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• W2034129394 abstract "Eleven bacteria capable of utilizing cyanophycin (cyanophycin granule polypeptide (CGP)) as a carbon source for growth were isolated. One isolate was taxonomically affiliated asPseudomonas anguilliseptica strain BI, and the extracellular cyanophycinase (CphE) was studied because utilization of cyanophycin as a carbon source and extracellular cyanophycinases were hitherto not described. CphE was detected in supernatants of CGP cultures and purified from a corresponding culture of strain BI employing chromatography on the anion exchange matrix Q-Sepharose and on an arginine-agarose affinity matrix. The mature form of the inducible enzyme consisted of one type of subunit withM r = 43,000 and exhibited high specificity for CGP, whereas proteins and synthetic polyaspartic acid were not hydrolyzed or were only marginally hydrolyzed. Degradation products of the enzyme reaction were identified as aspartic acid-arginine dipeptides (β-Asp-Arg) by high performance liquid chromatography and electrospray ionization mass spectrometry. The corresponding gene (cphE, 1254 base pairs) was identified in subclones of a cosmid gene library of strain BI by heterologous active expression inEscherichia coli, and its nucleotide sequence was determined. The enzyme exhibited only 27–28% amino acid sequence identity to intracellular cyanophycinases occurring in cyanobacteria. Analysis of the amino acid sequence of cphE revealed a putative catalytic triad consisting of the motif GXSXG plus a histidine and most probably a glutamate residue. In addition, the strong inhibition of the enzyme by Pefabloc® and phenylmethylsulfonyl fluoride indicated that the catalytic mechanism of CphE is related to that of serine type proteases. Quantitative analysis on the release of β-Asp-Arg dipeptides from C-terminal labeled CGP gave evidence for an exo-degradation mechanism. Eleven bacteria capable of utilizing cyanophycin (cyanophycin granule polypeptide (CGP)) as a carbon source for growth were isolated. One isolate was taxonomically affiliated asPseudomonas anguilliseptica strain BI, and the extracellular cyanophycinase (CphE) was studied because utilization of cyanophycin as a carbon source and extracellular cyanophycinases were hitherto not described. CphE was detected in supernatants of CGP cultures and purified from a corresponding culture of strain BI employing chromatography on the anion exchange matrix Q-Sepharose and on an arginine-agarose affinity matrix. The mature form of the inducible enzyme consisted of one type of subunit withM r = 43,000 and exhibited high specificity for CGP, whereas proteins and synthetic polyaspartic acid were not hydrolyzed or were only marginally hydrolyzed. Degradation products of the enzyme reaction were identified as aspartic acid-arginine dipeptides (β-Asp-Arg) by high performance liquid chromatography and electrospray ionization mass spectrometry. The corresponding gene (cphE, 1254 base pairs) was identified in subclones of a cosmid gene library of strain BI by heterologous active expression inEscherichia coli, and its nucleotide sequence was determined. The enzyme exhibited only 27–28% amino acid sequence identity to intracellular cyanophycinases occurring in cyanobacteria. Analysis of the amino acid sequence of cphE revealed a putative catalytic triad consisting of the motif GXSXG plus a histidine and most probably a glutamate residue. In addition, the strong inhibition of the enzyme by Pefabloc® and phenylmethylsulfonyl fluoride indicated that the catalytic mechanism of CphE is related to that of serine type proteases. Quantitative analysis on the release of β-Asp-Arg dipeptides from C-terminal labeled CGP gave evidence for an exo-degradation mechanism. cyanophycin granule polypeptide (cyanophycin) cyanophycin synthetase cyanophycinase extracellular cyanophycinase bovine serum albumin mass spectrometry electrospray ionization mass spectrometry high performance liquid chromatography Cyanophycin (cyanophycin granule polypeptide, CGP)1 is a naturally occurring poly(amino acid), which is synthesized in most cyanobacteria as nitrogen, carbon, and energy storage compound in the early stationary growth phase (1Mackerras A.H. DeChazal N.M. Smith G.D. J. Gen. Microbiol. 