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At least 19 recordsLinked to original sources
PubMed2005

Substrate uptake tests and quantitative FISH show differences in kinetic growth of bulking and non-bulking activated sludge.

The competition between filaments and floc formers in activated sludge has been historically described using kinetic selection. However, recent studies have suggested that bacterial storage may also be an important factor in microbial selection, since the dynamic nature of substrate flows into wastewater treatment plants elicit transient responses from microorganisms. Respirometry-based kinetic selection should thus be reevaluated by considering cell storage, and a more reliable method should be developed to include bacterial storage in the analysis of growth of filaments and floc formers in activated sludge. In this study, we applied substrate uptake tests combined with metabolic modeling to determine the growth rates, yields and maintenance coefficients of bulking and non-bulking activated sludge developed in lab scale reactors under feast and famine conditions. The results of quantitative fluorescence in situ hybridization (FISH) showed that the filaments Eikelboom Type 1851, Type 021N, and Thiothrix nivea were dominant in bulking sludge, comprising 42.0 % of mixed liquor volatile suspended solids (MLVSS), with 61.6% of the total filament length extending from flocs into bulk solution. Only low levels of Type 1851 filament length (4.9% of MLVSS) occurred in non-bulking sludge, 83.0% of which grew inside the flocs. The kinetic parameters determined from the substrate uptake tests were consistent with those from respirometry and showed that filamentous bulking sludge had lower growth rates and maintenance coefficients than non-bulking sludge. These results provide support for growth kinetic differences in explaining the competitive strategy of filamentous bacteria.

Bacteria↗
PubMed1988

The Vitreoscilla hemoglobin gene: molecular cloning, nucleotide sequence and genetic expression in Escherichia coli.

Vitreoscilla hemoglobin is involved in oxygen metabolism of this bacterium, possibly in an unusual role for a microbe. We have isolated the Vitreoscilla hemoglobin structural gene from a pUC19 genomic library using mixed oligodeoxy-nucleotide probes based on the reported amino acid sequence of the protein. The gene is expressed in Escherichia coli from its natural promoter as a major cellular protein. The nucleotide sequence, which is in complete agreement with the known amino acid sequence of the protein, suggests the existence of promoter and ribosome binding sites with a high degree of homology to consensus E. coli upstream sequences. In the case of at least some amino acids, a codon usage bias can be detected which is different from the biased codon usage pattern in E. coli. The downstream sequence exhibits homology with the 3' end sequences of several plant leghemoglobin genes. E. coli cells expressing the gene contain greater than fivefold more heme than controls.

Amino Acid Sequence↗
PubMed1981

Mixotrophic and heterotrophic growth of Beggiatoa alba in continuous culture.

Beggiatoa alba strain B18LD was grown in continuous culture under heterotrophic conditions on acetate or acetate and asparagine and under mixotrophic conditions on acetate plus either 1 mM sodium sulfide or 1 mM sodium thiosulfate. Considerable differences were observed between the yields and the cell compositions of heterotrophic and mixotrophic cultures at all dilution rates tested. The dry weight yield per gram acetate utilized was approximately three times higher in the acetate-sulfide mixotrophic culture than in the acetate heterotrophic culture, whereas the poly-beta-hydroxybutyric acid and carbohydrate contents were much higher in the heterotrophic cultures. The high yields (0.52--0.75, corrected for the weight of the sulfur) obtained with the mixotrophic cultures imply that the acetate was utilized mainly for biosynthesis. Thus, the oxidation of sulfide supplied energy. The addition of catalase to the chemostat cultures increased yields slightly, but it was insufficient to explain the differences between the heterotrophic and the mixotrophic cultures.

Bacterial Proteins↗
PubMed1987

Identification of b, c, and d cytochromes in the membrane of Vitreoscilla.

