Eur J Biochem, 1995 Mar 15, 228(3), 1009 - 19 The major N-linked carbohydrate chains from human urokinase . The occurrence of 4-O-sulfated, (alpha 2-6)-sialylated or (alpha 1-3)-fucosylated N-acetylgalactosamine(beta 1-4)-N-acetylglucosamine elements; Bergwerff AA et al.; The primary structure of the major N-linked carbohydrate chains attached to Asn302 of urinary-type plasminogen activator (urokinase) have been determined . Urokinase was completely deglycosylated with peptide-N4-(N-acetyl-beta-glucosaminyl)asparagine amidase F from Flavobacterium meningosepticum . Released oligosaccharides were separated from the remaining protein using gel-permeation chromatography on Bio-Gel P-100, and then on Bio-Gel P-6 . Fractionation of the oligosaccharides was achieved by a combination of FPLC anion-exchange chromatography on Mono Q HR 5/5 and amine-adsorption HPLC on LiChrospher 100-NH2 . Analysis by 1H-NMR spectroscopy demonstrated that the collection of N-glycans comprises di-, tri-, and tri'-antennary structures . The glycans contain predominantly GalNAc beta 1-4GlcNAc beta instead of Gal beta 1-4GlcNAc beta elements . The GalNAc residue is mainly sulfated at O4, or to a lesser extent it bears N-acetylneuraminic acid at O6; alternatively the GlcNAc residue can be fucosylated at O3 . The major component, which accounts for more than 30 mol/100 mol of the total oligosaccharide pool, consists of an (alpha 1-6)-fucosylated diantennary N-linked carbohydrate chain with (SO4-)-4GalNAc beta 1-4GlcNAc beta 1-2 antennae. Appl Microbiol Biotechnol, 1995 Mar, 42(6), 844 - 52 Enzymatic peptide synthesis by the recombinant proline-specific endopeptidase from Flavobacterium meningosepticum and its mutationally altered Cys-556 variant; Krieg F et al.; Proline-specific endopeptidase (PSE) (EC 3.4.21.26) was investigated for its potential as a catalyst in peptide synthesis . Using an activated peptide ester or a peptide amide as the acyl component, the enzyme catalyzed kinetically controlled aminolysis and transpeptidation respectively, with various amino acid amides as acyl acceptors . To a certain extent the nucleophile preference reflected the amino acid preference in the S1'-position of the enzyme in peptide hydrolysis: the highest fractions of aminolysis were obtained using amino acid amides with hydrophobic side-chains (e.g . Leu-NH2, Phe-NH2) . PSE also catalyzed the thermodynamically controlled condensation of short peptides with a free carboxyterminus and various amino acid amides . This enabled us to examine the acceptance of different acyl components in the substrate-binding site of the enzyme with regard to their amino acid composition: In the S1 position proline was clearly favored, but alanine was also accepted, whereas the S2 subsite accepted various amino acids rather unspecifically . Since PSE was shown to be extremely sensitive against water-miscible organic solvents, an alternative approach was used to increase yields in enzymatic peptide synthesis: a derivative of PSE in which the catalytic Ser-556 is converted to a Cys was constructed by protein engineering . This mutant (PSEcys) exhibited a dramatically increased peptide ligase activity in aqueous solution. Wei Sheng Wu Xue Bao, 1995 Feb, 35(1), 50 - 7 {Studies on elastase from Flavobacterium . I . Strain screening and enzyme purification}; Yan Z et al.; 132 strains bacteria secreting extracellular elastase were isolated from soil samples, 5 of them possessed considerably high elastiolytic activity of more than 100 u/ml . The highest-yield strain No . 17-87 was characterized as Flavobacterium odoratum, studies on the condition of elastase production revealed that its optimum carbohydrate and nitrogen source were glucose and casein respectively, and that it could utilize fowl ferther meal and wheat bran to give 80% relative yield . The culture exhibited maximum elastase activity at 26 degrees C for 21 hours, the productivity could be increased when the aeration was improved . The PAGE-homogenous elastase preparation was obtained from the culture broth by (NH4)2SO4 fractionation, DEAE-cellulose column chromatography and gel filtration on Sephadex G-75 . The molecular weight was determined to be 21380 by SDS-PAGE, the elastiolytic activity was optimal at pH7.4 and 50 degrees C . The enzyme was stable over the range of pH4.5-9.5 and below 40 degrees C, but the activity was inhibited completely by Fe3+, Zn2+, Cu2+, Cr3+, Co2+, Ni2+, Hg2+, Ag+. Arch Biochem Biophys, 1995 Jan 10, 316(1), 399 - 406 Molecular cloning and sequence analysis of Flavobacterium meningosepticum glycosylasparaginase: a single gene encodes the alpha and beta subunits; Tarentino AL et al.; A full-length insert for the Flavobacterium meningosepticum N4-(N-acetyl-beta-glucosaminyl)-L-asparagine amidase gene was located on a 2500-bp HindIII fragment and cloned into the plasmid vector pBluescript . DNA sequencing revealed an open reading frame of 1020 nucleotides encoding a putative 45-amino-acid leader sequence and a deduced precursor polypeptide of 295 amino acids . In F . meningosepticum this precursor polypeptide undergoes proteolytic processing by an as yet unknown mechanism to generate an alpha-subunit and a beta-subunit, which constitute the active form of the heterodimeric mature glycosylasparaginase . The Flavobacterium glycosylasparaginase gene was expressed in Escherichia coli and found to be enzymatically active . The recombinant enzyme was purified from crude lysates and shown by sodium dodecyl sulfate-polyacrylamide gel electrophoresis to consist of the typical alpha- and beta-subunits . The recombinant beta-subunit cross-reacted to antibody specific for the rat liver beta-subunit, and Edman analysis demonstrated that its amino-terminus corresponded exactly to that of the mature native glycosylasparagine beta-subunit . A comparison of the Flavobacterium glycosylasparaginase with a mammalian glycosylasparaginase revealed 30% structural identity and 60% overall similarity between the prokaryotic and eukaryotic forms of the enzyme . Even more striking was the conservation of the amino acid sequence in both proteins where the post-translational cleavage to generate the active enzyme occurs . Our data demonstrate that deglycosylation of asparagine-linked glycans via hydrolysis of the AspNHGlcNAc linkage is an important reaction which has been preserved during evolution. Biol Neonate, 1995, 68(2), 153 - 6 Intrathecal immune response in neonatal Flavobacterium meningosepticum meningitis; Abrahamsen TG et al.; Neonates are predisposed to serious infections such as meningitis, probably due to their immature host reaction to the pathogens . We have studied the intrathecal immune response in 2 newborns with Flavobacterium meningosepticum meningitis . They showed a significant elevation of immunoglobulin indices ( >1.10), also after the CSF had become sterile with a normalized cell count . In addition, an intrathecal increase and subsequent decrease of both C3dg and TCC (terminal complement complex) were observed in 1 patient . We conclude that immunoglobulin production and complement activation may occur in neonatal CSF. Am J Nephrol, 1995, 15(1), 82 - 4 Right-sided bacterial endocarditis due to Flavobacterium odoratum in a patient on chronic hemodialysis; Ferrer C et al.; Bacterial endocarditis, particularly involving the left side, has been shown to occur in patients in regular hemodialysis . We report a case of right-sided endocarditis characterized by a very torpid evolution . Although the diagnosis was suspected early in the course, confirmation was obtained 2 months after the onset . Flavobacterium odoratum was identified in the fourth month of evolution and only after multiple blood cultures had been obtained . We believe the very low infectivity of F . odoratum and its very slow growth in culture media prevented an early diagnosis. Microbios, 1995, 82(331), 109 - 13 Bacterial contamination of aerosol solutions containing antibiotics; Oie S et al.; In an investigation of microbial contamination of aerosol solutions containing antibiotics (hospital pharmaceutical preparations, no preservatives added), five of six residual solutions after multiple use for 7 days were contaminated at a concentration of 10(6) viable counts/ml . Contaminants were glucose-nonfermenting Gram-negative bacilli such as Pseudomonas cepacia and Flavobacterium meningosepticum . The major contaminant, P . cepacia, multiplied rapidly in the aerosol solution under simulated actual-use conditions . The contamination seemed to have been caused by storage, at room temperature instead of in a refrigerator, of the multiple-dose solutions and by frequent re-use of syringes to measure the solutions . Prompt refrigerator storage after each use of the solutions and abandonment of the syringes within 24 h eliminated bacterial contamination of the solutions . Sufficient attention is not paid to prevent bacterial contamination of aerosol solutions containing antibiotics because of the fact that they contain antibiotics . Thus aerosol solutions containing antibiotics should be handled with great care to prevent bacterial contamination. Am J Gastroenterol, 1994 Dec, 89(12), 2245 - 6 Septicemia occurring after colonoscopic polypectomy in a splenectomized patient taking corticosteroids; Lee M et al.; Although a transient bacteremia may occur in approximately 4% of patients after colonoscopic procedures, clinically significant bacteremia or endocarditis is exceedingly rare . To our knowledge, the case we describe herein is the first reported case of septicemia due to Flavobacterium meningosepticum and Escherichia coli after colonoscopic polypectomy . Our patient was probably immunocompromised and, therefore, predisposed to bacteremia after the procedure, because she was asplenic, diabetic, and receiving long-term steroid therapy for her chronic autoimmune disorders . Several recent reports have suggested that immunocompromised patients are more likely to develop septicemia after endoscopic procedures. Biodegradation, 1994 Dec, 5(3-4), 277 - 88 Molecular analysis of pentachlorophenol degradation; Orser CS et al.; A limited number of microorganisms have been described for their ability to partially degrade pentachlorophenol (PCP), or to completely mineralize it . Several years ago we chose one of these microorganisms, Flavobacterium sp . strain ATCC 39723, for use in a detailed molecular analysis of the catabolism of PCP . This strain was chosen because it had previously been studied in great detail for its growth characteristics in relation to degradation of PCP . In this paper we provide an overview of the degradation pathway of PCP to 2,6-dichloro-p-hydroquinone by Flavobacterium . The specific biochemical reactions and the genes encoding the enzymes are reviewed . The successful transformation and site specific mutagenesis of Flavobacterium, as well as the discovery of two new pcp alleles is also presented. Biodegradation, 1994 Dec, 5(3-4), 185 - 94 The nylon oligomer biodegradation system of Flavobacterium and Pseudomonas; Negoro S et al.; This review article is a compendium of the available information on the degradation of a man-made compound, 6-aminohexanoate-oligomer, in Flavobacterium and Pseudomonas strains, and discusses the molecular basis for adaptation of microorganisms toward these xenobiotic compounds . Three plasmid-encoded enzymes, 6-aminohexanoate-cyclic-dimer hydrolase (EI), 6-aminohexanoate-dimer hydrolase (EII), and endo-type 6-aminohexanoate-oligomer hydrolase (EIII) are responsible for the degradation of the oligomers . Two repeated sequences, designated RS-I and RS-II, are found on plasmid pOAD2, which is involved in 6-aminohexanoate degradation in Flavobacterium . RS-I appears 5 times on the pOAD2, and all copies have the same sequences as insertion sequence IS6100 . RS-II appears twice on the plasmid . RS-IIA contains the gene encoding EII, while RS-IIB contains the gene for the analogous EII' protein . Both EII and EII' are polypeptides of 392 amino acids, which differ by 46 amino acid residues . The specific activity of the EII enzyme is 200-fold higher than that of EII' . Construction of various hybrid genes demonstrated that only the combination of two amino acid residues in the EII' enzyme can enhance the activity of the EII' to the same level as that of EII enzyme. Biochemistry, 1994 Nov 29, 33(47), 13989 - 96 Crystal structure of endo-beta-N-acetylglucosaminidase F1, an alpha/beta-barrel enzyme adapted for a complex substrate; Van Roey P et al.; Endo-beta-N-acetylglucosaminidase F1 (Endo F1) is an endoglycosidase, secreted by Flavobacterium meningosepticum, that cleaves asparagine-linked oligosaccharides after the first N-acetylglucosamine residue . The enzyme is selective for high-mannose oligosaccharide chains . The crystal structure of Endo F1 has been determined at 2.0-A resolution . The molecular fold consists of a highly irregular alpha/beta-barrel, a commonly observed motif consisting of a cyclic 8-fold repeat of beta-strand/loop/alpha-helix units with an eight-stranded parallel beta-barrel at the center . Endo F1 lacks two of the alpha-helices, those of units 5 and 6 . Instead, the links after beta-strands 5 and 6 consist of a short turn followed by a section in an extended conformation that replaces the helix and a long loop at the bottom of the molecule . The absence of any excursion on top of the molecule following beta-strands 5 and 6 results in a pronounced depression in the rim of the barrel . This depression forms one end of a shallow cleft that runs across the surface of the molecule, over the core of the beta-barrel to the area between the loops of units 1 and 2 . The active site residues, Asp130 and Glu132, are located at the carboxyl end of beta-strand 4 and extend into this cleft . These residues are surrounded by several tyrosine residues . The cleft area formed by loops 1 and 2 is lined with polar residues, mainly asparagines . The latter area is thought to be responsible for oligosaccharide binding and recognition while the protein moiety of the substrate would be located outside the molecule but adjacent to the area of loops 5 and 6.