Spirochaeta aurantia has diacetyl chloramphenicol esterase activity

The free-living spirochete Spirochaeta aurantia was nearly as susceptible to diacetyl chloramphenicol, the product of chloramphenicol acetyltransferase, as it was to chloramphenicol itself. This unexpected susceptibility to diacetyl chloramphenicol was wholly or partly the consequence of intrinsic carboxylesterase activity, as indicated by high-performance liquid chromatography, thin-layer chromatography, and microbiological assays. The esterase converted the diacetate to chloramphenicol, thus inhibiting spirochete growth. The esterase activity was cell associated, reduced by proteinase K, eliminated by boiling, and independent of the presence of either chloramphenicol or diacetyl chloramphenicol. S. aurantia extracts also hydrolyzed other esterase substrates, and two of these, alpha-napthyl acetate and 4-methylumbelliferyl acetate, identified an esterase of approximately 75 kDa in a nondenaturing gel. Carboxylesterases occur in Streptomyces species, but in this study their activity was weaker than that of S. aurantia. The S. aurantia esterase could reduce the effectiveness of cat as either a selectable marker or a reporter gene in this species.     

An investigation of the divergence of major ampullate silk fibers from Nephila clavipes and Argiope aurantia

The major ampullate fiber of both Nephila clavipes and Argiope aurantia is composed of two different proteins, MaSp1 and MaSp2. Each of these proteins has a highly conserved pattern of silk-associated amino acid motifs. The GPGXX motif is the only source of proline and is unique to MaSp2. On the basis of the percent of proline, Nephila clavipes major ampullate silk was calculated to consist of 19% MaSp2 and 81% MaSp1, while Argiope aurantia was calculated to have a significantly higher MaSp2 content of 59% with MaSp1 comprising the remaining 41%. To investigate the functional implications of the difference in protein composition, major ampullate silk fibers from Nephila clavipes and Argiope aurantia were mechanically tested and compared. Stress-strain curves produced from polynomial regression show that the two significant differences between major ampullate silk fibers from Nephila clavipes and Argiope aurantia are the average peak load stress and Young's modulus, with Argiope higher for both.     

Relationship between cell coiling and motility of spirochetes in viscous environments.

The lowest viscosity that stops translational motility of cells (minimum immobilizing viscosity [MIV] was determined for various spirochetes. The viscous agent used was polyvinylpyrrolidone, The MIV for either Spirochaeta halophila P1 or Spirochaeta aurantia J4T was approximately 1,000 centipoise (cp), and for Leptospira interrogans (biflexa) B16 the MIV was greater than 500 cp. In comparison, the MIV for the flagellated bacteria Escherichia coli and Spirillum serpens was 60 cp. MIV values for two S. halophila mutant strains lacking the characteristic cell coiling (Hel-mutants) were 70 and 120 cp, approximately one-tenth the MIV for the wild-type strain. MIV values for cells of S. aurantia strains with fewer coils than comparably long cells of S. aurantia J4T were 300 to 600 cp. The average velocity of strains of S. aurantia and S. halophila decreased at viscosities higher than 2 to 3 cp. At 2 cp the average velocity of S. halophila P1 was 16 micron/s, whereas the average velocities of Hel-mutant strains were 7 to 9 micron/s. This study indicates that the coiling of spirochetes plays a role in their ability to move through environments of realtively high viscosity. Among the spirochetes we investigated, this ability is greater in the more extensively coiled strains.     

Chemotaxis in Spirochaeta aurantia.

Cell of Spirochaeta aurantia M1 suspended in isotropic buffer solution swam in nearly straight lines and appeared to spin around their longitudinal axis. Occasionally, cells stopped and flexed, and then resumed translational motility, usually in a different direction. The average cell velocity was 26 micron/s. A quantitative assay for chemotaxis was used to test various chemicals for their ability to attract S. aurantia M1. The cells exhibited a tactic response toward 5 X 10(-2) M D-glucose between 10 and 35degree C; the optimum response was at 25degree C. At 5 degree C motility was not impaired, but D-glucose taxis was abolished. Chemotaxis toward D-glucose was stimulated by L-cysteine (2 X 10(-4) M). D-Glucose, 2-deoxy-D-glucose, alpha-methyl-D-glucoside, D-galactose, D-fucose, D-mannose, D-fructose, D-xylose, maltose, cellobiose, and D-glucosamine were effectve attractants for S. aurantia M1. D-Galactose taxis and D-fucose taxis were induced by the presence of D-galactose in the growth medium. The amino acids tested did not serve as attractants, tgrowing cells of S. aurantia M1 exhibited an aerotactic response.     

