative of pTB46 where a 1.0-kb KpnI-XmaI fragment was deleted, ApRThis studypTB48Derivative of pMF9.2 where a 3.3-kb Acc65I-SpeI fragment was deleted, ApRThis studypTB49Derivative of pMF9.2 in which a single nucleotide substitution resulted in a BspHI site overlapping the psmE start codon, ApRThis studypTB50Derivative of pTB47 where a single nucleotide substitution resulted in a BspHI site overlapping the psmE start codon, ApRThis studypTB51Derivative of pTB48 where the 640-bp EcoRI-NcoI fragment was substituted with the corresponding DNA fragment in pTB50, ApRThis studypTB53Derivative of pMF9.2 where a 4.9-kb KpnI-SacI fragment from pMT9.2 was ligated into the corresponding sites of pMF9.2, ApRThis studypTB54Derivative of pTB53 where the 640-bp EcoRI-NcoI fragment was substituted with the corresponding DNA fragment in pTB50, ApRThis study Open table in a new tab Standard Recombinant DNA Technology—Standard recombinant DNA procedures were performed according to Sambrook and Russell (33Sambrook J. Russell D. Molecular Cloning: A Laboratory Manual. Third Ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY2001Google Scholar), whereas transformations utilizing rubidium chloride were performed according to a Northwest Fisheries Science Center protocol (available at micro.nwfsc.noaa.gov/protocols/rbcl.html). Plasmids were isolated using the Qiagen midi kit (Qiagen) or the Wizard plus SV minipreps kit (Promega). All cloning was done in Escherichia coli DH5α, and the expression plasmids were later transferred to E. coli ER2566. DNA sequencing was performed using the ABI Prism Dye Primer cycle sequencing kit (PerkinElmer Life Sciences) on an ABI 373A apparatus. Construction and Screening of a P. syringae pv. glycinea PG4180 Gene Library—For library construction, genomic DNA of P. syringae pv. glycinea PG4180 was isolated as described by Staskawicz et al. (34Staskawicz B.J. Dahlbeck D. Keen N.T. Proc. Natl. Acad. Sci. U. S. A. 1984; 81: 6024-6028Crossref PubMed Scopus (236) Google Scholar) and purified on CsCl-EtBr gradients (35Sambrook J. Fritsch E.F. Maniatis T. Molecular Cloning: A Laboratory Manual. 2nd Ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY1989Google Scholar). A PG4180 genomic library was constructed in pRK7813 as described previously (36Barta T.M. Kinscherf T.G. Willis D.K. J. Bacteriol. 1992; 174: 3021-3029Crossref PubMed Google Scholar), and Tcr E. coli transfectants were screened by colony hybridization. Plasmid p561 was provided by Dr. Matthias Ullrich (International University Bremen, Bremen, Germany). An 835-bp DNA probe was amplified from p561 by PCR amplification using the following primers: 5′-ATACAGCAGCCATTCAGGCCACTA-3′ and 5′-TGCTCAGGGTGTTATCAAAGACATCCAC-3′. The amplified DNA fragment was isolated from agarose gels and labeled with digoxigenin using the Genius Labeling and Detection kit (Roche Applied Science) or with [α-32P]dCTP using the Rad Prime DNA Labeling System (Invitrogen). Hybridization and post-hybridization washes to the PG4180 cosmid library were conducted using high stringency conditions. Sequence Analysis—Sequence manipulations, amino acid alignments, phenograms, and restriction maps were constructed using the Sci Ed Central Clone Manager Professional Suite. Data base searches were performed with the BLAST service of the National Center for Biotechnology Information. Preliminary genomic sequence data were obtained for P. syringae pv. tomato DC3000 from The Institute for Genomic Research (www.tigr.org/), and for P. syringae pv. syringae B728a from the Department of Energy Joint Genome Institute (www.jgi.doe.gov/). Fold recognition was done by using the Structure Prediction Meta Server (bioinfo.pl/Meta/). This web server combines the output from several different prediction methods through a jury system (3D-Jury) (37Ginalski K. Elofsson A. Fischer D. Rychlewski L. Bioinformatics. 