(ABSTRACT TRUNCATED AT 250 WORDS) Structure, 1994 Nov 15, 2(11), 1049 - 59 The three-dimensional structure of PNGase F, a glycosylasparaginase from Flavobacterium meningosepticum; Norris GE et al.; BACKGROUND: Peptide:N-glycosidase F (PNGase F) is an enzyme that catalyzes the complete removal of N-linked oligosaccharide chains from glycoproteins . Often called an endoglycosidase, it is more correctly termed an amidase or glycosylasparaginase as cleavage is at the asparagine-sugar amide linkage . The enzyme is widely used in structure-function studies of glycoproteins . RESULTS: We have determined the crystal structure of PNGase F at 1.8 A resolution . The protein is folded into two domains, each with an eight-stranded antiparallel beta jelly roll configuration similar to many viral capsid proteins and also found, in expanded form, in lectins and several glucanases . Two potential active site regions have been identified, both in the interdomain region and shaped by prominent loops from one domain . Exposed aromatic residues are a feature of one site . CONCLUSIONS: The finding that PNGase F is based on two jelly roll domains suggests parallels with lectins and other carbohydrate-binding proteins . These proteins either bind sugars on the concave face of the beta-sandwich structure (aided by loops) or amongst the loops themselves . Further analysis of the function and identification of the catalytic site should lead to an understanding of both the specificity of PNGase F and possibly also the recognition processes that identify glycosylation sites on proteins. Infect Immun, 1994 Nov, 62(11), 4938 - 47 An endo-acting proline-specific oligopeptidase from Treponema denticola ATCC 35405: evidence of hydrolysis of human bioactive peptides; Makinen PL et al.; An endo-acting proline-specific oligopeptidase (prolyl oligopeptidase {POPase}, EC 3.4.21.26) was purified to homogeneity from the Triton X-100 extracts of cells of Treponema denticola ATCC 35405 (a human oral spirochete) by a procedure that comprised five successive fast protein liquid chromatography steps . The POPase is a cell-associated 75- to 77-kDa protein with an isoelectric point of ca . 6.5 . The enzyme hydrolyzed (optimum pH 6.5) the Pro-pNA bond in carbobenzoxy-Gly-Pro-p-nitroanilide (Z-Gly-Pro-pNA) and bonds at the carboxyl side of proline in several human bioactive peptides, such as bradykinin, substance P, neurotensin, angiotensins, oxytocin, vasopressin, and human endothelin fragment 22-38 . The minimum hydrolyzable peptide size was tetrapeptide P3P2P1P'1, while the maximum substrate size was ca . 3 kDa . An imino acid residue in position P1 was absolutely necessary . The hydrolysis of Z-Gly-Pro-pNA was potently inhibited by the following, with the Ki(app) (in micromolar) in parentheses: insulin B-chain (0.7), human endothelin-1 (0.5), neuropeptide Y (1.7), substance P (32.0), T-kinin (4.0), neurotensin (5.0), and bradykinin (16.0) . Chemical modification and inhibition studies suggest that the POPase is a serine endopeptidase whose activity depends on the catalytic triad of COOH .. . Ser .. . His but not on a metal . The amino acid sequence around the putative active-site serine is Gly-Gly-Ser-Asn-Pro-Gly . The enzyme is suggested to contain a reactive cysteinyl residue near the active site . Amino acid residues 4 to 24 of the first 24 N-terminal residues showed a homology of 71% with the POPase precursor from Flavobacterium meningosepticum and considerable homology with the Aeromonas hydrophila POPase . The ready hydrolysis of human bioactive peptides at bonds involving an imino acid residue suggests that enzymes like POPase may contribute to the chronicity of periodontal infections by participating in the peptidolytic processing of those peptides. J Clin Microbiol, 1994 Oct, 32(10), 2398 - 403 Flavobacterium meningosepticum, a pathogen in birds; Vancanneyt M et al.; Five bacterial isolates were recovered from various diseased birds (chickens, a pigeon, and a zebra finch) and were identified as Flavobacterium meningosepticum . Four of them were isolated in pure or nearly pure culture of samples from internal organs, and one strain was isolated in mixed culture of a tarsal joint fluid sample . Except for the last case, there was no evidence of other disease agents . By using phenotypic, chemotaxonomic, and genomic methods, the strains were taxonomically characterized and could not be differentiated from the human clinical reference strains of the species . Two avian strains were different in their phenotypic behaviors and constituted another genotypic subgroup . In general, all F . meningosepticum strains constituted a single species which was easily differentiated from biochemically similar species and phylogenetically closely related taxa. Zhonghua Nei Ke Za Zhi, 1994 Sep, 33(9), 608 - 10 {Antibiotic treatment for pneumonia caused by Flavobacterium meningosepticum}; Wang JX et al.; Two cases of critical nosocomial pneumonia caused by flavobacterium meningosepticum (FM) were reported and both of them were successfully cured . There were 2 other cases of FM pneumonia reported in Chinese literature previously, but none of them survived . It has been found that the treatment for FM respiratory infection was very difficult because of its resistance to majority of antibiotics, including the third generation cephalosporins . The symptoms of FM pneumonia are similar to those of other gram-negative bacillus pneumonias, such as Klebsiella pneumoniae pneumonia . Definite diagnosis depends principally on etiological examination and clinical manifestations of pneumonia . Cefoperazone, Cefsulodin, Astreonam and Ciprofloxacin are valuable drugs in saving the lives of patients with FM Pneumonia. J Mol Biol, 1994 Aug 26, 241(4), 624 - 6 Purification and crystallization of the endoglycosidase PNGase F, a peptide:N-glycosidase from Flavobacterium meningosepticum; Norris GE et al.; The endoglycosidase peptide: N-glycosidase, secreted by the Gram-negative bacterium Flavobacterium meningosepticum (PNGase F), has been isolated, purified to homogeneity and crystallized from polyethylene glycol solutions using vapour diffusion and seeding techniques . The crystals are orthorhombic, space group P2(1)2(1)2, with unit cell dimensions a = 85.07 A, b = 85.14 A, c = 48.50 A, and are suitable for high resolution X-ray structure analysis. Glycobiology, 1994 Aug, 4(4), 535 - 44 Structural studies on the oligosaccharides isolated from bovine kidney heparan sulphate and characterization of bacterial heparitinases used as substrates; Sugahara K et al.; We prepared a series of oligosaccharides from commercial bovine kidney heparan sulphate after limited digestion with heparitinase I from Flavobacterium heparinum, and determined the structures of eight tetrasaccharides and a hexasaccharide by enzymatic analysis, fast atom bombardment mass spectrometry and 500 MHz 1H NMR spectroscopy . The tetrasaccharides share the common core structure delta 4,5HexA alpha 1-4GlcN alpha 1-4HexA1-4GlcN (where delta 4,5HexA is 4-deoxy-alpha-L-threo-hex-4-enopyranosyluronic acid and HexA is hexuronic acid), with zero, one or two sulphate groups . Seven of them contain non-sulphated glucuronic or iduronic acid, and the other, 2-O-sulphated iduronic acid at the internal position . Although they contain ordinary structures which should be widely distributed in the relatively low-sulphated region of heparan sulphate, five of the tetrasaccharides were isolated for the first time as discrete structures . The structure of the hexasaccharide was determined as delta 4,5HexA alpha 1-4GlcNAc alpha 1-4GlcA beta 1-3Gal beta 1-3Gal beta-1-4 Xyl and is derived from the carbohydrate-protein linkage region of the heparan sulphate chains . The hexasaccharide seems to have been released by the alkaline treatment used to prepare the heparan sulphate . The Gal residues were non-sulphated as are those in the porcine intestinal heparin chains, but in contrast to the sulphated Gal structures previously demonstrated in the carbohydrate-protein linkage region of chondroitin sulphate chains . These oligosaccharides were used to investigate the substrate specificity of heparitinases I and II from F . heparinum . The results revealed that heparitinase I cleaves hexosaminidic bonds linked to non-sulphated glucuronic or iduronic acid residues . The glucosaminidic linkage of the hexasaccharide was sensitive to heparitinase I, but resistant to heparitinase II, demonstrating the differential specificity of these enzymes towards the carbohydrate-protein linkage region. Biochem Biophys Res Commun, 1994 Jul 29, 202(2), 809 - 15 Isolation of prolylendopeptidase-inhibiting peptides from bovine brain; Ohmori T et al.; The peptides inhibiting prolylendopeptidase were screened and isolated from bovine brain by monitoring the inhibition of prolylendopeptidase produced by Flavobacterium meningosepticum . The amino acid sequence of the peptide having the highest inhibitory activity was determined as Met-Pro-Pro-Pro-Leu-Pro-Ala-Arg-Val-Asp-Phe-Ser-Leu-Ala-Gly-Ala-Leu-Asn . This peptide showed the IC50 and Ki values of 38.4 and 8.6 microM, respectively, for prolylendopeptidase isolated from bovine brain. Int J Syst Bacteriol, 1994 Jul, 44(3), 447 - 53 Flavobacterium scophthalmum sp . nov., a pathogen of turbot (Scophthalmus maximus L.); Mudarris M et al.; Fifty orange-pigmented, gram-negative, rod-shaped isolates were recovered from healthy and diseased turbot and from coastal waters (collected in Scotland) . On the basis of the results of an examination of 125 phenotypic characteristics and the results of DNA-DNA and DNA-rRNA hybridization experiments, we concluded that these isolates are members of a new species in the genus Flavobacterium, for which the name Flavobacterium scophthalmum is proposed . The type strain is CCM 4109 (= LMG 13028). Plant Cell Physiol, 1994 Jun, 35(4), 665 - 75 A prolyl endoproteinase that acts specifically on the extrinsic 18-kDa protein of photosystem II: purification and further characterization; Kuwabara T et al.; An endoproteinase, which specifically cleaves the Pro12-Leu13 bond of the extrinsic 18-kDa protein of PSII, was purified from PSII membranes of spinach . The presence of 0.05% (w/v) Tween 20 and 1 M NaCl was essential for maintenance of proteolytic activity during the purification . The molecular mass of the enzyme was estimated to be 95 kDa by gel-filtration chromatography . Active fractions contained a polypeptide of 165 kDa that was converted into diffusely stained polypeptides of 54 kDa upon reduction with dithiothreitol . The Km of the 18-kDa protein in the proteolytic reaction was 0.3 microM . Inhibition of the proteolysis by compounds that contain prolyl bonds revealed that both a prolyl bond and a positive charge are necessary for interaction with the proteinase, but some other structural factor(s) must also be involved in the high-affinity interaction between the proteinase and the 18-kDa protein . Reconstitution of NaCl-treated PSII membranes with the 23-kDa protein and/or the 18-kDa protein revealed that the 18-kDa protein was not cleaved by the proteinase when the substrate protein was functionally associated with the membranes . A comparison of the properties of the proteinase with those of a proline-specific endopeptidase from Flavobacterium suggests that these enzymes are quite different in terms of substrate specificity. Glycobiology, 1994 Jun, 4(3), 289 - 96 Action pattern of polysaccharide lyases on glycosaminoglycans; Jandik KA et al.; The action pattern of polysaccharide lyases on glycosaminoglycan substrates was examined using viscosimetric measurements and gradient polyacrylamide gel electrophoresis (PAGE) . Heparin lyase I (heparinase, EC 4.2.2.7) and heparin lyase II (no EC number) both acted on heparin in a random endolytic fashion . Heparin lyase II showed an ideal endolytic action pattern on heparan sulphate, while heparin lyase I decreased the molecular weight of heparan sulphate more slowly . Heparin lyase III (heparitinase, EC 4.2.2.8) acted endolytically only on heparan sulphate and did not cleave heparin . Chondroitin ABC lyase (chondroitinase ABC, EC 4.2.2.4) from Proteus vulgaris acted endolytically on chondroitin-6-sulphate (chondroitin sulphate C) and dermatan sulphate at nearly identical initial rates, but acted on chondroitin-4-sulphate (chondroitin sulphate A) at a reduced rate, decreasing its molecular weight much more slowly . Two chondroitin AC lyases (chondroitinase AC, both EC 4.2.2.5) were examined towards chondroitin-4- and -6-sulphates . The exolytic action of chondroitin AC lyase A from Arthrobacter aurescens on both chondroitin-4- and -6-sulphates was demonstrated viscosimetrically and confirmed using both gradient PAGE and gel permeation chromatography . Chondroitin AC lyase F from Flavobacterium heparinum (Cytophagia heparinia) acted endolytically on the same substrates . Chondroitin B lyase (chondroitinase B, no EC number) from F.heparinum acted endolytically on dermatan sulphate giving a nearly identical action pattern as observed for chondroitin ABC lyase acting on dermatan sulphate. Arch Biochem Biophys, 1994 May 15, 311(1), 127 - 32 Purification and characterization of a neutral zinc endopeptidase secreted by Flavobacterium meningosepticum; Grimwood BG et al.; Flavobacterium meningosepticum, Elder strain (ATCC 33958), secretes into the medium a neutral zinc endoprotease as a major component of the extracellular proteins . The enzyme was purified to homogeneity in a simple two-step procedure involving ammonium sulfate precipitation and hydrophobic interaction chromatography . The molecular weight of this metalloprotease was determined to be about 