1990; 136: 2057-2065Crossref Scopus (86) Google Scholar, 2Liotenberg S. Campbell D. Rippka R. Houmard J. deMarsac N.T. Microbiology. 1996; 142: 611-622Crossref PubMed Scopus (91) Google Scholar). The water-insoluble CGP is accumulated intracellularly in the form of membraneless granules (3Allen M.M. Weathers P. J. Bacteriol. 1980; 141: 959-962Crossref PubMed Google Scholar) and is degraded by the cells when growth is resumed. The backbone of this unique biopolymer consists of α-amino-α-carboxyl-linked l-aspartic acid monomers. Most of the β-carboxylic groups are covalently bound to the α-amino groups of l-arginine residues (4Simon R.D. Weathers P. Biochim. Biophys. Acta. 1976; 420: 165-176Crossref PubMed Scopus (137) Google Scholar, 5Simon R.D. Lawry N.H. McLendon G.L. Biochim. Biophys. Acta. 1980; 626: 277-281Crossref PubMed Scopus (24) Google Scholar); in recombinantEscherichia coli expressing cyanobacterial CGP-synthesizing enzymes (see below), a significant fraction of arginine is replaced by lysine (6Ziegler K. Diener A. Herpin C. Richter R. Deutzmann R. Lockau W. Eur. J. Biochem. 1998; 254: 154-159Crossref PubMed Scopus (155) Google Scholar). Although much information has been obtained concerning the non-ribosomal biosynthesis of CGP, which is catalyzed by the cyanophycin synthetase (CphA; see Ref. 7Oppermann-Sanio F.B. Steinbüchel A. Naturwissenschaften. 2002; 89 (,): 11-22Crossref PubMed Scopus (172) Google Scholar and cited references therein), only a few reports are available on the intracellular degradation of CGP. Intracellular CGP degradation was first observed in crude extracts of soluble proteins prepared from cells of Anabaena cylindrica (5Simon R.D. Lawry N.H. McLendon G.L. Biochim. Biophys. Acta. 1980; 626: 277-281Crossref PubMed Scopus (24) Google Scholar). The corresponding enzyme, cyanophycinase (CphB), was purified from a recombinant E. coli harboring thecphB gene from Synechocystis sp. PCC6803 and characterized in detail (8Richter R. Hejazi M. Kraft R. Ziegler K. Lockau W. Eur. J. Biochem. 1999; 263: 163-169Crossref PubMed Scopus (96) Google Scholar). Dipeptides consisting of arginine plus aspartic acid and free arginine were identified as products of CGP degradation in addition to small amounts of aspartic acid (8Richter R. Hejazi M. Kraft R. Ziegler K. Lockau W. Eur. J. Biochem. 1999; 263: 163-169Crossref PubMed Scopus (96) Google Scholar). In contrast to intracellular degradation, nothing is known about the extracellular decomposition of this biopolymer by bacteria or other microorganisms. In this study, we demonstrate for the first time that CGP can be easily degraded and utilized as the sole carbon source for growth by a variety of non-cyanobacterial eubacteria isolated from different habitats. Because it is known that CGP is resistant to a wide range of commercially available proteases (4Simon R.D. Weathers P. Biochim. Biophys. Acta. 1976; 420: 165-176Crossref PubMed Scopus (137) Google Scholar, 9Simon R.D. Fay P. van Baalen C. The Cyanobacteria. Elsevier Science Publishers B.V., Amsterdam1987: 199-225Google Scholar), these bacteria must possess an enzyme specialized for CGP degradation. We report on the isolation of a strain of the species Pseudomonas