Selective Growth Inhibition of Sphaerotilus natans and Beggiatoa sp. by Nucleosides

9-beta-d-Arabinofuranosyladenine (ara-A) has been found to specifically inhibit the growth of Sphaerotilus natans and Beggiatoa sp. at a low concentration (0.78 mug/ml). The nucleoside had no antimicrobial activity against various microorganisms other than Candida albicans at 1,000 mug/ml. 3'-Deoxyadenosine, 2'-deoxyadenosine, formycin, and some derivatives of ara-A also showed inhibitory activity against Sphaerotilus natans. The growth of Beggiatoa sp. was also inhibited by 9-beta-arabinofuranosylhypoxanthine, 3'-deoxyadenosine, 2'-deoxyadenosine, formycin, toyocamycin, tubercidin, and some derivatives of ara-A. ara-A was quite stable in water and had no harmful effect on fish at 200 mug/ml. The possible uses of ara-A and some nucleosides in controlling the proliferation of S. natans and Beggiatoa sp. in the environment are discussed.     

Factors Affecting Filamentous Growth of Sphaerotilus natans

Filamentous growth in cultures of Sphaerotilus natans can be measured and compared with total growth by a standardized procedure of winding filaments around an inoculating needle. Filaments and residual growth are then separately washed on Millipore filters, dried, and weighed. This method has been used to study changes in the growth habit of S. natans elicited by changes in the concentration of nutrients in the medium. The concentration of peptone, in a medium containing a sugar, phosphate buffer, and inorganic salts, has a much greater effect on the proportion of filamentous growth than does the nature or concentration of the carbon source or the concentration of phosphate buffer. Filament formation is significantly inhibited by concentrations of peptone greater than 0.25%; further increases in peptone concentration stimulate the production of large amounts of capsular material. Increasing the concentration of phosphate buffer to 0.05 M almost completely inhibits growth of S. natans.     

Growth of Sessile Sphaerotilus natans in a Tubular Recycle System

The growth of sessile Sphaerotilus natans was measured in a continuous-flow recycle system. Four methods were used to confirm that the growth of the biofilm was a linear function that increased with time. Of the methods used, one monitored in situ biofouling (increased fluid frictional resistance), two indicated biomass (ATP and DNA per square centimeter), and one allowed the direct observation of replaceable test surfaces by scanning electron microscopy. The filamentous growth of S. natans caused an increase in the fluid frictional resistance. The increase in biofouling was directly proportional to the increase in biomass.     

Attached Growth of Sphaerotilus natans in Continuous-Flow Apparatus and Its Growth Inhibition by 9-beta-d-Arabinofuranosyladenine

Sphaerotilus natans grew at the maximum specific growth rate (mu(max)) of 0.43/h when cultivated on PGY medium at 25 degrees C. The organism mainly grew attached to inside of the culture vessels when the culture medium was fed to the completely mixed continuous-flow apparatus at a dilution rate above mu(max) and the attached growth was directly related to the dilution rate. When a low concentration of the medium was supplied to the apparatus, almost all of the cells grown were filamentous and attached to the inside of the vessels. When a high concentration of the medium was fed, the organism grew as single cells or short chains and flowed out into the effluent. The attached growth of S. natans in the continuous-flow apparatus was inhibited by the minimal inhibitory concentration of 0.5 to 1.0 mug of 9-beta-d-arabinofuranosyladenine per ml. 9-beta-d-Arabinofuranosyladenine showed bacteriocidal activity against S. natans at a concentration of 50 to 100 mug/ml.     

The structure of the lipid A component of Sphaerotilus natans

The lipopolysaccharide of Sphaerotilus natans afforded a ladder-like pattern of bands in sodium deoxycholate-polyacrylamide gel electrophoresis, indicating the presence of a S-form lipopolysaccharide. The chemical analysis showed neutral sugars (rhamnose, glucose, l-glycero-d-manno-heptose), 3-deoxy-octulosonic acid (Kdo), amino compounds (glucosamine, glucosamine phosphate, ethanolamine and ethanolamine phosphate), and phosphorus. The lipid A fraction contained saturated and unsaturated capric, lauric, and myristic acids, and 3-hydroxy capric acid (3-OH-10:0). Its chemical structure was consisting of a glucosamine disaccharide, glycosidically substituted by a phosphomonoester, and substituted at C-4 by a pyrophosphodiester esterified with ethanolamine. The amino groups of both glucosamines are acylated by 3-hydroxy capric acids and these in turn are substituted by saturated and unsaturated capric, lauric, and myristic acids. Hydroxyl groups of the backbone disaccharide at C-3 and C-3 were also esterified by 3-hydroxy capric acid, those at C-4 and C-6 were unsubstituted. The latter provides the attachment site for Kdo.Abbreviations Kdo 3-deoxy-d-manno-octulosonic acid - 3-OH-10:0 3-hydroxy capric acid - DOC-PAGE deoxycholate-polyacrylamide gel electrophoresis - GC-MS gas chromatography/mass spectrometry - LD-MS laser desorption mass spectrometry - LPS lipopolysaccharide - PS polysaccharide     

