Flavobacterium

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 the amino acid residue preceding the prolyl residue in the substrate . The enzyme required a minimum of two amino acid residues toward the N terminus from the scissile bond, but further elongation of the peptide chain by up to six amino acid residues caused only a threefold increase in the rate of hydrolysis . Attempts to cleave at the prolyl residues in oxidized RNase failed, indicating that the enzyme does not hydrolyze long peptides, a peculiar property it shares with other proline-specific endopeptidases.

J Biol Chem, 1992 Feb 25, 267(6), 3868 - 72
Multiple endoglycosidase (Endo) F activities expressed by Flavobacterium meningosepticum . Endo F1: molecular cloning, primary sequence, and structural relationship to Endo H; Tarentino AL et al.; A full-length insert for the endo-beta-N-acetylglucosaminidase (Endo) F1 gene was located on a 2,200-base pair EcoRI fragment of genomic DNA and cloned into the plasmid vector Bluescript . Transformed Escherichia coli cells expressed Endo F1 activity very well, but the enzyme apparently was not processed and secreted into the medium as it normally is in Flavobacterium meningosepticum . DNA sequencing revealed an open reading frame of 1,017 nucleotides encoding a putative 50-amino acid signal sequence, and a mature protein (31,667 Da) of 289 amino acids . The deduced amino acid sequence was verified by direct Edman microsequencing of 88% of the purified protein as tryptic and V8 protease peptides . Alignment of Endo F1 (289 amino acids) with the established amino acid sequence of Streptomyces plicatus Endo H (271 amino acids) revealed a 32% structural identity over the entire sequence and a high degree of conservative replacements . Potential catalytic domains identified in other proteins that hydrolyze the beta 1,4 glycosidic linkage between N-acetylglucosamine residues are also conserved for amino acid identity and relative spacing in Endo F1.

J Biol Chem, 1992 Mar 5, 267(7), 4859 - 69
Isolation and characterization of heparan sulfate proteoglycans produced by cloned rat microvascular endothelial cells; Kojima T 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) . HSact and HSinact were also isolated by a combination of ion-exchange chromatography, treatment with protease and chondroitin ABC lyase, and affinity fractionation with AT III . HSact and HSinact have molecular sizes of about 25-30 kDa with the same overall composition of monosaccharides except that HSact exhibits about nine glucuronsyl 3-O-sulfated glucosamines/chain whereas HSinact possesses about three glucuronsyl 3-O-sulfated glucosamines/chain . Direct isolation of the AT III-binding site of HSact by exposing carbohydrate chains to Flavobacterium heparitinase in the presence of protease inhibitor revealed only a single interaction site which contained two to three glucuronsyl 3-O-sulfated glucosamine residues . The core proteins of HSPGact and HSPGinact were isolated by treatment with Flavobacterium heparitinase and purification by ion-exchange chromatography . The molecular sizes of the core proteins were established by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and their primary structures were examined by cleavage with trypsin or endopeptidase Glu-C as well as separation of peptides by reverse-phase high performance liquid chromatography . The results showed that both sets of core proteins exhibited three major components with molecular sizes of 50, 30, and 25 kDa, respectively . The 25-kDa species appears to be a proteolytic degradation product of the 30-kDa species . The peptide mapping revealed that HSPGact and HSPGinact possess extremely similar core proteins.

J Biochem Biophys Methods, 1992 Mar, 24(1-2), 71 - 9
Flavobacterium meningosepticum peptide:N-glycosidase: influence of ionic strength on enzymatic activity; Gosselin S et al.; Flavobacterium meningosepticum peptide:N-glycosidase-mediated deglycosylation of N-linked glycan strands of glycoproteins has been found to be strongly influenced by the ionic strength of the assay medium . By use of a modification of a previously published assay procedure for quantitative analysis of glycan release we have been able to improve reproducibility and thus to compare the extent of deglycosylation achieved under a variety of conditions of ionic strength . We have observed that enzyme activity is adversely affected by high ionic strength buffers such as those recommended for deglycosylation of various glycoproteins and recommend the use of low ionic strength buffers for routine use.

Appl Environ Microbiol, 1992 Feb, 58(2), 502 - 6
Biodegradation of the herbicide bromoxynil (3,5-dibromo-4-hydroxybenzonitrile) by purified pentachlorophenol hydroxylase and whole cells of Flavobacterium sp . strain ATCC 39723 is accompanied by cyanogenesis; Topp E et al.; A pentachlorophenol (PCP)-degrading Flavobacterium sp . (strain ATCC 39723) degraded bromoxynil with the production of bromide and cyanide . No aromatic intermediates were detected in the spent culture fluid . The cyanide produced upon bromoxynil metabolism was inhibitory to the Flavobacterium sp . Whole cells degraded PCP more rapidly than they did bromoxynil . Bromoxynil metabolism and PCP metabolism were coinduced, either substrate serving as the inducer . Purified PCP hydroxylase degraded bromoxynil with stoichiometric accumulation of cyanide and without bromide production . A product accumulated which was more hydrophilic than bromoxynil upon high-pressure liquid chromatographic analysis and which, when analyzed by gas chromatography-mass spectrometry, had a mass spectrum consistent with that expected for dibromohydroquinone . PCP hydroxylase consumed NADPH, oxygen, and bromoxynil in a 2:1:1 molar ratio, producing 1 mol of cyanide per mol of bromoxynil degraded . We propose a pathway by which bromoxynil is metabolized by the same enzymes which degrade PCP . The initial step in the pathway is the conversion of bromoxynil to 2,6-dibromohydroquinone by PCP hydroxylase . In addition to its utility for decontaminating PCP-polluted sites, the Flavobacterium sp . may be useful for decontaminating bromoxynil spills . This is the first report of cyanide production accompanying the metabolism of a benzonitrile derivative.

Appl Environ Microbiol, 1992 Feb, 58(2), 461 - 70
Effect of different holding regimens on the intestinal microflora of herring (Clupea harengus) larvae; Hansen GH et al.; The aerobic intestinal microflora of 2-week-old herring (Clupea harengus) larvae was characterized by using conventional microbiological methods and electron microscopy . Larvae were hatched and kept in filtered seawater or in seawater with penicillin and streptomycin . The gastrointestinal tract of herring larvae is essentially a straight tube divided into two compartments . Light microscopy revealed bacteria present in a progressively increasing amount throughout the length of the gastrointestinal tract from esophagus to anus . The posterior region of the intestinal lumen appeared completely occluded with bacteria . The intestinal microflora consisted mainly of members of the genera Pseudomonas and Alteromonas in the larvae incubated in filtered seawater, whereas Flavobacterium spp . dominated in larvae exposed to antibiotics . The intestinal microflora of untreated fish larvae was sensitive to all tested antibiotics, whereas multiple resistance was found in the intestinal microflora of the group given antibiotics . Thus, a dramatic change in the microflora resulted from incubation with antibiotics . Nonpigmented yeasts were detected in both larval groups . Ciliated epithelial cells were observed in the midgut, probably propeling bacteria towards the hindgut, where endocytosis of bacteria has been demonstrated . These findings suggest that transport and sequestering mechanisms resembling those of invertebrates may be found in the gut of fish larvae . The possible significance for larval health and nutrition is discussed.

Biochem Biophys Res Commun, 1992 Jan 15, 182(1), 361 - 6
Glutathione is the reducing agent for the reductive dehalogenation of tetrachloro-p-hydroquinone by extracts from a Flavobacterium sp; Xun L et al.; Tetrachloro-p-hydroquinone is the first intermediate during pentachlorophenol degradation by Flavobacterium sp . strain ATCC 39723, a strict aerobe . We report here that tetrachlorohydroquinone was reductively dehalogenated to 2,3,6-trichloro-p-hydroquinone and subsequently to 2,6-dichloro-p-hydroquinone under anaerobic conditions by the cell extract from Flavobacterium . The reducing agent was identified to be the reduced form of glutathione . This is the first time glutathione has been identified as the reducing agent for reductive dehalogenation.

Eur J Biochem, 1991 Dec 5, 202(2), 531 - 41
Heparinase II from Flavobacterium heparinum . HPLC analysis of the saccharides generated from chemically modified heparins; Moffat CF et al.; Saccharides produced by the action of heparinase II on native pig mucosal heparin (heparin IS), de-N-sulphated heparin (heparin IH), N-acetylheparin (heparin IA), de-N/O-sulphated heparin (heparin IVH), de-O-sulphated heparin (heparin IVS) and de-O-sulphated N-acetylheparin (heparin IVA) were analysed by reversed-phase HPLC using Spherisorb ODS2 . Fractions obtained by gel filtration with Bio-Gel P-4 were similarly examined . Heparin IS gave delta UA-2S----GlcNS-6S (IS) as the major unsaturated disaccharide and lesser amounts of delta UA----GlcNS-6S (IIS), delta UA-2S----GlcNS (IIIS), delta UA----GlcNS (IVS), delta UA-2S----GlcNAc-6S (IA), delta UA----GlcNAc-6S (IIA), delta UA-2S----GlcNAc (IIIA) and delta UA----GlcNAc (IVA) . Heparins IA, IVA and IVS gave as the predominant unsaturated disaccharide that corresponding to the major repeat structure of the polymer . These were respectively delta UA-2S----GlcNAc-6S (IA), delta UA-GlcNAc (IVA) and delta UA----GlcNS (IVS) . Minor disaccharides from the heterogeneous structure in native pig heparin and from residual O-sulphates after the de-O-sulphating process were detected . Heparin IH was degraded more slowly than any of the N-substituted heparins . The predominant unsaturated disaccharide was IH, which was derived from the major repeating unit . In addition, disaccharides IIH, IIIH, IA, IIA and IVA were detected . Heparin IVH showed little degradation, the unsaturated disaccharide IVH not being detected after 24 h . Disaccharide IVA was obtained from the heterogeneous sequence in heparin IVH . Several higher oligosaccharides were identified in the gel-filtration fractions including saccharides from the linkage region (for heparin IS and IVA) and the anti-thrombin binding site (for heparin IS only) . A tetrasaccharide and hexasaccharide, with the structures delta UA----GlcNAc----UA----GlcNAc and delta UA----GlcNAc----UA----GlcNAc----UA----GlcNAc, were present in the HPLC profiles of heparins IA and IVA.

J Biochem (Tokyo), 1991 Dec, 110(6), 873 - 8
Prolyl endopeptidase from Flavobacterium meningosepticum: cloning and sequencing of the enzyme gene; Yoshimoto T et al.; The prolyl endopeptidase {EC 3.4.21.26} gene of Flavobacterium meningosepticum was cloned in Escherichia coli with the aid of an oligonucleotide probe which was prepared based on the amino acid sequence . The hybrid plasmid, pFPEP1, with a 3.5 kbp insert at the HincII site of pUC19 containing the enzyme gene, was subcloned into pUC19 to construct plasmid pFPEP3 . The whole nucleotide sequence of an inserted HincII-BamHI fragment of plasmid pFPEP3 was determined by the dideoxy chain-terminating method . The purified prolyl endopeptidase was labeled with tritium DFP, and the sequence surrounding the reactive serine residue was found to be Ala (551)-Leu-Ser-Gly-Arg-*Ser-Asn(557) . Ser-556 was identified as a reactive serine residue . The enzyme consists of 705 amino acid residues as deduced from the nucleotide sequence and has a molecular weight of 78,705, which coincides well with the value estimated by ultra centrifugal analysis . The amino acid sequence was 38.2% homologous to that of the porcine brain prolyl endopeptidase {Rennex et al . (1991) Biochemistry 30, 2195-2203} and 24.5% homologous to E . coli protease II, which has substrate specificity for basic amino acids {Kanatani et al . (1991) J . Biochem . 110, 315-320}.

FEMS Microbiol Lett, 1991 Dec 1, 68(3), 239 - 44
High level expression in Escherichia coli of isopenicillin N synthase genes from Flavobacterium and Streptomyces, and recovery of active enzyme from inclusion bodies; Landman O et al.; A T7 promoter-based vector was used to express the isopenicillin N synthase (IPNS) genes of Flavobacterium sp . 12,154 and Streptomyces jumonjinensis in Escherichia coli . Most of the IPNS synthesized at 37 degrees C, and representing some 22% and 51% of the total cell protein respectively, occurred in an insoluble, enzymatically inactive form . Active IPNS was recovered in a rapid and simple two-step procedure in which the insoluble material was first denatured in 5 M urea and then refolded by passing the solubilized IPNS through a G-25 Sephadex sizing column . Further chromatography on DEAE-Sepharose resulted in highly active IPNS preparations . This procedure was found to be well suited for scaling up to produce large amounts of IPNS.

Eur J Biochem, 1991 Nov 15, 202(1), 175 - 80
Complete amino acid sequence of endo-beta-N-acetylglucosaminidase from Flavobacterium sp; Takegawa K et al.; The complete amino acid sequence of endo-beta-N-acetylglucosaminidase from Flavobacterium sp . has been determined by analysis of peptides after cleavage with lysyl endopeptidase, pepsin and chymotrypsin . The protein consists of a single polypeptide chain consisting of 267 amino acid residues and a molecular mass of 27972 Da . The sequence of Flavobacterium endo-beta-N-acetylglucosaminidase is very close to that of the Streptomyces enzyme (endo-H), having 60% similarity and very similar hydropathy profiles . Similarities were also found between Flavobacterium endo-beta-N-acetylglucosaminidase and chitinases from Bacillus circulans, Serratia marcescens and Phaseolus vulgaris.

Eur J Biochem, 1991 Nov 1, 201(3), 585 - 92
Purification and characterisation of 3-hydroxyphenylacetate 6-hydroxylase: a novel FAD-dependent monooxygenase from a Flavobacterium species; Van Berkel WJ et al.; 3-Hydroxyphenylacetate 6-hydroxylase was purified 70-fold from a Flavobacterium sp . grown upon phenylacetic acid as its sole carbon and energy source . The presence of FAD and dithiothreitol during purification is essential for high recovery of active enzyme . SDS/PAGE of purified enzyme reveals a single band with a minimum molecular mass of 63 kDa . Analytical gel-filtration, sedimentation-equilibrium and sedimentation-velocity experiments indicate that the purified enzyme exists in solution mainly as a dimer, containing 1 molecule non-covalently bound FAD/subunit . 3-Hydroxyphenylacetate 6-hydroxylase utilizes NADH and NADPH as external electron donors with similar efficiency . The enzyme shows a narrow substrate specificity . Only the primary substrate 3-hydroxyphenylacetate is hydroxylated efficiently, yielding 2,5-dihydroxyphenylacetate as a product . During turnover, the substrate analogues 3,4-dihydroxyphenylacetate and 4-hydroxyphenylacetate are partially hydroxylated, exclusively at the 6' (2') position . The physiological product 2,5-dihydroxyphenylacetate acts as an effector, strongly stimulating NAD(P)H oxidation . The activity of 3-hydroxyphenylacetate 6-hydroxylase is severely inhibited by chloride ions, competitive to the aromatic substrate . In the native state of enzyme, two sulfhydryl groups are accessible to 5,5'-dithiobis(2-nitrobenzoate) . Titration with stoichiometric amounts of either 5,5'-dithiobis(2-nitrobenzoate) or mercurial reagents completely blocks enzyme activity . Inactivation by cysteine reagents is inhibited by the substrate 3-hydroxyphenylacetate . The original activity is fully restored by treatment of the modified enzyme with dithiothreitol . The N-terminal amino acid sequence of the enzyme lacks the consensus sequence GXGXXG, found at the N-termini of all flavin-dependent external monooxygenases sequenced so far . The amino acid composition of 3-hydroxyphenylacetate 6-hydroxylase is also presented.

Can J Microbiol, 1991 Nov, 37(11), 885 - 8
Polyamine distributions in the Falvobacterium-Cytophaga-Sphingobacterium complex; Hamana K et al.; Homospermidine was found as the major polyamine in one newly described species of Flavobacterium (F . indologenes), in three species of Sphingobacterium (S . mizutae, S . multivorium, and S . spiritivorum), and in 10 species of Cytophaga (C . aquatilis, C . arvensicola, C . heparina, C . hutchinsonii, C . johnsonae, "C . keratolytica," C . lytica, C . marinoflava, C . uliginosa, and "C . xantha") . These bacteria also all contain putrescine and agmatine as minor components . Flavobacterium indologenes and C . johnsonae contain an unusual diamine, 2-hydroxyputrescine, as a major polyamine . The polyamine distributions of four other species originally included in Flavobacterium (F . acidurans, "F . dormitator," "F . tirrenicum," and Halomonas halmophila), whose taxonomic positions are or were uncertain, were different from the group mentioned above . They either contain spermidine as the major polyamine or lack any polyamine . These results suggest that homospermidine can serve as a chemotaxonomic marker to delineate true members of the Flavobacterium-Cytophaga-Sphingobacterium complex.

FEMS Microbiol Lett, 1991 Oct 1, 67(2), 127 - 9
B-lymphocyte mitogenicity and adjuvanticity of an ornithine-containing lipid or a serine-containing lipid; Kawai Y et al.; An ornithine-containing lipid (Orn-L) or a serine-containing lipid (Ser-L) from Flavobacterium meningosepticum exhibited strong mitogenicity for the splenocytes from both LPS-responder C3H/HeSlc and LPS-low-responder C3H/HeJ mice . The potency of the lipoamino acids was the same as that of LPS for responder mice . The lipoamino acids were B-lymphocyte mitogens . Furthermore, Orn-L or Ser-L exhibited strong adjuvanticity . Compared with the adjuvanticity of LPS, the activity of Orn-L was rather high . Based on these data, together with the previously reported data of macrophage activation, we propose that the lipoamino acids are non-toxic, potent immunoactivators.

Biochem Biophys Res Commun, 1991 Sep 30, 179(3), 1281 - 8
Proteoglycan and glycosaminoglycan free chain expression in keratinocytes, endothelium, and mesenchymal cells; Piepkorn M et al.; Cultured fibroblasts, bovine aortic endothelial cells, and human keratinocytes synthesize both proteoglycans and glycosaminoglycan free chains, the proportions varying between cell types . The major metabolic labeling is in proteoglycans, except for keratinocytes with approximately 60% of product as free chains . The proteoglycans range from approximately 50- greater than 1000 kDa, and the glycosaminoglycan side chains derived by alkaline elimination are approximately 30- greater than 100 kDa . The glycosaminoglycan free chains, in contrast, are smaller, from approximately 7-40 kDa in mass . The proteoglycans are both medium and cell layer constituents, whereas the glycosaminoglycan free chains are essentially confined to cells . The cellular proteoglycans and a portion of the free chains are accessible to in situ digestion by Flavobacterial glycosaminoglycan lyases, presumably reflecting localization to the cell surface . Collectively, the data show the free chains to be a common feature of all cells studied and to be partly expressed on cell surfaces . We hypothesize that the processing that creates these free chains occurs on cell surfaces, in which location they could serve ligand receptor functions.

Ann Pediatr (Paris), 1991 Sep, 38(7), 491 - 5
{Purulent meningitis due to flavobacterium meningosepticum in Cameroonian children}; Kokindombo PO et al.; Following a number of reports of purulent CSF specimens positive for Flavobacterium meningosepticum in pediatric patients in Yaounde, a prospective study was carried out in the Department of Pediatrics of the Central Yaounde Hospital from December 1988 through December 1989 . The goals of this study were to determine the incidence of Flavobacterium meningosepticum among infants and children with purulent meningitis, to discover the origin of this pathogen, and to examine its susceptibility to antimicrobial agents . Flavobacterium meningosepticum (18.4% of cases) was second by order of incidence, after pneumococci (50%) . Incidences were low for the other pathogens usually described in purulent meningitis (H . influenzae, meningococcus...) . All the pneumococcus strains recovered were susceptible to ampicillin . In contrast, 21.43% of strains of Flavobacterium meningosepticum were resistant to both ampicillin and chloramphenicol (the combination currently used as first line therapy in the Department), and 14.25% of strains were resistant to cefotaxime . The origin of the Flavobacterium meningosepticum strains found remains to be discovered . The low incidence of H . influenzae deserves to be reevaluated over the next few years.

