Structural determination of the sheath-forming polysaccharide of Sphaerotilus montanus using thiopeptidoglycan lyase which recognizes the 1,4 linkage between α-d-GalN and ß-d-GlcA.

Sphaerotilus natans is a filamentous sheath-forming bacterium commonly found in activated sludge. Its sheath is assembled from a thiolic glycoconjugate called thiopeptidoglycan. S. montanus ATCC-BAA-2725 is a sheath-forming member of stream biofilms, and its sheath is morphologically similar to that of S. natans. However, it exhibits heat susceptibility, which distinguishes it from the S. natans sheath. In this study, chemical composition and solid-state NMR analyses suggest that the S. montanus sheath is free of cysteine, indicating that disulfide linkage is not mandatory for sheath formation. The S. montanus sheath was successfully solubilized by N-acetylation, allowing solution-state NMR analysis to determine the sugar sequence. The sheath was susceptible to thiopeptidoglycan lyase prepared from the thiopeptidoglycan-assimilating bacterium, Paenibacillus koleovorans. The reducing ends of the enzymatic digests were labeled with 4-aminobenzoic acid ethyl ester, followed by HPLC. Two derivatives were detected, and their structures were determined. We found that the sheath has no peptides and is assembled as follows: [→4)-ß-d-GlcA-(1→4)-ß-d-Glc-(1→3)-ß-d-GalNAc-(1→4)-α-d-GalNAc-(1→4)-α-d-GalN-(1→]n (ß-d-Glc and α-d-GalNAc are stoichiometrically and substoichiometrically 3-O-acetylated, respectively). Thiopeptidoglycan lyase was thus confirmed to cleave the 1,4 linkage between α-d-GalN and ß-d-GlcA, regardless of the peptide moiety. Furthermore, vital fluorescent staining of the sheath demonstrated that elongation takes place at the tips, as with the S. natans sheath.

Uranium(VI) Scavenging by Amorphous Iron Phosphate Encrusting Sphaerotilus natans Filaments.

U(VI) sorption to iron oxyhydroxides, precipitation of phosphate minerals, as well as biosorption on bacterial biomass are among the most reported processes able to scavenge U(VI) under oxidizing conditions. Although phosphates significantly influence bacterially mediated as well as iron oxyhydroxide mediated scavenging of uranium, the sorption or coprecipitation of U(VI) with poorly crystalline nanosized iron phosphates has been scarcely documented, especially in the presence of microorganisms. Here we show that dissolved U(VI) can be bound to amorphous iron phosphate during their deposition on Sphaerotilus natans filamentous bacteria. Uranium LIII-edge EXAFS analysis reveals that the adsorbed uranyl ions share an equatorial oxygen atom with a phosphate tetrahedron of the amorphous iron phosphate, with a characteristic U-P distance of 3.6 Å. In addition, the uranyl ions are connected to FeO6 octahedra with U-Fe distances at ~3.4 Å and at ~4.0 Å. The shortest U-Fe distance corresponds to a bidentate edge-sharing complex often reported for uranyl adsorption onto iron oxyhydroxides, whereas the longest U-Fe and U-P distances can be interpreted as a bidentate corner-sharing complex, in which two adjacent equatorial oxygen atoms are shared with the vertices of a FeO6 octahedron and of a phosphate tetrahedron. Furthermore, based on these sorption reactions, we demonstrate the ability of an attached S. natans biofilm to remove uranium from solution without any filtration step.

Investigation of iron-containing products from natural and laboratory cultivated Sphaerotilus-Leptothrix bacteria.