Isolation and Chemical Composition of the Sheath of Sphaerotilus natans

A sheathed bacterium, Sphaerotilus natans, was cultured with vigorous shaking in a medium containing peptone. Then the biomass was harvested and treated with lysozyme, sodium dodecyl sulfate, and protease. With treatment, 1.6 mg of sheaths was obtained from 15 mg of biomass. For the preparation of sheaths of high purity, cultivation must be in the absence of glucose with sufficient aeration to prevent poly(3-hydroxybutyrate) accumulation. Carbohydrate (54.1%), protein (12.2%), and lipid (1-3%) were detected in the sheaths by colorimetric reactions and solvent extraction. Gas-liquid chromatography showed glucose and galactosamine to be present in the molar ratio of 1:4. The most abundant amino acids in the sheath protein were glycine (49.2 mol%) and cysteine (24.6 mol%). The sheaths were resistant to agents that reduce disulfide bonds (dithiothreitol and 2-mercaptoethanol) and to protease. However, sheathes were degraded completely by hydrazine, and a heteropolysaccharide composed of glucose and galactosamine (1:4) was released. The weight-average molecular weight of the polysaccharide was estimated to be 1.2×10⁵ by gel filtration chromatography with a low-angle laser-light scattering photometer and a rotation index detector. A ladder of 1.5-kDa peptides separable by sodium dodecyl sulfate gel electrophoresis was obtained by partial hydrolysis of sheaths, suggesting the sheath protein has repeating units of 1.5 kDa.     

Biosynthesis of poly-3-hydroxybutyrate by Sphaerotilus natans

A sheathed bacterium Sphaerotilus natans could not survive at 4°C for 2 months, and mutants that exhibited different colony phenotypes were obtained only by repeating the short period of storage at 4 °C. The ability of these mutants and the parent strain to produce poly-3-hydroxybutyrate (PHB) was compared in batch cultures. The parent strain accumulated 30% (w/w) PHB, while one of the mutants defective in sheath formation, designated as T2, accumulated over 50% PHB. Because T2 did not require strict air or nitrogen limitation for polymer accumulation, its production was growth-associated, allowing one-stage fermentation. In a pH-controlled fermentation using a jar fermentor, 10 g/l glucose was converted into 2.0 g/l PHB in 24 h.     

Prevalence of Broad-Host-Range Lytic Bacteriophages of Sphaerotilus natans, Escherichia coli, and Pseudomonas aeruginosa

Two bacteriophage collections were examined with regard to their ability to form plaques on multiple bacterial host species. Nine of 10 phages studied were found to be broad-host-range bacteriophages. These phages fell into two groups. Group 1, the SN series, was isolated from sewage treatment plant samples with Sphaerotilus natans ATCC 13338 as a host. The DNAs of these bacteriophages contained modified bases and were insensitive to cleavage by type I and II restriction endonucleases. The efficiency of plating of these bacteriophages was changed only slightly on the alternate host. Group 2, the BHR series, was isolated by a two-host enrichment protocol. These bacteriophages were sensitive to restriction, and their efficiency of plating was dramatically reduced on the alternate host. Our results suggest that a multiple-host enrichment protocol may be more effective for the isolation of broad-host-range bacteriophages by avoiding the selection bias inherent in single-host methods. At least two of the broad-host-range bacteriophages mediated generalized transduction. We suggest that broad-host-range bacteriophages play a key role in phage ecology and gene transfer in nature.     

Lysis of Sphaerotilus natans Swarm Cells by Bdellovibrio bacteriovorus

Six strains of Sphaerotilus natans (smooth form) were lysed by five parasitic strains of Bdellovibrio bacteriovorus. The possible use of Bdellovibrio to control the proliferation of S. natans in the environment was hypothesized.     

Composition of an Extracellular Polysaccharide Produced by Sphaerotilus natans

The capsular polysaccharide of Sphaerotilus natans has been isolated, purified, and analyzed. Chromatographic and chemical analyses performed on acid hydrolyzates of the purified material have shown that the major components are fucose, galactose, glucose, and glucuronic acid in approximately equimolar amounts. Glucose and glucuronic acid are believed to occur as an aldobiuronic acid unit.     