APMIS, 1991 Sep, 99(9), 780 - 6
Genotypic heterogeneity of Flavobacterium group IIb and Flavobacterium breve demonstrated by DNA-DNA hybridization; Ursing J et al.; DNA-DNA hybridization studies on 42 stains presumptively identified as members of Flavobacterium group IIb and Flavobacterium breve indicated pronounced genotypic heterogeneity within these taxa . Three large groups highly related to the type strains of F . gleum, F . indologenes and F . breve respectively, and eight small groups were found . The group containing the type strain of F . breve was phenotypically indistinguishable from another genomic group, and these two groups were significantly separated from the other flavobacteria studied . The other nine genomic groups, representing Flavobacterium group IIb, could not with certainty be differentiated from each other by phenotypic characteristics, and there is no evidence indicating that these genomic groups differ from each other with respect to pathogenicity or ecology . Thus, it is suggested that for the time being the name "Flavobacterium group IIb" rather than specific epithets continue to be used for these bacteria.

Eur J Biochem, 1991 Aug 15, 200(1), 165 - 9
Amino acid alterations essential for increasing the catalytic activity of the nylon-oligomer-degradation enzyme of Flavobacterium sp; Kato K et al.; The structural genes of two homologous enzymes, 6-aminohexanoate-dimer hydrolase (EII; nylB) and its evolutionally related protein EII' (nylB') of Flavobacterium sp . KI72 have an open reading frame encoding a peptide of 392 amino acids, of which 47 are different, and conserved restriction sites . The specific activity of EII towards 6-aminohexanoate dimer is about 1000-fold that of EII' . Construction of various hybrid genes obtained by exchanging fragments flanked by conserved restriction sites of the two genes demonstrated that two amino acid replacements in the EII' enzyme, i.e . Gly181----Asp (EII type) and His266----Asn (EII type), enhanced the activity toward 6-aminohexanoate dimer 1000-fold.

Appl Biochem Biotechnol, 1991 Aug, 30(2), 137 - 48
The release of heparinase from the periplasmic space of Flavobacterium heparinum by three-step osmotic shock; Zimmermann JJ et al.; Heparinase was released from the periplasmic space of Flavobacterium heparinum by three-step osmotic shock procedure . The procedure involves resuspending exponentially growing cells consecutively into (1) 40% sucrose, (2) 10 mM sodium phosphate, 2 mM magnesium chloride, pH 7, and (3) 10 mM sodium phosphate, 300 mM sodium chloride, 2 mM magnesium chloride, pH 7 . Typically, 50-75% of the total heparinase activity is recovered by this procedure with an observed 7-15-fold increase in purity . The majority of heparinase activity is released in the final step of the procedure allowing for resolution from cytoplasmic and nonspecific periplasmic material . F . heparinum cells can be stored in 40% sucrose at 4 degrees C for up to one week without significant losses in recovery yields.

Infect Immun, 1991 Aug, 59(8), 2560 - 6
Protection of mice from lethal endotoxemia by use of an ornithine-containing lipid or a serine-containing lipid; Kawai Y et al.; The effects of an ornithine-containing lipid {alpha-N-(3-acyloxyacyl)-ornithine (Orn-L)} or a serine-containing lipid {alpha-N-(3-acyloxyacyl)-serine (Ser-L)} from Flavobacterium meningosepticum on lethal endotoxemia in mice were examined . When 500 micrograms of Orn-L was intravenously administered 1 h before intravenous administration of a lethal dose of endotoxin, none of the mice died . The protective effect of Ser-L was weaker than that of Orn-L . Light and electron microscopic studies demonstrated that necrosis of hepatocytes caused by endotoxin was prevented by pretreatment with Orn-L . Furthermore, Kupffer cells were activated morphologically 1 h after the administration of Orn-L or Ser-L, and the liposomes of the lipoamino acids were incorporated into phagolysosomes in activated Kupffer cells . The activity of tumor necrosis factor in sera of endotoxin-treated mice was decreased markedly by pretreatment of mice with Orn-L . In vitro, the lipoamino acids suppressed endotoxin-induced tumor necrosis factor generation but did not suppress tumor necrosis factor generation induced by zymosan and whole cells of Staphylococcus aureus . These results suggested that Orn-L and Ser-L can be used as specific blocking agents against endotoxin . The blocking mechanism may be antagonistic, because of the structural similarities between the lipoamino acids and endotoxin lipid A.

Eur J Biochem, 1991 Aug 1, 199(3), 647 - 52
Peptide-N4-(N-acetyl-beta-glucosaminyl)asparagine amidase F cannot release glycans with fucose attached alpha 1----3 to the asparagine-linked N-acetylglucosamine residue; Tretter V et al.; The ability of peptide-N4-(N-acetyl-beta-glucosaminyl)asparagine amidase F (PNGase F) from Flavobacterium meningosepticum and PNGase A from sweet almonds to deglycosylate N-glycopeptides and N-glycoproteins from plants was compared . Bromelain glycopeptide and horseradish peroxidase-C glycoprotein, which contain xylose linked beta 1----2 to beta-mannose and fucose linked alpha 1----3 to the innermost N-acetylglucosamine, were used as substrates . In contrast to PNGase A, the enzyme from F . meningosepticum did not act upon these substrates even at concentrations 100-fold higher than required for complete deglycosylation of commonly used standard substrates . After removal of alpha 1----3-linked fucose from the plant glycopeptide and glycoprotein by mild acid hydrolysis, they were readily degraded by PNGase F at moderate enzyme concentrations . Hence we conclude that alpha 1----3 fucosylation of the inner N-acetylglucosamine impedes the enzymatic action of PNGase F . Knowledge of this limitation of the deglycosylation potential of PNGase F may turn it from a pitfall into a useful experimental tool.

Zhonghua Jie He He Hu Xi Za Zhi, 1991 Aug, 14(4), 221 - 2, 255
{Flavobacterium pneumonia . Report of a case and review of literature}; Chen EZ; A case of nosocomial pneumonia caused by Flavobacter meningosepticum (FM) is reported . The genus has been identified as causes of neonatal meningitis and treatment is difficult because of its resistance to many antibiotics . Recently several outbreaks of FM respiratory infections in the intensive care unit have been reported.

J Bacteriol, 1991 Jul, 173(14), 4447 - 53
Purification and properties of pentachlorophenol hydroxylase, a flavoprotein from Flavobacterium sp . strain ATCC 39723; Xun L et al.; A pentachlorophenol (PCP) hydroxylase which catalyzed the conversion of PCP to 2,3,5,6-tetrachlorohydroquinone and released iodide from triiodophenol in the presence of NADPH and oxygen was identified . The enzyme was purified by protamine sulfate precipitation, ammonium sulfate precipitation, hydrophobic chromatography, anion-exchange chromatography, gel filtration chromatography, and crystallization . The enzyme was a monomer with a molecular weight of 63,000 . Under certain conditions, dimer and multimer conformations were also observed . The pI of the enzyme was pH 4.3 . The optimal conditions for activity were a pH of 7.5 to 8.5 and a temperature of 40 degrees C . Each enzyme molecule contained one flavin adenine dinucleotide molecule . The Km for PCP was 30 microM and the Vmax was 16 mumol/min/mg of protein . The enzymatic reaction required 2 mol of NADPH per mol of halogenated substrate . On the basis of the data we present, it is likely that PCP hydroxylase is a flavoprotein monooxygenase . The addition of flavins to the reaction mixture did not stimulate the enzymatic reaction; however, we identified the photodegradation of triiodophenol and tribromophenol, but not PCP, by flavin mononucleotide or riboflavin and light.

J Biochem (Tokyo), 1991 Jul, 110(1), 17 - 21
Microbial endo-beta-N-acetylglucosaminidases acting on complex-type sugar chains of glycoproteins; Kadowaki S et al.; The activity and substrate specificity of endo-beta-N-acetylglucosaminidase {glycopeptide-D-mannosyl-N4-(N-acetyl-D-glucosaminyl)2-asparagine 1,4-N-acetyl-beta-glucosamino-hydrolase, EC 3.2.1.96} obtained from Mucor hiemalis (Endo-M) was compared with that of the enzyme obtained from Flavobacterium meningosepticum (Endo-F), which is the only enzyme available that acts on the complex oligosaccharides of asparagine-linked sugar chains in glycoproteins . They showed almost the same activities toward DNS-ovalbumin glycopeptide containing high-mannose and hybrid asparagine-linked oligosaccharides . However, Endo-M showed high activity towards DNS-asialotransferrin and DNS-transferrin glycopeptides, which contain complex biantennary oligosaccharides . Endo-M could weakly act even on DNS-asialofetuin glycopeptide containing complex triantennary oligosaccharides, while Endo-F could not . SDS-denatured asialotransferrin was deglycosylated by both enzymes in the presence of non-ionic detergent (NP-40) and EDTA, and the deglycosylated protein migrated to a lower molecular weight position than asialotransferrin on SDS-PAGE . However, even in the absence of detergent, Endo-M deglycosylated native asialotransferrin and transferrin . Deglycosylation of asialotransferrin was confirmed by means of Con A-Sepharose 4B column chromatography and SDS-PAGE.

Glycobiology, 1991 Jun, 1(3), 257 - 63
Purification of the oligosaccharide-cleaving enzymes of Flavobacterium meningosepticum; Plummer TH Jr et al.; Four oligosaccharide chain-cleaving enzymes, including two new endoglycosidases distinct from endo-beta-acetylglucosaminidase (Endo) F1, have been identified and purified to homogeneity from cultural filtrates of Flavobacterium meningosepticum . FPLC-directed hydrophobic-interaction chromatography in conjunction with high-resolution ion-exchange chromatography provided a more simple, rapid method for the isolation of endoglycosidase F1, F2 and F3, and the amidase, peptide-N4-N-acetyl-beta-D-glucosaminyl)-asparagine amidase (PNGase F), in greater than 50% yield . The specificity of PNGase F and Endo F1 are well established . Endo F2 and Endo F3 represent new distinct endoglycosidases that prefer complex as compared to high-mannose asparagine-linked glycans . Endo F2 cleaved biantennary oligosaccharides, whereas Endo F3 cleaved both bi- and triantennary oligosaccharides.

Can J Microbiol, 1991 Jun, 37(6), 440 - 4
Biodegradation of pentachlorophenol in soil: the response to physical, chemical, and biological treatments; Seech AG et al.; The effects of physical, chemical, and biological treatments on biodegradation of pentachlorophenol (PCP) were studied in a silt-loam soil contaminated with 175 mg PCP/kg and uniformly 14C-labelled PCP . Biodegradation of 14C-labelled PCP and technical-grade PCP were monitored over 210 days incubation . Mineralization of labelled PCP was significantly (p=0.05) influenced by soil treatments . Negligible biodegradation occurred in either the sterile control soil or the uninoculated control soil, with less than 1% of added 14C recovered as 14 CO2 . Inoculation of unamended soil with a strain of Flavobacterium (ATCC 39723) known to degrade PCP increased biodegradation of PCP; approximately 60% of the {14C}PCP was recovered as 14CO2 . Increased soil water content (60% versus 30% w/w) enhanced biodegradation (67% recovery of 14C as CO2), while increased chloride ion concentration and anoxic conditions were inhibitory (20 and 1% recoveries, respectively) . Residual soil PCP concentrations were also influenced by various treatments . In the sterile control soil and noninoculated control, after 210 days incubation, concentrations of PCP were 143 and 1223 mg/kg, respectively, while the PCP concentration in the inoculated soil was 21 mg/kg . When soil organic matter was increased by adding finely ground red clover leaf and stem material, the residual PCP concentration was reduced to 6 mg/kg after 210 days . Increased soil water content resulted in a residual PCP concentration of 5 mg/kg . High-pressure liquid chromatography of soil extracts revealed no accumulation of partial PCP degradation products . The results indicated that biodegradation of PCP in soil was significantly influenced by various soil amendments.

J Cell Physiol, 1991 Jun, 147(3), 455 - 9
Proteoglycan and glycosaminoglycan synthesis by cultured rat mesangial cells; Groggel GC et al.; The synthesis of metabolically labeled proteoglycans and glycosaminoglycans from medium, cell layer and substrate attached material by rat glomerular mesangial cells in culture was characterized . The cellular localization of the labeled proteoglycans and glycosaminoglycans was determined by treating the cells with Flavobacterial heparinase . Of the total sulfated glycosaminoglycans, 33% were heparan sulfate; 55% of the cell layer material was heparan sulfate; 80% of sulfated proteins in the medium were chondroitin sulfate/dermatan sulfate . Putative glycosaminoglycan free chains of heparan sulfate and chondroitin sulfate were found in both the medium and cell layer; 95% of total proteoglycans and most (90%) of the putative heparan sulfate free chains were removed from the cell layer by the heparinase, whereas only 50% of the chondroitin sulfate and 25% of dermatan sulfate were removed . Large amounts of hyaluronic acid labeled with 3H glucosamine were found in the cell layer . In summary, approximately 60% of total sulfated glycoproteins was in the form of putative glycosaminoglycan free chains . Thus rat mesangial cells may synthesize large amounts of putative glycosaminoglycan free chains, which may have biological functions in the glomerulus independent of proteoglycans.

Am J Perinatol, 1991 May, 8(3), 161 - 3
Intravenous immunoglobulin for Flavobacterium-induced thrombocytopenia in a premature infant; Amit Y et al.; Gram-negative septicemia at times causes morbidity and mortality in newborn nurseries . Prophylactic intravenous immunoglobulin results in a significant decrease in incidence and severity of septicemia resulting from infection in infants . The present case report describes the successful use of high-dose intravenous immunoglobulin in a Flavobacterium-induced thrombocytopenia in a premature infant.

Appl Environ Microbiol, 1991 May, 57(5), 1418 - 22
Cloning, sequencing, and expression of the N-acyl-D-mannosamine dehydrogenase gene from Flavobacterium sp . strain 141-8 in Escherichia coli; Yamamoto-Otake H et al.; The gene coding for N-acyl-D-mannosamine dehydrogenase (NAM-DH) from Flavobacterium sp . strain 141-8 was cloned and expressed under the control of a lac promoter in Escherichia coli JM109 . The DNA sequence of the gene was determined, and an open reading frame encoding a polypeptide composed of 272 amino acid residues (Mr, 27,473) was identified . The E . coli transformants which showed over 200-fold higher NAM-DH activity than did the Flavobacterium strain produced the enzyme as a protein fused with beta-galactosidase . Despite being a fusion, NAM-DH produced by E . coli transformants appeared unchanged in pH optimum, Km, and substrate specificity from Flavobacterium sp . strain 141-8 . This newly recombinant enzyme may be applicable to the quantitative determination of sialic acid in serum.

Zhonghua Min Guo Xiao Er Ke Yi Xue Hui Za Zhi, 1991 May-Jun, 32(3), 171 - 6
{Clinical observation of neonatal meningitis caused by flavobacterium meningosepticum}; Lin CH et al.; From January 1981 to December 1988, we collected 11 cases of neonatal meningitis caused by Flavobacterium meningosepticum . The 6 male and 5 female newborns ranged from 3 days to 20 days old . Birth body weight varied from 1100 gm to 3600 gm . Seven cases were premature or small for date . Nosocomial infection was noted in 7 of these 11 cases . Clinically, lethargy and poor activity were the most common symptoms . Cyanosis, fever and convulsion were the next . There were 9 cases showing pleocytosis, increased protein and decreased glucose level in the cerebrospinal fluid examination . The organisms isolated in all 11 cases were susceptible to piperacillin, resistant to ampicillin, aminoglycosides and cephalosporin . Five patients were treated with antibiotics other than piperacillin for 5 to 18 days . Three patients died; hydrocephalus was the cause of death in 2 of them . Two patients were discharged against advice . Among the remaining 6 cases we gave piperacillin for 3 weeks, one case developed hydrocephalus but eventually succumbed to K . pneumoniae sepsis . Out of five surviving cases, 3 developed hydrocephalus (VP shunt performed in two) . The other two patients were discharged without neurological deficit . In conclusion, neonatal Flavobacterium meningosepticum meningitis was more frequent in premature or small for date babies, and it usually appeared in nosocomial infection . The prognosis was poor and piperacillin was proved to be the drug of choice.

J Bacteriol, 1991 May, 173(9), 2920 - 6
Purification of a Flavobacterium pentachlorophenol-induced periplasmic protein (PcpA) and nucleotide sequence of the corresponding gene (pcpA); Xun LY et al.; A pentachlorophenol (PCP)-induced periplasmic protein (PcpA) with a molecular weight of 30,000 has been identified and purified from Flavobacterium sp . strain ATCC 39723 . Results suggest that PcpA may be involved in PCP mineralization . The induction of PcpA correlated with the induction of PCP-degrading activity . When PcpA was released from the periplasmic space by EDTA or osmotic shock treatment, PCP-degrading activity was arrested . Two PCP- mutants of ATCC 39723, which do not degrade PCP, did not produce PcpA following induction with PCP . The N terminus of PcpA was sequenced, and two degenerate primers were synthesized for use in DNA amplification of a 68-bp probe which hybridized to a genomic library clone containing pcpA . The nucleotide sequence of pcpA was determined and found to encode an open reading frame of 816 bp . pcpA was found to be part of a 1.35-kb transcript with a transcriptional start site 67 bp upstream of the translational start site . Data base search comparisons yielded no sequences of high similarity to pcpA or its protein product.

Biochim Biophys Acta, 1991 Apr 29, 1077(3), 265 - 72
Diabetic BB/Wor rat haptoglobin exhibits a probable structural abnormality in Asn-linked oligosaccharides; Chapman AE et al.; In this study we demonstrate that haptoglobin, a serum glycoprotein secreted by the liver, has altered structure in the BB/Wor diabetic rat . SDS-PAGE of haptoglobin (a tetramer composed of two glycosylated beta-chains each containing two sites for Asn-linked oligosaccharides connected by disulfide bonds with two nonglycosylated alpha-chains) clearly shows that the beta-chain of haptoglobin from diabetic rats is smaller than normal, with a molecular mass of 39 instead of 40 kDa . Both acute and chronic diabetic rats exhibit the defect . Defective haptoglobin appears in the serum within 4 days of onset of the disease, but insulin therapy prevents the defect . Removal of Asn-linked oligosaccharides with peptide: N-glycosidase F from Flavobacterium meningosepticum abolished the size difference between the beta-chains from normal and diabetic haptoglobin, with the molecular mass in both cases shifting to 30 kDa . Haptoglobin from both normal and diabetic rats was resistant to digestion by endoglycosidase H from Streptomyces griseus, which cleaves high mannose-type chains . Removal of sialic acid with neuraminidase treatment resulted in a reduction in the molecular mass in both cases, but without eliminating the size difference between the two . These results demonstrate that haptoglobin from diabetic BB/Wor rats contains a structural abnormality which correlates with onset of the disease . The defect is most likely due to an alteration in Asn-linked oligosaccharides, probably involving a change in the neutral sugars of complex-type oligosaccharide chains . This finding represents the first example of an altered Asn-linked oligosaccharides in diabetes.

Eur J Biochem, 1991 Apr 23, 197(2), 449 - 59
Heparinase II from Flavobacterium heparinum . Action on chemically modified heparins; Moffat CF et al.; Five chemically modified heparins were derived from native pig mucosal heparin (pig heparin Is) . These were de-N-sulphated heparin (heparin IH), N-acetylheparin (heparin IA), de-N/O-sulphated heparin (heparin IVH), de-O-sulphated heparin (heparin IVs) and de-O-sulphated N-acetyl-heparin (heparin IVA) . Their structures were studied by 13C-NMR spectroscopy at 90.56 MHz . Native heparin and the derivatives were incubated with Flavobacterium heparinase II at 25 degrees C . The progress of degradation was followed by the delta A235 and the final composition examined by gel filtration with Bio-Gel P-4 . Native heparin (Is) was readily degraded by heparinase II and, with the exception of heparin IVH for which degradation was negligible, the chemically modified derivatives were also degraded . Approximately 90% of the saccharides from heparins Is, IA, IVs and IVA were disaccharides and tetrasaccharides . For heparin IH, which was degraded more slowly, the proportion was 65% . Heparins Is, IVs and IVA underwent initial rapid degradation . The digestion of heparin Ia proceeded rapidly after an initial lag phase . The undegraded polymers produced similar elution profiles from Bio-Gel P-4 . Following the action of heparinase II on heparins Is, IA, IVs and IVA, the elution profiles revealed a major peak of disaccharides and minor peaks of higher oligomers . The profile of heparin IH revealed a greater proportion of intermediate-molecular-mass saccharides . Our results demonstrate a broad specificity for heparinase II . It is capable of lysing both N-acetylated and N-sulphated heparins independent of O-sulphation . Heparinase II will also degrade heparin derivatives that are non-N-substituted provided that they are O-sulphated.