27,000 (P27) by sodium dodecyl sulfate-polyacrylamide gel electrophoresis . P27 was comparable to thermolysin in the relative rates of elastin-orcein, azocasein, and azoalbumin hydrolysis . P27 and thermolysin hydrolyzed equally well 2,4-dinitrophenyl-Pro-Gln-Gly-Ile-Ala-Gly-Gln-D-Arg and 2,4-dinitrophenyl-Pro-Leu-Gly-Leu-Trp-Ala-D-Arg-NH2 at the same primary sites that are susceptible to cleavage by vertebrate collagenases, Gly-Ile, and Gly-Leu . P27 was also capable of partially hydrolyzing Type I acid-soluble calf skin collagen and slowly hydrolyzing N-{3-(2-furyl)acryloyl}-Leu-Gly-Pro-Ala, a bacterial collagenase substrate not cleaved by thermolysin . P27 was further differentiated from thermolysin from the inability of the former to hydrolyze N-{3-(2-furyl)acryloyl}-Gly-Leu-NH2 . In addition, a vertebrate elastase substrate succinyl-Ala-Ala-Ala-p-nitroanilide was hydrolyzed by P27 but not by thermolysin . P27 is a newly described and unique enzyme from the standpoint of substrate specificity and from the fact that it is resistant to inhibition by phosphoramidon, an inhibitor of a number of zinc endopeptidases, including thermolysin. Can J Microbiol, 1994 May, 40(5), 388 - 92 Effects of a chromated-copper-arsenate wood preservative on the bacterial degradation of pentachlorophenol; Wall AJ et al.; The effect of a chromated-copper-arsenate wood preservative on the degradation of pentachlorophenol by Flavobacterium sp . strain ATCC 53874 was examined in liquid culture . Both a commercially available and a laboratory-prepared formulation were tested . Each increased the lag time required for measurable pentachlorophenol degradation and the time required for complete degradation to nondetectable levels . This response was noted at all pentachlorophenol concentrations examined (10, 25, 50, 75, and 100 micrograms.mL-1) . The commercial formulation of chromated-copper-arsenate had the more significant impact on pentachlorophenol degradation . Inhibitory effects were evident at chromated-copper-arsenate component metal concentrations 0.1-0.5 mg.L-1 . These levels are thousands of times below those used commercially. Appl Microbiol Biotechnol, 1994 May, 41(3), 352 - 8 Expression of organophosphate hydrolase in the filamentous fungus Gliocladium virens; Dave KI et al.; The broad-spectrum organophosphate hydrolase (OPH; EC 3.1.8.1) encoded by the organophosphate-degrading gene (opd) from Pseudomonas diminuta MG and Flavobacterium sp . ATCC 27551 possesses capabilities of both P-O bond hydrolysis (e.g . paraoxon) and P-F bond hydrolysis {e.g . sarin and diisopropylfluorophosphate (DFP)} . In the present study a 9.4-kb plasmid, pCL1, was used to transform the saprophytic fungus Gliocladium virens . pCL1 was derived from pJS294 by placing the fungal promoter (prom1) from Cochliobolus heterostrophus upstream and the trpC terminator from Aspergillus nidulans down-stream of the opd gene . Southern analysis of restricted genomic DNA from various transformants indicated that integration occurred non-specifically at multiple sites . Western blot analysis of mycelial extracts from transformants confirmed the production of a processed form of the enzyme in the fungus . Maximal levels of OPH activity (rate of p-nitrophenol production from paraoxon) were observed after 168 h of culture and activity levels correlated with biomass production in mature vegetative growth. J Biochem (Tokyo), 1994 Apr, 115(4), 724 - 9 Molecular cloning and characterization of prolyl endopeptidase from human T cells; Shirasawa Y et al.; The prolyl endopeptidase (PEP) gene of human T cells was amplified by the PCR method and cloned in Escherichia coli . The complete gene consisted of 2,130 nucleotides corresponding to 710 amino acid residues with a calculated molecular mass of 80,750 . The nucleotide sequence of this clone revealed that T cell PEP DNA is 48, 50, and 91% homologous to those of Flavobacterium meningosepticum, Aeromonas hydrophila, and porcine brain PEP, respectively . This gene was fused to the lacZ sequence from E . coli and expressed as a fused protein in E . coli . This fused protein exhibited PEP activity, which was inhibited by Z-Pro-prolinal, a specific inhibitor of PEP . The fused protein was purified on a beta-galactosidase specific affinity column . A polyclonal antibody was raised against the purified protein . Immunological characterization suggested that this protein is different from cytosol-soluble PEP. Zentralbl Hyg Umweltmed, 1994 Apr, 195(4), 282 - 7 {Infections caused by Flavobacterium meningosepticum in patients in a neonatal intensive care unit}; Heeg P et al.; During a period of three and a half months 7 neonates from a neonatal intensive care unit became infected by F . meningosepticum, serotype B . The pathogens could be isolated from the tracheal secretions and--less frequently--from throat swabs, gastric juice and nose swabs . Environmental sampling led to the isolation of F . meningosepticum from the humidification fluid of the respirator, from vaporizers as well as from the artificial ventilation tubing . F . mengingosepticum was found in the water of humidifiers from 3 children, who developed neither a colonization nor an infection . In a number of cases the patients' environment was contaminated with F . meningosepticum prior to colinization or infection . Nearly identical resistance patterns against the antibiotics tested, could be demonstrated for the isolated strains . The primary source of infection could not be identified, it is supposed however that the index patient had been admitted to the hospital nine months before . A strict hygienic policy, like consequently performed hand hygiene, adequate processing of ventilation equipment and application of sterile tubings led to extinction of the infections . During subsequent environmental controls, F . meningosepticum could not be isolated. Proc Natl Acad Sci U S A, 1994 Mar 29, 91(7), 2527 - 31 Further perspective on the catalytic core and secondary structure of ribonuclease P RNA; Haas ES et al.; Phylogenetic comparative analyses of RNase P RNA-encoding gene sequences from Chlorobium limicola, Chlorobium tepidum, Bacteroides thetaiotaomicron, and Flavobacterium yabuuchiae refine the secondary structure model of the general (eu)bacterial RNase P RNA and show that a highly conserved feature of that RNA is not essential . Two helices, comprised of 2 base pairs each, are added to the secondary structure model and form part of a cruciform in the RNA . Novel sequence variations in the B . thetaiotaomicron and F . yabuuchiae RNA indicate the likelihood that all secondary structure resulting from canonical base-pairing has been detected: there are no remaining unpaired, contiguous, canonical complementarities in the structure model common to all bacterial RNase P RNAs . A nomenclature for the elements of the completed secondary structure model is proposed . The Chlorobium RNase P RNAs lack a stem-loop structure that is otherwise universally present and highly conserved in structure in other (eu)bacterial RNase P RNAs . The Chlorobium RNAs are nevertheless catalytic, with kinetic properties similar to those of RNase P RNAs of Escherichia coli and other Bacteria . Removal of this stem-loop structure from the E . coli RNA affects neither its affinity for nor its catalytic rate for cleavage of a precursor transfer RNA substrate . These results show that this structural element does not play a direct role in substrate binding or catalysis. J Neurochem, 1994 Mar, 62(3), 1126 - 30 Disaccharide composition of heparan sulfates: brain, nervous tissue storage organelles, kidney, and lung; Tekotte H et al.; We have characterized the structural properties of heparan sulfates from brain and other tissues after depolymerization with a mixture of three heparin and heparan sulfate lyases from Flavobacterium heparinum . The resulting disaccharides were separated by HPLC and identified by comparison with authentic standards . In rat, rabbit, and bovine brain, 46-69% of the heparan sulfate disaccharides are N-acetylated and unsulfated, and 17-21% contain a single sulfate residue in the form of a sulfoamino group . In rabbit, bovine, and 1-day postnatal rat brain, disaccharides containing both a sulfated uronic acid and N-sulfate account for an additional 10-14%, together with smaller and approximately equal proportions (5-9%) of mono-, di-, and trisulfated disaccharides having sulfate at the 6-position of the glucosamine residue . Kidney and lung heparan sulfates are distinguished by high concentrations of disaccharides containing 6-sulfated N-acetylglucosamine residues . In chromaffin granules, the catecholamine- and peptide-storing organelles of adrenal medulla, where heparan sulfate accounts for a minor portion (5-10%) of the glycosaminoglycans, we have determined that bovine chromaffin granule membranes contain heparan sulfate in which almost all of the disaccharides are either unsulfated (71%) or monosulfated (18%) . In sympathetic nerves, norepinephrine is stored in large dense cored vesicles that in biochemical composition and properties closely resemble adrenal chromaffin granules . However, in contrast to chromaffin granules, heparan sulfate accounts for approximately 75% of the total glycosaminoglycans in large dense-cored vesicles and more closely resembles heparin, insofar as it contains only 21% unsulfated disaccharides, 10% mono- and disulfated disaccharides, and 69% trisulfated disaccharides.(ABSTRACT TRUNCATED AT 250 WORDS) Glycobiology, 1994 Feb, 4(1), 69 - 78 Structural studies on the tri- and tetrasaccharides isolated from porcine intestinal heparin and characterization of heparinase/heparitinases using them as substrates; Yamada S et al.; We prepared a series of oligosaccharides from porcine intestinal heparin after extensive digestion with a mixture of Flavobacterium heparinase as well as heparitinases I and II . Previously, we reported the structures of the two glycoserines derived from the carbohydrate-protein linkage region {Sugahara et al., J . Biol . Chem., 267, 1528-1533 (1992)} and three tetrasaccharides derived from the antithrombin III-binding site {Yamada et al., J . Biol . Chem., 268, 4780-4787 (1993)} . In this study, we determined the structures of 10 other tetrasaccharides and a trisaccharide by enzymatic digestion, fast atom bombardment mass spectrometry and 500-MHz 1H NMR spectroscopy . These tetrasaccharides share the common disulphated structure, delta HexA alpha 1-4GlcN(N-sulphate)alpha 1-4IdoA(2-sulphate)alpha 1-4GlcN (where HexA is hexuronic acid and IdoA is L-iduronic acid), and their structural variations are based upon the positions of additional sulphate groups . Eight among the 10 have never been isolated as discrete structures . The structure of the trisaccharide is GlcN(N-sulphate)alpha 1-4IdoA(2-sulphate) alpha 1-4GlcN(N,6-disulphate) and is derived from the non-reducing terminus of heparin chains . This structure may represent the terminus of a biosynthetically formed native heparin chain or a newly formed non-reducing terminus exposed by a tissue endo-beta-glucuronidase which may be involved in the intracellular post-synthetic fragmentation of macromolecular heparin . The 11 structures characterized in the present study and 6 additional tetrasaccharides were used to investigate the substrate specificities of heparinase, as well as heparitinases I and II . The results indicate that modification of the adjacent glucosamine on the reducing side of the disaccharide cleavage site influences the enzymatic action of the lyases, whereas the adjacent uronic acid on the non-reducing side is not recognized by these enzymes. J Clin Microbiol, 1994 Feb, 32(2), 501 - 5 Ribotyping for differentiating Flavobacterium meningosepticum isolates from clinical and environmental sources; Colding H et al.; On the basis of DNA-DNA hybridization data, two main genomic relatedness groups (I and II) have been reported for a geographically varied collection of 52 strains of Flavobacterium meningosepticum . Herein, we have shown that genomic group II can be further divided into four subgroups (II:1 to II:4) . To examine the taxonomic relevance of the ribosomal patterns of the 52 F . meningosepticum strains, the patterns were compared with existing DNA-DNA hybridization data with restriction enzymes PstI and HindIII . Ribotyping of the 52 F . meningosepticum strains showed banding patterns that could identify them correctly to one of the five genomic groups or subgroups . To assess the value of ribotyping for the interpretation of epidemiological data, the discriminatory power of the method was investigated for the 52 F . meningosepticum strains . With one to four restriction enzymes (PstI, HindIII, ClaI, EcoRI), a discriminatory index of 0.95 to 0.97 was found . The value of ribotyping in an epidemiological setting was assessed for three clinical isolates of F . meningosepticum from an outbreak of meningitis and bacteremia in the neonatal intensive care unit, Rigshospitalet, Copenhagen, Denmark . The three clinical isolates were shown to belong to the same ribotype, characteristic of genomic subgroup II:1 . This ribotyping method will prove to be a useful tool for epidemiological studies concerning F . meningosepticum in the future. J Bacteriol, 1994 Feb, 176(4), 1197 - 200 Insertion sequence IS6100 on plasmid pOAD2, which degrades nylon oligomers; Kato K et al.; The nucleotide sequence of repeated sequence I, which appears in five regions on nylon oligomer-degrading plasmid pOAD2, harbored in Flavobacterium sp . strain K172, was determined . The five regions of repeated sequence I had 880 bp of identical sequence, and the sequence was identical to that of IS6100, an insertion sequence classified in the IS6 family, initially found in Mycobacterium fortuitum . Sequences homologous to that of IS6100 were found for another nylon oligomer-degrading plasmid, pNAD2, harbored in Pseudomonas sp . strain NK87, by Southern hybridization experiments. Nippon Hoigaku Zasshi, 1993 Dec, 47(6), 435 - 44 {Relationship between postmortem change and biological reaction}; Takatori T; In the adipocere which is one of postmortem changes, some specific fatty acids possessing higher melting points together with soap play an important role in the formation of adipocere . These fatty acids were clarified to be mainly 10-hydroxystearic and 10-hydroxypalmitic acids . Moreover, slight amounts of 10-oxostearic and 10-oxopalmitic acids, which have higher melting points than those of hydroxy fatty acids, exist in the adipocere as well . The substantial adipocere is formed and stabilized by these specific fatty acids together with the soap . The hydroxy fatty acid (OHFA) and oxo fatty acid (OXOFA) are biosynthesized by some enzymes from bacteria . Various aerobic and anaerobic bacteria are involved in the formation of adipocere . For example, microbial conversion of various unsaturated fatty acids to 10-OHFA by Micrococcus luteus was investigated . As a result, 10-OHFA was synthesized only from fatty acids possessing cis-9-unsaturation . It was also clarified that 10-OHFAs were converted to the corresponding 10-OXOFAs but the 10-OXO compounds were inactive as substrates . Furthermore, the enzyme preparations from Flavobacterium meningosepticum solubilized by sonication catalyzed not only hydration of oleic acid to produce 10-hydroxystearic acid but also dehydrogenation of this product in the presence of deuterium . On the other hand, we found out that there was 10-hydroxy-12-octadecenoic acid (10-OHLA) from linoleic acid in some kinds of adipocere . 