Enzymatic activities for interconversion of purines in spirochetes.

Enzymatic activities that catalyze the interconversion of purines and purine derivatives were detected in cell extracts of Spirochaeta aurantia, Spirochaeta stenostrepta, Treponema succinifaciens, and Treponema denticola. Phosphoribosyltransferase activities present in cell extracts of each of the four spirochete species functioned in the conversion of adenine, hypoxanthine, and guanine to AMP, IMP, and GMP, respectively. Nucleotidase activities in the extracts mediated the formation of nucleosides from nucleotides. The conversion of adenosine, inosine, and guanosine to the respective purine bases was catalyzed by nucleoside phosphorylase and, in some instances, by nucleoside hydrolase activities. Guanine deaminase activity was found in both S. aurantia and S. stenostrepta, whereas adenosine deaminase activity was detected only in S. aurantia. Adenine deaminase activity in T. succinifaciens extracts was sensitive to O2 and was relatively resistant to heating. Our results indicate that the four species of spirochetes studied possess a broad spectrum of purine interconversion enzymes. It is suggested that these enzymes may function in metabolic processes important for the survival of spirochetes in nutrient-poor natural environments.     

Morphology and physiology of Spirochaeta aurantia strains isolated from aquatic habitats

1. Seven strains of Spirochaeta aurantia were isolated from pond and swamp water by means of a selective technique which utilized the ability of these organisms to move through bacterial filters and to diffuse through agar media. Although most of the isolations were accomplished when enrichment media low in carbohydrates were used, all seven strains were found to be exclusively saccharolytic. 2. The isolates could be divided into two groups on the basis of cell morphology: a loosely coiled group, and a tightly coiled group with markedly smaller wave length and wave apmlitude. Spirochetes of the latter group possessed a slightly lower GC content in their DNA. The isolates were facultative anaerobes, synthesized carotenoid pigments which conferred an orange color to aerobic colonies, and utilized a variety of carbohydrates--but not amino acids--as energy sources. Exogenous thiamine was required by six isolates tested, riboflavin by four, and biotin by one. The major products of glucose fermentation were acetate, ethanol, CO2 and H2. Growth of the isolates was inhibited by a variety of antibiotics. Determinations of GC contents of DNA showed that strains of S. aurantia are phylogenetically distant from spirochetes classified in the genera Treponema and Leptospira. 3. S. aurantia populations inoculated in the center of agar medium plates migrated in the form of growth rings toward the periphery of the plates. The rate of migration of glucose-utilizing rings was greatest at low glucose concentrations (e.g., 0.02 g/100 ml). It was concluded that migration of cells present in these rings was mainly due to a chemotactic response to glucose which served both as the attractant and the substrate. Chemotaxis of S. aurantia toward glucose may be used as a selective factor in isolating this bacterium from natural environments. 4. The subspecific epithet stricta is proposed to recognize, taxonomically, the tightly coiled strains of S. aurantia.     

Spirochaeta aurantia, a Pigmented, Facultatively Anaerobic Spirochete

A strain of Spirochaeta aurantia was isolated from mud by a procedure involving migration of the organisms through cellulose ester filter discs (0.3-mum pore diameter) onto the surface of culture plates. The helical cells measured 0.3 by 10 to 20 mum during exponential growth. Electron microscopy showed the presence of two subterminally inserted axial fibrils partially overlapping in a 1-2-1 arrangement. An outer envelope, exhibiting a polygonal substructure, was observed. The spirochete grew either aerobically or anaerobically, with aerobic yields of 9.8 x 10(8) cells per ml and anaerobic yields of 3.0 x 10(8) cells per ml. The organism used carbohydrates, but not amino acids, as energy sources. Amino acids served as sole nitrogen sources, whereas inorganic ammonium salts did not. The presence of biotin and thiamine in the medium was required for growth. Growing cells fermented maltose mainly to carbon dioxide, hydrogen, ethyl alcohol, and acetic acid. Small amounts of formic and lactic acids, acetoin, and diacetyl were produced. Cells of S. aurantia growing aerobically produced a yellow-orange pigment. Chemical analysis indicated that the pigment was carotenoid in nature, its main component being lycopene or a similar compound. S. aurantia is not closely related to the leptospires, since it lacks both the hemolytic antigen and the hooked ends typical of the latter organisms. Furthermore, the guanine plus cytosine content in the deoxyribonucleic acid of S. aurantia (66.8 moles%) differs drastically from that of leptospires.     

Chemotaxis of Spirochaeta aurantia: involvement of membrane potential in chemosensory signal transduction.