Hokkaido Igaku Zasshi, 1991 Mar, 66(2), 142 - 50
{Production of hydroxy and oxo fatty acids by microorganisms as a model of adipocere formation}; Gotouda H; Microbial synthesis of hydroxy and oxo fatty acids was studied as one of the model of experimental adipocere formation . Conversion of various fatty acids into 10-hydroxy and 10-oxo fatty acids by Micrococcus luteus was also studied . Fatty acids possessing cis-9-unsaturated forms were converted into 10-hydroxy and 10-oxo fatty acids . On the other hand, enoic acids possessing trans-9-unsaturated form or the ones which do not have double bond at the 9 -carbon position were inactive as substrates . 10-Hydroxypalmitic and 10-hydroxystearic acids were converted into the corresponding 10-oxo fatty acids but the 10-oxo fatty acids were inactive as substrates . To study the mechanism of the formation of 10-hydroxy and 10-oxo fatty acids, the crude enzyme preparation from Flavobacterium meningosepticum solubilized by sonication was used . The mechanism of hydration and dehydrogenation was proved by gas chromatography-mass spectrometry of 10-hydroxy and 10-oxo fatty acids produced from oleic acid in the presence of D2O or H218O . These results indicate that oleic acid is hydrated to 10-hydroxystearic acid at first and then, the latter compound is dehydrogenated to 10-oxostearic acid.

Appl Environ Microbiol, 1991 Feb, 57(2), 610 - 1
Genetic and biochemical evidence for the lack of significant hydrolysis of soman by a Flavobacterium parathion hydrolase; Pogell BM et al.; Pure recombinant Flavobacterium parathion hydrolase (an organophosphorus acid anhydrase) from Streptomyces lividans was found to hydrolyze the toxic nerve agent soman at only 0.1% of the rate observed with parathion as substrate . Studies with wild-type and recombinant strains of S . lividans support the lack of significant soman breakdown by the hydrolase and also indicate the presence in S . lividans of other significant hydrolytic enzymatic activity towards soman.

J Biol Chem, 1991 Jan 25, 266(3), 1646 - 51
Identification of distinct endoglycosidase (endo) activities in Flavobacterium meningosepticum: endo F1, endo F2, and endo F3 . Endo F1 and endo H hydrolyze only high mannose and hybrid glycans; Trimble RB et al.; Flavobacterium meningosepticum endo-beta-acetyl-glucosaminidase F preparations have been resolved by hydrophobic interaction chromatography on TSK-butyl resin into at least three activities designated endo F1, endo F2 and endo F3 each with a unique substrate specificity . The 32-kDa endo F1 protein is the principle component representing in excess of 95% of most earlier and currently available commercial endoglycosidase preparations, the remainder being a mixture of five proteins from 32 to 43 kDa . Substrate specificity studies reveal endo F1 and endo H from Streptomyces plicatus to have nearly identical capacities to hydrolyze high-mannose oligosaccharides with a minimum Man1 alpha 3Man1 alpha 6Man1 beta 4GlcNAc1 beta 4GlcNAc structure . Although endo H will hydrolyze fucose-containing hybrid oligosaccharides at rates approaching comparable high-mannose forms, core-linked fucose reduces the hydrolysis rate of endo F1 by over 50-fold relative to high-mannose structures . Neither homogeneous endo F1 nor endo H hydrolyze complex multi-antennary glycans . The biantennary cleaving activity previously reported for endo F preparations (Tarentino, A . L., Gomez, C . M., and Plummer, T . H., Jr . (1985) Biochemistry 24, 4665-4671) is a characteristic of the contaminating endo F2 activity.

Biochem Biophys Res Commun, 1991 Jan 15, 174(1), 43 - 8
Biodegradation of triiodophenol by cell-free extracts of a pentachlorophenol-degrading Flavobacterium sp; Xun L et al.; Pentachlorophenol (PCP) degrading Flavobacterium sp . ATCC 39723 was found to degrade other polyhalogenated phenolic compounds, including triiodophenol, tribromophenol, and trichlorophenol . Each compound was able to induce the degradation of the other compounds . A PCP Flavobacterium sp . mutant, F-2, was unable to degrade any of the halogenated compounds . The results suggest that all of the polyhalogenated phenols were degraded by the same enzyme system . This observation led us to exploit the sensitive leuco crystal violet assay, which measures the iodide released from triiodophenol . Cell free extracts from PCP-induced cells were able to release iodide from triiodophenol . The reaction required NADPH and oxygen.

Adv Perit Dial, 1991, 7, 133 - 4
Response of Weeksella virosa peritonitis to imipenem/cilastin; Faber MD et al.; CAPD peritonitis is most commonly due to gram positive infection . Gram negative bacillary infection is less frequent but is often seen in hospitalized patients or in those on antibiotics . Weeksella virosa (formerly known as Flavobacterium II F) has been isolated from the vaginal secretions and urine of normal women . As gram negative colonization typically proceeds from the perineal region, Weeksella virosa peritonitis might be expected in women at risk for gram negative peritonitis . A 33-year-old woman on CAPD developed multiply resistant Weeksella virosa peritonitis after prior hospitalization for pericarditis and antibiotic treatment for pneumonia . Cultures became negative and cell counts returned to normal during treatment with intravenous imipenem/cilastin . Curative treatment was completed with intraperitoneal imipenem/cilastin and oral ampicillin . Treatment was well tolerated despite theoretical concerns about the risk of seizures in patients with severe renal insufficiency not on hemodialysis.

J Enzyme Inhib, 1991, 5(1), 51 - 75
Synthesis and inhibitory activity of acyl-peptidyl-pyrrolidine derivatives toward post-proline cleaving enzyme; a study of subsite specificity; Saito M et al.; Several pyrrolidine derivatives have been synthesized and examined for their inhibitory activity on post-proline cleaving enzymes from Flavobacterium meningosepticum and bovine brain . Almost all the compounds tested in this study inhibited the activity of both enzymes at low IC50 values (from nM to microM) but a specificity difference was observed with alkylacyl-peptidyl-pyrrolidine derivatives which strongly inhibited only the bacterial enzyme . The most effective inhibitors have a proline residue on their P2 sites and a substituted or unsubstituted phenoxybutyryl moiety on their P3 sites . Thus phenoxybutyryl-prolyl-pyrrolidine is the most effective partial structure of the inhibitors . The best inhibitors found were: 4-(4-benzylphenoxy)butyryl-prolyl-pyrrolidine for bacterial enzyme (IC50 1.4 nM) and 4-phenylbutyryl-thioprolyl-pyrrolidine for bovine brain enzyme (IC50 67 nM) . In the passive avoidance test, using amnesic rats experimentally induced with scopolamine, the pyrrolidine derivatives which had potent inhibitory activity toward post-proline cleaving enzymes also showed strong anti-amnesic activities at doses of 1-5 mg/kg, i.p.

Appl Environ Microbiol, 1990 Nov, 56(11), 3593 - 4
Specific plate assay for bacterial heparinase; Zimmermann JJ et al.; A procedure was developed for detecting heparinase activity on heparin agar plates . The method is based on the differential precipitation of heparin and heparinase-generated heparin fragments by protamine sulfate . Heparinase activity is detected by the presence of clear zones against a white background . This method can be used to screen for the expression of recombinant heparinase and to identify Flavobacterium heparinum mutants expressing heparinase constitutively.

J Biol Chem, 1990 Oct 5, 265(28), 16807 - 13
Purification and substrate specificity of heparitinase I and heparitinase II from Flavobacterium heparinum . Analyses of the heparin and heparan sulfate degradation products by 13C NMR spectroscopy; Nader HB et al.; The purification of two heparitinases and a heparinase, in high yields from Flavobacterium heparinum was achieved by a combination of molecular sieving and cation-exchange chromatography . Heparinase acts upon N-sulfated glucosaminido-L-iduronic acid linkages of heparin . Substitution of N-sulfate by N-acetyl groups renders the heparin molecule resistant to degradation by the enzyme . Heparitinase I acts on N-acetylated or N-sulfated glucosaminido-glucuronic acid linkages of the heparan sulfate . Sulfate groups at the 6-position of the glucosamine moiety of the heparan sulfate chains seem to be impeditive for heparitinase I action . Heparitinase II acts upon heparan sulfate producing disulfated, N-sulfated and N-acetylated-6-sulfated disaccharides, and small amounts of N-acetylated disaccharide . These and other results suggest that heparitinase II acts preferentially upon N,6-sulfated glucosaminido-glucuronic acid linkages . The total degradation of heparan sulfate is only achieved by the combined action of both heparitinases . The 13C NMR spectra of the disaccharides formed from heparan sulfate and a heparin oligosaccharide formed by the action of the heparitinases are in accordance to the proposed mode of action of the enzymes . Comparative studies of the enzymes with the commercially available heparinase and heparitinase are described.

Agric Biol Chem, 1990 Oct, 54(10), 2675 - 80
Cloning and expression of endo-beta-1,3-glucanase gene from Flavobacterium dormitator in Escherichia coli and characterization of the gene product; Nagata S et al.; The beta-1,3-glucanase (1,3-beta-D-glucan glucanohydrolase, EC 3.2.1.6) gene from Flavobacterium dormitator var . glucanolyticae was cloned into Escherichia coli C600 with a vector plasmid, pBR322 . The E . coli cells carrying a recombinant plasmid, pKU beta G1 (8.2 kb), showed a high beta-1,3-glucanase activity and a lytic activity on viable yeast cells . These activities were found in the periplasmic space of E . coli clone cells . Southern hybridization analysis showed that the cloned gene was derived from F . dormitator chromosomal DNA . The gene products were purified from the periplasmic fraction of E . coli by ammonium sulfate fractionation and ion-exchange chromatography . The purified enzymes were demonstrated to be identical with a lytic endo-beta-1,3-glucanase II and a nonlytic endo-beta-1,3-glucanase I from F . dormitator from their enzymological and immunological properties . In the E . coli cells, endo-beta-1,3-glucanase I was also formed by a proteolytic digestion of endo-beta-1,3-glucanase II during the cultivation as in F . dormitator . Thus, the only endo-beta-1,3-glucanase II was coded for in the cloned gene.

J Biol Chem, 1990 Sep 15, 265(26), 15606 - 10
Molecular cloning and heterologous expression of N-glycosidase F from Flavobacterium meningosepticum; Lemp D et al.; N-Glycosidase F (peptide-N4-(N-acetyl-beta-glycosaminyl)asparagine amidase; EC 3.5.1.52) catalyzes the cleavage of N-glycosidically linked carbohydrate chains between N-acetylglucosamine and asparagine . The structural gene was isolated by screening a Flavobacterium meningosepticum genomic DNA library in lambda gt10 with oligonucleotides, deduced from partial amino acid sequences of the protein . A clone with an open reading frame of 1062 bases was obtained . The amino acid sequence reveals a 42-residue-long leader peptide, which shows similarities to the endoglycosidase H-leader with respect to the cleavage site of the signal peptide, but is distinct from the ones known from other Gram-positive or -negative bacteria . The molecular weight of the native protein, derived from the DNA sequence, is in agreement with the molecular weight of the purified protein on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (35,000) . Escherichia coli, transformed with a plasmid containing this DNA sequence, expresses N-glycosidase F activity . The enzyme with its natural Flavobacterium promoter and leader peptide is not secreted in E . coli but seems to be associated with cell membranes.

J Biol Chem, 1990 Aug 15, 265(23), 13609 - 17
Purification and characterization of a novel heparinase; Bohmer LH et al.; A unique heparinase was isolated from a recently discovered Gram-negative soil bacterium . The enzyme (heparinase III) was purified by hydroxylapatite chromatography, chromatofocusing, and gel permeation chromatography . The enrichment was 48x, and the specific activity of catalytically pure heparinase was 127 IU/mg of protein . Similar to the heparinase I from Flavobacterium heparinum, heparinase III also degrades heparin to mainly disaccharide fragments . It is specific for heparin and also breaks down heparan sulfate, but not hyaluronic acid and chondroitin sulfate . Heparinase III, however, differs markedly from heparinase I in several other aspects: it has a higher molecular mass (94 versus 43 kDa), pI (9.2 versus 8.5), its Km and kcat are different, and it has a higher energy of activation (15.6 versus 6.3 kcal/mol) . Optimal activity was also found at higher pH (7.6 versus 6.5) and temperature (45 versus 37 degrees C) . Furthermore, the amino acid composition of heparinase III is quite different from that of heparinase I.

Gaoxiong Yi Xue Ke Xue Za Zhi, 1990 Aug, 6(8), 418 - 21
{Bacterial flora of the external ear canal . 1 . Cancer patients}; Juan KH et al.; The bacterial flora of the external ear canal was investigated in a group of 64 patients with head and neck cancer, to observe why otitis externa frequently occurs in cancer patients . Staphylococcus epidermidis (48.5%), Pseudomonas sp . (18.8%), and Flavobacterium sp . (16.8%) were the prominent isolates from ear samples, and Gram-negative organisms which rarely existed in normal canal, were found to be markedly increased (59.4%) . Moreover, about half of all non-sterile ear canals were found to have more than one Gram-negative flora . We have concluded that cancer patients probably suffer from external otitis more frequently because of enhanced colonization by Gram-negative pathogens.

Appl Microbiol Biotechnol, 1990 Jul, 33(4), 395 - 400
Improved production of heterologous protein from Streptomyces lividans; Payne GF et al.; Protein-secreting procaryotic host organisms are currently being sought as alternatives to Escherichia coli for recombinant processing . In this study we examined how manipulation of the cultivation conditions can enhance heterologous protein production by Streptomyces lividans . The recombinant S . lividans used in this study expressed and excreted a Flavobacterium enzyme capable of hydrolyzing organophosphates . Initial shake-flask studies demonstrated that supplementing Luria-Bertani medium with moderate amounts of glucose (30 g/l), led to improved enzyme production . In fermentor studies with controlled pH, a further twofold increase in production was observed when glucose was fed continuously as compared to batch cultivation . This improved production in the glucose-fed culture may be related to a reduced accumulation of acids . Continuous feeding of both glucose and tryptone led to a further sixfold increase in production . In addition to enhancing production 25-fold, the efficiency of enzyme production and the specific activity of the excreted enzyme were also improved by glucose and tryptone feeding . These results demonstrate that in addition to genetic manipulations, optimization of cultivation conditions can lead to significant improvements in the production of heterologous proteins from Streptomyces.

J Med Chem, 1990 Jun, 33(6), 1639 - 45
Oligosaccharide mapping of low molecular weight heparins: structure and activity differences; Linhardt RJ et al.; Low molecular weight heparins from a variety of commercial sources were examined . These had been prepared by several methods including peroxidative cleavage, nitrous acid cleavage, chemical beta-elimination, enzymatic beta-elimination, and chromatographic fractionation . The molecular weight and polydispersity of these low molecular weight heparins showed greater differences than were observed for typical commercial heparin preparations . Considerable differences were also observed in the antithrombin III mediated anti factor Xa activity, the heparin cofactor II mediated antifactor IIa activity, and the USP activity of these low molecular weight heparins . An oligosaccharide-mapping technique (comparable to the peptide mapping of proteins) was applied to these low molecular weight heparins in an effort to understand the structural features responsible for their activity differences . Heparin lyase from Flavobacterium heparinum was first used to depolymerize the low molecular weight heparin into its constituent oligosaccharides . The oligosaccharides present in the resultant mixture were identified and quantitated by using standard oligosaccharides of defined structure on gradient polyacrylamide gel electrophoresis and strong anion exchange high pressure liquid chromatography . Six of the oligosaccharide products have been identified and represent nearly 90 wt % of heparin's mass . Even though all the low molecular weight heparins showed these six oligosaccharide components, their content in each varied greatly, accounting for 20 to over 90% of their mass . The antithrombin III mediated anti factor Xa activities of the low molecular weight heparins correlated only poorly to the concentration of a hexasaccharide containing a portion of heparin's antithrombin III binding site . The heparin cofactor II mediated antifactor IIa activity, however, could not be correlated to these six oligosaccharides of known structure nor to the molecular weight or charge density of these low molecular weight heparins . The low molecular weight heparins prepared by different methods each showed a new distinctive oligosaccharide in their maps . Their isolation and structural characterization, which included two-dimensional NMR and fast atom bombardment mass spectrometry, indicated that these unusual oligosaccharides result from end-sugar modification during chemical depolymerization . Both gel electrophoresis and high-pressure liquid chromatography mapping techniques showed a greater structural diversity between low molecular weight heparins than had previously been observed between similarly analyzed commercial heparins.

Agric Biol Chem, 1990 Jun, 54(6), 1453 - 7
Cloning and expression of the creatinase gene from Flavobacterium sp . U-188 in Escherichia coli; Koyama Y et al.; The gene coding for creatinase (creatine amidinohydrolase, EC 3.5.3.3) was isolated from Flavobacterium sp . U-188 . The primary structure of creatinase deduced from the nucleotide sequence showed a protein (molecular weight, 42, 651) composed of 378 amino acids . The creatinase gene was over-expressed in Escherichia coli under the control of the lac promoter and the amount of this enzyme was over 20% of the soluble protein in the cell.

Chem Pharm Bull (Tokyo), 1990 May, 38(5), 1419 - 20
Chemical modification by 2,4,6-trinitrobenzenesulfonic acid (TNBS) of an essential amino group in 3-ketovalidoxylamine A C-N lyase; Takeuchi M et al.; 3-Ketovalidoxylamine A C-N lyase of Flavobacterium saccharophilum is a monomeric protein with a molecular weight of 36000, and contains 32 amino groups and no cysteine or cystine residues . The enzyme was inactivated by 2,4,6-trinitrobenzenesulfonic acid (TNBS) following pseudo-first order kinetics . Substrate of the lyase, p-nitrophenyl-3-ketovalidamine, protected the enzyme against the inactivation, suggesting that the modification occurred at or near the active site . Although several amino groups were modified by TNBS, a plot of log (reciprocal of the half-time of inactivation) versus log (concentration of TNBS) suggested that one amino group has an essential role in catalysis.

J Clin Microbiol, 1990 May, 28(5), 1079 - 81
Recovery of an unusual Flavobacterium group IIb-like isolate from a hand infection following pig bite; Goldstein EJ et al.; An unusual gram-negative rod (RMA 1571) was isolated from a hand infection following a pig bite . This unclassified isolate was characterized by growth requirements, microscopic examination, biochemical characteristics, antimicrobial susceptibility tests, and cellular fatty acid analysis . It was indole positive and produced yellow-pigmented growth, which placed it in the genus Flavobacterium, but its other features, including cellular fatty acid analysis, did not appear to be those of a named species.

Appl Microbiol Biotechnol, 1990 May, 33(2), 213 - 6
Isolation and characterization of a 2-(2,4-dichlorophenoxy) propionic acid-degrading soil bacterium; Horvath M et al.; The 2-(2,4-dichlorphenoxy)propionic acid (2,4-DP)-degrading bacterial strain MH was isolated after numerous subcultivations of a mixed culture obtained by soil-column enrichment and finally identified as Flavobacterium sp . Growth of this strain was supported by 2,4-DP (maximum specific growth rate 0.2 h-1) as well as by 2,4-dichlorophenoxyacetic acid (2,4-D), 4-(2,4-dichlorophenoxy)butyric acid (2,4-DB), and 2-(4-chloro-2-methylphenoxy)propionic acid (MCPP) as sole sources of carbon and energy under aerobic conditions . 2,4-DP-Grown cells (10(8} of strain MH degraded 2,4-dichlorophenoxyalkanoic acids, 2,4-dichlorophenol (2,4-DCP), and 4-chlorophenol at rates in the range of 30 nmol/h . Preliminary investigations indicate that cleavage of 2,4-DP results in 2,4-DCP, which is further mineralized via ortho-hydroxylation and ortho-cleavage of the resulting 3,5-dichlorocatechol.