Cytochromes b, c, d, and o were identified by spectroscopic analysis of respiratory membrane fragments from Vitreoscilla sp., strain C1. Carbon monoxide difference spectra of the reduced membranes had absorption maxima at 416, 534, and 571 nm (ascribed to cytochrome o) and 632 nm (cytochrome d). Derivative spectra of the pyridine hemochromogen spectra of the membranes identified the presence of b- and c-type cytochromes in Vitreoscilla. The cyanide binding curve of the membranes was biphasic with dissociation constants of 2.14 mM and 10.7 which were assigned to cytochrome o and cytochrome d, respectively. Membranes bound carbon monoxide with dissociation constant 3.9 microM, which was assigned to cytochrome o. Cytochrome c556 and a NADH-p-iodonitrotetrazolium violet reductase component were partially purified from Vitreoscilla membranes.

Carbon Monoxide↗
PubMed1994

Sequence of the region downstream of the Vitreoscilla hemoglobin gene: vgb is not part of a multigene operon.

The 1668 base pairs (bp) downstream of the Vitreoscilla hemoglobin gene were sequenced in the hope of finding related genes that might be part of an operon. Instead, a sequence was found that constituted an open reading frame (ORF) of 569 amino acids (apparently the carboxy-terminal part of a larger ORF), in the direction opposite to the hemoglobin gene. This sequence was found to have 64% similarity with the 1685 bp at the 3' end of the Escherichia coli uvrA gene. The inferred amino acid sequence of the Vitreoscilla DNA has 69% similarity with the corresponding sequence of the E. coli uvrA protein, with similarities of 90, 100, and 85% in the helix-turn-helix, C-terminal ATP binding, and C-terminal zinc finger domains, respectively. The distance between the 3' ends of the Vitreoscilla hemoglobin and uvrA genes is 63 bp.

Adenosine Triphosphatases↗
PubMed1991

Studies on the bacterial hemoglobin from Vitreoscilla. Redox properties and spectroscopic characterization of the different forms of the hemoprotein.

Vitreoscilla contained a homodimeric bacterial hemoglobin (VtHb). The purification of this protein yielded VtmetHb which exhibited electronic and electron paramagnetic resonance (EPR) spectra, showing that it existed predominantly in a high-spin ferric form, both axial and rhombic components being present. The preparations also contained variable amounts of low-spin components. There was no evidence that these high-spin and low-spin forms were in equilibrium. The former were reducible by NADH catalyzed by the NADH-metVtHb reductase, and the latter were not. High ionic strength and high pH led to the formation of low-spin metVtHb; both treatments were reversible. Cyanide and imidazole liganded to VtHb resulted in the conversion of high-spin to low-spin ferric heme centers, each with characteristic electronic and EPR spectra. Some preparations of VtHb exhibited EPR signals consistent with a sulfur ligand bound to the ferric site. When VtHb was treated with NADH plus the reductase in the presence of oxygen, the intensity of the high-spin EPR signals decreased significantly. No reduction occurred in the absence of oxygen, suggesting a possible role for the superoxide anion. Dithionite treatment of VtHb resulted in a slow reduction, but the main product of the reaction of dithionite-reduced VtHb with oxygen was VtmetHb, not VtHbO2. EPR spectra of whole cells of Vitreoscilla exhibited a variety of intense signals at low and high magnetic field, the g-values being consistent with the presence of high-spin ferric heme proteins, in addition to an iron-containing superoxide dismutase (FeSOD) and iron-sulfur proteins. EPR spectra of the cytosol fraction of Vitreoscilla showed the expected resonances for VtmetHb and FeSOD.

Bacterial Proteins↗
PubMed2006

Community structure of bacteria associated with sheaths of freshwater and brackish thioploca species.