10-OHFA existing in adipocere has been thought not to exist in a living body . However, recently 10-epoxy-12-octadecenoic acid (leukotoxin, LTx) which is one of lipid peroxides was found not only in rice plants but in polymorphonuclear leukocytes . It was also clarified that these polymorphonuclear leukocytes produced the same 10-OHLA as the compound found in adipocere . Since LTx was found from leukocytes related to inflammatory response, it has been interested in involvement of not only the basic mechanism of biological defense but also the mechanism of shock as a vasoactive substance . A postmortem change itself is little associated with a phenomenon on a living body . However, 10-OHLA found in adipocere existed also in polymorphonuclear leukocytes, suggesting that this compound metabolized from LTx is closely related to a biological reaction. Biochem Biophys Res Commun, 1993 Nov 30, 197(1), 179 - 86 The first demonstration of a procaryotic glycosylasparaginase; Tarentino AL et al.; Glycosylasparaginase was purified to near homogeneity from intracellular lysates of Flavobacterium meningosepticum . The enzyme is a heterodimer with an estimated molecular weight of 38 kDa and consists of one alpha-subunit (18 kDa) and one beta-subunit (16 kDa) . The beta-subunit of the Flavobacterium enzyme has a direct evolutionary relationship to the beta-subunit of mammalian glycosylasparaginases as evidenced by: (1) strong cross-reactivity with antibodies made to the denatured rat beta-subunit, (2) a high degree of homology with the amino-terminus of the corresponding eukaryotic enzymes, and (3) irreversible inactivation with 5-diazo-4-oxo-L-norvaline, a reagent known to react with the catalytic amino-terminal threonine residue on the beta-subunit of a mammalian glycosylasparaginase. Appl Environ Microbiol, 1993 Nov, 59(11), 3978 - 80 Nylon oligomer degradation gene, nylC, on plasmid pOAD2 from a Flavobacterium strain encodes endo-type 6-aminohexanoate oligomer hydrolase: purification and characterization of the nylC gene product; Kakudo S et al.; A new type of nylon oligomer degradation enzyme (EIII) was purified from an Escherichia coli clone harboring the EIII gene (nylC) . This enzyme hydrolyzed the linear trimer, tetramer, and pentamer of 6-aminohexanoate by an endo-type reaction, and this specificity is different from that of the EI (nylA gene product) and EII (nylB gene product) . Amino acid sequencing and sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the purified EIII demonstrated that the enzyme is made of two polypeptide chains arising from an internal cleavage between amino acid residues 266 and 267. Arch Biochem Biophys, 1993 Nov 1, 306(2), 461 - 8 Substrate specificity of the heparin lyases from Flavobacterium heparinum; Desai UR et al.; A detailed knowledge about the substrate specificities of the heparin lyases is necessary when using these enzymes as tools for elucidating the sequence of heparin and heparan sulfate . The substrate specificity of heparin lyases I, II, and III have been profiled with structurally defined, heparin-derived oligosaccharides . The primary substrate specificities of heparin lyases I and III require the presence of 2-O-sulfated alpha-L-idopyranosyluronic acid and beta-D-glucopyranosyluronic acid residues, respectively, at the linkages being cleaved . Heparin lyase II demonstrates an intriguingly broad primary specificity for oligosaccharides, acting at linkages containing alpha-L-idopyranosyluronic and beta-D-glucopyranosyluronic acid as well as at linkages containing alpha-L-galactopyranosyluronic acid residues . In addition to their primary specificities, each lyase also demonstrates secondary specificities under forcing conditions . Differences in the sulfation pattern within uronic acid residues and sulfation of adjacent residues has profound impact on the ease of lyase cleavage of a glycosidic linkage . Specifically, heparin lyases I and III exhibit secondary specificity for oligosaccharides containing an unsulfated alpha-L-idopyranosyluronic acid residue . The lack of sulfation on residues adjacent to the linkage undergoing cleavage increases the action of heparin lyase III on a glycosidic linkage . In contrast, reduced sulfation on adjacent residues make glycosidic linkage resistant to heparin lyase I . The primary and secondary specificity can be rationalized on the basis of most favorable solution conformation of the uronic acid residues. Yakugaku Zasshi, 1993 Oct, 113(10), 683 - 97 {Microbial enzymes and their inhibitors}; Tsuru D; Several proteolytic enzymes and dehydrogenases of microbial origin were studied with special regard to structure-activity relationship . Enzyme genes of Zn-proteases, subtilisin and pyroglutamyl aminopeptidase from genus Bacillus, prolyl endopeptidases from Flavobacterium and Aeromonas, and of protease II from E . coli were cloned, sequenced and overproduced in E . coli, their active site structures being elucidated by chemical modification as well as by site-directed mutagenesis . Homology analysis revealed that there is a prolyl endopeptidase family as a new family of serine endopeptidases . In addition, enzymatic properties and the primary structures of glutathione-independent formaldehyde dehydrogenase of Pseudomonas putida and 7 alpha-hydroxysteroid dehydrogenase from E . coli were elucidated . Amino acid sequences deduced from the nucleotide sequences of their genes indicated that the former enzyme should be classified into a long-chain metallo alcohol dehydrogenase family, and the latter belongs to a member of the short-chain nonmetallo-alcohol dehydrogenase family. Int J Syst Bacteriol, 1993 Oct, 43(4), 768 - 76 Riemerella anatipestifer gen . nov., comb . nov., the causative agent of septicemia anserum exsudativa, and its phylogenetic affiliation within the Flavobacterium-Cytophaga rRNA homology group; Segers P et al.; The phylogenetic position of the causative agent of septicemia anserum exsudativa, now most often referred to as {Moraxella} anatipestifer (brackets indicate a generically misnamed taxon) or "{Pasteurella} anatipestifer," was established by performing rRNA cistron similarity studies . {Moraxella} anatipestifer belongs to rRNA superfamily V, together with the genera Flavobacterium, Cytophaga, Flexibacter, Weeksella, Capnocytophaga, and Sphingobacterium . The detailed structure of rRNA superfamily V, which now contains five major rRNA homology groups, is described . An analysis of various phenotypic parameters, including new data (cellular proteins and fatty acids) and previously published data (respiratory quinones, enzyme activities, and classical phenotypic features), revealed that {Moraxella} anatipestifer differs in many aspects from its closest relatives, Flavobacterium indologenes, Flavobacterium gleum, Flavobacterium indoltheticum, Flavobacterium balustinum, Flavobacterium meningosepticum, and Weeksella zoohelcum . The combined genotypic and phenotypic data indicate that this organism should be placed in a separate genus; the name Riemerella anatipestifer gen . nov., comb . nov . is proposed for this bacterium . The specific epithet anatipestifer is kept in order to avoid nomenclatural confusion . However, it should be emphasized that the illness caused by this organism is a septicemic disease which is not restricted to ducks. Appl Microbiol Biotechnol, 1993 Oct, 40(1), 90 - 7 Cloning of proline-specific endopeptidase gene from Flavobacterium meningosepticum: expression in Escherichia coli and purification of the heterologous protein; Diefenthal T et al.; Proline-specific endopeptidase (PSE) (EC 3.4.21.26) from Flavobacterium meningosepticum was subjected to partial amino acid sequencing . According to the peptide sequences obtained, oligonucleotides were used to amplify a PSE-specific DNA fragment of 930 bp from F . meningosepticum genomic DNA, employing the polymerase chain reaction technique . This fragment served as a molecular probe to isolate the respective gene . DNA sequencing revealed that the PSE gene consists of 2118 bp coding for a 78,634 Da protein of 705 amino acids . The coding region was cloned in different expression vectors of Escherichia coli . Transformed E . coli cells overproduce an active prolyl endopeptidase of 75,000 relative molecular mass, which is delivered to the bacterial periplasmic space . Up to 1.6 units of active prolyl endopeptidase were obtained from 1 mg E . coli cells . Furthermore, the efficient purification of active prolyl endopeptidase from the periplasm of recombinant E . coli cells is described. J Clin Microbiol, 1993 Oct, 31(10), 2751 - 7 Cultivation of cilia-associated respiratory bacillus in artificial medium and determination of the 16S rRNA gene sequence; Schoeb TR et al.; Cilia-associated respiratory (CAR) bacillus, an unclassified gliding bacterium associated with respiratory disease in rats, mice, and rabbits, has previously been cultivated only in embryonated chicken eggs, cell culture, or cell culture medium supplemented with conditioned medium from cultured tracheas . A reference strain of CAR bacillus, originally isolated in eggs, grew in cell culture flasks as adherent individual bacilli and ropy, whorled fascicles in cell culture media supplemented only with fetal calf serum . Using Dulbecco's minimal essential medium, we isolated CAR bacillus from naturally infected rats and a naturally infected rabbit and from experimentally inoculated mice and rats . Isolates were maintained for up to 20 passages . Isolates from rats were similar in morphology to the reference strain, but most were more actively motile and formed pincushion-like aggregates . The rabbit bacilli were smaller and formed fewer aggregates . DNAs of rat isolates differed only slightly in restriction fragment patterns from that of the reference strain, whereas that of the rabbit isolate was distinctly different . Cultures of CAR bacilli of all strains from rats contained Mycoplasma fermentans, Mycoplasma pulmonis, or both, and cultures of the CAR bacillus from the rabbit contained an unidentified arginine-utilizing mycoplasma . The sequence of the 16S rRNA gene of the reference strain was determined by amplification by polymerase chain reaction, cloning of the product, and sequencing by the dideoxynucleotide chain termination method . Comparison of the sequence with sequences in the GenBank data base indicated that CAR bacillus is a unique organism most closely related to Flavobacterium ferrugineum and Flexibacter sancti. J Biol Chem, 1993 Sep 25, 268(27), 20590 - 7 Phosphorylation and reaction intermediates of the prokaryotic Ca(2+)-ATPase; Gambel AM et al.; A P-type Ca(2+)-ATPase activity has been identified previously in membranes of the Gram-negative bacteria Flavobacterium odoratum . The reaction mechanism of the prokaryotic Ca(2+)-ATPase was examined by isolation of reaction cycle intermediates formed during reversal of the normal cycle using inorganic P(i) . The eukaryotic ATPase can be directly phosphorylated by P(i) only in the absence of calcium, and half-maximal inhibition is observed at 1-2 microM calcium . The F . odoratum Ca(2+)-ATPase is also phosphorylated by P(i) in the absence of calcium, but in contrast to the eukaryotic pump, a 30-35-fold increase in phosphoenzyme (E-P) formation is observed when the enzyme is preincubated in millimolar calcium . Furthermore, the half-maximal activation of E-P formation is observed at approximately 100 microM calcium, similar to the value for low-affinity calcium binding in the sarcoplasmic reticulum Ca(2+)-ATPase . The data suggest that the prokaryotic Ca(2+)-ATPase forward reaction is ordered and that the prokaryotic enzyme does not appear to bind calcium at the high-affinity site until ATP binds . Thus, calcium does not inhibit phosphoenzyme formation by P(i) because the high-affinity Ca2+ binding site is not exposed on the free enzyme (E1). Int J Biochem, 1993 Sep, 25(9), 1219 - 25 Structure of heparan sulfate from the fresh water mollusc Anomantidae sp: sequencing of its disaccharide units; Ferreira TM et al.; 1 . The disaccharide sequences of a heparan sulfate isolated from Anomantidae sp . was determined with the aid of heparitinase I, heparitinase II from Flavobacterium heparinum, mollusc beta-glucuronidase and alpha-N-acetylglucosaminidase besides nitrous acid degradation and chemical analyses . 