2003; 19: 1015-1018Crossref PubMed Scopus (656) Google Scholar). Classification of 3D structures into fold classes were based on the database Structural Classification of Protein (38Lo Conte L. Brenner S.E. Hubbard T.J. Chothia C. Murzin A.G. Nucleic Acids Res. 2002; 30: 264-267Crossref PubMed Google Scholar, 39Murzin A.G. Brenner S.E. Hubbard T. Chothia C. J. Mol. Biol. 1995; 247: 536-540Crossref PubMed Scopus (5587) Google Scholar). Structure data were retrieved from the Protein Data Bank (40Berman H.M. Westbrook J. Feng Z. Gilliland G. Bhat T.N. Weissig H. Shindyalov I.N. Bourne P.E. Nucleic Acids Res. 2000; 28: 235-242Crossref PubMed Scopus (27313) Google Scholar), and fold-related alignments were generated with ClustalX (41Thompson J.D. Gibson T.J. Plewniak F. Jeanmougin F. Higgins D.G. Nucleic Acids Res. 1997; 25: 4876-4882Crossref PubMed Scopus (35409) Google Scholar) and Alscript (42Barton G.J. Protein Eng. 1993; 6: 37-40Crossref PubMed Scopus (1110) Google Scholar). In Vitro Mutagenesis—The QuikChange™ site-directed mutagenesis kit (Stratagene) was used according to the manufacturer's instructions. Two primers were used to introduce a BspHI site comprising the start codon; forward: 5′-CCGGAGTAACATCATGATATTAAACAC-3′ and reverse: 5′-GTGTTTAATATCATGATGTTACTCCGG -3′. Changed nucleotides are underlined, and the resulting BspHI sites are shown in bold. Alginate Substrates—5-3H-Labeled, chemically deacetylated alginate and unlabeled, O-acetylated alginate were prepared from P. aeruginosa (17Rehm B.H. Ertesvåg H. Valla S. J. Bacteriol. 1996; 178: 5884-5889Crossref PubMed Google Scholar) and contained less than 7% G residues. The 1-13C-labeled and unlabeled mannuronan (100% M, chemically deacetylated) were prepared from an epimerase-defective (algG) P. fluorescens mutant (18Gimmestad M. Sletta H. Ertesvåg H. Bakkevig K. Jain S. Suh S.J. Skjåk-Bræk G. Ellingsen T.E. Ohman D.E. Valla S. J. Bacteriol. 2003; 185: 3515-3523Crossref PubMed Scopus (117) Google Scholar, 43Hartmann M. Duun A.S. Markussen S. Grasdalen H. Valla S. Skjåk-Bræk G. Biochim. Biophys. Acta. 2002; 1570: 104-112Crossref PubMed Scopus (23) Google Scholar). Alginate containing alternating MG residues (MG-alginate) was produced in vitro from mannuronan by using recombinantly produced AlgE4 (23Høidal H.K. Ertesvåg H. Skjåk-Bræk G. Stokke B.T. Valla S. J. Biol. Chem. 1999; 274: 12316-12322Abstract Full Text Full Text PDF PubMed Scopus (68) Google Scholar). Alginate from the leaves of Laminaria hyperborea and LF 10/60, which is an L. hyperborea stripe alginate, were obtained from FMC Biochemicals, Drammen, Norway. Alginate from Macrocystis pyrifera was obtained from Sigma. Measurement of Epimerase Activity by Radiolabeling—For enzyme purification, E. coli ER2566 cells containing selected plasmids were grown in a medium containing 30 g/liter Tryptone, 15 g/liter yeast extract, and 5 g/liter NaCl. Enzyme extracts were prepared and partially purified by fast protein liquid chromatography as described previously (22Ertesvåg H. Høidal H.K. Schjerven H. Svanem B.I. Valla S. Metab. Eng. 1999; 1: 262-269Crossref PubMed Scopus (55) Google Scholar). Crude extracts for which activities are indicated were prepared by growing the cells for 3 h at 37 °C in medium supplemented with 5 mm CaCl2. The temperature was lowered to 18 °C, and cultures then induced with isopropyl-1-thio-β-d-galactopyranoside (0.25 mm) and grown at the low temperature for 16 h before harvesting. 20 mm MOPS (pH 6.8, containing 4 mm CaCl2) was used for cell disruption and 20 mm MOPS (pH 6.8, containing 1 mm CaCl2) for protein purification. In the assay 50 mm MOPS with varying pH and CaCl2 concentrations was used. Epimerase activities were quantified by measuring the liberation of tritium from [5-3H]alginate to water as described previously (44Ertesvåg H. Skjåk-Bræk G. Bucke C. Methods in Biotechnology 10, Carbohydrate Biotechnology Protocols. Humana Press Inc., Totowa, NJ1999: 71-78Google Scholar). One unit is defined as the amount of enzyme needed to epimerize 1 μmol of substrate (sugar residues