Bacterial communities associated with sheaths of Thioploca spp. from two freshwater lakes (Lake Biwa, Japan, and Lake Constance, Germany) and one brackish lake (Lake Ogawara, Japan) were analyzed with denaturing gradient gel electrophoresis (DGGE) of 16S rRNA gene fragments. The comparison between the DGGE band patterns of bulk sediment and Thioploca filaments of Lake Biwa suggested the presence of specific bacterial communities associated with Thioploca sheaths. As members of sheath-associated communities, bacteria belonging to Bacteroidetes were detected from the samples of both freshwater lakes. A DGGE band from Thioploca of Lake Biwa, belonging to candidate division OP8, was quite closely related to another DGGE band detected from that of Lake Constance. In contrast to the case of freshwater lakes, no bacterium of Bacteroidetes or OP8 was detected from Thioploca of Lake Ogawara. However, two DGGE bands from Lake Ogawara, belonging to Chloroflexi, were quite closely related to a DGGE band from Lake Constance. Two DGGE bands obtained from Lake Biwa were closely related to phylogenetically distant dissimilatory Fe(III)-reducing bacteria. Cloning analyses for a dissimilatory sulfite reductase gene were performed on the same samples used for DGGE analysis. The results of the analyses suggest that sheaths of freshwater/brackish Thioploca have little ecological significance for the majority of sulfate reducers.

Animals↗
PubMed2003

Effect of dissolved oxygen concentration on sludge settleability.

This laboratory study presents a detailed evaluation of the effects of dissolved oxygen concentration and accumulation of storage polymers on sludge settleability in activated sludge systems with an aerobic selector. The oxygen and substrate availability regime were simulated in laboratory sequencing batch reactor systems. The experiments showed that low dissolved oxygen concentration (< or =1.1 mg O2 l(-1)) had a strong negative effect on sludge settleability, leading to the proliferation of filamentous bacteria (Thiothrix spp., Type 021N and Type 1851). This negative effect was stronger at high chemical oxygen demand loading rate. This indicates that a compartmentalised (plug flow) aerobic contact tank, designed at short hydraulic residence time to guarantee a strong substrate gradient, with low dissolved oxygen concentration, might be worse for sludge settleability than an "overdesigned" completely mixed contact tank. Contrary to the general hypothesis, the maximum specific acetate uptake rate, poly-beta-hydroxybutyrate production rate, and resistance to short starvation periods are similar in both poor- and well-settling sludge. The results of this study support our previous hypothesis on the importance of substrate gradients for the development of filamentous structures in biological flocs, from soluble organic substrate gradients to dissolved oxygen gradients in sludge flocs.

Acetates↗
PubMed1986

Cyanide- and carbon monoxide-resistant mutants of Vitreoscilla: altered cytochromes and respiratory properties.

Two respiratory mutants of the aerobic bacterium, Vitreoscilla, have been studied: a CO-resistant mutant that can grow in 50% CO-50% oxygen, and a cyanide-resistant mutant that can grow in 1 mM KCN. Wild-type cells are unable to grow under either condition. This report presents evidence that the resistance of the CO mutant is due to an altered membrane-bound cytochrome o [cytochrome o(m)], and that of the cyanide mutant is due to the presence of an increased amount of cytochrome d, which has a lower affinity for cyanide than cytochrome o(m). The evidence was obtained from spectral studies on the three types of intact cells as well as enzymatic and ligand-binding techniques on the cytoplasmic cytochromes o[cytochrome o(s)] and the respiring membrane vesicles isolated from these cells. Carbon monoxide difference spectra of intact cells revealed a 5-nm shift in an absorption maximum of a CO-binding pigment in the CO mutant relative to that of the wild type. The formation of oxygenated cytochrome o(s) and its conversion to the reduced form when the cells became anaerobic due to cellular respiration were inhibited when 1 mM KCN was added to a cell suspension of wild-type cells; the cyanide mutant cells showed resistance to cyanide in this experiment. Cytochrome o(s) purified from all three cell types had identical physical, electron transferring, and ligand binding properties within experimental error. Respiring membrane vesicles isolated from the two mutants showed more resistance to inhibition by cyanide and carbon monoxide than those from the wild type. Carbon monoxide difference spectra of these membrane vesicles revealed that there was a fivefold increase in the amount of cytochrome d in the cyanide mutant relative to the wild type. A CO absorption band of the membrane-bound cytochrome o in the CO mutant membrane vesicles showed a 5-nm shift relative to that of the wild type.