The effects of valinomycin and nigericin on sugar chemotaxis in Spirochaeta aurantia were investigated by using a quantitative capillary assay, and the fluorescent cation, 3,3'-dipropyl-2,2'-thiodicarbocyanine iodide was used as a probe to study effects of chemoattractants on membrane potential. Addition of a chemoattractant, D-xylose, to cells in either potassium or sodium phosphate buffer resulted in a transient membrane depolarization. In the presence of valinomycin, the membrane potential of cells in potassium phosphate buffer was reduced, and the transient membrane depolarization that resulted from the addition of D-xylose was eliminated. Although there was no detectable effect of valinomycin on motility, D-xylose taxis of cells in potassium phosphate buffer was completely inhibited by valinomycin. In sodium phosphate buffer, valinomycin had little effect on membrane potential or D-xylose taxis. Nigericin is known to dissipate the transmembrane pH gradient of S. aurantia in potassium phosphate buffer. This compound did not dissipate the membrane potential or the transient membrane depolarization observed upon addition of D-xylose to cells in either potassium or sodium phosphate buffer. Nigericin did not inhibit D-xylose taxis in either potassium or sodium phosphate buffer. This study indicates that the membrane potential but not the transmembrane pH gradient of S. aurantia is somehow involved in chemosensory signal transduction.     

Motility and chemotaxis of Spirochaeta aurantia: computer-assisted motion analysis.

A computer program has been designed to study behavior in populations of Spirochaeta aurantia cells, and this program has been used to analyze changes in behavior in response to chemoattractants. Three kinds of behavior were distinguished: smooth swimming, flexing, and reversals in direction of swimming after a short pause (120 ms). Cell populations exposed to chemoattractants spent, on average, 66, 33, and 1% of the time in these modes, respectively. After the addition of a chemoattractant, behavior was modified transiently--smooth swimming increased, flexing decreased, and reversals were suppressed. After addition of D-xylose (final concentration, 10 mM), the adaptation time (the time required for the populations to return to the unmodified behavior) for S. aurantia was 1.5 to 2.0 min. A model to explain the behavior of S. aurantia and the response of cells to chemoattractants is described. This model includes a coordinating mechanism for flagellar motor operation and a motor switch synchronizing device.     

Enhancement of chemotaxis in Spirochaeta aurantia grown under conditions of nutrient limitation.

Spirochaeta aurantia M1 cells were grown in a chemostat under conditions of energy and carbon source limitation. The chemotactic responses of the chemostat-grown cells were compared with those of S. aurantia cells grown in batch culture in the presence of excess energy and carbon source. Chemotactic responses were measured by determining the number of cells that entered a capillary tube containing a solution of attractant. S. aurantia cells grown in the chemostat under energy and carbon source limitation exhibited enhanced chemotactic responses and detected lower concentrations of attractant, as compared with cells grown in batch culture. The chemotactic response toward an attractant was specifically enhanced when that attractant was the growth-limiting energy and carbon source. The medium used contained either D-glucose or D-xylose as the sole energy and carbon source. Cells had the greatest chemotactic response toward glucose when grown at a dilution rate (D) of 0.045 h-1 under glucose limitation and toward xylose when grown at D = 0.06 h-1 under xylose limitation. When cells were grown under glucose limitation (D = 0.045 h-1), they sensed concentrations of attractant (glucose) ca. 1,000 times lower than those sensed by batch-grown cells. A similar enhancement of sensing ability (toward xylose) was observed in cells grown under xylose limitation. The results indicated that S. aurantia cells are able to regulate their chemosensory system in response to nutrient limitation. Maximum enhancement of chemotaxis occurs in cells growing at very low concentrations of energy and carbon source. Most likely, this property provides the spirochetes with competitive advantages when the availability of nutrients becomes severely limited in their habitats.     

Nucleotide sequence and analysis of a gene encoding anthranilate synthase component I in Spirochaeta aurantia.

A Spirochaeta aurantia DNA fragment containing the trpE gene and flanking chromosomal DNA was cloned, and the sequence of the trpE structural gene plus 870 bp upstream and 1,257 bp downstream of trpE was determined. The S. aurantia trpE gene codes for a polypeptide of 482 amino acid residues with a predicted molecular weight of 53,629 that showed sequence similarity to TrpE proteins from other organisms. The S. aurantia TrpE polypeptide is not more closely related to the other published spirochete TrpE sequence (that of Leptospira biflexa) than to TrpE polypeptides of other bacteria. Two additional complete open reading frames and one partial open reading frame were identified in the sequenced DNA. One of the complete open reading frames and the partial open reading frame are upstream of trpE and are encoded on the DNA strand opposite that containing trpE. The other open reading frame is downstream of trpE and on the same DNA strand as trpE. On the basis of the results of a protein sequence data base search, it appears that trpE is the only tryptophan biosynthesis gene in the sequenced DNA. This is in contrast to L. biflexa, in which trpE is separated from trpG by only 64 bp.     