anguillisepticaand describe the substrate utilization capabilities of this bacterium and the purification of an extracellular cyanophycinase (extracellular CGPase (CphE)) from culture supernatants of cells grown on CGP. Furthermore, CphE was biochemically characterized to reveal the degradation mechanism and to identify the cleavage products. In addition, the CGPase gene (cphE) of the isolated P. anguilliseptica strain BI was cloned and characterized. CGP-degrading bacteria isolated in this study are listed in Table I. These strains were either grown on Standard I complex medium (Merck) or grown on basic inorganic medium B (10Claus D. Walker N. J. Gen. Microbiol. 1964; 36: 107-122Crossref PubMed Scopus (46) Google Scholar) for CGP degradation and substrate utilization experiments. The concentrations of CGP and other carbon sources added to the medium are indicated in the text. All isolates were grown at 30 °C. The following microorganisms were used as reference strains in substrate utilization assays on solid CGP medium (see below) with 0.05% (w/v) glucose as an additional carbon source:E. coli K12 (wild type), Pseudomonas putidaKT2440 (11Worsey M.J. Williams P.A. J. Bacteriol. 1975; 124: 7-13Crossref PubMed Google Scholar), Micrococcus luteus (DSMZ 348), Bacillus subtilis 168+ (DSMZ 402), and Bacillus megaterium (DSMZ 319). For CGP production, an E. coliDH1 strain harboring plasmid pMa/c5–914::cphAexpressing cphA from Synechocystis PCC6803 (12Frey, K. M., Oppermann-Sanio, F. B., Schmidt, H., and Steinbüchel, A. (2002) Appl. Environ. Microbiol.DOI:10.1128/AEM.68.7.000-000.2002Google Scholar) was employed (see below). E. coli strains were usually grown at 37 °C in Luria-Bertani (LB) medium or terrific broth (TB) complex medium (13Sambrook J. Fritsch E.F. Maniatis T. Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1989: A1-A2Google Scholar).Table IGrowth of CGP-degrading bacteria on compounds related to cyanophycin or other polyamide substratesIsolateStrainGrowth on carbon source1-aND, taxonomically not determined.1-bGrowth was qualitatively estimated as follows: –, no growth; +, poor growth; ++, moderate growth; +++, good growth; PGA, poly(γ-d-glutamic acid). Strains were grown on basic inorganic medium B with concentrations of 0.25% of each of the applied carbon sources.BSAPGAAspArgAsp + ArgCitrateAVNND––+++++++++BI P. anguilliseptica–––+++++++BIIND––+++++++++DIII P. alcaligenes––++++++++++++DIV P. alcaligenes––++++++++++++DXND––++++++++DxiND––++++++++DXI1ND––+++++++++DXIIIND–++++++++++++++BE21-cStrain BE2 was isolated in a screening for poly(γ-D-glutamic acid) degrading bacteria (33). P. alcaligenes++++++++++++++PAS IND+++––+++++++– B. subtilis+–––+++++Strain AVN was isolated from Baltic sea water; strains BI and BII were obtained from pond sediments and the strains of the D-series were isolated from sewage sludge. B. subtilis 168+was used as a control.1-a ND, taxonomically not determined.1-b Growth was qualitatively estimated as follows: –, no growth; +, poor growth; ++, moderate growth; +++, good growth; PGA, poly(γ-d-glutamic acid). Strains were grown on basic inorganic medium B with concentrations of 0.25% of each of the applied carbon sources.1-c Strain BE2 was isolated in a screening for poly(γ-D-glutamic acid) degrading bacteria (33Oppermann F.B Pickartz S. Steinbüchel A. Polym. Degrad. Stabil. 1998; 59: 337-344Crossref Google Scholar). Open table in a new tab Strain AVN was isolated from Baltic sea water; strains BI and BII were obtained from pond sediments and the strains of the D-series were isolated from sewage sludge. B. subtilis 168+was used as a control. Samples from different sources were spread on solid basic inorganic medium B (10Claus D. Walker N. J. Gen. Microbiol. 