2 . Like the mammalian heparan sulfates the mollusc heparan sulfate is composed of different oligosaccharide blocks of N-acetylated disaccharides, N-sulfated disaccharides and N,6-sulfated disaccharides and has in its nonreducing end the monosaccharide glucosamine 2,6-disulfate . 3 . The oligosaccharides produced by heparitinase I degradation contain at their reducing ends a N-acetylated, 6-sulfated disaccharide . 4 . These and other results lead to the conclusion that the general structure of the heparan sulfate is maintained through evolution. Biochemistry, 1993 Aug 17, 32(32), 8140 - 5 Specificity studies on the heparin lyases from Flavobacterium heparinum; Desai UR et al.; An understanding of the substrate specificity study of the heparin lyases (heparinase and heparitinases) is crucial for elucidation of the sequence of heparin and heparan sulfate . Four chemically modified heparins have been used to study the substrate specificity of the three heparin lyases . These modified heparins include the N- and O-desulfated and then specifically N-sulfated or N-acetylated derivatives of heparin and a modified heparin containing L-galactopyranosyluronic acid residues . These chemically modified heparins were degraded to various extents by the three heparin lyases . Differences in degree of sulfation have profound impact on the ease of cleavage of glycosidic linkages . Heparin lyase I (EC 4.2.2.7) is selective in cleaving highly sulfated polysaccharide chains containing linkages to 2-O-sulfated alpha-L-idopyranosyluronic acid residues . Heparin lyase III (EC 4.2.2.8) cleaves linkages that have reduced density of sulfation and that contain beta-D-glucopyranosyluronic acid residues . The ability of heparin lyase III to act on linkages to unsulfated alpha-L-idopyranosyluronic acid residues is observed for the first time . Heparin lyase II (no assigned EC number) demonstrates an unparalleled, wide specificity for substrates comprised of linkages containing both alpha-L-idopyranosyluronic and beta-D- glucopyranosyluronic acid residues . Heparin lyase II can also act on substrates containing linkages to unnatural alpha-L-galactopyranosyluronic acid residues . The high level of specificity of heparin lyase I makes it particularly suitable for use in the sequencing of heparin and heparan sulfate, while caution must be exercised in using heparin lyases II and III to sequence heparin and heparan sulfate because of their relatively broad specificity. Clin Infect Dis, 1993 Aug, 17(2), 185 - 7 Flavobacterial sepsis in massively burned pediatric patients; Sheridan RL et al.; An experience with wound sepsis due to Flavobacterium meningosepticum in two pediatric burn patients is described . This organism, which is typically found in water and soil, generally has low pathogenicity but may become clinically important in immunocompromised hosts . It is typically resistant to antibiotics prescribed for infections caused by aerobic, gram-negative bacteria . When flavobacteria are suspected as pathogens, initial therapy should begin with ciprofloxacin, trimethoprim-sulfamethoxazole, or clindamycin. J Nat Prod, 1993 Aug, 56(8), 1304 - 12 Immobilization of isochorismate hydroxymutase . Comparison of native versus immobilized enzyme; Schaaf PM et al.; Partially purified isochorismate hydroxymutase (isochorismate synthase, E.C . 5.4.99.6) from Flavobacterium K3-15, a vitamin K overproducer, was immobilized on CNBr-activated Sepharose 4B, alkylamine glass substituted with glutardialdehyde, and aminohexyl Sepharose 4B substituted with glutardialdehyde . The immobilized enzyme exhibited a lower specific activity but a broader pH tolerance and a higher thermostability than the soluble enzyme . The stability of the enzyme was greatly increased by immobilization . Isochorismic acid, which is not commercially available, was prepared by a constant flow incubation. Biochem Mol Biol Int, 1993 Jul, 30(3), 579 - 87 Activation of a heparin-degrading enzyme by a 'protein matrix' effect; Khan MY et al.; An unusual activating effect of protein on Flavobacterium heparinase is described . The phenomenon is nonselective with respect to protein species, but does not occur with other biomolecules such as nucleic acids, polysaccharides, or free amino acids . We show that protein activates heparinase over broad ranges of temperature and ionic strength, and stabilizes the enzyme against both reversible and irreversible structural changes . The nonselective activation of an inducible enzyme by protein may be an important regulatory mechanism in microenvironments in which the concentration of organic material may vary. Int J Syst Bacteriol, 1993 Jul, 43(3), 555 - 64 Proposal of six new species in the genus Aureobacterium and transfer of Flavobacterium esteraromaticum Omelianski to the genus Aureobacterium as Aureobacterium esteraromaticum comb . nov; Yokota A et al.; Twelve strains placed in the genera Flavobacterium, Pseudomonas, and Aureobacterium, including soil isolates, were characterized taxonomically . On the basis of morphological, physiological, and chemotaxonomic data, as well as DNA-DNA hybridization data, we propose that 11 of these strains should be classified in the genus Aureobacterium as new combinations or new species, as follows: Aureobacterium esteraromaticum comb . nov . (type strain, IFO 3751 {= ATCC 8091}), Aureobacterium arabinogalactanolyticum sp . nov . (type strain, IFO 14344), Aureobacterium keratanolyticum sp . nov . (type strain, IFO 13309), Aureobacterium luteolum sp . nov . (type strain, IFO 15074 {= DMS 20143}), Aureobacterium schleiferi sp . nov . (type strain, IFO 15075 {= DMS 20489}), Aureobacterium terrae sp . nov . (type strain, IFO 15300), and Aureobacterium trichothecenolyticum sp . nov . (type strain, IFO 15077 {= JCM 1358}) . Whereas the peptidoglycan type of members of this genus is considered to be B2beta, the new species A . keratanolyticum was shown to have a new peptidoglycan type, murein variation B2alpha . An emended description of the genus Aureobacterium is presented. Chem Biol Interact, 1993 Jun, 87(1-3), 55 - 68 Characterization of organophosphorus hydrolases and the genetic manipulation of the phosphotriesterase from Pseudomonas diminuta; Dave KI et al.; There are a variety of enzymes which are specifically capable of hydrolyzing organophosphorus esters with different phosphoryl bonds from the typical phosphotriester bonds of common insecticidal neurotoxins (e.g . paraoxon or coumaphos) to the phosphonate-fluoride bonds of chemical warfare agents (e.g . soman or sarin) . These enzymes comprise a diverse set of enzymes whose basic architecture and substrate specificities vary dramatically, yet they appear to be ubiquitous throughout nature . The most thoroughly studied of these enzymes is the organophosphate hydrolase (opd gene product) of Pseudomonas diminuta and Flavobacterium sp . ATCC 27551, and the heterologous expression, post-translational modification, and genetic engineering studies undertaken with this enzyme are described. J Biochem (Tokyo), 1993 Jun, 113(6), 790 - 6 Prolyl endopeptidase from Aeromonas hydrophila: cloning, sequencing, and expression of the enzyme gene, and characterization of the expressed enzyme; Kanatani A et al.; A strain of Aeromonas hydrophila was found to show prolyl endopeptidase activity . The enzyme gene was cloned and expressed in Escherichia coli JM83 . A 12 kbp EcoRI fragment containing the enzyme gene was subcloned at the HincII site of pUC19 to construct plasmid pAPEP-3 with a 3.5 kbp insert . E . coli JM83 transformed with this plasmid showed about 100-fold higher activity than the parent Aeromonas . Analysis of the nucleotide sequence of the insert revealed that the mature enzyme-encoding sequence starts just after the ATG initiation codon of the open reading frame . The enzyme was a single polypeptide composed of 689 amino acid residues with a molecular weight of 76,383 . It showed properties very similar to those of Flavobacterium prolyl endopeptidase, except that the isoelectric point was 5.5 . The amino acid sequence was 56 and 41% homologous to those of Flavobacterium and porcine brain prolyl endopeptidases, respectively . From a survey of sequence homology with other members of the prolyl endopeptidase family, the amino acid residues involved in the catalytic triad were deduced to be Ser-537, His-656, and Asp-512 (or Asp-621). J Gen Microbiol, 1993 Jun, 139 ( Pt 6), 1155 - 61 Phylogenetic diversity of the genus Cytophaga revealed by 16S rRNA sequencing and menaquinone analysis; Nakagawa Y et al.; To clarify the intra- and intergeneric relationships of the genus Cytophaga, 16S rRNA sequences and respiratory isoprenoid quinones were determined for the type strains of the 21 validly published species and one isolate in the genus Cytophaga . The sequence analysis revealed extreme heterogeneity of this genus, which diverged into nine distinct lines of descent . Each lineage of Cytophaga was characterized by possessing either menaquinone-6 (MK-6) or MK-7 . The MK-6-possessing species were located in the two lineages that were remote from MK-7 species . One of the MK-6 lineages was composed only of terrestrial species and the other only of marine species . Flavobacterium aquatile, the type species of the genus Flavobacterium, was located in the MK-6 terrestrial lineage . The terrestrial Cytophaga species with MK-6 should be transferred to the genus Flavobacterium . The marine facultative anaerobes with MK-7 were located in the bacteroides branch, and possessed signature sequences with features intermediate between the bacteroides and the flavobacteria subdivisions . Cytophaga hutchinsonii, the type species of the genus Cytophaga, had a close relationship only with Cytophaga aurantiaca . The genus Cytophaga should be restricted to these two cellulose-degrading species . The genus Cytophaga is so heterogeneous that it should be divided into several genera and higher taxa in accordance with the phylogenetic relationships. Nucleic Acids Res, 1993 May 11, 21(9), 2193 - 9 Unusually biased nucleotide sequences on sense strands of Flavobacterium sp . genes produce nonstop frames on the corresponding antisense strands; Ikehara K et al.; From investigation of eight Flavobacterium sp . genes encoding enzyme proteins, it was found that six genes had nonstop frames (NSFs) on the antisense strands, and base sequences of the genes are mainly composed of repeating triplet sequence(s), 5'-GNC-3' (where G and C are guanine and cytosine, and N is either of the four bases), in the reading frames . Thus, we concluded that the biased nucleotide sequences on the sense strands produce NSFs on the corresponding antisense strands . Furthermore, from the precise alignments of both nucleotide and amino acid sequences of two related Flavobacterium sp . genes, nyIB and nyIB', it was found that base replacements might have occurred symmetrically in the codons . That is, transversions between G and C were observed at high frequencies at the first and third positions of codons, but not at the second positions . At the first position, AG base transitions were observed much more than similar CT transitions, whereas CT transitions were found at the third positions at a relatively high frequency . These suggest that symmetrical base replacements in codons might be the main contribution to evolution in Flavobacterium sp . genes. J Biol Chem, 1993 May 5, 268(13), 9702 - 8 Multiple endoglycosidase F activities expressed by Flavobacterium meningosepticum endoglycosidases F2 and F3 . Molecular cloning, primary sequence, and enzyme expression; Tarentino AL et al.; The genes for Flavobacterium meningosepticum Endo (endoglycosidase) F2 and Endo F3 were cloned, and their nucleotide sequences were determined . The deduced amino acid sequences were verified independently to a large extent by direct peptide microsequencing of 66 and 84% of native Endo F2 and Endo F3, respectively . Structurally, the Endo F2 and Endo F3 genes code for a typically long leader sequence of 45 and 39 amino acids, respectively, and, in both cases, a mature protein of 290 amino acids . Comparative structural analysis demonstrated minimum overall homology (15-30%) between Endo F1, Endo F2, and Endo F3, but revealed distinct clusters of identical residues distributed throughout the entire sequence, which represent motifs for binding and hydrolysis of beta 1,4-di-N-acetylchitobiosyl linkages in complex carbohydrates . The mobility of native Endo F2 and Endo F3 on SDS-polyacrylamide gel electrophoresis, unlike Endo F1, did not correlate with the molecular weights determined from the coding region of the corresponding genes . Mass spectrometry confirmed that Endo F2 and Endo F3 were heterogeneous and contained approximately 4000 and 1200 daltons of mass not accounted for in the gene structure . We presume that Endo F2 and Endo F3 are variably post-translationally modified during secretion by possible linkage to the hydroxyl of serine. J Bacteriol, 1993 May, 175(9), 2734 - 42 Low-molecular-weight thiols in streptomycetes and their potential role as antioxidants; Newton GL et al.; The intracellular low-molecular-weight thiols present in five gram-positive Streptomyces species and one Flavobacterium species were analyzed by high-performance liquid chromatography after fluorescence labeling with monobromobimane . Bacteria were chosen to include penicillin and cephalosporin beta-lactam producers and nonproducers . No significant amount of glutathione was found in any of the streptomycetes . Major intracellular thiols in all strains examined were cysteine, coenzyme A, sulfide, thiosulfate, and an unknown thiol designated U17 . Those streptomycetes that make beta-lactam antibiotics also produce significant amounts of delta-(L-alpha-aminoadipyl)-L-cysteinyl-D-valine (ACV), a key intermediate in their