J Biol Chem, 1990 Apr 25, 265(12), 6967 - 72
Cloning and expression of peptide-N4-(N-acetyl-beta-D-glucosaminyl)asparagine amidase F in Escherichia coli; Barsomian GD et al.; The peptide-N4-(N-acetyl-beta-D-glucosaminyl) asparagine amidase F (PNGase F) gene from Flavobacterium meningosepticum was cloned into a high copy number Escherichia coli plasmid . Levels of PNGase F activity produced in cultures of the recombinant strain were up to 100-fold higher than those obtained in cultures of F . meningosepticum . The complete PNGase F gene sequence was determined . Comparison of the predicted amino acid sequence of pre-PNGase F to the N-terminal sequence of the native mature enzyme indicates that the protein is synthesized with a 40-amino acid signal sequence that is removed during secretion in F . meningosepticum . The recombinant PNGase F produced in E . coli is a mixture of products comprised predominantly of two proteins with molecular masses of 36.3 and 36.6 kDa . These proteins have a higher apparent molecular mass than the 34.7-kDa native enzyme . N-terminal amino acid sequencing demonstrated that these higher molecular mass products result from cleavage of the pre-PNGase F in E . coli upstream of the native N terminus . The PNGase F gene was engineered to encode a preenzyme that was processed in E . coli to give an N terminus identical to that of the native enzyme . Purified preparations of this form of recombinant PNGase F were shown to be suitable for glycoprotein analyses since they possess no detectable endo-beta-N-acetylglucosaminidase F, exoglycosidase, or protease activity.

J Biol Chem, 1990 Apr 25, 265(12), 6961 - 6
Molecular cloning and amino acid sequence of peptide-N4-(N-acetyl-beta-D-glucosaminyl)asparagine amidase from flavobacterium meningosepticum; Tarentino AL et al.; A 3,000-base pair EcoRI fragment containing the Flavobacterium meningosepticum gene for peptide-N4-(N-acetyl-beta-glucosaminyl)asparagine amidase was cloned into the Bluescript plasmid vector and expressed in Escherichia coli . The gene consists of an open reading frame of 1,062 base pairs coding for a 354-amino acid protein; the first 40 amino acids are presumed to be the natural secretory signal sequence, with the remaining 314 amino acids (34,779 Da) representing the catalytically active protein . The deduced amino acid sequence was verified independently by direct microsequencing of over 94% of the pure protein (Flavobacterium peptide-N4-(N-acetyl-beta-glucosaminyl)asparagine amidase) as tryptic and cyanogen bromide peptides . Peptide-N4-(N-acetyl-beta-glucosaminyl)asparagine amidase was not secreted by E . coli; molecular weight analysis of the partially purified recombinant enzyme suggested incomplete processing of the putative leader sequence.

J Histochem Cytochem, 1990 Apr, 38(4), 589 - 93
Glycosaminoglycans of bovine aorta endothelial cells: identification and localization by use of a platelet factor 4-fluorescein probe; Silbert DI et al.; We utilized platelet factor 4 (PF4) conjugated to fluorescein to stain the proteoglycans of permeabilized fixed bovine aorta endothelial cells in monolayer culture . Treatment of the monolayers with chondroitin ABC lyase and/or a preparation from Flavobacterium heparinum was used to remove chondroitin sulfate and/or heparan sulfate before staining, with resultant separate identification and partial localization of these glycosaminoglycans . When PF4-fluorescein was utilized with untreated control monolayers, fairly uniform reticular, perinuclear, and cell surface fluorescence was seen . After treatment with chondroitin ABC lyase, fluorescence was retained only on the cell surface . In contrast, treatment with the F . heparinum preparation resulted in the loss of all cell surface fluorescence . Use of both glycosaminoglycan lyases together resulted in loss of essentially all the fluorescence . The cell surface heparan sulfate observed by fluorescence after removal of cell surface chondroitin sulfate appeared to be unevenly distributed, with a heavier accumulation at one pole of each cell . This technique offers a specific method for identification and partial localization of cell surface heparan sulfate.

Int J Syst Bacteriol, 1990 Apr, 40(2), 154 - 9
Evidence that Bacteroides nodosus belongs in subgroup gamma of the class Proteobacteria, not in the genus Bacteroides: partial sequence analysis of a B . nodosus 16S rRNA gene; La Fontaine S et al.; The taxonomic status of the anaerobe Bacteroides nodosus has for some time been uncertain . To resolve this uncertainty, the distal portion of a 16S rRNA gene from this important ovine pathogen was cloned, mapped, and sequenced . A comparison of the sequence with the sequences of 16S rRNA molecules from other bacteria indicated that B . nodosus is more closely related to Escherichia coli and other members of the class Proteobacteria than to Bacteroides fragilis or the bacteroides-flavobacterium-cytophaga phylum . The evidence from the comparison of sequence signatures suggests that B . nodosus is not a member of the genus Bacteroides but that it belongs in subgroup gamma of the class Proteobacteria.

Biochemistry, 1990 Mar 13, 29(10), 2611 - 7
Examination of the substrate specificity of heparin and heparan sulfate lyases; Linhardt RJ et al.; We have examined the activities of different preparations of heparin and heparan sulfate lyases from Flavobacterium heparinum . The enzymes were incubated with oligosaccharides of known size and sequence and with complex polysaccharide substrates, and the resulting degradation products were analyzed by strong-anion-exchange high-performance liquid chromatography and by oligosaccharide mapping using gradient polyacrylamide gel electrophoresis . Heparinase (EC 4.2.2.7) purified in our laboratory and a so-called Heparinase I (Hep I) from a commercial source yielded similar oligosaccharide maps with heparin substrates and displayed specificity for di- or trisulfated disaccharides of the structure----4)-alpha-D-GlcNp2S(6R)(1----4)-alpha-L-IdoAp2S( 1----(where R = O-sulfo or OH) . Oligosaccharide mapping with two different commercial preparations of heparan sulfate lyase {heparitinase (EC 4.2.2.8)} indicated close similarities in their depolymerization of heparan sulfate . Furthermore, these enzymes only degraded defined oligosaccharides at hexosaminidic linkages with glucuronic acid:----4)-alpha-D-GlcNpR(1----4)-beta-D-GlcAp(1----(where R = N-acetamido or N-sulfo) . The enzymes showed activity against solitary glucuronate-containing disaccharides in otherwise highly sulfated domains including the saccharide sequence that contains the antithrombin binding region in heparin . A different commercial enzyme, Heparinase II (Hep II), displayed a broad spectrum of activity against polysaccharide and oligosaccharide substrates, but mapping data indicated that it was a separate enzyme rather than a mixture of heparinase and heparitinase/Hep III . When used in conjunction with the described separation procedures, these enzymes are powerful reagents for the structural/sequence analysis of heparin and heparan sulfate.

EMBO J, 1990 Mar, 9(3), 741 - 7
Beta-lactam antibiotic biosynthetic genes have been conserved in clusters in prokaryotes and eukaryotes; Smith DJ et al.; A cosmid clone containing closely linked beta-lactam antibiotic biosynthetic genes was isolated from a gene library of Flavobacterium sp . SC 12,154 . The location within the cluster of the DNA thought to contain the gene for delta-(L-alpha-aminoadipyl)-L-cysteinyl-D-valine synthetase (ACVS), the first step in the beta-lactam antibiotic biosynthetic pathway, was identified by a novel method . This DNA facilitated the isolation, by cross-hybridization, of the corresponding DNA from Streptomyces clavuligerus ATCC 27064, Penicillium chrysogenum Oli13 and Aspergillus nidulans R153 . Evidence was obtained which confirmed that the cross-hybridizing sequences contained the ACVS gene . In each case the ACVS gene was found to be closely linked to other beta-lactam biosynthetic genes and constituted part of a gene cluster.

Appl Environ Microbiol, 1990 Feb, 56(2), 541 - 4
Degradation of pentachlorophenol by a Flavobacterium species grown in continuous culture under various nutrient limitations; Topp E et al.; A Flavobacterium sp . was grown in continuous culture limited for growth with ammonium, phosphate, sulfate, glucose, glucose + pentachlorophenol (PCP) (0.065 h -1), or PCP . Cells ere harvested, washed, and suspended to 3 x 10(7) cells ml (-1) in shake flasks containing a complete mineral salts medium without added carbon or supplemented with 50 mg of PCP ml(-1) or 50 mg of PCP ml(-1) + 100 mg of glucose ml(-1) . The PCP concentration and the viable cell density were determined periodically . Cells that were grown under phosphate, glucose, or glucose + PCP limitation were more sensitive to PCP and took longer to degrade 50 mg of PCP ml(-1) than did cells that very were grown under ammonium, sulfate, or PCP limitation . Glucose stimulated viability and PCP degradation in all cases except when the cells were grown under carbon limitation with glucose and PCP added together as the carbon source . These results indicate that there is a relationship between nutrient limitation, phenotypic variation, and the sensitivity to and degradation of PCP by this organism.

Microbiol Immunol, 1990, 34(1), 73 - 6
Genotypic and phenotypic differentiation of Flavobacterium indologenes Yabuuchi et al 1983 from Flavobacterium gleum Holmes et al 1984; Yabuuchi E et al.; The type and eight strains of Flavobacterium indologenes were clearly differentiated from the type and two reference strains of Flavobacterium gleum by deoxyribonucleic acid-deoxyribonucleic acid homology data and phenotypic characteristics . Phenotypic characteristics useful to differentiate the two species are presented.

Intensive Care Med, 1990, 16(8), 511 - 2
Pressure transducers: an overlooked source of sepsis in the intensive care unit; Hekker TA et al.; Between January 1988 and May 1989 twenty cases of bacteremia due to Flavobacterium sp . occurred in 17 patients admitted to a surgical intensive care unit . Epidemiologic studies disclosed that the source of the Flavobacterium bacteremias was contaminated reusable pressure transducers . Despite the use of disposable domes spread of the bacteria from the contaminated transducer heads to the fluids given to the patients occurred . An indirect contamination by hands at the time the equipment was initially assembled must have been the mode of transmission . Reinstitution of routine disinfection of the transducer heads controlled the outbreak . Disposable domes failed to prevent septicemia from contaminated pressure transducers.

J Antimicrob Chemother, 1990 Jan, 25 Suppl A, 15 - 8
In-vitro activity of azithromycin against various Gram-negative bacilli and anaerobic bacteria; Kitzis MD et al.; The MICs of azithromycin, erythromycin and roxithromycin were determined (by an agar dilution method) for 65 strains of Gram-negative bacteria responsible for endocarditis and gastrointestinal infections, for 20 strains of non-fermenting Gram-negative bacteria and for 16 strains of anaerobic bacteria . The MICs of azithromycin were up to eight times lower than those of erythromycin and (except in the case of Flavobacterium spp.) up to 16 times lower than those of roxithromycin . Azithromycin was ineffective against strains showing a high degree of erythromycin resistance.

Glycoconj J, 1990, 7(4), 279 - 86
Optimized deglycosylation of glycoproteins by peptide-N4-(N-acetyl-beta-glucosaminyl)-asparagine amidase from Flavobacterium meningosepticum; Nuck R et al.; Peptide-N4-(N-acetyl-beta-glucosaminyl)asparagine amidase F(PNGase F) from Flavobacterium meningosepticum is a highly useful enzyme for the structural analysis of N (asparagine)-linked carbohydrate chains derived from glycoproteins . The enzyme was enriched using a published procedure {Tarentino AL, Gomez CM, Plummer TH, Jr (1984) Biochemistry 1985:4665-71; Tarentino AL, Plummer TH, Jr (1987) Methods Enzymol 138:770-78} and further purified by hydrophobic interaction HPLC on a weak hydrophobic TSK-Ether column from which it was eluted by a decreasing gradient of 1.7 M ammonium sulphate in 100 mM sodium phosphate, pH 7.0, containing 5 mM EDTA . To determine the optimal conditions for a complete deglycosylation of glycoproteins by PNGase F, experiments were performed with human alpha 1-acid glycoprotein, because the five complex type carbohydrate chains are quite resistant to enzymic hydrolysis . The influence of different detergents on the enzyme reaction was studied . Complete deglycosylation of human alpha 1-acid glycoprotein was achieved by the use of 60 mU/ml PNGase F in 0.25 M sodium phosphate buffer, pH 8.6, containing 0.2% (w/v) SDS, 20 mM mercaptoethanol and 0.5% Mega-10.

J Enzyme Inhib, 1990, 3(3), 163 - 78
Synthesis and inhibitory activity of acyl-peptidyl-prolinalderivatives toward post-proline cleaving enzyme as nootropic agents; Saito M et al.; Several prolinal derivatives were synthesized and examined for their inhibitory activity on post-proline cleaving enzymes from Flavobacterium meningosepticum and bovine brain and their possible properties as nootropic agents . Almost all the compounds tested inhibited the activity of both enzymes at low IC50 values of the order of nM, but a specificity difference was observed with alkylacyl-prolinal derivatives which strongly inhibited only the bacterial enzyme . Prolyl-prolinal derivatives were the most effective inhibitors for both enzymes . In the passive avoidance test using amnesic rats experimentally induced with scopolamine, the prolinal derivatives that have potent inhibitory activity toward post-proline cleaving enzymes showed also strong anti-amnesic activities at dose of 10-1000 micrograms/kg, i.p . Some of the compounds showed a bell-shape dose dependency . These results suggest that the post-proline cleaving enzymes play an important role in the regulation of learning and memory consolidation in the brain and inhibitors of these enzymes are suggested as possible candidates for nootropic agents, particularly for an anti-amnesic drug.

J Bacteriol, 1989 Dec, 171(12), 6740 - 6
Parathion hydrolase specified by the Flavobacterium opd gene: relationship between the gene and protein; Mulbry WW et al.; The sequence of a 1,693-base-pair plasmid DNA fragment from Flavobacterium sp . strain ATCC 27551 containing the parathion hydrolase gene (opd) was determined . Within this sequence, there is only one open reading frame large enough to encode the 35,000-dalton membrane-associated hydrolase protein purified from Flavobacterium extracts . Amino-terminal sequence analysis of the purified Flavobacterium hydrolase demonstrated that serine is the amino-terminal residue of the hydrolase protein . The amino-terminal serine corresponds to a TCG codon located 87 base pairs downstream of the presumptive ATG initiation codon in the nucleotide sequence . The amino acid composition of the purified protein agrees well with that predicted from the nucleotide sequence, using serine as the amino-terminal residue . These data suggest that the parathion hydrolase protein is processed at its amino terminus in Flavobacterium sp . Construction in Escherichia coli of a lacZ-opd gene fusion in which the first 33 amino-terminal residues of opd were replaced by the first 5 residues of lacZ resulted in the production of an active hydrolase identical in molecular mass to the hydrolase isolated from Flavobacterium sp . E . coli cells containing the lacZ-opd fusion showed higher levels of hydrolase activity than did cells containing the parent plasmid.

Eur J Biochem, 1989 Nov 20, 185(3), 521 - 4
Determination of the active-site serine of 6-aminohexanoate-dimer hydrolase; Negoro S et al.; Diisopropylfluorophosphate, an inhibitor of serine proteinase, was used to label 6-aminohexanoate-dimer hydrolase, a nylon oligomer degradative enzyme of Flavobacterium sp . K172 . More than 95% of the enzyme activity was lost upon incorporation of 1-1.5 molecules inhibitor/subunit of the enzyme . The tryptic peptide of the labeled enzyme was purified by HPLC (reverse-phase partition) and its amino acid sequence was identified . Radioactivity was found to be incorporated into an 8-amino-acid peptide (108His-Leu-Leu-Met-Ser-Val-Ser-Lys115) . Amino acid alteration from Ser to Ala at the position 112 by site-directed mutagenesis caused loss of enzyme activity to below the detection threshold (1% of the activity of the parental enzyme) . These results indicate that Ser112 is essential for the activity.

Dev Biol, 1989 Nov, 136(1), 97 - 103
The mechanism of precartilage mesenchymal condensation: a major role for interaction of the cell surface with the amino-terminal heparin-binding domain of fibronectin; Frenz DA et al.; Using low magnification Hoffman Modulation Contrast microscopy to rapidly identify precartilage mesenchymal condensations in chick limb bud cultures, we have determined the effect on condensation number of treatments disruptive of the interaction of cell surface components with endogenously produced fibronectin . A monoclonal antibody directed against the amino-terminal heparin-binding domain of fibronectin reduced the number of condensations by more than 50%, as did the oligopeptide gly-arg-gly, which is a repeated motif in that fibronectin domain . In contrast, monoclonal antibodies directed against the collagen- and integrin-binding domains of fibronectin, or oligopeptides containing the fibronectin integrin-recognition sequence arg-gly-asp-ser, had no significant effect on condensation number . Addition of Flavobacterium heparinase to cultures also reduced condensation number by more than 50% . Alcian blue staining of sulfated proteoglycan was greatly reduced in differentiated cultures that had been exposed to treatments that reduced condensation number . Taken together with the accompanying study, which directly demonstrates an adhesive interaction between the amino-terminal domain of extracellular fibronectin and heparin-like molecules on the surfaces of latex bead probes, the data presented here strongly indicate a major role for the corresponding cell-matrix interaction in mediating precartilage condensation in limb mesenchyme.

J Appl Bacteriol, 1989 Nov, 67(5), 551 - 9
The taxonomic relationship of certain environmental flavobacteria to the genus Weeksella; Botha WC et al.; Forty environmental strains and reference cultures of Flavobacterium, Cytophaga and Weeksella spp . were examined by numerical taxonomy . Twenty-seven strains were recovered in four phena . Phena 1A and 1B comprised 48% of the strains and were sufficiently similar to the genus Weeksella as to suggest possible inclusion in this genus . They could not be accommodated in the existing species W . virosa and W . zoohelcum . Strains from phenon 2 appear to belong neither in the Flavobacterium or the Weeksella genus . Although no reference strains were included in phena 3 and 4 they appear phenotypically to be most similar to F . breve and F . odoratum respectively.

J Clin Microbiol, 1989 Oct, 27(10), 2309 - 15
Methods for identification of flavobacteria; Pickett MJ; Published reports disagree on the best features for detecting and distinguishing between Flavobacterium meningosepticum (biovar IIa) and Flavobacterium species CDC group IIb (biovar IIb; Flavobacterium indologenes) . This report discloses that at least some of these disagreements may reflect the methods used . To detect production of indole, a modified Kovacs reagent (not Ehrlich) and a buffered tryptophan medium were optimal, but not all strains of these two biovars produced indole . To distinguish the two biovars, hydrolysis of corn starch was preferable to that of soluble potato starch . Both biovars may hydrolyze DNA; the differentiation achieved varied with the methods used . Both biovars presented pigmented growth; only IIb, however, was obviously pigmented on a 2-day blood agar plate . Acidification of D-arabinose definitively distinguished these two biovars; several additional features were useful but not definitive.

J Cell Physiol, 1989 Sep, 140(3), 584 - 92
Extracellular matrix heparan sulfate proteoglycans modulate the mitogenic capacity of acidic fibroblast growth factor; Gordon PB et al.; Confluent cultures of human endothelial cells deposit into extracellular matrix (ECM) distinct heparan sulfate proteoglycans (HSPG) which modulate acidic fibroblast growth factor's (aFGF) ability to stimulate human endothelial cell mitogenic capacity . Extracellular matrix 35S-HSPG were isolated from cultures metabolically labelled with Na235SO4 by DEAE-Sepharose, Sepharose CL-4B, and aFGF-Affi-Gel 15 column chromatography and identified by resistance to chondroitinase ABC and sensitivity to nitrous acid . Fifty to sixty percent of the 35S-HSPG deposited into ECM do not bind aFGF . The bound 35S-HSGP (40-50% of the total counts applied) eluted from the aFGF-Affi-Gel column after the addition of buffer containing 2 M NaCl . aFGF-binding and aFGF-nonbinding 35S-HSPG were individually pooled and further purified by Sepharose CL-4B column chromatography . 35S-HSPG which bind aFGF, designated HSPGP, were 100-fold superior to heparin in augmenting the mitogenic efficacy of aFGF in sparse proliferating cultures . In contrast, however, 35S-HSPG, which did not bind aFGF, designated HSPG1, inhibited aFGF-stimulated proliferation in both sparse and subconfluent endothelial cell cultures . The majority of the biological activity of both aFGF-potentiating HSPGP and aFGF-inhibitory HSPG1 was contained in the glycosaminoglycan chains released by alkaline borohydride treatment of intact HSPGP or HSPG1, respectively . 3H-Core protein derived from HSPGP or HSPG1 contained only minor biological activity . The ability of heparitinase or heparinase (Flavobacterium heparinum) to abolish biological activity differed, depending upon the HSPG tested, also suggested that these are two distinct HSPGs.