1964; 36: 107-122Crossref PubMed Scopus (46) Google Scholar) supplemented with trace element solution SL 7 (14Widdel F. Pfennig N. Arch. Microbiol. 1981; 129: 395-400Crossref PubMed Scopus (607) Google Scholar) and overlaid with 0.5% (w/v) agar containing 0.2% (w/v) of CGP. For the preparation of the overlay agar, diethyl ether-sterilized CGP was first dissolved in 0.1 nHCl and then added to sterile medium under vigorous stirring to avoid the formation of inhomogeneous CGP precipitates. For adjustment of the pH value, an equal volume of 0.1 n NaOH was added before pouring plates. For isolation of plasmid DNA, the lithium preparation method was applied (15He M. Wilde A. Kaderbhai M.A. Nucleic Acids Res. 1990; 18: 1660Crossref PubMed Scopus (66) Google Scholar). Total genomic DNA of P. anguilliseptica strain BI was isolated according to the method of Rao et al. (16Rao R.N. Richardson M.A. Kuhstoss S. Methods Enzymol. 1987; 153: 166-198Crossref PubMed Scopus (74) Google Scholar). After partial digestion with the endonuclease PstI, genomic DNA fragments were ligated to the cosmid vector pHC79 (17Hohn B. Collins J. Gene (Amst.). 1980; 11: 291-298Crossref PubMed Scopus (489) Google Scholar), and E. colistrain S17–1 (18Simon R. Priefer U. Pühler A. Bio/Technology. 1983; 1: 784-790Crossref Scopus (5643) Google Scholar) was used as a recipient for transduction of the cosmid library. A Gigapack® III XL packaging extract (Stratagene, La Jolla, CA) was employed for the packaging of DNA and subsequent infection of strain S17–1 as described by the manufacturer.E. coli strain XL1-Blue (Stratagene) was used in combination with pBluescript SK− (Stratagene) for cloning of a PstI restriction subfragment (2600 bp), sequence analysis of cphE, and heterologous production of the enzyme. 5′-infrared fluorescence dye 800-labeled synthetic oligonucleotides (MWG-Biotech, Ebersberg, Germany) were used as primers, and a Sequi Therm EXCEL TM II long-read cycle sequencing kit (Epicentre Technologies, Madison, WI) was employed for DNA-sequencing according to the “primer-hopping strategy” (19Strauss E.C. Kobori J.A. Siu G. Hood L.E. Anal. Biochem. 1986; 154: 353-360Crossref PubMed Scopus (167) Google Scholar). Analysis was done in 6% (w/v) acrylamide gels using Sequagel XR®(acrylamide/urea), Complete® (buffer reagent) solutions (National Diagnostics, Atlanta, GA), and buffer containing 89 mm Tris, 89 mm boric acid, and 2 mm EDTA in a LI-COR 4000L automatic sequencing apparatus (MWG-Biotech). Nucleic acid sequence data and deduced amino acid sequences were analyzed with the sequence analysis software CAP (Contig Assembly Program; (20Huang X. Genomics. 1992; 14: 18-25Crossref PubMed Scopus (234) Google Scholar)), ClustAlX 1.8 (21Thompson J.D. Gibson T.J. Plewniak F. Jeanmougin F. Higgins D.G. Nucleic Acids Res. 1997; 25: 4876-4882Crossref PubMed Scopus (35497) Google Scholar), and Genamics Expression 1.1. The 16-S rRNA gene was amplified from total DNA (see above) using oligonucleotide primers as described before (22Rainey F.A. Ward-Rainey N. Kroppenstedt R.M. Stackebrandt E. Int. J. Syst. Bacteriol. 1996; 46: 1088-1092Crossref PubMed Scopus (947) Google Scholar). After purification of the PCR products with a NucleoTrap®CR kit (Macherey-Nagel, Düren, Germany), their nucleotide sequences were determined as described above. The 16-S rDNA sequence was aligned with published sequences from representative Pseudomonas species from the National Center for Biotechnology Information (NCBI) data base. The nucleotide and amino acid sequence data reported here for cphE have been submitted to the NCBI data base under accession number AY065671. The 16-S rRNA gene sequence data of P. anguilliseptica strain BI were deposited in the NCBI data base under accession numberAF439803. For production of native CGP,Synechocystis sp. strain PCC6308 was cultivated in full-strength BG11 medium (23Rippka R. Deruelles J. Waterbury J.B. Herdman M. Stanier R.Y. J. Gen. Microbiol. 