in deacetylated mannuronan) in 1 min. The amounts of protein in the samples were estimated by using the Bio-Rad Coomassie Brilliant Blue-based protein assay (Bio-Rad). Measurement of Epimerase and Acetyl Hydrolase Activities by 1H NMR—In general low molecular weight mannuronan with a degree of polymerization, DPn, of ∼30 was used as substrate in the epimerase assay. In one experiment a high molecular weight mannuronan was used. Other alginate substrates were epimerized under similar conditions. In the acetylhydrolase assay, a high molecular weight P. aeruginosa alginate with an initial fraction of O-acetyl groups of 0.7 and an initial fraction of G, FG, of 0.07 was used as the substrate. The reactions were performed in a total volume of 6 ml, containing 20 mm Mops (pH 6.8), 1 mm CaCl2 (3 mm CaCl2 in the acetylhydrolase experiments), and 7.5 mg of alginate. Different reaction levels were achieved by varying the amount of enzyme or the incubation time. High molecular weight samples were partially hydrolyzed prior to the 1H NMR recordings as described previously (44Ertesvåg H. Skjåk-Bræk G. Bucke C. Methods in Biotechnology 10, Carbohydrate Biotechnology Protocols. Humana Press Inc., Totowa, NJ1999: 71-78Google Scholar). NMR spectra were recorded on a Bruker DPX 300 (300 MHz) spectrometer, and FG, FGG, FMM, FGM,MG, FMGM, and FGGG were calculated from the integrated spectra as described by Grasdalen (45Grasdalen H. Carbohydr. Res. 1983; 118: 255-260Crossref Scopus (424) Google Scholar). The degree of acetylation was calculated from the integrated spectra as described by Skjåk-Bræk et al. (12Skjåk-Bræk G. Grasdalen H. Larsen B. Carbohydr. Res. 1986; 154: 239-250Crossref PubMed Scopus (241) Google Scholar). Portions of these samples were chemically deacetylated (44Ertesvåg H. Skjåk-Bræk G. Bucke C. Methods in Biotechnology 10, Carbohydrate Biotechnology Protocols. Humana Press Inc., Totowa, NJ1999: 71-78Google Scholar) prior to the 1H NMR recordings to permit calculation of the degree of epimerization. Time-resolved 13C NMR Spectroscopy—Prior to the NMR recording, the epimerase was partially purified by fast protein liquid chromatography, dialyzed against a low ionic strength buffer, and lyophilized. Individual solutions (final concentrations are shown) in D2O of the different components, Tris-HCl (10 mm, pH 7.4 at 37 °C), [1-13C]mannuronan (9 mg), CaCl2 (2.5 mm), NaCl (20 mm), and epimerase (1 mg of lyophilized powder), were prepared separately, and calculated volumes were then transferred into an NMR sample tube (0.5-ml volume). Spectra were recorded on a Bruker DPX 300 (75 MHz) spectrometer. To monitor the progress of a single epimerization experiment, a series of 60 successive 13C NMR spectra were recorded as described by Hartmann et al. (43Hartmann M. Duun A.S. Markussen S. Grasdalen H. Valla S. Skjåk-Bræk G. Biochim. Biophys. Acta. 2002; 1570: 104-112Crossref PubMed Scopus (23) Google Scholar). Each spectrum was calculated from 400 scans, which represents an average of 18 min. We chose to set the time for each result as the end-time for each uptake. The scanning for the first spectrum was started 13 min after the addition of the enzyme. The reaction was continued until the fraction of G residues no longer increased. The annotation of the signals and the calculation of FG, FM, FGG, FMG, FMM, and FGM from the integrated spectra was done according to Grasdalen et al. (46Grasdalen H. Larsen B. Smidsrød O. Carbohydr. Res. 1981; 89: 179-191Crossref Scopus (298) Google Scholar). Cloning of the Putative Mannuronan C-5-epimerase from P. syringae—The 2.0-kb insert in plasmid p561, previously reported to contain an algE2-like region (32Ullrich M.S. Schergaut M. Boch J. Ullrich B. Microbiology. 