Appl Environ Microbiol, 1989 Sep, 55(9), 2113 - 8
Degradation of pentachlorophenol by polyurethane-immobilized Flavobacterium cells; O'Reilly KT et al.; Polyurethane-immobilized Flavobacterium cells (ATCC 39723) degraded pentachlorophenol (PCP) at initial concentrations as high as 300 mg liter-1 . The reversible binding of PCP to the polyurethane was shown to be important in the protection of the cells from inhibition of PCP degradation . The degradation activity of the bacteria was monitored for 150 days in semicontinuous batch reactors . The degradation rate dropped by about 0.6% per day . PCP was degraded in a continuous-culture bioreactor at a rate of 3.5 to 4 mg g of foam-1 day-1 for 25 days . Electron micrographs of the polyurethane suggested that the cells were entrapped within 50- to 500-microns-diameter pockets in the foam.

Gene, 1989 Aug 15, 80(2), 193 - 208
Nucleotide sequence of the FokI restriction-modification system: separate strand-specificity domains in the methyltransferase; Looney MC et al.; The genes for FokI, a type-IIS restriction-modification system from Flavobacterium okeanokoites (asymmetric recognition sequence: 5'-GGATG/3'-CCTAC), were cloned into Escherichia coli . Recombinants carrying the fokIR and fokIM genes were found to modify their DNA completely, and to restrict lambdoid phages weakly . The nt sequences of the genes were determined, and the probable start codons were confirmed by aa sequencing . The FokI endonuclease (R.FokI) and methyltransferase (M.FokI) are encoded by single, adjacent genes, aligned in the same orientation, in the order M then R . The genes are large by the standards of type-II systems, 1.9 kb for the M gene, and 1.7 kb for the R gene . Preceding each gene is a pair of FokI recognition sites; it is conceivable that interactions between the sites and the FokI proteins could regulate expression of the genes . The aa sequences of the N- and C-terminal halves of M.FokI are similar to one another, and to certain other DNA-adenine methyltransferases, suggesting that the enzyme has a 'tandem' structure, such as could have arisen by the fusion of a pair of adjacent, ancestral M genes . Truncated derivatives of M . FokI were constructed by deleting the 5'- or 3'-ends of the fokIM gene . Deleting most of the C-terminus of M.FokI produced derivatives that methylated only the top (GGATG) strand of the recognition sequence . Conversely, deleting most of the N-terminus produced derivatives that methylated only the bottom (CATCC) strand of the recognition sequence . These results indicate that the domains in M.FokI for methylating the two strands of the recognition sequence are largely separate.

Gene, 1989 Aug 15, 80(2), 209 - 16
Purification and characterization of the FokI restriction endonuclease; Kaczorowski T et al.; The restriction endonuclease FokI from Flavobacterium okeanokoites was purified to homogeneity . Based on gel filtration, sedimentation and sodium dodecyl sulfate-polyacrylamide-gel electrophoresis, the following properties of the enzyme were determined: FokI exists in one active monomeric form, and has an Mr of 64-65.4 x 10(3).FokI is a strongly basic protein with an isoelectric point of 9.4 . The enzyme exhibits restriction activity in the pH range 5.0 to 10.5 (maximum level at pH 7.0-8.5) and its divalent cation requirement is satisfied not only by Mg2+, but also by Co2+, Mn2+, Ni2+, Cd2+, Zn2+ and Fe2+.

Malays J Pathol, 1989 Aug, 11, 49 - 52
Activity of imipenem against clinical isolates; Lim VK; Imipenem is a new carbapenem antibiotic which is highly active against both Gram positive and Gram negative bacteria . The purpose of this study is to establish the in-vitro activity of imipenem against recent clinical isolates of bacteria obtained from patients at the Kuala Lumpur General Hospital . Minimum inhibitory concentrations of the antibiotic against these isolates were determined using an agar dilution method . With the exception of Flavobacterium sp, some Pseudomonas sp and certain other non-fermentative Gram negative bacilli, imipenem was found to be active against a wide range of both Gram positive and Gram negative organisms . Imipenem could be a valuable alternative in cases of hospital infection caused by multiply resistant organisms.

Biochem Cell Biol, 1989 Aug, 67(8), 460 - 4
Effect of deglycosylation of N-linked sugar chains on glucose oxidase from Aspergillus niger; Takegawa K et al.; Endo-beta-N-acetylglucosaminidase from Flavobacterium sp . released about 30% of the N-linked sugar chains from the glucose oxidase of Aspergillus niger . To elucidate the role of the carbohydrate moiety, the enzymatic properties of native and carbohydrate-depleted glucose oxidases were compared . It was found that their catalytic activities and thermal and pH stabilities were identical . However, the carbohydrate-depleted glucose oxidase was more rapidly precipitated by the addition of trichloroacetic acid and ammonium sulfate than the native enzyme . These results show that the N-linked sugar chains of the glucose oxidase contributed to the high solubility of the enzyme in water.

APMIS, 1989 Jul, 97(7), 591 - 4
Crossed immunoelectrophoretic analysis of Flavobacterium meningosepticum DNA hybridization groups I and II; Hoiby N et al.; By means of crossed immunoelectrophoresis and crossed-line immunoelectrophoresis, antigens were compared from four strains and five strains, respectively, of Flavobacterium meningosepticum DNA hybridization groups I and II . Two reference antisera raised against the pooled antigens of each of the two DNA groups were used . The two reference antigen-antibody systems produced 81 and 51 immunoprecipitates, respectively, and within each group nearly all antigens were common to all strains, giving matching coefficients of 0.98-1.00 . Group specific antigens were not detected . When antigens from individual strains from each group were compared to individual strains of the other group, however, 5-15 of the antigens were not cross-reactive, giving matching coefficients of 0.83-0.94 . The antigenic relatedness between strains belonging to the two different groups was, therefore, just below the level commonly found for strains belonging to the same species . The results thus confirm the previously published differences detected by a DNA hybridization technique.

J Clin Microbiol, 1989 Jul, 27(7), 1446 - 8
Pneumonia and meningitis caused by a new nonfermentative unknown gram-negative bacterium; Casalta JP et al.; Seven isolates of an unclassified bacterium resembling Flavobacterium spp . were characterized by growth requirements, microscopic examination, biochemical characteristics, antimicrobial susceptibility tests, protein profile analysis, and serologic data . The unclassified isolates were differentiated from Flavobacterium meningosepticum, Flavobacterium odoratum, Flavobacterium balustinum, Flavobacterium strain IIb, Chromobacterium violaceum, Aquaspirillum serpens, and Pseudomonas spp . The bacterium was a gram-negative rod with a polar flagellum . Protein profile analysis demonstrated two major protein bands present in the unclassified isolates that were absent from the Flavobacterium and Pseudomonas controls but present in the Aquaspirillum and Chromobacterium controls . However, no serologic cross-reactions were observed . Our results showed that the unclassified bacterium was distinct from any previously known genus of bacterium.

J Bacteriol, 1989 Jun, 171(6), 3187 - 91
High homology between 6-aminohexanoate-cyclic-dimer hydrolases of Flavobacterium and Pseudomonas strains; Tsuchiya K et al.; The nucleotide sequences of the genes for 6-aminohexanoate-cyclic-dimer hydrolases of Flavobacterium sp . strain K172 (F-nylA) and Pseudomonas sp . NK87 (P-nylA), enzymes essential for the degradation of a by-product of the nylon-6 industry, were obtained by the dideoxynucleotide chain-termination method . A 1,479-base-pair open reading frame starting at a GTG and terminating at a TGA was found for the both of the genes . The P-nylA and F-nylA genes encoded polypeptides of 493 amino acids and had only 10 base substitutions in the coding region, which caused seven amino acid substitutions.

J Bacteriol, 1989 Jun, 171(6), 3181 - 6
Plasmid dependence of Pseudomonas sp . strain NK87 enzymes that degrade 6-aminohexanoate-cyclic dimer; Kanagawa K et al.; A bacterial strain, Pseudomonas sp . strain NK87, that can use 6-aminohexanoate-cyclic dimer as the sole source of carbon and nitrogen was newly isolated from wastewater of a factory which produces nylon-6 . Two responsible enzymes, 6-aminohexanoate-cyclic-dimer hydrolase (P-EI) and 6-aminohexanoate-dimer hydrolase (P-EII), were found in the NK87 strain, as is the case with Flavobacterium sp . strain KI72, another 6-aminohexanoate-cyclic-dimer-metabolizing bacterium (H . Okada, S . Negoro, H . Kimura, and S . Nakamura, Nature {London} 306:203-206, 1983) . The P-EI enzyme is immunologically identical to the 6-aminohexanoate-cyclic-dimer hydrolase of KI72 (F-EI) . However, antiserum against the 6-aminohexanoate-dimer hydrolase purified from KI72 (F-EII) did not react with cell extracts of NK87, indicating that the F-EII and P-EII enzymes are immunologically different . Restriction endonuclease analyses show that the NK87 strain harbors at least six plasmids ranging in size from 20 to 80 kilobase pairs (kbp) . The P-EI and P-EII genes were cloned in Escherichia coli . Both the P-EI and F-EI probes strongly hybridized with a 23-kbp plasmid in Southern hybridization analyses . The P-EII probe hybridized specifically with an 80-kbp plasmid, but the F-EII probe hybridized with none of the plasmids harbored in NK87 . These results indicate that the P-EI gene and P-EII gene are encoded on the 23-kbp and 80-kbp plasmids, respectively.

Am J Infect Control, 1989 Jun, 17(3), 121 - 5
Outbreak of nosocomial Flavobacterium meningosepticum respiratory infections associated with use of aerosolized polymyxin B; Brown RB et al.; Flavobacterium meningosepticum is an uncommon cause of adult nosocomial infection . On a medical/surgical intensive care unit we recently encountered an adult outbreak of respiratory colonization and infection caused by this organism, which was associated with the prophylactic use of aerosolized polymyxin B that had been used in an attempt to abort an outbreak of infection caused by highly resistant strains of Pseudomonas aeruginosa . Twenty isolates (95% from respiratory secretions) of F . meningosepticum from nine persons were identified during a 2 1/2-month period . No environmental source has been identified to date . Pneumonia developed in five patients, and two deaths associated with this organism occurred . All isolates were sensitive to ciprofloxacin; none were sensitive to other antibiotics tested, including third-generation cephalosporins, aminoglycosides, erythromycin, trimethoprim-sulfamethoxazole, antipseudomonal penicillins, aztreonam, and imipenem/cilastatin . Two patients with nosocomial pneumonia were successfully treated with oral ciprofloxacin . F . meningosepticum may emerge as an important pathogen if prophylactic use of polymyxin B becomes more widespread . Ciprofloxacin may become the agent of choice for treatment of this organism.

Eur J Clin Microbiol Infect Dis, 1989 Jun, 8(6), 509 - 14
Flavobacterium meningosepticum infection in a neonatal ward; Bruun B et al.; An outbreak of infections due to Flavobacterium meningosepticum type C in a neonatal intensive care unit is described . During a period of two weeks, two infants developed meningitis and a third was colonized in the respiratory tract and had transient bacteremia . The two meningitis patients were treated with clindamycin, rifampicin and cefotaxime systemically, plus rifampicin intraventricularly . Bacteriological eradication was achieved within 48 h, and both infants recovered from the meningitis without apparent neurological sequelae; however, one infant died two months later of unrelated causes . Environmental surveillance cultures failed to demonstrate a reservoir for the epidemic strain, but other Flavobacterium strains were recovered . Two clinically healthy infants were found to be colonized in the nasopharynx with strains that were extremely difficult to differentiate phenotypically from the epidemic strain . Extensive characterization of strains is necessary in order to differentiate between strains and subsequently to determine a certain source of infection.

Blood, 1989 May 1, 73(6), 1534 - 9
Interaction of platelet factor four with cultured vascular endothelial cells; Rybak ME et al.; Platelets secrete a low-molecular-weight protein, platelet factor four (PF-4), which binds to and neutralizes heparin and related sulfated glycosaminoglycans (GAGs) . To examine the interactions of PF-4 with the GAGs present on endothelial cell surfaces, we incubated 125I-PF-4 with cell suspensions derived from confluent monolayers of cultured bovine aortic endothelium . Binding of 125I-PF-4 was inhibited by a 100-fold excess of nonradioactive PF-4 and varied with duration and temperature of incubation . At 4 degrees C, binding reached equilibrium at 20 minutes with kd = 2.87 mumol/L and Bmax of 63.83 pmol/10(5) cells . Binding capacity was reduced 83.4% by brief incubation of endothelial cells with trypsin and 46.67% by incubation with Flavobacterium heparinase, but was unchanged by chondroitin-ABCase treatment . At 37 degrees C, PF-4 was internalized by confluent monolayer of bovine aortic endothelial cells primarily through low-affinity adsorptive endocytosis . The internalized PF-4 was degraded to amino acids and small peptides with 50% conversion after 18-hour incubation . These studies demonstrate that a secreted platelet protein can bind to and enter endothelial cells . Binding may explain the rapid clearance of released PF-4 from plasma and could have important local effects on endothelial structure and function.

Zhonghua Yu Fang Yi Xue Za Zhi, 1989 May, 23(3), 147 - 9
{Bacteriologic investigation of the hospital air-conditioning water}; Zhou GM; We have isolated a pathogenic strain of LDB from the water of the hospital cooling-water storage tank for air-conditioning system . At the same time, other bacteria were also isolated with Pseudomonas and Flavobacterium being predominant, most of these organisms in the water of air-conditioning system were resistant to antibacterial drugs . A proposal to improve architectural design and to sterilize air-conditioning water was suggested.

Gene, 1989 Apr 15, 77(1), 1 - 10
M.FokI methylates adenine in both strands of its asymmetric recognition sequence; Landry D et al.; M.FokI, a type-IIS modification enzyme from Flavobacterium okeanokoites, was purified, and its activity was characterized in vitro . The enzyme was found to be a DNA-adenine methyltransferase and to methylate both strands of the asymmetric FokI recognition sequence: (formula; see text) M.FokI does not methylate single-stranded DNA, nor does it methylate double-stranded DNA at sequences other than FokI sites.

J Biol Chem, 1989 Apr 5, 264(10), 5751 - 6
The fokI restriction-modification system . I . Organization and nucleotide sequences of the restriction and modification genes; Kita K et al.; A DNA fragment that carried the genes coding for FokI endonuclease and methylase was cloned from the chromosomal DNA of Flavobacterium okeanokoites, and the coding regions were assigned to the nucleotide sequence by deletion analysis . The methylase gene was 1,941 base pairs (bp) long, corresponding to a protein of 647 amino acid residues (Mr = 75,622), and the endonuclease gene was 1,749 bp long, corresponding to a protein of 583 amino acid residues (Mr = 66,216) . The assignment of the methylase gene was further confirmed by analysis of the N-terminal amino acid sequence . The endonuclease gene was downstream from the methylase gene in the same orientation, separated by 69 bp . The promoter site, which could be recognized by Escherichia coli RNA polymerase, was upstream from the methylase gene, and the sequences adhering to the ribosome-binding sequence were identified in front of the respective genes . Analysis of the gene products expressed in E . coli cells by gel filtration and sodium dodecyl sulfate-polyacrylamide gel electrophoresis indicated that the molecular weights of both enzymes coincided well with the values estimated from the nucleotide sequences, and that the monomeric forms were catalytically active . No significant similarity was found between the sequences of the two enzymes . Sequence comparison with other related enzymes indicated that FokI methylase contained two copies of a segment of tetra-amino acids which is characteristic of adenine-specific methylase.

Singapore Med J, 1989 Apr, 30(2), 177 - 83
Management of Flavobacterium meningitis in the neonates: experience with 18 consecutive cases; Boo NY et al.; 18 neonates with bacteriologically confirmed Flavobacterium meningitis and ventriculitis were treated with various antibiotic regimens, including the use of intraventricular antibiotics . During the course of treatment, four patients died . 8/14 patients developed progressive hydrocephalus which required insertion of ventriculo-peritoneal shunts . The remainder 6/14 patients had normal ventricles or only mild ventriculomegaly . 5/8 patients with progressive hydrocephalus and 5/6 patients with normal or mildly dilated ventricles were followed up for at least 24 months . 4/5 of the patients with progressive hydrocephalus had severe bilateral hearing loss and delayed milestones . All the 5 patients with normal or mildly dilated ventricles had normal hearing although 2 of them had gross motor delay due to spastic paraplegia . Patients with progressive hydrocephalus received effective antibiotic treatment more than 8 days after the onset of infection while those with normal or mildly dilated ventricles within 8 days of infection . Onset of ventricular dilatation was associated with ventriculitis . Daily ultrasound scanning of the ventricles in the early stage helped to determine the need for early instillation of intraventricular antibiotics . Combined use of intravenous rifampicin, moxalactam and piperacillin showed promise as an effective antibiotic regimen in treating patients with normal or mildly dilated ventricles . Once significant ventriculomegaly has occurred, concomitant intravenous and intraventricular administration of antibiotics, to which the organisms were sensitive, was necessary to eradicate the infection.

J Antibiot (Tokyo), 1989 Apr, 42(4), 585 - 90
Preparation of 3-amino-3-deoxy derivatives of trehalose and sucrose and their activities; Asano N et al.; The 3-keto derivatives of trehalose and sucrose respectively were prepared by a one-step enzymatic route using D-glucoside 3-dehydrogenase from Flavobacterium saccharophilum . Reductive amination of 3-ketotrehalose with ammonium acetate and sodium cyanoborohydride gave three compounds, 3-amino-3-deoxy-alpha-D-allopyranosyl alpha-D-glucopyranoside, 3-amino-3-deoxy-alpha-D-glucopyranosyl alpha-D-glucopyranoside (3-trehalosamine) and 3-amino-3-deoxy-alpha-D-mannopyranosyl alpha-D-glucopyranoside . From the reductive amination of 3-ketosucrose, 3-amino-3-deoxy-alpha-D-allopyranosyl beta-D-fructofuranoside and 3-amino-3-deoxy-alpha-D-glucopyranosyl beta-D-fructofuranoside were obtained . The antibiotic and glycohydrolase inhibitory activities of these 3-amino-3-deoxy derivatives were determined.

Bioorg Khim, 1989 Mar, 15(3), 417 - 8
{A study of substrate specificity of DNA-methylases from Flavobacterium okeanokoites}; Belavin PA et al.; The specificity of DNA methylase M . FokI towards oligonucleotides containing sequence 5'...GGATG.../3'...CCTAC.. . was investigated, and N6-methyladenine in the GGATG chain was shown to be the only product of the modification.

Appl Environ Microbiol, 1989 Feb, 55(2), 289 - 93
Purification and characterization of three parathion hydrolases from gram-negative bacterial strains; Mulbry WW et al.; Three unique parathion hydrolases were purified from gram-negative bacterial isolates and characterized . All three purified enzymes had roughly comparable affinities for ethyl parathion and had broad temperature optima at ca . 40 degrees C . The membrane-bound hydrolase of Flavobacterium sp . strain ATCC 27551 was composed of a single subunit of approximately 35,000 daltons (Da) and was inhibited by sulfhydryl reagents such as dithiothreitol (DTT) and by metal salts such as CuCl2 . The cytosolic hydrolase of strain B-1 was composed of a single subunit of approximately 43,000 Da and was stimulated by DTT and inhibited by CuCl2 . The membrane-bound hydrolase of strain SC was composed of four identical subunits of 67,000 Da and was inhibited by DTT and stimulated by CuCl2 . The substrate ranges of the three enzymes also differed, as evidenced by their relative affinities for parathion and the related organophosphate insecticide O-ethyl-O-4-nitrophenyl phenylphosphonothioate (EPN) . The B-1 hydrolase displayed equal affinity for both compounds, the Flavobacterium enzyme showed twofold-lower affinity for EPN than for parathion, and the SC hydrolase displayed no activity toward EPN . The range in characteristics of these three enzymes can be exploited in different waste disposal strategies.