1979; 111: 1-61Crossref Google Scholar) in an 80-liter closed tubular glass photobioreactor as described before (24Hai T. Ahlers H. Gorenflo V. Steinbüchel A. Appl. Microbiol. Biotechnol. 2000; 53: 383-389Crossref PubMed Scopus (45) Google Scholar). Also, a recombinantE. coli DH1 harboring plasmid pMa/c5–914::cphA (see above) with a temperature-sensitive inducible promoter was employed for the production of CGP. A 42-liter Biostat UD30 stainless steel bioreactor (B. Braun Biotech International, Melsungen, Germany) with TB complex medium was used for production as described previously (12Frey, K. M., Oppermann-Sanio, F. B., Schmidt, H., and Steinbüchel, A. (2002) Appl. Environ. Microbiol.DOI:10.1128/AEM.68.7.000-000.2002Google Scholar). After cell harvest, CGP was isolated according to the method of Simon (25Simon R.D. Proc. Natl. Acad. Sci. U. S. A. 1971; 68: 265-267Crossref PubMed Google Scholar), which was modified by applying only one washing step at each Triton X-100®concentration and two additional centrifugation steps of the acidic and neutralized suspensions, respectively. The purity of CGP was controlled both by SDS-PAGE with subsequent Coomassie staining (26Weber K. Osborn M. J. Biol. Chem. 1969; 244: 4406-4417Abstract Full Text PDF PubMed Google Scholar) and by HPLC analysis after acid hydrolysis of the polymer and subsequent derivatization of amino groups with o-phtaldialdehyde reagent (27Aboulmagd E. Oppermann-Sanio F.B. Steinbüchel A. Arch. Microbiol. 2000; 174: 297-306Crossref PubMed Scopus (93) Google Scholar). Motility and Gram behavior were determined as described before. Oxidase (Bactident® Oxidase test strips from Merck) and catalase tests were performed according to standard protocols. Further determinations were done by using the API 20NE test kit (BioMérieux, Marcy-l'Etoile, France). Reversed phase HPLC was used to determine the products of enzymatic CGP degradation as described by the method for the quantitative determination of amino acids (27Aboulmagd E. Oppermann-Sanio F.B. Steinbüchel A. Arch. Microbiol. 2000; 174: 297-306Crossref PubMed Scopus (93) Google Scholar). Electrospray ionization mass spectrometry (ESI) was applied for identification of the final degradation product of CGP by mass determination and structural analysis (28-Google Scholar). All measurements were performed employing a Quattro LCZ system (Micromass, Manchester, UK) with a nanospray inlet. A cell-free supernatant from a CGP culture was obtained by sedimentation of the cells in the late exponential growth phase by centrifugation and subsequent filtration of the supernatant through a 0.2-μm nitrocellulose membrane. All steps were carried out at 4 °C and in the presence of 50 mmsodium phosphate buffer (pH 8.3). Further components added to the buffer are mentioned below. After concentration in an ultrafiltration chamber (Amicon, Beverly, MA) using a YM10 membrane, the buffered solution was applied onto a MonoQ HR5/5 anion exchange column (AmershamBiosciences). After washing the column with 2 bed volumes of buffer, CGPase was eluted with a linear NaCl gradient (0–1 m) employing an increase of NaCl concentration of 17 mm/ml and a total flow rate of 1 ml/min. Active fractions (1 ml) were detected after the transfer of 10 μl of the respective eluates onto CGP overlay plates (see above) by the occurrence of halos after 5–40 min of incubation at 30 °C. Fractions with high activity were combined, desalted by ultrafiltration (see above), and applied onto an arginine-agarose column (5-ml bed volume; Sigma). For selective elution of the enzyme, an arginine gradient (0–1 m) was applied. To avoid nonspecific protein binding and to prevent the enzyme from binding in the presence of high arginine concentrations, the buffer in addition contained 100 mm NaCl. SDS-PAGE of active enzyme fractions or CGP samples was performed in 11.5% polyacrylamide gels according to standard protocols (29Laemmli U.K. Nature. 