Bacterial biomass collected from sheath-forming bacteria of the genera Sphaerotilus and Leptothrix was collected from a high-mountain natural stream water source. The elemental constitution and oxide phases of the products after selective cultivation of the bacteria on two different elective media using neutron activation analysis (NAA), electron microscopy (SEM, TEM), and X-ray diffraction (XRD) were studied. A high enrichment level of iron was revealed by the NAA technique in cultivated isolates as compared to the reference sample from nature. Three types of iron oxide compounds were established after cultivation in Adler's medium: lepidocrocite (γ-FeOOH), magnetite (Fe3O4), and goethite (α-FeOOH). The cultivation in the Isolation medium yielded a single phase, that of goethite, excluding one sample with a distinguishable amount of lepidocrocite. XRD and EM investigations show that the biogenic oxides are nanosized. Our study exemplifies the possibilities of the biotechnology approach for obtaining, under artificial conditions, large quantities of iron-containing by-products that could be of further used in appropriate nano- and biotechnologies.

Conformational analysis of an extracellular polysaccharide produced by Sphaerotilus natans.

Sphaerotilus natans is a filamentous sheath-forming bacterium, commonly found in bulking activated sludge. The bulky nature of this bacterium is caused by an extracellular polysaccharide (EPS). EPS is a linear acidic polysaccharide with the following chemical structure: [ → 4)-α-D-Glcp-(1 → 2)-ß-D-GlcpA-(1 → 2)-α-L-Rhap-(1 → 3)-ß-L-Rhap-(1 → ](n). (1)H-(1)H and (1)H-(13)C correlation nuclear magnetic resonance (NMR) experiments were performed to acquire nuclear Overhauser effect signals, which were used for conformational elucidation. Molecular mechanics calculations were performed on each disaccharide unit of the EPS building blocks. On the basis of the results of the calculations, the conformation of a pentasaccharide fragment was estimated. After confirmation of the coincidence between the NMR data and the predicted conformation of the pentasaccharide fragment, the conformation of a heptadecasaccharide fragment was estimated using the same procedure. The heptadecasaccharide was found to form a (12/1) helix and take locally folded chain, which is attributed to a triangular arrangement formed by a series of residues, α-D-Glcp-(1 → 2)-ß-D-GlcpA-(1 → 2)-α-L-Rhap. This arrangement is caused by the peculiar consecutive (1 → 2) linkages and reinforced by a hydrogen bond between the α-glucosyl and α-rhamnosyl residues. Considering the steric hindrance due to this triangular arrangement, EPS molecules are not supposed to form double helix in an aqueous environment. We propose the name 'sphaeran' to refer to this unique chemical structure and properties of EPS.

[Obtaining homogenous preparations of succinate dehydrogenase isoforms from the D-507 strain of Sphaerotilus natans].

Enzymatic preparations of two isoforms of succinate dehydrogenase (SDG) with specific activity of 22.00 E/mg of protein were obtained from the colorless sulfur bacterium Sphaerotilus natans D-507 cultured organotrophically. Both SDG forms were shown to be heteromers with subunit molecular masses of 70.8, 35.0, 31.8, and 16.2 kDa. The K(m) values for the first and the second forms of SDG were evaluated as 0.615 and 0.531 mM, respectively, with an optimal pH value of 7.2. It was found that the Cl- ion has an activating effect on the SDG activity that can be explained by the specific chemical modification of the enzyme molecule. The results suggest that the isolated enzyme forms are included in different multienzyme complexes, which provide the functioning of the tricarboxylic acid cycle, and SDG preparations can be used for the investigation of other enzyme systems or in vitro modeling of supramolecular cellular structures.

Study of a novel glycoconjugate, thiopeptidoglycan, and a novel polysaccharide lyase, thiopeptidoglycan lyase.