biosynthesis . In Streptomyces clavuligerus, a potent producer of beta-lactams, the level of ACV was low during the early phase of growth and increased rapidly toward the end of exponential growth, paralleling that of antibiotic production . These and other observations indicate that ACV does not function as a protective thiol in streptomycetes . U17 may have this role since it was the major thiol in all streptomycetes and appeared to occur at levels about 10-fold higher than those of the other thiols measured, including ACV . Purification and amino acid analysis of U17 indicated that it contains cysteine and an unusual amine that is not one of the common amino acids . This thiol is identical to an unknown thiol found previously in Micrococcus roseus and Streptomyces griseus . A high level of ergothioneine was found in Streptomyces lactamdurans, and several unidentified thiols were detected in this and other streptomycetes. J Bacteriol, 1993 May, 175(9), 2640 - 4 Characterization of a Flavobacterium glutathione S-transferase gene involved reductive dechlorination; Orser CS et al.; The gene pcpC, encoding tetrachloro-p-hydroquinone (TeCH) reductive dehalogenase, was cloned from Flavobacterium sp . strain ATCC 39723 and sequenced . The gene was identified by hybridization with a degenerate oligonucleotide designed from the N-terminal sequence of the purified protein . An open reading frame of 747 nucleotides was found, which predicts a translational product of 248 amino acids having a molecular weight of 28,263, which agrees favorably with the sodium dodecyl sulfate-polyacrylamide gel electrophoresis-determined molecular weight of 30,000 reported for the purified protein . The predicted translational product of pcpC matched the N-terminal sequence of the purified protein exactly . From the nucleotide sequence, the protein appears to have a processed formylmethionyl . An Escherichia coli pcpC overexpression clone was shown to produce dichlorohydroquinone and trichlorohydroquinone from TeCH . Protein data base searches grouped the predicted translational sequence of pcpC with two previously reported plant glutathione S-transferases but less significantly with any of the mammalian glutathione S-transferases or the glutathione-utilizing, hydrolytic dechlorinating enzyme from Methylobacterium sp . strain DM4. Proc Natl Acad Sci U S A, 1993 Apr 15, 90(8), 3660 - 4 Cloning and expression of heparinase I gene from Flavobacterium heparinum; Sasisekharan R et al.; Heparinases, enzymes that cleave heparin and heparin sulfate, are implicated in physiological and pathological functions ranging from wound healing to tumor metastasis and are useful in deheparinization therapies . We report the cloning of the heparinase I (EC 4.2.2.7) gene from Flavobacterium heparinum using PCR . Two degenerate oligonucleotides, based on the amino acid sequences derived from tryptic peptides of purified heparinase, were used to generate a 600-bp probe by PCR amplification using Flavobacterium genomic DNA as the template . This probe was used to screen a Flavobacterium genomic DNA library in pUC18 . The open reading frame of heparinase I is 1152 bp in length, encoding a precursor protein of 43.8 kDa . Eleven of the tryptic peptides (approximately 35% of the total amino acids) mapped onto the open reading frame . The amino acid sequence reveals a consensus heparin binding domain and a 21-residue leader peptide with a characteristic Ala-(Xaa)-Ala cleavage site . Recombinant heparinase was expressed in Escherichia coli as a soluble protein, using the T7 polymerase pET expression system . The recombinant heparinase cleavage of heparin was identical to that of native heparinase. J Gen Microbiol, 1993 Apr, 139 ( Pt 4), 787 - 95 Characterization of the 6-aminohexanoate-dimer hydrolase from Pseudomonas sp . NK87; Kanagawa K et al.; The DNA base sequence of the Pseudomonas sp . NK87 gene (P-nylB) for 6-aminohexanoate-dimer hydrolase (P-EII), a xenobiotic-compound-degrading enzyme, was determined . It has an open reading frame of 1188 bp, initiated by ATG and terminated by TAG, and coding for 396 amino acids . The base sequence of the open reading frame has 53% sequence similarity to that of the gene for the same enzyme of Flavobacterium sp . KI72 (F-nylB) and 35% sequence similarity with respect to the deduced amino acid sequence . The P-EII enzyme was purified from an Escherichia coli clone in which the P-EII gene was highly expressed . The P-EII enzyme was inhibited by a serine protease inhibitor, diisopropyl fluorophosphate, as was the F-EII enzyme . Double reciprocal plots obtained from various concentrations of 6-aminohexanoate-dimer indicated that the kcat value of the P-EII enzyme (9.2 s-1) was approximately half that of the F-EII enzyme (19 s-1), and the P-EII enzyme had higher affinity toward this substrate (Km for P-EII, 0.6 mM; Km for F-EII, 15 mM) . The P-EII enzyme had a temperature optimum of 48 degrees C, and a pH optimum of 7.5 . It is speculated that since the P-nylB and F-nylB genes are more diverged from each other than the corresponding nylA genes, the latter may have evolved more recently. J Biol Chem, 1993 Mar 5, 268(7), 4780 - 7 Structural studies on the bacterial lyase-resistant tetrasaccharides derived from the antithrombin III-binding site of porcine intestinal heparin; Yamada S et al.; Three discrete tetrasaccharide structures which are resistant to Flavobacterium heparinase and heparitinases I and II were isolated from porcine intestinal heparin after exhaustive digestion with a mixture of all the above enzymes, and the tri-, tetra-, and penta-sulfated structures were determined by negative ion mode fast atom bombardment mass spectrometry and 500-MHz 1H NMR analysis as delta 4,5GlcA beta 1-4GlcNAc (6-sulfate)alpha 1-4GlcA beta 1-4GlcN(N,3-disulfate), delta 4,5 GlcA beta 1-4GlcNAc(6-sulfate)alpha 1-4GlcA beta 1-4GlcN (N,3,6-trisulfate), and delta 4,5GlcA beta 1-4GlcN (N,6-disulfate)alpha 1-4GlcA beta 1-4GlcN(N,3,6-trisulfate) . The three components share the 3-O-sulfated reducing GlcN and the 6-O-sulfated internal GlcN, indicating that they are structural variants derived from the nonreducing portion of the minimal pentasaccharide sequence required for binding to antithrombin III . Isolation of the pentasulfated component has never been reported . Their unexpected resistance to heparitinases I and II indicates that 3-O-sulfation of the reducing GlcN contributes to the resistant nature of these tetrasaccharides to the enzymes . The present study demonstrates that the nonreducing trisaccharide portion of the structural variants of the antithrombin III-binding pentasaccharide sequence can be isolated in tetrasaccharides resistant to heparinase/heparitinases I and II, while the rest of the repeating region is degraded into disaccharide units . The lyase treatment is applicable to evaluation of heparin/heparan sulfate preparations in terms of the presence or absence of the specific structure containing the 3-O-sulfated GlcN representing biosynthetic precursors, intermediates or final products of the binding site. Haemostasis, 1993 Mar, 23 Suppl 1, 161 - 76 Isolation and characterization of ryudocan and syndecan heparan sulfate proteoglycans, core proteins, and cDNAs from a rat endothelial cell line; Shworak NW et al.; We have isolated heparan sulfate proteoglycans (HSPGs) from cloned rat microvascular endothelial cells using a combination of ion-exchange chromatography, affinity fractionation with antithrombin III (AT III), and gel filtration in denaturing solvents . The anticoagulantly active heparan sulfate proteoglycans (HSPGact) which bind tightly to AT III bear mainly anticoagulantly active heparan sulfate (HSact) whereas the anticoagulantly inactive heparan sulfate proteoglycans (HSPGinact) possess mainly anticoagulantly inactive heparan sulfate (HSinact) . The core proteins of HSPGact and HSPGinact were isolated by treatment with Flavobacterium heparitinase and purification by ion-exchange chromatography . SDS-PAGE showed that both sets of core proteins exhibited three major components with M(r) of 25-, 30-, and 50-kD, respectively . Peptide mapping revealed that HSPGact and HSPGinact possess extremely similar core proteins . The primary sequences of internal peptides obtained from HSPGinact core proteins and the NH2-terminal sequence analyses of the 25-kD component from the HSPGinact core proteins demonstrate that the 30-kD component is a previously unidentified species--designated as ryudocan--with the 25-kD component representing a proteolytic degradation product; while the 50-kD component is the rat homolog of syndecan {Saunders S, Jalkanen M, O'Farrell S, Bernfield M: J Cell Biol 1989; 108:1547-1556} . Specific oligonucleotide probes were obtained for ryudocan and syndecan by PCR, and the corresponding cDNAs were isolated from a RFP-EC library . The cDNAs encode type I integral membrane proteins of 202 and 313 amino acids, respectively, which have homologous transmembrane and intracellular domains but very distinct extracellular regions . In particular, ryudocan exhibits only 3 potential glycosaminoglycan (GAG) attachment sites within the extracellular region while syndecan has 5 GAG attachment sites within the same domain . The levels of ryudocan and syndecan mRNA were measured by quantitative PCR in primary microvascular endothelial cells and associated non-endothelial cells isolated by cell sorting . Ryudocan and syndecan mRNAs were abundantly expressed in both populations representing about 0.1-0.5% of mRNA. Int J Biochem, 1993 Mar, 25(3), 331 - 6 Monoclonal antibodies prepared against heparin lyase I and their reactivity toward heparin lyase I, II and III; Gu K et al.; 1 . Six different monoclonal IgG mouse antibodies to heparin lyase I from Flavobacterium heparinum were prepared . 2 . The monoclonal antibodies were used to detect heparin lyases I, II and III by dot-blotting immunoassay and by Western blotting . 3 . Individual antibodies showed different reactivity toward the three heparin lyases . 4 . The reactivity of two of the monoclonal antibodies was destroyed by exposing heparin lyases to sodium dodecyl sulfate . 5 . The antibodies can be used to rapidly distinguish between the three heparin lyases. Appl Theor Electrophor, 1993, 3(6), 297 - 303 Capillary electrophoresis to measure sulfoesterase activity on chondroitin sulfate and heparin derived disaccharides; Pervin A et al.; Capillary electrophoresis was used to assay sulfoesterase activity on sulfated disaccharides derived from chondroitin sulfate, dermatan sulfate and heparin . The three sulfoesterases studied were chondro-4-O-sulfatase (EC 3.1.6.9) and chondro-6-O-sulfatase (EC 3.1.6.10) from Proteus vulgaris and heparo-2-O-sulfatase from Flavobacterium heparinum . Capillary electrophoresis was used to analyse sulfated disaccharide before and after sulfoesterase treatment and a change in migration time was indicative of the presence of sulfoesterase activity . This assay was used both on purified sulfoesterases and on minor sulfoesterase contaminants present in other enzyme preparations . The high sensitivity of capillary electrophoresis permits the elimination of 35S-radiolabeled substrates normally required to assay sulfoesterases . The high resolution of capillary electrophoresis allows the use of this assay on impure enzyme preparations containing high protein concentrations. Int J Syst Bacteriol, 1993 Jan, 43(1), 77 - 83 Direct sequencing of the polymerase chain reaction-amplified 16S rRNA gene of Flavobacterium gondwanense sp . nov . and Flavobacterium salegens sp . nov., two new species from a hypersaline Antarctic lake; Dobson SJ et al.; Phenotypic data and phospholipid ester-linked fatty acid profiles indicate that pigmented bacterial strains isolated from a hypersaline Antarctic lake are members of the "flavobacterium-bacteroides" phylum and may represent new taxa . Nearly complete 16S rRNA sequences were obtained for representative strains by directly sequencing the polymerase chain reaction-amplified 16S rRNA gene . Sequence signatures confirmed that these organisms were members of the flavobacterium-bacteroides phylum . A phylogenetic analysis, in which the sequences of the Antarctic strains were compared with a large number of sequences available for members of the flavobacterium-bacteroides phylum, showed that the Antarctic strains were phylogenetically distinct . The new species cluster with a group of organisms that contains the type species of the genus Flavobacterium, Flavobacterium aquatile . Two new species are described, for which the names Flavobacterium gondwanense and Flavobacterium salegens are proposed; strains ACAM 44 (= DSM 5423) and ACAM 48 (= DSM 5424) are the type strains of F . gondwanense and F . salegens, respectively. J Bacteriol, 1993 Jan, 175(2), 411 - 6 Cloning, sequence analysis, and expression of the Flavobacterium pentachlorophenol-4-monooxygenase gene in Escherichia coli; Orser CS et al.; The pcpB gene of Flavobacterium sp . strain ATCC 39723 was cloned by using a degenerate primer designed from the N-terminal sequence of the purified enzyme . The nucleotide sequence of pcpB was determined and found to encode an open reading frame of 1,614 nucleotides, yielding a predicted translation product of 538 amino acids, in agreement with the estimated size of the purified protein analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis . The transcriptional start of pcpB was found to be 80 bp upstream of the translational start, and the transcript was found to be induced in Flavobacterium sp . strain ATCC 39723 by the presence of pentachlorophenol but to be constitutive in the Escherichia coli pcpB clone . DNA hybridizations with genomic DNAs from Arthrobacter sp . strain ATCC 33790 and Pseudomonas sp . strain SR3 revealed a similar-size 3.0-kb EcoRI fragment, whereas there was no positive hybridization with genomic DNA from Rhodococcus chlorophenolicus . Cell extracts from an E . coli pcpB overexpression strain, as well as the whole cells, were proficient in the dechlorination of pentachlorophenol to tetrachlorohydroquinone . Protein data base comparisons of the predicted translation products revealed regions of homology with other microbial monooxygenases, including phenol-2-monooxygenase and tryptophan-2-monooxygenase. J Biol Chem, 1992 Dec 5, 267(34), 24347 - 55 Purification and characterization of heparin lyases from Flavobacterium heparinum; Lohse DL et al.; Heparin lyase I has been purified from Flavobacterium heparinum and has been partially characterized (Yang, V . C., Linhardt, R . J., Berstein, H., Cooney, C . L., and Langer, R . (1985) J . Biol . Chem . 