J Appl Bacteriol, 1989 Feb, 66(2), 137 - 52
A numerical taxonomic study of proteolytic and lipolytic psychrotrophs isolated from caprine milk; Cox JM et al.; Lipolytic and proteolytic psychrotrophs were isolated from raw and pasteurized goats' milk, which had been stored at 5 degrees C for 7 d . The 241 strains isolated and 20 reference strains were examined by 149 biochemical, physiological, and morphological tests . The results yielded 195 characters suitable for taxonomic analysis . Computer-assisted complete linkage analysis, using the Jaccard coefficient, produced 22 phenons at 75% S . The results showed that Pseudomonas fluorescens was the predominant psychrotrophic bacterium, but that Pseudomonas fragi was dominant in some milk samples . Strains of Serratia liquefaciens and Flavobacterium balustinum were also identified.

Biochim Biophys Acta, 1989 Jan 27, 990(1), 98 - 100
Primary structure of an N-linked sugar chain derived from glucoamylase of Rhizopus niveus; Takegawa K et al.; The primary structure of the N-linked sugar chain of Rhizopus niveus glucoamylase (major component) was investigated . The carbohydrate moiety was released from the polypeptide backbone by Flavobacterium sp . endo-beta-N-acetylglucosaminidase digestion . Studies using the method of exoglycosidase digestion of the fluorescent pyridylamino derivative, gel-permeation chromatography on Bio-Gel P-4 and 400-MHz 1H-NMR spectroscopy revealed that the most abundant structure is (Man)8-GlcNac-ol.

Acta Paediatr Scand, 1989 Jan, 78(1), 51 - 5
Flavobacterium meningosepticum infections in a neonatal intensive care unit; Abrahamsen TG et al.; An outbreak of nosocomial infections by Flavobacterium meningosepticum in a neonatal intensive care unit is described . During a period of eleven weeks two patients presented with septicaemia and meningitis . In addition, nine patients were found to be colonized and three of these neonates developed septicaemia . The infected patients were treated with clindamycin and piperacillin . All the patients survived, but the neonates with meningitis developed hydrocephalus . An extensive bacteriological screening of the staff was negative, but in the ward environment, F . meningosepticum was found around sinks, on rubber stoppers for milk bottles and on "cleaned" teats . Several infection control measures were instituted . Established routines were revised, with particular emphasis on the handling of objects containing or in regular contact with water.

Arch Microbiol, 1989, 152(4), 407 - 10
Superoxide dismutase in strains of the genus Flavobacterium: isolation and characterization; Sanchez-Moreno M et al.; Two electrophoretically different forms of superoxide dismutase, one of them containing manganese-protein and the other iron-protein, were detected in eleven different strains of the genus Flavobacterium . The activities of the different strains were similar to those described for other bacteria . The two molecular forms of the enzyme differed clearly with regard to activity, electrophoretic behaviour, sensitivity to cyanide and peroxide, and NaCl requirement . Both molecular forms were isolated from Flavobacterium halmephilum . Molecular mass absorption spectra, metal content, optimum pH, heat-sensitivity and stability were described.

Appl Environ Microbiol, 1989 Jan, 55(1), 137 - 41
Associated bacterial flora, growth, and toxicity of cultured benthic dinoflagellates Ostreopsis lenticularis and Gambierdiscus toxicus; Tosteson TR et al.; The growth, toxicity, and associated bacterial flora of 10 clonal cultures of the toxic benthic dinoflagellates Ostreopsis lenticularis and Gambierdiscus toxicus isolated from the coastal waters of southwest Puerto Rico have been examined . Clonal cultures of O . lenticularis grew more rapidly and at broader temperature ranges than those of G . toxicus . All five Ostreopsis clones were toxic, while only one of the five Gambierdiscus clones was poisonous . The degree of toxicity among poisonous clones was highly variable . The number of associated bacterial genera and their frequency of occurrence were quite variable among clones of both dinoflagellate genera . Bacterial isolates represented six genera (Nocardia, Pseudomonas, Vibrio, Aeromonas, Flavobacterium, and Moraxella) in addition to coryneform bacteria . Extracts of dinoflagellate-associated bacteria grown in pure culture were not toxic . Gambierdiscus clones were characterized by the frequent presence of Pseudomonas spp . (four of five clones) and the absence of coryneforms . In O . lenticularis, only one of five clones showed the presence of Pseudomonas spp., and Moraxella sp . was absent altogether . Detailed analyses of toxicity and associated microflora in a selected Ostreopsis clone, repeatedly cultivated (four times) over a period of 160 days, showed that peak cell toxicities developed in the late static and early negative culture growth phases . Peak Ostreopsis cell toxicities in the stationary phase of culture growth were correlated with significant increases in the percent total bacteria directly associated with these cells . Changes in the quantity of bacteria directly associated with microalgal cell surfaces and extracellular matrices during culture growth may be related to variability and degree of toxicity in these laboratory-cultured benthic dinoflagellates.

Appl Environ Microbiol, 1989 Jan, 55(1), 229 - 32
Enzyme-capture assay for rapid detection of Escherichia coli in oysters; Holt SM et al.; Enzyme-capture assays (ECAs) for Escherichia coli beta-D-glucuronidase (GUD) were performed directly from 24-h gas-positive lauryl tryptose broth (LTB) fermentation tubes that had been inoculated with oyster homogenate seeded with E . coli . The LTB-ECA method yielded results in 1 day that were equivalent to those obtained in 2 days by an LTB and EC-4-methylumbelliferyl-beta-D-glucuronide (EC-MUG) method . Overall, 62 of 64 (97%) positive EC-MUG broths from which E . coli was isolated were correctly identified by ECA . Of 61 LTB tubes identified as GUD negative by ECA, 59 were confirmed to be free of E . coli by using EC-MUG; thus, the false-negative rate was approximately 3% . Polyclonal antibodies prepared against E . coli GUD reacted only with GUDs of E . coli, Escherichia vulneris, and Shigella sonnei . The antibodies did not react with GUDs from Flavobacterium spp., Staphylococcus spp., Yersinia enterocolitica, shellfish, or bovine liver . The GUD ECA test, when used in conjunction with the most-probable-number technique, was a rapid method for E . coli enumeration in oysters.

J Biochem Biophys Methods, 1989, 20(1), 53 - 68
Peptide: N-glycosidase F: studies on the glycoprotein aminoglycan amidase from Flavobacterium meningosepticum; Mussar KJ et al.; Peptide: N-glycosidase from Flavobacterium meningosepticum was isolated in a homogeneous state and its physico-chemical characterization was accomplished . The reliability of the previously recorded assay procedures was assessed . Using an octaglycopeptide derived from ovomucoid a rapid and sensitive FPLC method was developed for the assay of enzymatic activity . Peptide: N-glycosidase was found to effect deglycosylation of glycoproteins bearing complex and/or multiantennary glycans even in their native state . In contrast, glycoproteins with high mannose and/or hybrid carbohydrates required denaturation to become susceptible to deglycosylation by the enzyme.

Appl Environ Microbiol, 1989 Jan, 55(1), 256 - 8
Cyanase-mediated utilization of cyanate in Pseudomonas fluorescens NCIB 11764; Kunz DA et al.; Pseudomonas fluorescens NCIB 11764 was capable of utilizing cyanate (OCN-) as a sole nitrogen source for growth . Crude cell extracts from cells grown on cyanate, but not on ammonium sulfate, were induced for an enzyme catalyzing cyanate conversion to ammonia . Enzymatic activity was shown to be bicarbonate dependent and specific for cyanate as a substrate, suggesting that cyanate utilization in this organism is facilitated by an enzyme resembling cyanase (cyanate amidohydrolase; EC 3.5.5.3), as described previously in Escherichia coli and Flavobacterium sp.

Appl Environ Microbiol, 1988 Oct, 54(10), 2586 - 9
Dissimilar plasmids isolated from Pseudomonas diminuta MG and a Flavobacterium sp . (ATCC 27551) contain identical opd genes; Harper LL et al.; The opd (organophosphate-degrading) gene derived from a 43-kilobase-pair plasmid (pSM55) of a Flavobacterium sp . (ATCC 27551) has a sequence identical to that of the plasmid-borne gene of Pseudomonas diminuta . Hybridization studies with DNA fragments obtained by restriction endonuclease digestion of plasmid DNAs demonstrated that the identical opd sequences were encoded on dissimilar plasmids from the two sources.

Appl Environ Microbiol, 1988 Oct, 54(10), 2452 - 9
Influence of readily metabolizable carbon on pentachlorophenol metabolism by a pentachlorophenol-degrading Flavobacterium sp; Topp E et al.; The influence of high concentrations of pentachlorophenol (PCP) and readily metabolizable carbon on the activity and viability of a PCP-degrading Flavobacterium sp . was examined in a mineral salts medium . Lags preceding PCP removal by glutamate-grown Flavobacterium cells were greatly attenuated by the addition of glutamate, aspartate, succinate, acetate, glucose, or cellobiose . The effect of these supplementary carbon sources on the apparent lag was not mediated entirely through the stimulation of growth since PCP metabolism accompanied the onset of growth . The specific activity of PCP-degrading cells in the absence of supplementary carbon was 1.51 x 10(-13) +/- 0.08 x 10(-13) g of PCP per cell per h and in the presence of supplementary carbon was 0.92 x 10(-13) +/- 0.09 x 10(-13) g of PCP per cell per h . Glutamate in combination with glucose or cellobiose partially repressed PCP metabolism . PCP removal by PCP-induced, glutamate-grown cells suspended in the presence of 4 g of sodium glutamate per liter was sensitive to shock loads of PCP, with a Ki of about 86.8 micrograms/ml . Subsequent removal rates, however, were more resistant to PCP . Optimal stimulation of PCP removal by sodium glutamate required 3.0 g/liter, about the same concentration as that which saturated growth in the absence of PCP . PCP removal rates decayed within minutes following the transfer of PCP-induced, glutamate-grown cells to media containing PCP without supplementary carbon, and increasing PCP concentrations accelerated the decay.(ABSTRACT TRUNCATED AT 250 WORDS)

J Biochem (Tokyo), 1988 Oct, 104(4), 622 - 7
Studies on prolyl endopeptidase from shakashimeji (Lyophyllum cinerascens): purification and enzymatic properties; Yoshimoto T et al.; High prolyl endopeptidase (post-proline cleaving enzyme) {EC 3.4.21.26} activity was detected in fruit bodies of shakashimeji (Lyophyllum cinerascens), tsukuritake (mushroom: Agaricus bisporus), hirohachichitake (Lactarius hygrophoroides), and yaburebenitake (Russula lepida) which belong to the genus Basidiomycetes . Cell-free extract of shakashimeji showed high activities of proline iminopeptidase and arylamidase as well as prolyl endopeptidase . The prolyl endopeptidase was purified from the extract of shakashimeji by sequential chromatographies on DEAE-Toyopearl, DEAE-Sephadex and hydroxyapatite, and high-performance liquid chromatography with a DEAE-5PW column . The purified enzyme was homogeneous as judged by disc gel electrophoresis . The enzyme was most active at pH 6.8 as checked with Z-Gly-Pro-beta-naphthylamide as a substrate and was stable in the range of pH 5.8-7.4 . The isoelectric point of the enzyme was 5.2 and the molecular weight was estimated to be 76,000 by gel filtration on Sephadex G-150 and by sodium dodecyl sulfate (SDS) gel electrophoresis, suggesting that the enzyme was a monomer . The enzyme was completely inhibited by diisopropyl fluorophosphate (DFP), Z-Gly-Pro-CH2Cl, and Z-Pro-prolinal, while it was not inhibited by p-chloromercuribenzoate (PCMB), phenylmethylsulfonyl fluoride (PMSF), or metal chelators . It was estimated that at least five subsites were concerned with the enzyme-substrate binding . Among them, the S1, S2, and S1' sites showed high stereospecificity, as in mammalian, microbial, and plant enzymes . The enzyme hydrolyzed TRH at the carboxyl side of the proline residue . The mushroom enzyme, that was sensitive to DFP, Z-Pro-prolinal, and Z-Gly-Pro-CH2Cl, but not to PCMB, were quite similar in characteristics to the Flavobacterium enzyme.(ABSTRACT TRUNCATED AT 250 WORDS)

J Bacteriol, 1988 Oct, 170(10), 4954 - 7
31P nuclear magnetic resonance studies of effects of some chlorophenols on Escherichia coli and a pentachlorophenol-degrading bacterium; Steiert JG et al.; A Flavobacterium sp . that mineralizes pentachlorophenol degrades some, but not all, of the other chlorinated phenols . Whole-cell 31P nuclear magnetic resonance was used to compare and observe transmembrane pH gradients and nucleotide pools in the Flavobacterium sp . and Escherichia coli after pentachlorophenol and 3,4,5-trichlorophenol were added to the cell suspensions . The data suggest that those chlorinated phenols which are not degraded by the Flavobacterium sp . may be resistant to degradation because they act as proton dissipators.

J Biol Chem, 1988 Sep 15, 263(26), 13090 - 6
Homogeneous, structurally defined heparin-oligosaccharides with low anticoagulant activity inhibit the generation of the amplification pathway C3 convertase in vitro; Linhardt RJ et al.; This paper demonstrates that heparin-oligosaccharides with low anticoagulant activity have a high capacity to inhibit activation of the amplification pathway of complement in vitro . We prepared heparin-oligosaccharides by partial depolymerization of heparin using purified flavobacterial heparinase . The resulting oligosaccharide mixture was then fractionated using strong anion exchange-high pressure liquid chromatography to produce individual oligosaccharide components of this mixture, with degree of polymerization ranging from 2 to 16 . These heparin-oligosaccharides were examined for both their anticoagulant activity and capacity to inhibit activation of the amplification pathway of complement . Although there was little difference among commercial heparins, a correlation between molecular weight and activity to inhibit convertase generation was clearly established for heparin-oligosaccharides between degree of polymerization 2 through 16 . Heparin-oligosaccharides of degree of polymerization 10-16 (Mr 3888-5320) demonstrated up to 54% of heparin's activity on a molar basis (and up to 163% of heparin's activity on a weight basis) in inhibiting the amplification pathway of complement in vitro while showing almost no anticoagulant activity . These studies, for the first time, completely separate heparin's ability to inhibit complement activation from its anticoagulant activity.

J Biochem (Tokyo), 1988 Sep, 104(3), 466 - 71
Purification and properties of N-acyl-D-mannosamine dehydrogenase from Flavobacterium sp . 141-8; Horiuchi T et al.; A new enzyme, N-acyl-D-mannosamine dehydrogenase, was purified to apparent homogeneity from a cell-free extract of Flavobacterium sp . 141-8 and some of its properties were investigated . The enzyme showed optimum activity at pH 8.0-9.5 . N-Acetyl- and N-glycolyl-D-mannosamine were oxidized but other commonly existing sugars, such as N-acetylglucosamine, N-acetylgalactosamine, amino sugars, neutral hexoses, and pentoses, were not oxidized . NAD+ was specifically utilized as an effective hydrogen acceptor . The apparent Km values for N-acetyl- and N-glycolyl-D-mannosamine, and NAD+ were 1.0, 13.3, and 0.41 mM, respectively . The stoichiometry data showed that 1 mol each of N-acetyl-D-mannosamine and NAD+ were converted to 1 mol each of N-acetyl-D-mannosaminic acid and NADH, respectively . Although the formation of lactone was detected in the enzyme reaction mixture, the reverse reaction of the enzyme, the reduction of N-acetyl-D-mannosamino-lactone, was not observed . The enzyme activity was strongly inhibited by Hg2+ and SDS, but metal-chelating reagents and sulfhydryl-group-blocking reagents had almost no effect . The molecular weight of the enzyme was estimated to be 120,000 on gel filtration and 29,000 on SDS-polyacrylamide gel electrophoresis . Its isoelectric point was at pH 4.8 . On trial application of the enzyme, it was indicated that N-acetylneuraminic acid can be determined quantitatively with the combined enzyme system involving the new enzyme and N-acetylneuraminic acid aldolase.

Antimicrob Agents Chemother, 1988 Aug, 32(8), 1143 - 8
Comparative activities of cefuroxime, amoxicillin-clavulanic acid, ciprofloxacin, enoxacin, and ofloxacin against aerobic and anaerobic bacteria isolated from bite wounds; Goldstein EJ et al.; We studied the comparative in vitro activities of 10 oral antimicrobial agents against 147 aerobic and 61 anaerobic bacteria making up species in 13 genera (Staphylococcus aureus, streptococci, Eikenella corrodens, Pasteurella multocida, Haemophilus-Actinobacillus spp., M-5, EF-4, Moraxella spp., Flavobacterium IIb, Bacteroides melaninogenicus, Bacteroides spp., Fusobacterium spp., and Peptostreptococcus spp.) that were isolated from bite wounds . Cefuroxime was generally greater than fourfold more active than cephalexin and cefadroxil against all aerobic isolates, including Pasteurella multocida . The fluoroquinolones were highly active against most aerobic isolates but were less active against anaerobic isolates . Ciprofloxacin was generally more active than either enoxacin or ofloxacin . Discrepancies of greater than 30% in the interpretation of susceptibilities between break points suggested by the National Committee for Clinical Laboratory Standards and those related to oral dose peak levels (one-half to one-quarter of maximum achievable concentrations) were noted in 14% (18 of 130) of the instances.

Ann Inst Pasteur Microbiol, 1988 Jul-Aug, 139(4), 411 - 9
Identification of Flavobacterium strains by gas liquid chromatographic analysis of volatile fatty acids produced in culture; Rasoamananjara D et al.; The classification previously established for 74 Flavobacterium strains by gas liquid chromatographic (GLC) analysis of volatile fatty acids (VFA) produced in culture allowed the recovery of 9 groups (J . gen . Microbiol., 1986, 132, 2723-2732) . Since graphic representation of the strains based on the first 3 factors obtained by principal component analysis (PCA) clearly separated these groups, we tried to identify 80 new strains by comparing their positions with those of the 9 groups, on the basis of both hierarchical classification and PCA methods . Of the 153 strains studied, only 12 were not allocated to a group corresponding to their original biochemical identification . Thus, on the whole, this characterization method by GLC analysis seemed satisfactory, although it could not be established whether the method was adequate for routine identification, or would serve merely as a complement.

Appl Environ Microbiol, 1988 Jun, 54(6), 1414 - 9
Bacterial metabolism of carbofuran; Chaudhry GR et al.; Fifteen bacteria capable of degrading carbofuran (2,3-dihydro-2,2-dimethyl-7-benzofuranyl methylcarbamate) were isolated from soil samples with a history of pesticide application . All isolates were gram negative and were oxidase- and catalase-positive rods; they occurred singly or as short chains . All of the identified isolates belonged to one of two genera, Pseudomonas and Flavobacterium . They were separated into three groups based on their mode of utilization of carbofuran . Six isolates were placed in group I; these isolates utilized carbofuran as a sole source of nitrogen . Seven isolates were placed in group II; these isolates utilized the pesticide as a sole source of carbon . Isolates of both groups I and II hydrolyzed carbofuran to carbofuran phenol . Two isolates, designated group III, also utilized carbofuran as a sole source of carbon . They degraded the pesticide more rapidly, however, so up to 40% of {14C}carbofuran was lost as 14CO2 in 1 h . The results suggest that these isolates degrade carbofuran by utilizing an oxidative pathway.

Forensic Sci Int, 1988 Jun, 37(4), 249 - 57
The mechanism of experimental adipocere formation: hydration and dehydrogenation in microbial synthesis of hydroxy and oxo fatty acids; Gotouda H et al.; The enzyme preparations from Flavobacterium meningosepticum solubilized by sonication catalyzed not only hydration of oleic acid to produce 10-hydroxystearic acid but dehydrogenation of this product . The mechanism of the hydration and dehydrogenation was proved by gas chromatography-mass spectrometry of 10-hydroxy and 10-oxostearic acids produced in the presence of D2O or H2(18)O . The activity of these enzymes was increased by preincubating Flavobacterium meningosepticum with oleic acid.