A typical filamentous bacterium, Sphaerotilus natans, secretes a thiolic glycoconjugate which is assembled into a microtube, so called sheath. The glycoconjugate is known to consist of a pentasaccharide-dipeptide repeating unit, but its chemical structure has not been completely elucidated. In order to determine its chemical structure, the sheath was broken down by performic acid oxidation. The released sulfonated derivative was water soluble which was suitable for detailed NMR analysis. The data exhibited the presence of two stoichiometric and one substoichiometric (relative abundance was about 0.5) acetylations, suggesting that the glycoconjugate is composed of two equimolar pentasaccharide-dipeptide repeating units each having either two or three acetyl groups. However, the position of substoichiometric acetylation could not be defined. To determine the position, the sheath was derivatized with a thiol selective fluorescent reagent followed by digestion with a specific polysaccharide lyase prepared from a sheath-degrading bacterium, Paenibacillus koleovorans. As expected, two fluorescent digests were recovered by reverse-phase HPLC and were subjected to NMR analysis. The data revealed that both digests are pentasaccharide-dipeptides which have unsaturated glucuronic acid and galactosamine residues at their reducing and non-reducing ends, respectively. It was also confirmed that one digest has 3-O-acetylated glucose residue while the other has non-derivatized glucose residue. The substoichiometric acetylation was thus identified with the 3-O-acetylation, and structural determination of the thiolic glycoconjugate was completed. By virtue of the clarification of the two digests' structures, the cleavage site was specified as (1→4)-α-galactosaminic bond to glucuronic acid. Based on the present and earlier findings, we propose a novel glycoconjugate category named thiopeptidoglycan and a novel polysaccharide lyase named thiopeptidoglycan lyase.

Ionic strength effect on copper biosorption by Sphaerotilus natans: equilibrium study and dynamic modelling in membrane reactor.

Biosorption of copper by Sphaerotilus natans in different conditions of ionic strength and pH was studied by performing sorption tests in batch and membrane reactors. Equilibrium batch tests evidenced the negative effect of ionic strength and the positive effect of pH on biosorption performances: the highest determined value for copper specific uptake, q, was about 60 mg/g at pH 6 and about 15 mg/g at pH 4. A competitive equilibrium model was successfully fitted to experimental data at different ionic strength levels to account for copper-sodium competition. In membrane reactor tests, experimental profiles of copper concentration in the permeate vs. time did not evidence a significant effect of ionic strength at low pH values (4 and 5). On the other hand a more remarkable effect of ionic strength on copper concentration in the permeate was observed at pH 6. Experimental profiles of continuous biosorption in the membrane reactors were successfully simulated by developing a dynamic model accounting for Cu-Na competition and for binding ability of cells fragments.

Biosorption of binary heavy metal systems onto Sphaerotilus natans cells confined in an UF/MF membrane reactor: dynamic simulations by different Langmuir-type competitive models.

Continuous heavy metal biosorption in membrane reactor apparatus using binary metal solutions was here considered. A dynamic model was developed to simulate biosorption performances on the base of metal mass balances in the system considering biosorption as an equilibrium process. The effect of three Langmuir-type competitive models on dynamic simulations of biosorption was then studied by using predictive equilibrium models (whose adjustable parameters were determined only by single metal system biosorption data) and not predictive equilibrium models (adjustable parameters directly by binary biosorption data). Predictive competitive models can give simulation profiles that are different from those obtained using non-predictive models. This detachment is due to the non-ideal competition among metals in solution which cannot be predicted only on the base of biosorption data in single metal systems. The dynamic model for multi-component biosorption here proposed was compared with experimental results reported in the literature and obtained using a biomass in a similar membrane reactor apparatus with ternary metal systems. The simulated profiles (obtained by using predictive equilibrium models) can reproduce qualitatively the specific adsorbent selectivity and the overshoot regions in the permeate concentrations of the metals with the minor affinity.

A novel gene encoding an enzyme that degrades a polysaccharide from the sheath of Sphaerotilus natans.

A gene encoding an enzyme that is able to depolymerize the basic polysaccharide prepared from the sheath of Sphaerotilus natans was identified in a sheath-degrading bacterium, Paenibacillus koleovorans. The gene was constructed from 2217 bp coding for 738 amino acids, including the signal sequence of 34 amino acids. No closely related protein or gene was indicated by a homology search. The gene was expressed in Escherichia coli as a glutathione S-transferase fusion protein. The fusion protein depolymerized the sheath polysaccharide into an oligosaccharide, introducing an unsaturated sugar residue, suggesting that the gene codes for a polysaccharide lyase acting on a basic polysaccharide.