Structural Analysis of the Extracellular Polysaccharide Produced by Sphaerotilus natans

An extracellular polysaccharide (EPS) was recovered and purified from the culture fluid of a sheathed bacterium, Sphaerotilus natans. Glucose, rhamnose, and aldobiouronic acid were detected in the acid hydrolysate of EPS by thin-layer chromatography (TLC). The aldobiouronic acid was found to be composed of glucuronic acid and rhamnose by TLC and gas-liquid chromatography analyses of the corresponding neutral disaccharide. The structure of EPS was identified by methylation linkage analysis and nuclear magnetic resonance. Additionally, partial acid hydrolysates of EPS were prepared and put through fast atom bombardment-mass spectrometry to determine the sugar sequence of EPS. The resulting data showed that EPS produced by S. natans is a new gellan-like polysaccharide constructed from a tetrasaccharide repeating unit, as shown below.

→4)-α-D-Glcp-(1→2)-β-D-GlcAp-(1→2)-α-L-Rhap- (1→3)-β-L-Rhap-(1→     

Structural analysis of the extracellular polysaccharide produced by Sphaerotilus natans

An extracellular polysaccharide (EPS) was recovered and purified from the culture fluid of a sheathed bacterium, Sphaerotilus natans. Glucose, rhamnose, and aldobiouronic acid were detected in the acid hydrolysate of EPS by thin-layer chromatography (TLC). The aldobiouronic acid was found to be composed of glucuronic acid and rhamnose by TLC and gas-liquid chromatography analyses of the corresponding neutral disaccharide. The structure of EPS was identified by methylation linkage analysis and nuclear magnetic resonance. Additionally, partial acid hydrolysates of EPS were prepared and put through fast atom bombardment-mass spectrometry to determine the sugar sequence of EPS. The resulting data showed that EPS produced by S. natans is a new gellan-like polysaccharide constructed from a tetrasaccharide repeating unit, as shown below. -->4)-alpha-D-Glcp-(1-->2)-beta-D-GlcA p-(1-->2)-alpha-L-Rha p-(1-->3)-beta-L-Rha p-(1-->.     

球衣菌对工业废水中Cu2+吸附条件的研究

利用球衣菌FQ32制备生物吸附剂,研究其对Cu^2＋的耐受能力,并以连江某工厂含Cu^2＋废水为样液,对其进行吸附条件试验,分析了影响吸附率和吸附量的因素,结果表明,球衣菌FQ32对Cu^2＋的耐受能力大于150mg／L。在工业废水中Cu^2＋的浓度为23．1mg／L,吸附剂用量为0．2g／L,30℃,pH值6．0,吸附时间10min的条件下,该吸附剂对Cu^2＋的吸附率达到85．2％,吸附量达91．21mg／g。     

Commensal relationship between a sheath-forming bacterium, Sphaerotilus natans, and a sheath-degrading bacterium, Paenibacillus sp

Paenibacillus sp. strain TB is capable of degrading the sheath prepared from a sheathed bacterium, Sphaerotilus natans. S. natans was able to grow alone on casamino acids but strain TB was not. Cocultivation of strain TB and S. natans was examined in a medium supplemented with casamino acids as a growth substrate. The growth of strain TB was observed when the sheath was supplied to the medium or in cocultivation with S. natans. The phospholipid amount reached a maximum after 24 h of cocultivation and subsequently kept almost the same level for 96 h. The sheath amount also reached a maximum after 24 h and then gradually declined. The cell concentration of strain TB increased throughout the cocultivation. By competitive PCR targeted for amplification of a part of 16S rDNA, the abundance ratio (S. natans/strain TB) of 6.7 was obtained at 72 h. Almost no growth of strain TB was detected in a coculture with a sheath-less mutant of S. natans. The evidence allows the conclusion that strain TB grew by utilizing the intact sheath in coculture with S. natans.     

Structure of the polysaccharide isolated from the sheath of Sphaerotilus natans

A polysaccharide was isolated from the sheath of a sheathed bacterium, Sphaerotilus natans. The sheath polysaccharide (SPS) was composed of D-glucose and D-(N-acetyl)galactosamine in molar ratios of 1:4. Methylation linkage analysis revealed the presence of the residues of 4-linked glucose, 4-linked (N-acetyl)galactosamine, and 3-linked (N-acetyl)galactosamine in molar ratios of 1:3:1. The oligomer of SPS was prepared with an SPS-specific degrading enzyme from a sheath-degrading bacterium, Paenibacillus koleovorans. The oligomer was derivatized and subjected to fast atom bombardment-mass spectrometry to investigate the monosaccharide sequence of SPS. The structure of SPS was confirmed by nuclear magnetic resonance. The resulting data showed that SPS is a straight-chained basic polysaccharide constructed of a pentasaccharide repeating unit.