1970; 227: 680-685Crossref PubMed Scopus (207233) Google Scholar). Proteins were visualized by the Coomassie staining method (26Weber K. Osborn M. J. Biol. Chem. 1969; 244: 4406-4417Abstract Full Text PDF PubMed Google Scholar). An “in-gel” renaturation method described for activity staining with proteases after SDS-PAGE (30Salamone P.R. Wodzinski R.J. Appl. Microbiol. Biotechnol. 1997; 48: 317-324Crossref PubMed Scopus (62) Google Scholar) was used to obtain reactivated CGPase after separation of enzyme subunits according to their apparent molecular mass under denaturating conditions. The ability of reactivated CGPase to form degradation halos was tested by the application of a thin CGP-agar layer (see above) on top of buffer-pretreated gels. Protein concentrations were determined by the procedure of Bradford (31Bradford M.M. Anal. Biochem. 1976; 72: 248-254Crossref PubMed Scopus (216440) Google Scholar). For determination of the substrate specificity of the CGPase, the purified recombinant enzyme was incubated at 30 °C in 1 ml of 50 mm sodium phosphate buffer (pH 8.3) with various polypeptide substrates. Each reaction contained 1 mg of the respective substrate and 1.6 μg of enzyme. The reaction was stopped after 120 min by incubation at 70 °C for 5 min. After centrifugation, 100-μl aliquots of supernatant were incubated at 95 °C for 5 min in the presence of 1.25% ninhydrin (Merck) in 1 ml of total reaction volume. Subsequently, they were assayed photometrically at 570 nm for the presence of released hydrolysis products. Bovine casein (Hammersten grade) was from Merck, bovine serum albumin (BSA) was from Roth (Karlsruhe, Germany), and poly(α,β-d/l-aspartic acid) (M r = 11,000) was obtained from Bayer (Leverkusen, Germany). Labeling experiments were performed by enzymatic elongation of the C terminus of a CGP primer (32Berg H. Ziegler K. Piotukh K. Baier K. Lockau W. Volkmer-Engert R. Eur. J. Biochem. 2000; 267: 5561-5570Crossref PubMed Scopus (107) Google Scholar).l-[U-14C]arginine was incorporated into the polymer chain using purified cyanophycin synthetase fromSynechocystis sp. strain PCC6308 heterologously produced inE. coli (27Aboulmagd E. Oppermann-Sanio F.B. Steinbüchel A. Arch. Microbiol. 2000; 174: 297-306Crossref PubMed Scopus (93) Google Scholar). Labeled CGP was incubated at 30 °C with 1.6 μg of CGPase in 50 mm sodium phosphate buffer (pH 8.3) under vigorous shaking. For heat inactivation of the enzyme, 50-μl samples were transferred to test tubes containing 500 μl of water preincubated at 70 °C. After 5 min of inactivation, the samples were transferred onto ice for 5 min to allow for CGP reprecipitation. After subsequent centrifugation, 50 μl of supernatant were mixed with 500 μl of Hydroluma® Scintillation mixture (J. T. Baker, Inc.). Radioactivity was measured with a model LS 6500 scintillation counter (Beckman Instruments (27Aboulmagd E. Oppermann-Sanio F.B. Steinbüchel A. Arch. Microbiol. 