N-terminal amino acid sequences and amino acid compositions of the Spirochaeta aurantia flagellar filament polypeptides.

The amino-terminal sequences and amino acid compositions of the three major and two minor polypeptides constituting the filaments of Spirochaeta aurantia periplasmic flagella were determined. The amino-terminal sequence of the major 37.5-kDa outer layer polypeptide is identical to the sequence downstream of the proposed signal peptide of the protein encoded by the S. aurantia flaA gene. However, the amino acid composition of the 37.5-kDa polypeptide is not in agreement with that inferred from the sequence of flaA. The 34- and 31.5-kDa major filament core polypeptides and the 33- and 32-kDa minor core polypeptides show a striking similarity to each other, and the amino-terminal sequences of these core polypeptides show extensive identity with homologous proteins from members of other genera of spirochetes. An additional 36-kDa minor polypeptide that occurs occasionally in preparations of S. aurantia periplasmic flagella appears to be mixed with the 37.5-kDa outer layer polypeptide or a degradation product of this polypeptide.     

A lectin from an ascomycete mushroom, Melastiza chateri: no synthesis of the lectin in mycelial isolate

Using an affinity adsorbent prepared from L-fucose and starch, a lectin was isolated from fruit bodies of an ascomycete mushroom, Melastiza chateri. The lectin was found to cross-react with antiserum against Aleuria aurantia lectin (AAL), that had been obtained from another ascomycete mushroom. The N-terminal amino acid sequence was analyzed, and among 20 residues 12 were the same as AAL. The molecular mass of the lectin estimated by SDS-PAGE was approximately 40 kDa, which is larger than that of AAL. Mycelial isolate was obtained from M. chateri by germinating ascospores, and identified by analyzing restriction fragment length polymorphisms (RFLP) of DNA. The isolate from M. chateri did not synthesize the lectin, although the isolate from A. aurantia had been known to synthesize AAL as much as the fruit body.     

A Lectin from an Ascomycete Mushroom,Melastiza chateri: No Synthesis of the Lectin in Mycelial Isolate

Using an affinity adsorbent prepared from L-fucose and starch, a lectin was isolated from fruit bodies of an ascomycete mushroom, Melastiza chateri. The lectin was found to cross-react with antiserum against Aleuria aurantia lectin (AAL), that had been obtained from another ascomycete mushroom. The N-terminal amino acid sequence was analyzed, and among 20 residues 12 were the same as AAL. The molecular mass of the lectin estimated by SDS-PAGE was approximately 40 kDa, which is larger than that of AAL. Mycelial isolate was obtained from M. chateri by germinating ascospores, and identified by analyzing restriction fragment length polymorphisms (RFLP) of DNA. The isolate from M. chateri did not synthesize the lectin, although the isolate from A. aurantia had been known to synthesize AAL as much as the fruit body.     

Relationship between proton motive force and motility in Spirochaeta aurantia.

The effects of various metabolic inhibitors on the motility of Spirochaeta aurantia were investigated. After 15 min in sodium arsenate buffer, 90% of cells remained motile even though adenosine triphosphate levels dropped from 5.6 to 0.1 nmol/mg (dry weight) of cells. After 70 min in sodium arsenate, 5% of cells were motile. Addition of phenazine methosulfate plus ascorbate at this time resulted in motility of 95% of cells, but adenosine triphosphate levels remained at 0.1 nmol/mg of cell dry weight. Carbonyl cyanide-m-chlorophenyl hydrazone rapidly (within 1 min) and completely inhibited motility of metabolizing cells in potassium phosphate buffer. However, after 15 min in the presence of carbonyl cyanide m-chlorophenyl hydrazone the cellular adenosine triphosphate level was 3.4 nmol/mg (dry weight) of cells, and the rate of oxygen uptake was 44% of the rate measured in the absence of carbonyl cyanide m-chlorophenyl hydrazone. Cells remained motile under conditions where either the electrical potential or the pH gradient across the membrane of S. aurantia was dissipated. However, if both gradients were simultaneously dissipated, motility was rapidly inhibited. This study indicates that a proton motive force, in the form of either a transmembrane electrical potential or a transmembrane pH gradient, is required for motility in S. aurantia. Adenosine triphosphate does not appear to directly activate the motility system in this spirochete.     