Biomed Environ Sci, 1988 Jun, 1(1), 105 - 14
Studies on fermented corn flour food poisoning in rural areas of China . II . Isolation and identification of causal microorganisms; Meng Z et al.; Using potato dextrose agar medium, 40 strains of microorganisms were isolated from leftover fermented corn flour samples involved in outbreaks of food poisoning . All strains produced powerful toxins which caused the same intoxication to mice, dogs, and monkeys as the leftover food samples . On the basis of results obtained from the morphology of this bacteria and its colony, from biochemical tests, and from the G-C mole percentage in DNA, the bacteria was identified as Flavobacterium farinofermentans nov . sp . (Meng, Z . and Wang, D.).

J Biochem (Tokyo), 1988 Jun, 103(6), 938 - 43
Physiological role of glucoside 3-dehydrogenase and cytochrome c551 in the sugar oxidizing system of Flavobacterium saccharophilum; Takeuchi M et al.; Flavobacterium saccharophilum cytoplasmic membranes contain several cytochromes linked to the respiratory chain . The presence of c-type cytochrome, cytochrome o, and a small amount of a-type cytochrome was proved . Cytochrome c551 was purified to electrophoretic homogeneity by ion-exchange chromatography and gel filtration from a membrane fraction of F . saccharophilum and its properties determined . Cytochrome c551 possessed absorption peaks at 407 nm in the oxidized form, and at 415, 521, 551 nm in the reduced form . The cytochrome c551 had a molecular weight of 15,500 as determined by sodium dodecyl sulfate polyacrylamide gel electrophoresis . Glucoside 3-dehydrogenase of F . saccharophilum reduced the cytochrome c551 with methyl-alpha-D-glucoside, D-glucose, sucrose, or validoxylamine A . When the purified glucoside 3-dehydrogenase was incubated with methyl-alpha-D-glucoside and purified ferricytochrome c551, methyl-alpha-D-3-ketoglucoside was formed as indicated by GC-MS analysis . The addition of a substrate to the membrane fraction caused an increase in the rate of oxygen uptake and an abrupt reduction in cytochrome c551 . The electron transfer in the 3-keto sugar forming system may be as follows: sugars----glucoside 3-dehydrogenase----cytochrome c551----cytochrome oxidase----O2 . Thus, the electron acceptor of glucoside 3-dehydrogenase is possibly connected to the membrane-bound cytochrome system.

Biochim Biophys Acta, 1988 May 18, 954(2), 161 - 9
Mechanism of proline-specific proteinases: (I) Substrate specificity of dipeptidyl peptidase IV from pig kidney and proline-specific endopeptidase from Flavobacterium meningosepticum; Heins J et al.; The substrate specificity of dipeptidyl peptidase IV (dipeptidyl peptide hydrolase, EC 3.4.14.5) from pig kidney and proline-specific endopeptidase from Flavobacterium meningosepticum, was investigated with a series of N-terminal unprotected (dipeptidyl peptidases IV) and succinylated dipeptidyl-p-nitroanilides (proline-specific endopeptidase) . Both enzymes are specific for the S configuration of the amino-acid residue in P1 and P2 position if the penultimate residue is proline . In the case of alanine substrates (Ala in P1, dipeptidyl peptidase IV hydrolyzes such compounds where the configuration of the P2 residue is R . The penultimate residue with dipeptidyl peptidase IV can be, beside proline and alanine, dehydroproline, hydroxyproline and pipecolic acid . Proline substrates (Pro in P1) with an R configuration in P2 are inhibitors of the hydrolysis of proline substrates with an S,S configuration in an uncompetitive (dipeptidyl peptide IV) or mixed inhibition type (proline-specific endopeptidase) . Derivatives of Gly-Pro-pNA where the N-terminal amino group is methylated are hydrolyzed by dipeptidyl peptidase IV.

J Clin Microbiol, 1988 May, 26(5), 1070 - 1
Purification and properties of Bacteroides heparinolyticus heparinase (heparin lyase, EC 4.2.2.7); Nakamura T et al.; Heparinase (heparin lyase, EC 4.2.2.7) was isolated from the cell extract of an oral bacterium, Bacteroides heparinolyticus . It was a basic protein with an isoelectric point of 9.5 . Its molecular weight was 63,000 . The enzyme was the most active against heparin among the tested mucopolysaccharides . Catalytic properties may be similar to those of heparinase of Flavobacterium heparinum, since the enzymatic degradation products obtained by using the two enzymes were the same on the basis of paper chromatography.

Jpn J Antibiot, 1988 May, 41(5), 557 - 62
{A case of neonatal meningitis due to Flavobacterium meningosepticum successfully treated with cefmetazole and cefotaxime}; Takeda A et al.; A 3-day-old male infant, weighing 3,413 g and a gestational age of 38 weeks developed neonatal meningitis due to Flavobacterium meningosepticum . Treatment with cefmetazole and cefotaxime led him to a complete recovery without neurologic deficit . Of 82 previously published cases under 1 year old, 41 cases died and 16 of survivors developed hydrocephalus because the organism was resistant to many antibiotics . Therapy of meningitis due to the organism has not been standardized but the early laboratory identification and the choice of effective and safe antibiotics determined by antimicrobial sensitivity test improve the outcome.

Ann Inst Pasteur Microbiol, 1988 Mar-Apr, 139(2), 159 - 70
{Composition of the complex lipids of Flavobacterium meningosepticum}; Asselineau J et al.; The free lipids of Flavobacterium meningosepticum were separated by thin layer chromatography, and the main lipid fractions were analysed by FAB (fast atom bombardment) mass spectrometry . The major products were di-iso-C15- and iso-C15-iso-C17-phosphatidylethanolamine, and two ninhydrin + and phosphorus- fractions . The structures of the latter two fractions were established as ornithine lipids by using MIKE (mass ions kinetic energy) mass spectrometry, GC/MS (gas chromatography coupled with mass spectrometry) and conventional methods . The presence of small amounts of sphingolipids with C17- and C16-sphinganines was demonstrated . F . meningosepticum can be distinguished from F . multivorum and F . spiritivorum by easy characterization of the ornithine lipids by thin layer chromatography.

Appl Environ Microbiol, 1988 Feb, 54(2), 428 - 33
Metabolism of 2,6-dimethylnaphthalene by flavobacteria; Barnsley EA; Flavobacteria that were able to grow on 2,6-dimethylnaphthalene (2,6-DMN) were isolated from soil . Most were able to oxidize a broad range of aromatic hydrocarbons after growth on 2,6-DMN at rates comparable to that of the oxidation of 2,6-DMN itself . One small group was neither able to grow on naphthalene nor able to oxidize this compound after growth on 2,6-DMN, but metabolized 2,6-DMN by a pathway which converged with that previously described for naphthalene metabolism in pseudomonads . These organisms could also grow on salicylate or methylsalicylate, and in so doing, early enzymes for 2,6-DMN metabolism were induced.

Appl Environ Microbiol, 1988 Feb, 54(2), 288 - 93
Isolation of a methyl parathion-degrading Pseudomonas sp . that possesses DNA homologous to the opd gene from a Flavobacterium sp; Chaudhry GR et al.; Two mixed bacterial cultures isolated by soil enrichment were capable of utilizing methyl parathion (O,O-dimethyl O-p-nitrophenylphosphorothioate) and parathion (O,O-diethyl O-p-nitrophenylphosphorothioate) as a sole source of carbon . Four isolates from these mixed cultures lost their ability to utilize the pesticides independently in transfers subsequent to the initial isolation . One member of the mixed cultures, a Pseudomonas sp., however, hydrolyzed the pesticides to p-nitrophenol but required glucose or another carbon source for growth . The crude cell extracts prepared from this bacterium showed an optimum pH range from 7.5 to 9.5 for the enzymatic hydrolysis . Maximum enzymatic activity occurred between 35 and 40 degrees C . The enzyme activity was not inhibited by heavy metals, EDTA, or NaN3 . Another isolate from the mixed cultures, a Flavobacterium sp., used p-nitrophenol for growth and degraded it to nitrite . Nitrite was assimilated into the cells under conditions during which the nitrogen source was excluded from the minimal growth medium . The hybridization data showed that the DNAs from a Pseudomonas sp . and from the mixed culture had homology with the opd (organophosphate degradation) gene from a previously reported parathion-hydrolyzing bacterium, Flavobacterium sp . The use of the opd gene as a probe may accelerate progress toward understanding the complex interactions of soil microorganisms with parathions.

Eur J Biochem, 1988 Jan 15, 171(1-2), 73 - 80
Various kinds of lipoamino acids including a novel serine-containing lipid in an opportunistic pathogen Flavobacterium . Their structures and biological activities on erythrocytes; Kawai Y et al.; Lipoamino acids were found to represent 60% of the total extractable cellular lipids in Flavobacterium meningosepticum and F . indologenes which are known to be opportunistic pathogens . The structures of the lipoamino acids were resolved by chemical and physicochemical methods . Three kinds of lipoamino acids were included in the lipid of Flavobacterium: 3-hydroxyisoheptadecanoic acid amide-linked to serine and esterified to isopentadecanoic acid, and 3-hydroxyisoheptadecanoic acid amide-linked to ornithine and esterified to isopentadecanoic acid or 2-hydroxyisopentadecanoic acid . This type of serine-containing lipid is a novel, rare substance and exhibited high hemagglutinating activity (minimum hemagglutinating concentration was 0.25-0.5 micrograms/ml) with very good, stable dispersion of its liposomes . We called the serine-containing lipid 'flavolipin', based on the genus name of the bacteria . Of the two types of the ornithine-containing lipids, the one that had a nonpolar terminal fatty acid showed higher hemagglutinating activity than the one that had a polar terminal fatty acid . The reconstituted liposomes whose lipid composition was similar to that of the original bacterial cell membrane exhibited definite hemagglutinating activity . As soon as a terminal fatty acid of the lipoamino acids was lost, their biological activity on erythrocytes changed from hemagglutinating to hemolytic, being accompanied by the disappearance of animal species specificity . The mechanism of both the hemagglutination and the hemolysis was discussed.

J Antibiot (Tokyo), 1988 Jan, 41(1), 81 - 5
Properties of a broad spectrum beta-lactamase isolated from Flavobacterium meningosepticum GN14059; Fujii T et al.; A broad substrate-spectrum beta-lactamase was purified from Flavobacterium meningosepticum GN14059 . The purified enzyme gave a single protein band on SDS-polyacrylamide gel electrophoresis . The molecular weight was estimated to be about 30,000 and the isoelectric point was 5.1 . The enzyme hydrolyzed various beta-lactam antibiotics including oxyiminocephalosporins and aztreonam . Relative rates, with cephaloridine as 100, were cephalothin 200, cefazolin 48, cefuroxime 153, cefotaxime 51, ceftazidime 20, ampicillin 26, carbenicillin 19, and aztreonam 20 . The enzyme activity was inhibited by clavulanic acid, sulbactam, imipenem and cephamycins.

J Cell Sci, 1987 Dec, 88 ( Pt 5), 623 - 9
Cell spreading on serum is not identical to spreading on fibronectin; McInnes C et al.; Adhesion and spreading of cell lines on dishes coated with serum-derived proteins were studied after removal of cell-surface proteoglycans . A mixture of glycosaminoglycans lyases from heparin-induced Flavobacterium heparinum removed 80% of the {35S}sulphate-labelled glycosaminoglycans from the surface of attached cells within 30 min, but this had little effect on cell morphology . The rate of cell attachment to dishes coated with serum was unaffected by prior treatment of cells with this mixture of glycosaminoglycan lyases . While a heparan sulphate lyase preparation abolished cell spreading in response to fibronectin there was no effect of the enzyme on the spreading mediated by vitronectin . These results suggest that, although heparan sulphate is required for spreading on purified fibronectin, the spreading stimulated by serum under routine culture conditions requires neither cellular heparan sulphate nor serum-derived fibronectin.

Mol Pharmacol, 1987 Nov, 32(5), 565 - 71
Glycosylation of the mammalian alpha 1-adrenergic receptor by complex type N-linked oligosaccharides; Sawutz DG et al.; The binding subunit of the alpha 1-adrenergic receptor has been identified as an Mr = 80,000 peptide in several tissues . Adsorption of the alpha 1-adrenergic receptor to a wheat germ agglutinin lectin-agarose resin suggests that the receptor protein is glycosylated . In this study, we investigated the nature of the carbohydrate chains linked to the alpha 1-adrenergic receptor peptide . The alpha 1-adrenergic receptor from DDT2 MF-2 smooth muscle cell and rat brain membranes was photolabeled with 125I-azido-prazosin {( 125I}CP65,526) and then treated with exoglycohydrolases prior to SDS-PAGE and autoradiography . Removal of terminal sialic acid residues by neuraminidase decreased the receptor Mr by 6,000; however, alpha-mannosidase was without effect, indicating complex type glycosylation of the receptor-protein . Similar results were observed for the rat hepatic membrane alpha 1-adrenergic receptor . Removal of N-linked carbohydrates at asparagine residues by peptide-N4{N-acetyl-beta-glucosaminyl}asparagine amidase (from Flavobacterium meningosepticum) resulted in a specifically labeled peptide at Mr = 50,000-55,000 in DDT1 MF-2 membrane and solubilized receptor preparations . Treatment of DDT1 MF-2 cells with swainsonine or (+)-1-deoxymannojirimycin, inhibitors of complex type carbohydrate chain biosynthesis, caused a reduction in the apparent molecular weight of the receptor (Mr = 60,000) but did not alter the number of alpha 1-adrenergic receptors per cell or their affinity for the radioligand {3H}prazosin . These findings indicate that the alpha 1-adrenergic receptor is heavily glycosylated, the major oligosaccharide moiety being of the complex type, N-linked to asparagine residues . The peptide backbone of the receptor has an Mr less than or equal to 55,000, consistent with the predicted molecular mass of other membrane neurotransmitter receptors based on sequence analysis of isolated cDNA clones.

Mol Gen Genet, 1987 Oct, 209(3), 570 - 4
Cloning of two type II methylase genes that recognise asymmetric nucleotide sequences: FokI and HgaI; Nwankwo D et al.; The modification genes of Flavobacterium okeanokoites and Haemophilus galinarum have been cloned into the vector pBR322 and expressed in Escherichia coli cells . FokI methylase gene is contained on a 3.80 kb piece of F . okeanokoites DNA . Plasmid constructs carrying this fragment of DNA are resistant to digestion by FokI restriction endonuclease but are sensitive to cleavage by HindIII, EcoRI and PstI . Unmodified lambda DNA molecules, exposed in vitro to cell extracts prepared from cells habouring this plasmid, became resistant to digestion by FokI . The smallest HgaI methylase clone carries the pBR322 plasmid containing a 3.50 kb piece of H . galinarum DNA . This plasmid is resistant to digestion by HgaI . Neither the FokI nor the HgaI restriction endonuclease was detected in either clone . This is the first report of cloning modification genes whose protein products recognise asymmetric nucleotide sequences.

Appl Environ Microbiol, 1987 Sep, 53(9), 2262 - 4
New selective and differential medium for Vibrio cholerae and Vibrio vulnificus; Massad G et al.; Thiosulfate-citrate-bile salts-sucrose agar has been routinely used for the isolation of pathogenic vibrios, although its selectivity for Vibrio cholerae and Vibrio vulnificus is inadequate . Therefore, a new plating medium, cellobiose-polymyxin B-colistin agar, was developed for the isolation of these two species . Cellobiose-polymyxin B-colistin agar demonstrated a significant advantage over other media designed for the isolation or differentiation of vibrios: of both the 136 strains representing 19 Vibrio species and the marine isolates of the genera Pseudomonas, Flavobacterium, and Photobacterium, only V . vulnificus and V . cholerae were able to grow . Furthermore, the fermentation of cellobiose by V . vulnificus allowed for the easy differentiation of these two species . This medium offers significant potential as a selective and differential medium for these two pathogenic vibrios.

Appl Biochem Biotechnol, 1987 Sep-Dec, 16, 35 - 50
Large scale preparation and characterization of mucopolysaccharase contamination free heparinase; Yang VC et al.; By a combination of hydroxylapatite chromatography and negative adsorption on QAE-Sephadex at pH 8.3, heparinase (E.C.4.2.2.7) can be successfully isolated from all the other mucopolysaccharase contaminants present in Flavobacterium heparinum . Hydroxylapatite isolates heparinase primarily from chondroitinases, hyaluronidase, and most glycuronidases . QAE-Sephadex chromatography at pH 8.3 further separates heparinase from heparitinases, sulfatases, and the remaining glycuronidases . The heparinase preparation thus obtained contains no statistically significant levels of other contaminating mucopolysaccharases except for heparitinases that are present at an apparent maximum level of 3.4% . Owing to the presence of a crossreaction of heparinase on heparitin sulfate at conditions employed for the assay of heparitinase, the heparitinase level of 3.4% could be misleading because of the action of heparinase on heparitin sulfate . Characterization of this heparinase preparation shows that the enzyme has an optimum salt concentration of 0.08M NaCl, an optimum pH of 6.5, an activation energy of 5 kcal/mol, and a Km of 7.95 X 10(-6) M . These parameters are almost identical to those displayed by a homogeneous heparinase preparation . The method described here is suitable for scale-up purposes using batch chromatographic procedures.

Plasmid, 1987 Sep, 18(2), 173 - 7
Physical comparison of parathion hydrolase plasmids from Pseudomonas diminuta and Flavobacterium sp; Mulbry WW et al.; Restriction maps of two plasmids encoding parathion hydrolase have been determined . pPDL2 is a 39-kb plasmid harbored by Flavobacterium sp . (ATCC 27551), while pCMS1 is a 70-kb plasmid found in Pseudomonas diminuta (strain MG) . Both plasmids previously have been shown to share homologous parathion hydrolase genes (termed opd for organophosphate degradation) as judged by DNA-DNA hybridization and restriction mapping . In the present study, we conducted DNA hybridization experiments using each of nine PstI restriction fragments from pCMS1 as probes against Flavobacterium plasmid DNA . The opd genes of both plasmids are located within a highly conserved region of approximately 5.1 kb . This region of homology extends approximately 2.6 kb upstream and 1.7 kb downstream from the opd genes . No homology between the two plasmids is evident outside of this region.

Acta Pathol Microbiol Immunol Scand {B}, 1987 Aug, 95(4), 245 - 52
Crossed immunoelectrophoretic analysis of Flavobacterium meningosepticum and allied Flavobacterium taxa; Bruun B et al.; A total of 55 different antigens were demonstrated in pooled lysate of six Flavobacterium meningosepticum strains by crossed immunoelectrophoresis against homologous rabbit antibody . The six strains used represented the two DNA relatedness groups within this species . All 52 strains of F . meningosepticum investigated were found to cross-react with 53 to 55 of these F . meningosepticum reference antigens . Using this F . meningosepticum reference system, cross-reactions between the F . meningosepticum reference antigen and antigens from representatives of other Flavobacterium taxa were also investigated . A positive correlation was demonstrated between the number of cross-reacting antigens and the taxonomic relatedness of the investigated taxa as determined by other classification methods, including DNA-DNA hybridization . The crossed immunoelectrophoretic techniques were not useful in differentiating the two main DNA groups of F . meningosepticum.

J Gen Microbiol, 1987 Aug, 133 ( Pt 8), 2115 - 22
Purification and characterization of chloramphenicol acetyltransferase from Flavobacterium CB60; Nolte G et al.; From the highly chloramphenicol-resistant cytophaga-like bacterium Flavobacterium CB60, which can both acetylate chloramphenicol and degrade it in co-metabolism, the chloramphenicol acetyltransferase (CAT) was purified to homogeneity and characterized . The purification included fractional precipitation with ammonium sulphate and two affinity chromatography steps, eluting CAT the first time with 5 mM-chloramphenicol and the second time with a linear gradient (0-10 mM) of chloramphenicol . The purification was 3979-fold . Properties of this CAT were investigated and compared with CATs from other bacteria . Although CAT from Flavobacterium CB60 shares some properties with the enzymes from Escherichia coli and other Gram-negative bacteria--especially with CATII and CATIII--it has distinct properties like extreme heat lability and the inability to produce diacetylchloramphenicol, so that it might be regarded as a new variant.