2000; 174: 297-306Crossref PubMed Scopus (93) Google Scholar)). To screen for CGP-degrading bacteria, samples from typical habitats of CGP-producing cyanobacteria were plated onto solid mineral medium containing CGP as the sole carbon source. Due to the insolubility of CGP at neutral pH, the agar was turbid. Colonies of CGP-degrading microorganisms were recognized because of the formation of degradation halos, which appeared after 12–18 h of incubation at 30 °C (Fig.1 A). Based on this feature, axenic cultures of nine bacterial strains were finally isolated from Baltic sea water, different pond sediments, and sewage sludge (Table I). In addition to the newly isolated CGP-degrading strains, other bacteria from our culture collection were also tested, and two additional strains with CGP degradation capability were detected (strains BE2 and PAS1, TableI). However, E. coli K12, P. putida KT2440, M. luteus, B. subtilis 168+, andB. megaterium were not able to cause formation of halos on CGP overlay agar plates, although some of these bacteria (e.g. B. subtilis) are known to use proteins as nutrients. All isolates tested (from Baltic sea water, pond sediment, and sewage sludge,i.e. “A, B, and D series”; Table I) were Gram-negative, oxidase- and catalase-positive rod-shaped bacteria. With the exception of isolate DXIII, all strains showed motility. Applying the API 20NE test kit, two isolates (DIII and DIV) revealed acceptable identification profiles. Both strains were taxonomically affiliated as strains of the species Pseudomonas alcaligenes (TableII). As listed in Table I, most strains isolated in this study showed growth on the amino acid constituents of CGP, i.e. on aspartic acid and arginine. For most strains, growth with arginine was faster than with aspartic acid. Only isolate BI showed no growth on aspartic acid. Strain PAS 1 from the culture collection of our institute also did not grow on aspartic acid as the sole carbon source. None of the isolates was able to grow on synthetic poly(α,β-d/l-aspartic acid) (data not shown). With poly(γ-d-glutamic acid) as the sole carbon source, only isolate DXIII and to some extent also P. alcaligenes strain BE2 (33Oppermann F.B Pickartz S. Steinbüchel A. Polym. Degrad. Stabil. 1998; 59: 337-344Crossref Google Scholar) showed growth (Table I). With bovine serum albumin as the sole carbon source, only strain PAS I and P. alcaligenes strain BE2 exhibited good or poor growth, respectively. Citrulline and ornithine, two putative degradation products of arginine, were not utilized as carbon sources for growth by any of the bacteria investigated in this study. The only exceptions were isolate AVN, which was able to grow on ornithine, and B. subtilis 168+, which utilized citrulline (data not shown).Table IISubstrate utilization of CGP-degrading bacteria in the API 20NE physiological determination assayIsolateStrainAPI 20 NE-Assays2-aND, taxonomically not determinable with this test; –, no growth; +, poor growth.2-bAPI20 NE tests: NO2, NO3 reduction → NO2; ADH, arginine dihydrolase; GEL, gelatinase; OX, oxidase assay; aerobic substrate utilization: MLT, malate; CIT, citrate; CAP, caprate.NO2ADHGELOXMLTCITCAPAVNND–––++++BI P. anguilliseptica2-cTaxonomically determined by 16-S rDNA sequence analysis.–+–++++DIII P. alcaligenes+–+++++DIV P. alcaligenes+–+++++Strain AVN was isolated from Baltic sea water, strain BI was obtained from pond sediments, and the strains of the D-series were isolated from sewage sludge. The following tests were negative for all four isolates and are not shown in the table: denitrification test; indole formation (tryptophan conversion); urease test; β-glucosidase test; β-galactosidase test; anaerobic utilization of glucose (fermentation); aerobic substrate utilization tests: glucose, arabinose, mannose, manitol, N-acetyl-glucosamine, maltose, gluconate, adipate, phenyl-acetate.2-a ND, taxonomically not determinable with this test; –, no growth; +, poor growth.2-b API20 NE tests: NO2, NO3 reduction → NO2; ADH, arginine dihydrolase; GEL, gelatinase; OX, oxidase assay; aerobic substrate utilization: MLT, malate; CIT, citrate; CAP, caprate.2-c Taxonomically determined by 16-S rDNA sequence