2000; 146: 2457-2468Crossref PubMed Scopus (22) Google Scholar), was fully sequenced, and found to be similar to the entire A module of algE2 and the corresponding parts of the six other known A. vinelandii algE genes. In silico translation of the sequence revealed an ORF encoding the putative A module beginning 346 bp from the mini-Tn5 insertion in p561; however, a stop codon was not identified within the 2.0-kb cloned fragment. Therefore, it was necessary to obtain a clone containing more of the 3′ part of the putative gene, and for this purpose a cosmid library of P. syringae pv. glycinea PG4180 was constructed in pRK7813. An 835-bp PCR fragment derived from the A module region of p561 was used to screen the library for clones containing the full-length ORF. Several cosmids were hybridized with the probe, and a clone designated pMF9 was chosen for further analysis. An 11-kb HindIII fragment from the insert in pMF9 was cloned in pET21a, forming pMT9.2. Sequence analysis of this insert revealed a stop codon in a 4830-bp ORF, which contained the putative epimerase. The gene represented by this ORF was designated psmE. Sequence Analysis of the Deduced psmE Product—Inspection of the deduced amino acid sequence of PsmE showed that it contains 1610 amino acids and has a modular structure that can be described as A-R1-R2-M-R3-N-RTX-S (Fig. 1A), where A and R refer to sequences sharing similarity with the A and R modules of AlgE1–7. S refers to a sequence at the C terminus, which is also similar to the corresponding ends of the AlgE epimerases. The putative A module in PsmE comprises 383 amino acids and terminates with the sequence FPLVT. This module shares 61–67% nucleotide and 54–61% amino acid sequence identity to the A. vinelandii AlgE A modules (Fig. 1B), clearly suggesting an evolutionary relationship (Fig. 2A). The next 150 amino acids (residues 384–533) comprise the R1 module in PsmE. With the exception of AlgE4, the A. vinelandii AlgE epimerases contain more R than A modules, and the diversity of their sequences is also somewhat broader than among the A modules (20Ertesvåg H. Høidal H.K. Hals I.K. Rian A. Doseth B. Valla S. Mol. Microbiol. 1995; 16: 719-731Crossref PubMed Scopus (118) Google Scholar, 21Svanem B.I. Skjåk-Bræk G. Ertesvåg H. Valla S. J. Bacteriol. 1999; 181: 68-77Crossref PubMed Google Scholar). The phenogram in Fig. 2B illustrates that the PsmE R1 module is more similar to the A. vinelandii AlgE R modules than to R2 and R3 (154 amino acids each) in PsmE.Fig. 2Phenograms constructed based on the multiple sequence alignments. A, phylogenetic tree showing relatedness of A-modules of PsmE epimerases from different P. syringae strains and those of AlgE1–7 and AlgY from A. vinelandii. B, phylogenetic tree as in panel A, for R-modules of PsmE and selected AlgE1–7 and AlgY R-modules. The A. vinelandii epimerase R-modules were chosen based on a phenogram presented by Svanem et al. (21Svanem B.I. Skjåk-Bræk G. Ertesvåg H. Valla S. J. Bacteriol. 1999; 181: 68-77Crossref PubMed Google Scholar) to represent the whole group of R-modules.View Large Image Figure ViewerDownload (PPT) The designation RTX (repeat in toxin) refers to a motif that is tandemly repeated and present in a family of proteins synthesized by a diverse group of Gram-negative bacteria (47Lally E.T. Hill R.B. Kieba I.R. Korostoff J. Trends Microbiol. 1999; 7: 356-361Abstract Full Text Full Text PDF PubMed Scopus (213) Google Scholar). This portion of PsmE (370 amino acids residues) contains the COG2931 motif characteristic of RTX toxins and related Ca2+-binding proteins (48Welch R.A. Curr. Top. Microbiol. Immunol. 2001; 257: 85-111PubMed Google Scholar) and shares significant similarity to the putative hemolysin-type Ca2+-binding RTX proteins in P. putida (39% sequence similarity) (49Nelson K.E. Weinel C. Paulsen I.T. Dodson R.J. Hilbert H. Martins dos" @default.
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• W2046167506 title "The Pseudomonas syringae Genome Encodes a Combined Mannuronan C-5-epimerase and O-Acetylhydrolase, Which Strongly Enhances the Predicted Gel-forming Properties of Alginates" @default.
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