260, 1849-1857) . There has been no report of the purification of the other polysaccharide lyases from this organism . Although all three of these heparin/heparan sulfate lyases are widely used, with the exception of heparin lyase I, there is no information on their purity or their physical and kinetic characteristics . The absence of pure heparin lyases and a lack of understanding of the optimal catalytic conditions and substrate specificity has stood in the way of the use of these enzymes as reagents for the specific depolymerization of heparin and heparan sulfate into oligosaccharides for structure and activity studies . This paper describes a single, reproducible scheme to simultaneously purify all three of the heparin lyases from F . heparinum to apparent homogeneity . Heparin lyase I (heparinase, EC 4.2.2.7), heparin lyase II (no EC number), and heparin lyase III (heparitinase, EC 4.2.2.8) have molecular weights (by sodium dodecyl sulfate-polyacrylamide gel electrophoresis) and isoelectric points (by isoelectric focusing) of M(r) 42,800, pI 9.1-9.2, M(r) 84,100, pI 8.9-9.1, M(r) 70,800, pI 9.9-10.1, respectively . Their amino acid analyses and peptide maps demonstrate that while these proteins are different gene products they are closely related . The kinetic properties of the heparin lyases have been determined as well as the conditions to optimize their activity and stability . These data should improve the application of these important enzymes in the study of heparin and heparan sulfate. J Bacteriol, 1992 Dec, 174(24), 8003 - 7 Purification and characterization of a tetrachloro-p-hydroquinone reductive dehalogenase from a Flavobacterium sp; Xun L et al.; Tetrachloro-p-hydroquinone (TeCH) is the first intermediate in pentachlorophenol (PCP) degradation by Flavobacterium sp . strain ATCC 39723 . We previously purified a PCP hydroxylase that oxidized PCP to TeCH . Subsequently, we identified the reductive dehalogenation of TeCH to 2,3,6-trichloro-p-hydroquinone and then to 2,6-dichloro-p-hydroquinone in a cell extract with the reduced form of glutathione as the reducing agent under anaerobic conditions . Here we report the purification of a TeCH reductive dehalogenase that reductively dehalogenated TeCH to trichlorohydroquinone and then to dichlorohydroquinone . The enzyme was purified by protamine sulfate treatment, ammonium sulfate fractionation, and phenyl-agarose, anion-exchange, and gel filtration column chromatographies . As determined by gel filtration and sodium dodecyl sulfate-polyacrylamide gel electrophoresis analyses, the protein has a molecular weight of about 30,000; nondenaturing polyacrylamide gel electrophoresis analysis suggests that the native enzyme exists as a dimer . The enzyme used glutathione but not NADPH, NADH, dithiothreitol, or ascorbic acid as the reducing agent . The optimal pH was close to neutral. J Bacteriol, 1992 Dec, 174(24), 7948 - 53 A new nylon oligomer degradation gene (nylC) on plasmid pOAD2 from a Flavobacterium sp; Negoro S et al.; Flavobacterium sp . strain KI725 harbors plasmid pOAD21, a derivative of nylon oligomer-degradative plasmid pOAD2, in which all of nylA (the gene for 6-aminohexanoate cyclic dimer hydrolase {EI}) was deleted but nylB (the gene for 6-aminohexanoate dimer hydrolase {EII}) was retained . KI725 showed no growth on unfractionated nylon oligomers (Nom1) obtained from a nylon factory as a sole carbon and nitrogen source (Nom1 minimum plate) . Extracts of KI725 cells possessed hydrolytic activity for Nom1 (approximately 5% of the activity of KI72), but pOAD2-cured strains (KI722 and KI723) showed no activity . KI725R strains which grew on the Nom1 minimum plate were spontaneously isolated from KI725 at a frequency of 10(-7) per cell . Activity toward Nom1 was enhanced in KI725R strains (10 to 30% of the activity of KI72) . This new Nom1 degrading enzyme (EIII, the nylC gene product) hydrolyzed not only Nom1 but also the N-carbobenzoxy-6-aminohexanoate trimer, a substrate which was not hydrolyzed by either EI or EII . Cloning and sequence analysis showed that the nylC gene is located close to nylB on pOAD21 and is a 1,065-bp open reading frame corresponding to 355 amino acid residues . The nucleotide sequence of the nylC gene and the deduced amino acid sequence of EIII had no detectable homology with the sequences of nylA (EI) and nylB (EII). J Am Vet Med Assoc, 1992 Dec 1, 201(11), 1766 - 70 Flavobacterium indologenes infection in leopard frogs; Olson ME et al.; An investigation of an epidemic of infectious disease in a frog (Rana pipiens) colony was conducted . Six of 40 frogs in a continuous (once through) water flow housing system had weight loss, swollen abdomen, corneal edema, uveitis, subcutaneous edema, petechial hemorrhage, incoordination, and respiratory distress . The frogs had lesions consistent with bacterial septicemia . A gram-negative, nonfermenting bacillus, Flavobacterium indologenes (Flavobacterium sp biovar IIb), was isolated in pure culture from tissues and blood . The clinical isolate was used to inoculate healthy frogs sc . An isolate identical to the one isolated from the sick frogs was recovered from tissues and blood of the inoculated frogs . Inoculation of the housing water in a nonflow-through system did not result in disease, despite proliferation of the Flavobacterium spp in the water; therefore, it is likely that establishment of infection requires the presence of the organism in sufficient numbers and a portal of entry into the body. Jpn J Pharmacol, 1992 Dec, 60(4), 377 - 80 Protective effect of eurystatins A and B, new prolyl endopeptidase inhibitors, on scopolamine-induced amnesia in rats; Kamei H et al.; Eurystatins A and B, which are produced by Streptomyces eurythermus R353-21, potently inhibited Flavobacterium prolyl endopeptidase (PED) with IC50 values of 0.004 and 0.002 micrograms/ml, respectively, while no inhibition was observed against another 5 proteases, even at 100 micrograms/ml . The protective effect of eurystatins A and B against scopolamine (3 mg/kg, i.p.)-induced amnesia in rats was evaluated by the step-through one-trial passive avoidance method . When administered i.p . 30 min prior to the acquisition trial, both eurystatins A, at 2-8 mg/kg, and B, at 4-8 mg/kg, significantly protected rats from the amnesic effect of scopolamine without behavioral side effects. Gene, 1992 Nov 2, 121(1), 149 - 53 The aryldialkylphosphatase-encoding gene adpB from Nocardia sp . strain B-1: cloning, sequencing and expression in Escherichia coli; Mulbry WW; Using degenerate oligodeoxyribonucleotides (oligos) derived from the N-terminal sequence of an aryldialkylphosphatase (ADPase) from Nocardia sp . strain B-1, an amplification reaction was used to isolate a DNA segment containing a 57-bp fragment from the adpB gene . Based on the nucleotide (nt) sequence of this fragment, a nondegenerate oligo was synthesized and used to screen a subgenomic library of strain B-1 DNA for fragments containing adpB . A 3.55-kb PstI fragment containing adpB was cloned into Escherichia coli, and the nt sequence of a 1600-bp region containing adpB was determined . Under control of the lac promoter of pUC19, adpB expression in E . coli cultures was approx . 15-fold higher than in strain B-1 under the native adpB promoter . Comparison of adpB with the Flavobacterium ADPase-encoding gene, opd, revealed no significant homology at the nt or aa levels. Nihon Kyobu Shikkan Gakkai Zasshi, 1992 Oct, 30(10), 1864 - 8 {A case of hypersensitivity pneumonitis caused by a humidifier}; Tsujino I et al.; A 58-year-old woman was admitted complaining of dry cough and exertional dyspnea . Physical findings, chest X-ray films, chest CT scan and respiratory function tests were suggestive of interstitial pneumonia . Transbronchial lung biopsy showed specific findings of hypersensitivity pneumonitis . As a result of positive provocation test using her home humidifier, a diagnosis of humidifier lung was made . Many microorganisms including Flavobacterium meningosepticum were cultured from the water left in the humidifier for one week . As both complement fixation test and precipitation test were positive to humidifier water and to extract of Flavobacterium meningosepticum, the humidifier and Flavobacterium meningosepticum were suggested to be causative in this case. Nippon Eiseigaku Zasshi, 1992 Oct, 47(4), 798 - 810 {Statistical study of the optimal conditions of the spiral plating method for counting bacterial numbers in river water}; Wada M; The application of the spiral plating method, a rapid and labor-saving technique for enumerating bacteria in food, to evaluate bacteria in river water, was examined . Standard plate counts and the numbers of heterotrophic bacteria, coliforms and Flavobacterium spp . in the water were tested by the spiral plating method, the results were compared with those of the conventional method and the optimal conditions for using the spiral plating method were considered and selected to fit the conventional methods . The optimal conditions selected were as follows: by laser colony counter after incubation at 35 degrees C for 38h . for standard plate counts; by laser colony counter after incubation at 25 degrees C for 48h . for the numbers of heterotrophic bacteria and Flavobacterium spp.; by colony viewer counting reddish colonies > or = 0.25mm in diameter after incubation at 35 degrees C for 22h . for the number of coliforms . The numbers of bacteria, except for coliforms, determined by this spiral plating method were found to be closely related to those from conventional methods (r > or = 0.91), and the replicating variances of both methods were not significant . The counts of bacteria by laser colony counter gave results similar to those by colony viewer counting by visual inspection . This spiral plating method saved time, money and labor in the evaluation of bacteria in river water as comparable to that in food . These results indicate that the spiral plating method can be used in place of conventional methods in evaluating the number of bacteria in river water. Appl Microbiol Biotechnol, 1992 Oct, 38(1), 94 - 100 The effect of signal sequences on the efficiency of secretion of a heterologous phosphotriesterase by Streptomyces lividans; Rowland SS et al.; A heterologous phosphotriesterase (parathion hydrolase) containing the native Flavobacterium species signal sequence was previously shown to be secreted by Streptomyces lividans . Western blot analysis of the recombinant phosphotriesterase produced by S . lividans demonstrated only the mature form extracellularly but both processed and unprocessed forms in cell-associated samples . To investigate the efficiency of secretion in Streptomyces, a construction was made that substituted a native Streptomyces beta-galactosidase signal sequence for the Flavobacterium signal sequence . This resulted in a higher proportion of hydrolase in the extracellular fluid and a lower proportion of parathion hydrolase remaining cell-associated . These results suggest that use of a native Streptomyces signal sequence may result in more efficient secretion of heterologous proteins. Eur J Biochem, 1992 Sep 15, 208(3), 669 - 76 Occurrence of alpha 1-2-fucosylation in membrane glycoproteins of Morris hepatoma 7777 but not in liver . Aberrant type of fucosylation in a malignant tissue; Nuck R et al.; A comparative study was undertaken to characterize the linkages of L-fucose in N-glycans of plasma membrane glycoproteins from Morris hepatoma 7777, host liver and kidney cortex, as well as from rat serum . After in-vivo radiolabelling of rats with L-{6-3H}fucose, the asparagine-linked carbohydrate chains were released from delipidated plasma membrane glycoproteins, as well as from serum glycoproteins, by enzymic digestion with peptide-N4-(N-acetyl-beta-glucosaminyl)asparagine amidase from Flavobacterium meningosepticum . They were then converted to their corresponding oligosaccharide alditols by reduction with sodium borohydride . Two specific alpha-L-fucosidases from almond emulsin and from Aspergillus niger, combined with affinity HPLC on immobilized Aleuria aurantia lectin were used to study the linkage of L-fucose in the oligosaccharide chains . Fucose alpha 1-2 linked to galactose, was present only in the plasma membrane of hepatoma 7777 (18% of total L-{3H}fucose in N-glycans), but was not expressed in host liver, kidney cortex and serum . None of the investigated sources contained an appreciable amount of fucose alpha 1-3/4 linked to N-acetyl-D-glucosamine . All the radioactively labelled oligosaccharides from host liver, kidney cortex and serum, but only 82% of these oligosaccharides from hepatoma, contained alpha-fucosyl residues linked at the C6 position of the proximal N-acetyl-D-glucosamine. J Bacteriol, 1992 Sep, 174(17), 5745 - 7 Confirmation of oxidative dehalogenation of pentachlorophenol by a Flavobacterium pentachlorophenol hydroxylase; Xun L et al.; Pentachlorophenol (PCP) hydroxylase purified from Flavobacterium sp . strain ATCC 39723 converted PCP or 2,3,5,6-tetrachlorophenol to tetrachloro-p-hydroquinone (TeCH) with the co-consumption of O2 and NADPH . The purified enzyme