Zentralbl Bakteriol Mikrobiol Hyg {A}, 1987 Aug, 266(1-2), 155 - 66
Concomitant infections of Anopheles stephensi with Plasmodium berghei and Serratia marcescens: additive detrimental effects; Seitz HM et al.; The mortality rate of Anopheles stephensi increased after infection with Plasmodium berghei and correlated negatively with temperature . Development of oocysts is inhibited at temperatures above 21 degrees C . We tested the hypothesis that microorganisms were involved in killing the mosquitoes . In fact we were able to demonstrate that in our A . stephensi colony great numbers of Serratia marcescens could be found in the midgut of the insects . The highest value was 2.3 x 10(7) cfu/ml . Other bacteria were rarely seen (1 out of 30 females had flavobacteria) . Serratia was neither found in larvae and pupae nor in the water of the breeding dishes . Moderate numbers were detectable in glucose solutions (for feeding of adult mosquitoes) as well as in jars where pupae emerged . Isolated Serratia strains grew faster at 25 degrees C than at 21 degrees C . In glucose solutions alone growth rates were low but they rose rapidly after the addition of blood . -In experimental infections of A . stephensi with S . marcescens (1 x 10(7) bacteria/ml glucose solution) the mortality increased at 25 degrees C . At 21 degrees C the effect of Serratia was insignificant whereas in P . berghei-infected A . stephensi the damaging effects of migrating ookinetes were obvious . Additive detrimental effects were observed at 25 degrees C in mosquitoes infected with P . berghei and Serratia concomitantly.

J Clin Microbiol, 1987 Jul, 25(7), 1285 - 90
Classification and identification of Flavobacterium species by carbon source utilization; Rasoamananjara D et al.; Carbon substrates used as the sole source of carbon and energy were tested for the classification and identification of species of Flavobacterium: Flavobacterium meningosepticum, F . breve, F . odoratum, F . multivorum, F . thalpophilum, and Flavobacterium sp . group IIb . Hierarchical classification and stepwise discriminant analysis revealed three F . meningosepticum, two F . breve, two F . odoratum, and two Flavobacterium sp . group IIb subgroups . Glucose, histidine, asparagine, tryptophan, maltose, citric acid, and glycine were selected as the most useful substrates to differentiate between the groups and subgroups.

Eur J Biochem, 1987 Jun 15, 165(3), 633 - 8
Flavobacterium heparinum sulphamidase for D-glucosamine sulphamate . Purification and characterisation; Bruce JS et al.; A novel assay has been developed for 2-deoxy-2-sulphamido-D-glucose (GlcNS) sulphamidase from Flavobacterium heparinum . This has enabled the 1930-fold purification of the enzyme from a soluble fraction of bacterial homogenate . From SDS/polyacrylamide gel electrophoresis the enzyme was shown to have a relative molecular mass of 81,500 . Ca2+ was essential for enzyme activity . Inorganic phosphate and sulphate inhibited activity by 28% and 29% respectively at 5 mmol dm-3 . The purified sulphamidase had a pH optimum of 7.0 and a Km of 8.32 mumol dm-3 for GlcNS . The degradation of 2-deoxy-2-sulphamido-6-O-sulpho-D-glucose (GlcNS-6S) was also re-investigated . The two sulphate groups were hydrolysed sequentially in a single non-bifurcate manner, in contrast to previous reports {Dietrich, C.P., Silva, M.E . and Michelacci, Y.M . (1973) J . Biol . Chem . 248, 6408-6415}.

Biochem J, 1987 Jun 15, 244(3), 515 - 22
Fractionation of heparin-derived oligosaccharides by gradient polyacrylamide-gel electrophoresis; Rice KG et al.; Heparin-derived oligosaccharides, prepared by using flavobacterial heparinase, having a high degree of heterogeneity (sequence variability) were resolved into sharp well-defined bands by using polyacrylamide gel electrophoresis (PAGE) . The use of a stacking gel and a high-density-pore-gradient resolving gel was primarily responsible for the success of this separation . Low-Mr standards of known structure and having a degree of polymerization (dp) 2-6 were used to establish that the separation on gradient PAGE was primarily dependent on molecular size . High-Mr oligosaccharides (dp 8-20) were prepared using strong-anion-exchange h.p.l.c . and were used to help characterize the gradient PAGE separation . Kinetic profiles were obtained for the depolymerization of heparin and heparan sulphate with heparinase and heparitinase respectively . The utility of this approach in sequencing oligosaccharides derived from glycosaminoglycans is discussed.

J Biol Chem, 1987 Jun 15, 262(17), 8262 - 7
Structure and immunochemistry of an oligosaccharide repeating unit of the capsular polysaccharide of type III group B Streptococcus . A revised structure for the type III group B streptococcal polysaccharide antigen; Wessels MR et al.; We have derived oligosaccharides from the capsular polysaccharide of type III group B Streptococcus by enzymatic hydrolysis of a specific backbone glycosidic bond utilizing an endo-beta-galactosidase from Flavobacterium keratolyticus . Enzymatic digestion of the polysaccharide produced oligosaccharide fragments of one or more pentasaccharide repeating units . On the basis of 13C NMR, 1H NMR, and methylation analyses, it was established that the smallest digestion fragment was alpha-D-NeupNAc-(2----3)-beta-D-Galp-(1----4)-{beta-D-Glcp-(1----6 )}- beta-D-GlcpNAc-(1----3)-beta-D-Gal . The isolation of this oligosaccharide is consistent with the susceptibility of the beta-D-Galp-(1----4)-beta-D-Glcp linkage in the backbone of the type III group B streptococcal polysaccharide and confirms that the polysaccharide is composed of a pentasaccharide repeating unit . High resolution 13C NMR spectroscopic studies indicated that, as in the case of the pentasaccharide, the terminal sialic acid residues of the type III group B streptococcal polysaccharide were linked to O-3 and not to O-6 of its branch beta-D-galactopyranosyl residues as had been previously reported (Jennings, H . J., Rosell, K.-G., and Kasper, D . L . (1980) Can . J . Chem . 58, 112-120) . This linkage was confirmed in an independent methylation analysis of the type III group B streptococcal polysaccharide . Thin layer chromatogram binding assay and radioactive antigen binding assays with radiolabeled oligosaccharides demonstrated the single repeating unit pentasaccharide oligosaccharide to be poorly antigenic . Increasing oligosaccharide size to a decasaccharide consisting of two repeating units resulted in an 8-fold increase in antigen binding in the direct radioactive antigen binding assay . The results suggest that a region of the immunodeterminant site critical for antibody binding is located in the backbone of the polysaccharide and involves the beta-D-galactopyranose-(1----4) beta-D-glucopyranose bond.

Appl Environ Microbiol, 1987 Jun, 53(6), 1322 - 6
Bacterial colonization and endotoxin content of a new renal dialysis water system composed of acrylonitrile butadiene styrene; du Moulin GC et al.; We measured endotoxin and bacterial levels in tap water, in water purified by reverse osmosis, and in dialysate samples over a 4-month period in a new 10-bed renal dialysis unit . Water treated by reverse osmosis is conducted to the 10 stations through 111 m of piping composed of acrylonitrile butadiene styrene (ABS) . All determinations were made prior to the opening of the unit and after the system was purged for 35 h with all bedside station taps open . Formaldehyde disinfection of the piping system was attempted with a recommended protocol after 11 weeks by feeding 2.5 liters of 37% formaldehyde (0.85%, vol/vol) into the delivery system . Prior to water purging, 24 ng of endotoxin per ml was detected . This level decreased to 2.0 ng of endotoxin after the purging . Levels of endotoxin remained below 1.0 ng of endotoxin per ml throughout the duration of the study . In contrast, the level of viable microorganisms recovered from the treated water was approximately 3.5 X 10(4) CFU/100 ml . Even after disinfection of the system, there was no significant decrease in culturable bacteria from the water even though endotoxin levels were lower . Species isolated from the renal dialysis system were predominately pseudomonads, whereas species isolated from the tap water were Bacillus and Flavobacterium species . ABS provides a surface suitable for long-term colonization and growth of bacteria . Currently recommended decontamination protocols are ineffective in removing potentially pathogenic bacteria from ABS pipes and thus constitute an increased risk to patients undergoing dialysis.

Proc Natl Acad Sci U S A, 1987 Jun, 84(11), 3565 - 9
Heparin sequences in the heparan sulfate chains of an endothelial cell proteoglycan; Nader HB et al.; The structure of the glycosaminoglycan chain of a heparan sulfate proteoglycan isolated from the conditioned medium of an endothelial cell line has been analyzed by using various degradative enzymes (heparitinase I, heparitinase II, heparinase, glycuronidase, sulfatases) from Flavobacterium heparinum . This proteoglycan inhibits the thromboplastin-activated pathway of coagulation; as a consequence, the catalytic conversion of prothrombin to thrombin is arrested . Heparitinase I (EC 4.2.2.8), an enzyme with specificity restricted to the heparan sulfate portion of the polysaccharide, releases fragments with the electrophoretic mobility and the structure of heparin . Conversely, an assessment of the size and distribution of the heparan sulfate regions has been provided by the use of heparinase (EC 4.2.2.7), which, by degrading the heparin sections of the chain, releases two segments that exhibit the structure of heparan sulfate . One of these segments is attached to the protein core . On the basis of these findings, the heparan sulfate chain can be defined as a copolymer containing heparin regions in its structure . The combined use of these enzymes has made it possible to establish the disaccharide sequence of parts of the glycosaminoglycan moiety of this proteoglycan.

Appl Environ Microbiol, 1987 May, 53(5), 907 - 10
Degradation of chlorinated phenols by a pentachlorophenol-degrading bacterium; Steiert JG et al.; A pentachlorophenol (PCP)-degrading Flavobacterium sp . was tested for its ability to dechlorinate other chlorinated phenols by using resting cells that had been grown in the presence or absence of PCP . Phenols with chlorine atoms at positions 2 and 6 of the phenol ring were dechlorinated completely by PCP-induced cells . Other chlorinated phenols were not significantly mineralized . When PCP was added to a culture growing on L-glutamate, there was a lag period before the start of PCP degradation . When similar cells were treated with chloramphenicol prior to the addition of PCP, they did not degrade added PCP, even after prolonged incubations . Thus, the enzymes necessary for PCP degradation appeared to be inducible . Suspensions of cells grown in the presence of 2,4,6-trichlorophenol or 2,3,5,6-tetrachlorophenol did not show a lag period for mineralization of PCP, 2,4,6-trichlorophenol, or 2,3,5,6-tetrachlorophenol, indicating that one enzyme system probably was induced for the biodegradation of all three compounds . Nondegradable chlorophenols were toxic toward the Flavobacterium sp., probably acting as uncouplers of oxidative phosphorylation.

Pathol Biol (Paris), 1987 May, 35(5), 648 - 51
{Treatment of febrile episodes in neutropenic children by ceftazidime combined with netilmicin . Results of a multicenter study apropos of 88 cases}; Leverger G et al.; Infection is the most important cause of mortality in leucopenic patients . A broad spectrum antibiotic therapy is imperative in febrile and neutropenic patients . In a multicentric study we have used ceftazidime (100 mg/kg/d) and netilmicin (6 mg/kg/d) in 88 children (fever greater than or equal to 38.5 degrees C, neutropenia less than 500/mm3) treated for acute leukemias (59), non Hodgkin lymphomas (13) or solid tumors (16) . Median age was 7 years (2 months-16 years) . In patients who continued to remain febrile, vancomycin (40 mg/kg/d) was added after 48 hours . The effective treatment was continued until a neutrophil count greater than 1,000/mm3 . The first combination (ceftazidime + netilmicin) was effective in 64 children (73%) and the second combination (ceftazidime + netilmicin + vancomycin) in 11 patients . Bacteria were isolated in 39 children: Escherichia coli: 9, Staphylococcus epidermidis: 9, Staphylococcus aureus: 8, Streptococcus: 6, Pseudomonas aeruginosa: 3, Streptococcus pneumoniae: 1, Haemophilus: 1, Klebsiella pneumoniae: 1, Proteus: 1, Serratia: 1, Flavobacterium: 1 . In these 39 patients, 30 became apyretic with ceftazidime and netilmicin and 6 after vancomycin . All blood culture were negative after the first combination . The median duration of antibiotic therapy was 14 days (5-9 days: 28, 10-20 days: 43, greater than 20 days: 17) . There were no death, no superinfection . Tolerance was good without kidney or liver or biological perturbation . We conclude that the combination ceftazidime and netilmicin is effective in neutropenic children.

Acta Pathol Microbiol Immunol Scand {B}, 1987 Apr, 95(2), 95 - 101
Antimicrobial susceptibility of Flavobacterium meningosepticum strains identified by DNA-DNA hybridization; Bruun B; Antimicrobial susceptibilities of 52 strains of Flavobacterium meningosepticum were determined by an agar plate dilution technique and by a tablet diffusion method . No useful susceptibility differences between the two main DNA relatedness groups within F . meningosepticum were found . All strains produced beta-lactamase and were resistant to most beta-lactam antibiotics . Ciprofloxacin, rifampicin and clindamycin appear to be the most promising for treating infections caused by F . meningosepticum.

Appl Environ Microbiol, 1987 Apr, 53(4), 791 - 6
Explanations for the acclimation period preceding the mineralization of organic chemicals in aquatic environments; Wiggins BA et al.; A study was conducted of possible reasons for acclimation of microbial communities to the mineralization of organic compounds in lake water and sewage . The acclimation period for the mineralization of 2 ng of p-nitrophenol (PNP) or 2,4-dichlorophenoxyacetic acid per ml of sewage was eliminated when the sewage was incubated for 9 or 16 days, respectively, with no added substrate . The acclimation period for the mineralization of 2 ng but not 200 ng or 2 micrograms of PNP per ml was eliminated when the compound was added to lake water that had been first incubated in the laboratory . Mineralization of PNP by Flavobacterium sp . was detected within 7 h at concentrations of 20 ng/ml to 2 micrograms/ml but only after 25 h at 2 ng/ml . PNP-utilizing organisms began to multiply logarithmically after 1 day in lake water amended with 2 micrograms of PNP per ml, but substrate disappearance was only detected at 8 days, at which time the numbers were approaching 10(5) cells per ml . The addition of inorganic nutrients reduced the length of the acclimation period from 6 to 3 days in sewage and from 6 days to 1 day in lake water . The prior degradation of natural organic materials in the sewage and lake water had no effect on the acclimation period for the mineralization of PNP, and naturally occurring inhibitors that might delay the mineralization were not present . The length of the acclimation phase for the mineralization of 2 ng of PNP per ml was shortened when the protozoa in sewage were suppressed by eucaryotic inhibitors, but it was unaffected or increased if the inhibitors were added to lake water.(ABSTRACT TRUNCATED AT 250 WORDS)

Kidney Int, 1987 Apr, 31(4), 981 - 5
Renal transplantation after prolonged dwell peritoneal dialysis in children; Malagon M et al.; Thirty-three children received a total of 38 renal transplants (18 living related donor, and 20 cadaveric) after being on CAPD and/or CCPD (PDPD) . Ten patients (12 transplants) were converted to hemodialysis pre-transplant in order to be free of the risk of peritonitis and off antibiotics, whereas 23 patients (26 transplants) were on PDPD at the time of transplant . The latter group of patients are described in greater detail . Within this group there was one episode of catheter colonization with Flavobacterium, and only three patients developed ascites post-transplant . Of the 26 transplants, catheters were removed at the time of transplant in the 13 LRD allografts but left in situ for a mean of 3.8 weeks in the 13 cadaveric transplant recipients . Peritoneal dialysis was required post-transplant in seven patients (two LRD recipients requiring a new catheter placement) without complications . Our policy of removing PD catheters at the time of transplant in LRD recipients and prior to hospital discharge in cadaveric transplant recipients has resulted in the avoidance of additional hospitalizations in 19 of the 26 transplants and avoided extra surgery in 11 of the 13 LRD transplants . We conclude that children who have been on PDPD are suitable candidates for renal transplantation and that the early removal of PD catheters, including removal at the time of transplantation in LRD recipients, is associated with a significant reduction in operative procedures for the patients.

Clin Chem, 1987 Mar, 33(3), 377 - 80
Comparison of Flavobacterium and Sphingobacterium species by enzyme profiles, with use of pattern recognition of two-dimensional fluorescence data; Pau CP et al.; Enzyme profiles of eight Flavobacterium species and one Sphingobacterium species were compared after using a two-dimensional fluorescence technique . Enzyme contents and corresponding activities were rapidly determined for whole-cell preparations after incubation with a mixture of preselected fluorogenic substrates . A two-dimensional fluorescence spectrum of the resulting product mixture, measured with a video fluorometer, provided a characteristic "fingerprint" for each organism . Comparison of fluorescent spectra was facilitated by a Fourier-transform-based pattern-recognition algorithm and by a clustering technique involving the Pearson product-moment correlation coefficient . F . multivorum, F . thalpophilum, and S . mizutae formed one cluster; F . indologenes, F . spiritivorum, F . odoratum, and F . balustinum formed a second . F . meningosepticum was intermediate between the first and second cluster, whereas F . breve was different from all other strains examined, based on their spectral dissimilarity indices and correlation coefficients.

Biochim Biophys Acta, 1987 Feb 20, 923(2), 291 - 301
Isolation and characterization of an induced chondroitinase ABC from Flavobacterium heparinum; Michelacci YM et al.; During the investigation of alternative methods for the large scale preparation of chondroitinases AC, B and C from Flavobacterium heparinum, a new chondroitinase activity was observed . This new enzyme, like the other chondroitinases, acts as an eliminase, forming unsaturated sulfated disaccharides from dermatan and chondroitin sulfates . In contrast to the chondroitinases previously described, which are endoglycosidases, this chondroitinase ABC cleaves the glycosidic linkages in an exolytic fashion, beginning at the reducing end of the substrate molecules . The oligosaccharides formed as transient products by the action of either chondroitinases or testicular hyaluronidase upon dermatan and chondroitin sulfates are also rapidly degraded by the chondroitinase ABC, regardless of their size or the presence of delta-4,5 unsaturation in the terminal uronic acid residue . The maximum activity of the chondroitinase ABC occurs at 30 degrees C and at pH 6.0-7.5 . Only 15% of the activity was observed at 37 degrees C, indicating that the enzyme is very sensitive to thermal denaturation . It is strongly inhibited by phosphate ions and is also inhibited by the unsaturated disaccharides formed.

Appl Environ Microbiol, 1987 Feb, 53(2), 292 - 7
Role of dissolution rate and solubility in biodegradation of aromatic compounds; Stucki G et al.; Strains of Moraxella sp., Pseudomonas sp., and Flavobacterium sp . able to grow on biphenyl were isolated from sewage . The bacteria produced 2.3 to 4.5 g of protein per mol of biphenyl carbon, and similar protein yields were obtained when the isolates were grown on succinate . Mineralization of biphenyl was exponential during the phase of exponential growth of Moraxella sp . and Pseudomonas sp . In biphenyl-supplemented media, Flavobacterium sp . had one exponential phase of growth apparently at the expense of contaminating dissolved carbon in the solution and a second exponential phase during which it mineralized the hydrocarbon . Phase-contrast microscopy did not show significant numbers of cells of these three species on the surface of the solid substrate as it underwent decomposition . Pseudomonas sp . did not form products that affected the solubility of biphenyl, although its excretions did increase the dissolution rate . It was calculated that Pseudomonas sp . consumed 29 nmol of biphenyl per ml in the 1 h after the end of the exponential phase of growth, but 32 nmol of substrate per ml went into solution in that period when the growth rate had declined . In a medium with anthracene as the sole added carbon source, Flavobacterium sp . converted 90% of the substrate to water-soluble products, and a slow mineralization was detected when the cell numbers were not increasing . Flavobacterium sp . and Beijerinckia sp . initially grew exponentially and then arithmetically in media with phenanthrene as the sole carbon source . Calculations based on the growth rates of these bacteria and the rates of dissolution of phenanthrene suggest that the dissolution rate of the hydrocarbon may limit the rate of its biodegradation.

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