An examination of the potential role of spider digestive proteases as a causative factor in spider bite necrosis

Tissue necrosis following spider bites is a widespread problem. In the continental United States, the brown recluse (Loxosceles reclusa), hobo spider (Tegenaria agrestis), garden spider (Argiope aurantia) and Chiracanthium species, among others, reportedly cause such lesions. The exact mechanism producing such lesions is controversial. There is evidence for both venom sphingomyelinase and spider digestive collagenases. We have examined the role of spider digestive proteases in spider bite necrosis. The digestive fluid of A. aurantia was assayed for its ability to cleave a variety of connective tissue proteins, including collagen. Having confirmed that the fluid has collagenases, the digestive fluid was injected into the skin of rabbits to observe whether it would cause necrotic lesions. It did not. The data do not support the suggestions that spider digestive collagenases have a primary role in spider bite necrosis.     

药用真菌金耳的rDNA ITS序列分析与鉴别

研究了两个居群的金耳Tremella aurantialba以及近似品的rDNAITS区碱基全序列的特征及其差异,首次报道了金耳的ITS和5.8SrDNA完整序列,序列总长度为467~468,长度变异较少。聚类分析表明两个居群金耳亲缘关系非常密切,金耳药材和近似品金黄银耳形成一个稳定的独立分支,近似品黄金银耳形成另一个分支。ITS序列的差异为金耳的鉴别提供了可靠的分子标记,为金耳菌类药材基原入药建立了遗传基础。     

Demonstration of rare protein in the outer membrane of Treponema pallidum subsp. pallidum by freeze-fracture analysis.

The surface of Treponema pallidum subsp. pallidum (T. pallidum), the etiologic agent of syphilis, appears antigenically inert and lacks detectable protein, as judged by immunocytochemical and biochemical techniques commonly used to identify the outer membrane (OM) constituents of gram-negative bacteria. We examined T. pallidum by freeze-fracture electron microscopy to visualize the architecture of its OM. Treponema phagedenis biotype Reiter (T. phagedenis Reiter), a nonpathogenic host-associated treponeme, and Spirochaeta aurantia, a free-living spirochete, were studied similarly. Few intramembranous particles interrupted the smooth convex and concave fracture faces of the OM of T. pallidum, demonstrating that the OM of this organism is an unusual, nearly naked lipid bilayer. In contrast, the concave fracture face of the OM of S. aurantia was densely covered with particles, indicating the presence of abundant integral membrane proteins, a feature shared by typical gram-negative organisms. The concentration of particles in the OM concave fracture face of T. phagedenis Reiter was intermediate between those of T. pallidum and S. aurantia. Similar to typical gram-negative bacteria, the OM convex fracture faces of the three spirochetes contained relatively few particles. The unique molecular architecture of the OM of T. pallidum can explain the puzzling in vitro properties of the surface of the organism and may reflect a specific adaptation by which treponemes evade the host immune response.     

Fractionation of L-fucose-containing oligosaccharides on immobilized Aleuria aurantia lectin

The carbohydrate-binding specificity of Aleuria aurantia lectin was investigated by analyzing the behavior of a variety of fucose-containing oligosaccharides on an A. aurantia lectin-Sepharose column. Studies with complex-type oligosaccharides obtained from various glycoproteins by hydrazinolysis and their partial degradation fragments indicated that the presence of the alpha-fucosyl residue linked at the C-6 position of the proximal N-acetylglucosamine moiety is indispensable for binding to the lectin column. Binding was not affected by the structures of the outer chain moieties nor by the presence of the bisecting N-acetylglucosamine residue. These results indicated that A. aurantia lectin-Sepharose is useful for the group separation of mixtures of complex-type asparagine-linked sugar chains. Studies of glycosylated Bence Jones proteins indicated that this procedure is also applicable to intact glycoproteins. The behavior of oligosaccharides isolated from human milk and the urine of patients with fucosidosis indicated that the oligosaccharides with Fuc alpha 1----2Gal beta 1----4GlcNAc and Gal beta 1----4(Fuc alpha 1----3)GlcNAc groups interact with the lectin, but less strongly than complex-type sugar chains with a fucosylated core. Lacto-N-fucopentaitol II, which has a Gal beta 1----3(Fuc alpha 1----4)GlcNAc group, interacts less strongly than the above two groups with the matrix. Oligosaccharides with Fuc alpha 1----2Gal beta 1----3GlcNAc and Gal beta 1----4GlcNAc beta 1----3Gal beta 1----4(Fuc alpha 1----3)GlcNAc groups showed almost no interaction with the matrix.