incorporated 18O from 18O2 but not from H218O into the reaction end product TeCH . The results clearly demonstrate that PCP is oxidatively converted to TeCH by a monooxygenase-type enzyme from Flavobacterium sp . strain ATCC 39723. J Clin Microbiol, 1992 Sep, 30(9), 2447 - 50 Acanthamoeba keratitis: synergy between amebic and bacterial cocontaminants in contact lens care systems as a prelude to infection; Bottone EJ et al.; We encountered a patient with Acanthamoeba keratitis whose contact lens care solution contained numerous trophozoites and cysts admixed with Xanthomonas maltophilia organisms, many of which were adherent to the trophozoite surface and internalized within endocytic vacuoles . Because of this finding, we investigated the role of bacterial cocontaminants in contact lens care systems as substrates for the growth of Acanthamoeba spp . Individual cocultivation of Acanthamoeba castellanii and A . polyphaga with X . maltophilia, Flavobacterium breve, and Pseudomonas paucimobilis showed better enhancement (1.5x) of ameba growth after 96 h than that obtained in the presence of Staphylococcus aureus, S . epidermidis, and Escherichia coli, the standard cocultivation species used for isolation of amebae from clinical specimens . Our data suggest that contamination of contact lens care systems with Acanthamoeba spp . and a bacterial species capable of supporting amebic growth may be the first step in the pathogenesis of ameba-induced keratitis by the provision of large inocula of amebae. J Biol Chem, 1992 Aug 5, 267(22), 15923 - 31 Characterization of a P-type Ca(2+)-ATPase from Flavobacterium odoratum; Gambel AM et al.; In most bacterial cell types studied, low intracellular free calcium is maintained by a variety of secondary exchangers which utilize transmembrane ion gradients . Prokaryotic calcium ATPases appear to be extremely uncommon, and none have been reported in Gram-negative organisms . We demonstrate ATP-dependent calcium uptake in everted membrane vesicles of Flavobacterium odoratum, a common Gram-negative soil and water bacterium . Calcium is transported with an apparent initial rate of 10 nmol/min mg of protein . It is inhibited by 20 microM orthovanadate, a specific P-type ATPase inhibitor, but significantly, it is unaffected by the addition of N-ethylmaleimide, N,N-dicyclohexylcarbodiimide, valinomycin, or nigericin . Because the Ca(2+)-ATPase makes up a high proportion of the total ATPase activity it is easily detected by a soluble ATP hydrolysis assay, with an initial rate for calcium-dependent ATPase activity in vesicles of 25-40 nmol/min.mg at pH 7.8 and 25 degrees C . The calcium-dependent activity is preferentially solubilized by the detergent C12E8 and can be precipitated at 55-80% ammonium sulfate in a fraction free of other contaminating ATPase activities . This partially purified fraction is enriched 15-fold and demonstrates an apparent Km for calcium of 2 microM, and for ATP of 130 microM . The IC50 for vanadate is 1.6 microM . These values are similar to those obtained for the eukaryotic sarcoplasmic reticulum calcium ATPase . The enzyme is rapidly phosphorylated by {gamma-32P}ATP in a calcium-dependent, vanadate-inhibitable manner . The phosphorylated species migrates with an apparent molecular mass of 60 kDa by NaDodSO4-polyacrylamide gel electrophoresis, and the phosphoryl group is sensitive to alkaline conditions, a characteristic of the acylphosphate linkage found in ATPases . These data demonstrate that the majority of calcium transport in F . odoratum is facilitated by a P-type ATPase. Glycoconj J, 1992 Aug, 9(4), 162 - 7 Use of resorufin-labelled N-glycopeptide in a high-performance liquid chromatography assay to monitor endoglycosidase activities during cultivation of Flavobacterium meningosepticum; Bourgerie S et al.; Peptide-N4-(N-acetyl-beta-glucosaminyl) asparagine amidase F (PNGase F) and endo-beta-N-acetyl glucosaminidase F (Endo F) activities were monitored during cultivation of Flavobacterium meningosepticum using a new fluorescence-HPLC procedure based on a commercially available substrate . The PNGase F activity reached a maximum level at the end of the log phase and remained constant during the stationary phase, while Endo F continuously increased until late stationary phase . PNGase F obtained at the end of the log phase was less contaminated by other proteins compared with late stationary phase. Biotechniques, 1992 Aug, 13(2), 226 - 30 Subcloning using simplified adaptor addition; Supak-Koslovsky JM et al.; A simplified procedure for the addition of synthetic oligonucleotide adaptors to subclone DNA fragments with incompatible ends is presented . An organophosphate degradation gene on a PstI fragment was cloned into the HindIII site of the fungal vector pH1S . The opd gene specifies parathion hydrolase and was first isolated from a Flavobacterium sp . The gene was present in 12% of the plasmids recovered and was inserted in either direction with similar frequencies: 53% with the opd start codon distal to the single SalI site of pH1S and 47% in the other orientation . All enzymatic steps were carried out in a single microconcentrator eliminating DNA loss through manipulation and transfer . Normally, during adaptor or linker addition, a larger number of oligonucleotides are attached at each end of the insert DNA and must be removed before cloning . The need for enzymatic digestion to remove excess adaptors was avoided . Traditional methods have utilized phenol/chloroform extraction, ethanol precipitation, gel filtration chromatography, spermine precipitation, or preparative gel electrophoresis . Eliminating these steps resulted in a simpler, more reliable procedure. Proc Natl Acad Sci U S A, 1992 May 15, 89(10), 4275 - 9 Functional domains in Fok I restriction endonuclease; Li L et al.; The PCR was used to alter transcriptional and translational signals surrounding the Flavobacterium okeanokoites restriction endonuclease (fokIR) gene, so as to achieve high expression in Escherichia coli . By changing the ribosome-binding site sequence preceding the fokIR gene to match the consensus E . coli signal and by placing a positive retroregulator stem-loop sequence downstream of the gene, Fok I yield was increased to 5-8% of total cellular protein . Fok I was purified to homogeneity with phosphocellulose, DEAE-Sephadex, and gel chromatography, yielding 50 mg of pure Fok I endonuclease per liter of culture medium . The recognition and cleavage domains of Fok I were analyzed by trypsin digestion . Fok I in the absence of a DNA substrate cleaves into a 58-kDa carboxyl-terminal and 8-kDa amino-terminal fragment . The 58-kDa fragment does not bind the DNA substrate . Fok I in the presence of a DNA substrate cleaves into a 41-kDa amino-terminal fragment and a 25-kDa carboxyl-terminal fragment . On further digestion, the 41-kDa fragment degrades into 30-kDa amino-terminal and 11-kDa carboxyl-terminal fragments . The cleaved fragments both bind DNA substrates, as does the 41-kDa fragment . Gel-mobility-shift assays indicate that all the protein contacts necessary for the sequence-specific recognition of DNA substrates are encoded within the 41-kDa fragment . Thus, the 41-kDa amino-terminal fragment constitutes the Fok I recognition domain . The 25-kDa fragment, purified by using a DEAE-Sephadex column, cleaves nonspecifically both methylated (pACYCfokIM) and nonmethylated (pTZ19R) DNA substrates in the presence of MgCl2 . Thus, the 25-kDa carboxyl-terminal fragment constitutes the Fok I cleavage domain. J Bacteriol, 1992 May, 174(9), 2898 - 902 Diverse substrate range of a Flavobacterium pentachlorophenol hydroxylase and reaction stoichiometries; Xun L et al.; An understanding of the enzymatic reactions catalyzing the degradation of substituted phenols, a major group of environmental pollutants, is required for the development of biological methods for the decontamination of halophenol-polluted sites . We found that a flavomonooxygenase, pentachlorophenol hydroxylase, isolated from a Flavobacterium sp., catalyzed a primary attack on a broad range of substituted phenols, hydroxylating the para position and removing halogen, nitro, amino, and cyano groups to produce halide, nitrite, hydroxylamine, and cyanide, respectively . Elimination of 1 mol of a halogen, nitro, or cyano group required 2 mol of NADPH, while only 1 mol of NADPH was required to remove 1 mol of an amino group or hydrogen. Lett Appl Microbiol, 1992 May, 14(5), 203 - 5 Rapid differentiation of Pseudomonas pseudomallei from Pseudomonas cepacia; Ashdown LR; The Minitek disc system was utilized for the differentiation of Pseudomonas pseudomallei, the causative agent of melioidosis, from Ps . cepacia . The system was simple to use, inexpensive, and furnished rapid, clear-cut test results after 4 h . This procedure is suitable for differentiating soil bacteria presumptively identified as Ps . pseudomallei, Ps . cepacia or flavobacteria, and for the rapid confirmation of the presumptive identification of either Ps . pseudomallei or Ps . cepacia obtained by commercial identification-kit systems in the clinical laboratory. J Biol Chem, 1992 Apr 25, 267(12), 8192 - 9 Characterization of a prolyl endopeptidase from Flavobacterium meningosepticum . Complete sequence and localization of the active-site serine; Chevallier S et al.; A prolyl endopeptidase was purified from Flavobacterium meningosepticum . It was digested with trypsin . Two oligonucleotides, based on tryptic peptide sequences and used in PCR experiments, amplified a 300-base pair (bp) fragment . A 2.4-kilobase EcoRI fragment that hybridized to the 300-bp probe was cloned in lambda ZAP and sequenced from both strands . It contains a reading frame of 2115 bp, encoding the complete protein sequence of 705 amino acids . Ion-spray mass spectrometry experiments demonstrated the presence of an NH2-terminal signal peptide: the periplasmic mature protease is 685 residues in length for a molecular mass of 76784 Da . The prolyl endopeptidase showed no general sequence homology with known protein sequences except with that of porcine brain prolyl endopeptidase . In order to identify the active-site serine, the prolyl endopeptidase was labeled with {3H}diisopropyl fluorophosphate . One labeled peptide was purified and sequenced . The active-site serine was located in position 536 within the sequence GRSNGG . This sequence is different from the active-site sequence of the trypsin (GDSGGP) and subtilisin (GTSMAS) families. J Environ Sci Health B, 1992 Apr, 27(2), 139 - 54 Characterization and growth response of bacteria in soil following application of carbofuran; Edwards DE et al.; Enhanced biodegradation of carbofuran (2, 3-dihydro-2, 2 dimethyl-7-benzofuranyl methyl carbamate) is an economically significant, but poorly understood, microbial phenomenon in soil . A series of experiments was conducted to examine short term changes in soil bacterial populations stimulated by carbofuran application at field rates . In the field experiment, commercially formulated carbofuran and butylate (S-ethyl diisobutyl carbamothioate) were applied at 5.6 kg ai ha-1 and 8.4 kg ai ha-1, respectively, on a soil (Putnam silt loam) exhibiting enhanced degradation of carbofuran . In laboratory studies, technical grade carbofuran (20 mg kg-1 soil) was applied to samples of the field soil . Bacterial populations were estimated using non-selective (tryptic soy agar) and selective media containing carbofuran or butylate . Largest population increases in pesticide-treated soil were observed between 7 and 15 days after treatment (DAT) compared to populations in non-treated soil . Significant increases (P less than 0.05) in total bacterial populations and presumed carbofuran-degraders due to carbofuran application were associated with increased populations of Pseudomonas spp . and Flavobacterium spp . Application of carbofuran appeared to provide a competitive advantage to these species over actinomycetes persisting beyond 20 DAT . Growth responses of bacteria to carbofuran in the Putnam soil were compared to those in a native prairie soil (Mexico silt loam), which exhibited a much slower rate of carbofuran degradation . Bacterial population response to carbofuran was measurable, but small and short-lived . Perpetuation of the enhanced degradation phenomenon may lie in a persistent pesticide-induced competitive advantage given to a very small segment of the microbial population . This advantage may not be detectable after 20 days using conventional plating techniques. J Bacteriol, 1992 Apr, 174(8), 2454 - 9 Proline-specific endopeptidases from microbial sources: isolation of an enzyme from a Xanthomonas sp; Szwajcer-Dey E et al.; An extensive screening among microorganisms for the presence of post-proline-specific endopeptidase activity was performed . This activity was found among ordinary bacteria from soil samples but not among fungi and actinomycetes . This result is in contrast to the previous notion that this activity is confined to the genus Flavobacterium . A proline endopeptidase was isolated from a Xanthomonas sp . and characterized with respect to physicochemical and enzymatic properties . The enzyme is composed of a single peptide chain with a molecular weight of 75,000 . The isoelectric point is 6.2 . It is inhibited by diisopropylfluorophosphate and may therefore be classified as a serine endopeptidase . The activity profile is bell shaped with an optimum at pH 7.5 . By using synthetic peptide substrates and intramolecular fluorescence quenching it was possible to study the influence of substrate structure on the rate of hydrolysis . The enzyme specifically hydrolyzed Pro-X peptide bonds . With Glu at position X, low rates of hydrolysis were observed; otherwise the enzyme exhibited little preference for particular amino acid residues at position X . A similar substrate preference was observed with respect to th