analysis. Open table in a new tab Strain AVN was isolated from Baltic sea water, strain BI was obtained from pond sediments, and the strains of the D-series were isolated from sewage sludge. The following tests were negative for all four isolates and are not shown in the table: denitrification test; indole formation (tryptophan conversion); urease test; β-glucosidase test; β-galactosidase test; anaerobic utilization of glucose (fermentation); aerobic substrate utilization tests: glucose, arabinose, mannose, manitol, N-acetyl-glucosamine, maltose, gluconate, adipate, phenyl-acetate. For several reasons, isolate BI was a good candidate for a more detailed investigation of CGP degradation. Therefore, the taxonomic position of the isolate was determined. Analysis of the 16-S rDNA sequence of isolate BI revealed 98% identity to the nucleotide sequence of all three P. anguillisepticastrains available at the NCBI data base including the P. anguilliseptica type strain NCIMB 1949. Maximum sequence identity to other species of the genus Pseudomonas was only in the range of 95–96% (Fig. 2). Therefore, the new isolate was referred to as P. anguilliseptica strain BI. Preliminary 16-S rDNA sequence data of strain PAS 1 (about 1000 bp) revealed that this strain most probably belongs to the genusStreptomyces. This finding corresponds well with the streptomycete-like habitus of this strain, e.g. the formation of exospores in aging colonies. The ability of P. anguilliseptica strain BI to grow on CGP as the sole carbon source was investigated in more detail. Therefore, growth of this strain on CGP and on its amino acid constituents as well as on the non-related substrate citrate was monitored over 24 h (Fig.3). Living cell counts for the cyanophycin culture revealed that growth of the cells started at about 4 h of incubation (data not shown) after inoculation from a citrate culture. The turbidity caused by suspended CGP particles disappeared visibly during incubation. Strain BI grew best with a combination of arginine and aspartic acid if these amino acids were provided at a molar ratio according to their proportional masses in the CGP molecule (248 Klett units). Growth on CGP led to a maximum optical density of 202 Klett units, which is in the range of the OD of the citrate culture. Slightly weaker growth was detected for the arginine culture (182 Klett units). No increase or change of the OD occurred in the control (sterile medium containing citrate) or in mineral salt medium containing aspartic acid as the sole carbon source (Fig. 3). During growth on CGP, 46% (w/w) of the polymer was converted into cellular dry matter by P. anguilliseptica strain BI. The extracellular CGPase of P. anguilliseptica was purified to electrophoretic homogeneity from CGP-grown cultures by the application of anion exchange chromatography on Q-Sepharose followed by l-arginine-agarose affinity chromatography (Fig. 1 C). The latter is usually used for different purposes, e.g. purification of transfer RNA molecules (34Jay F.T. Coultas C. Jones D.S. Nucleic Acids Res. 1976; 3: 177-190Crossref PubMed Scopus (6) Google Scholar). The l-arginine-agarose matrix was highly specific for the binding of CGPase under the employed conditions, revealing a high affinity of the enzyme to this matrix. Therefore, an arginine gradient (0–1 m) in sodium phosphate buffer was applied for the elution of the CGPase. To further reduce nonspecific binding of other pro" @default.
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• W2034129394 title "Isolation of Cyanophycin-degrading Bacteria, Cloning and Characterization of an Extracellular Cyanophycinase Gene (cphE) from Pseudomonas anguilliseptica Strain BI" @default.
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