Carbon Monoxide↗
PubMed1992

NADH-dependent methemoglobin reductase from the obligate aerobe Vitreoscilla: improved method of purification and reexamination of prosthetic groups.

The NADH-dependent methemoglobin reductase from the bacterium Vitreoscilla was purified using hydrophobic chromatography on a phenyl-Sepharose column. The new procedure resulted in a purer protein and increased the overall yield of the enzyme by a factor of approximately three. The active site of the enzyme was investigated by ultraviolet/visible, fluorescence, Mössbauer, and electron paramagnetic resonance spectroscopy (EPR) at 9.4 GHz. Prosthetic group analysis revealed the presence of one FAD per active enzyme molecule but no iron in contrast to earlier reports. The NADH-methemoglobin reductase activity of the pure enzyme was in the range of 1.1-1.25 units; its electronic and fluorescence spectra were typical of metal-free flavoproteins. No EPR signals were detected between 5 and 150 K over a field range 0.05-0.5 T, and there was no Mössbauer signal, consistent with the absence of iron. Methemoglobin reductase from Vitreoscilla was reduced by dithionite, NADH, and deazaflavin/EDTA upon illumination. The main species observed during these anaerobic oxidation-reduction experiments was the blue semiquinone radical with an EPR signal at g = 2.005, linewidth 1.5 mT. The fully reduced state of the enzyme, FlredH3, was also observed in the reaction with NADH. The reduction was fully reversible with ferricyanide. The observations reported here are consistent with a redox enzyme interacting both with a two-electron donating agent such as NADH and a one-electron accepting center such as the Fe(III)/Fe(II) couple of Vitreoscilla hemoglobin.

Aerobiosis↗
PubMed1992

Sodium-coupled ATP synthesis in the bacterium Vitreoscilla.

The bacterium Vitreoscilla generates an electrical potential gradient due to sodium ion (delta psi Na+) across its membrane via respiratory-driven primary Na+ pump(s). The role of the delta psi Na+ as a driving force for ATP synthesis was, therefore, investigated. In respiring starved cells pulsed with 100 mM external Na+ [( Na+]o) there was a 167% net increase in cellular ATP concentration over basal levels compared with 0, 56, 78, and 78% for no addition, choline, Li+, and K+ controls, respectively. Doubling the [Na+]o to 200 mM boosted the net increase to 244% but a similar doubling of the choline caused only an increase to 78%. When the initial condition was intracellular Na+ ([Na+]i) = [Na+]o = 100 mM, there was a 94% net increase in cellular ATP compared with only 18 and 11% for Li+ and K+ controls, respectively, indicating that Nai+ may be the only cation tested that the cells extruded to generate the electrochemical gradient required to drive ATP synthesis. The Na(+)-dependent ATP synthesis was inhibited completely by monensin (12 microM), but only transiently by the protonophore 3,5-di-tert-butyl-4-hydroxybenzaldehyde (100 microM), further evidence that the Na+ gradient and not a H+ gradient was driving the ATP synthesis. ATP synthesis in response to an artificially imposed H+ gradient (delta pH approximately 3) in the absence of an added cation, or in the presence of Li+, K+, or choline, yielded similar delta ATP/delta pH ratios of 0.98-1.22. In the presence of Na+, however, this ratio dropped to 0.23, indicating that Na+ inhibited H(+)-coupling to ATP synthesis and possibly that H+ and Na+ coupling to ATP synthesis share a common catalyst. The above evidence adds to previous findings that under normal growth conditions Na+ is probably the main coupling cation for ATP synthesis in Vitreoscilla.

Adenosine Triphosphatases↗
PubMed1992

The bacterial hemoglobin from Vitreoscilla can support the aerobic growth of Escherichia coli lacking terminal oxidases.

Two Escherichia coli mutants that lack both cytochrome o and d terminal oxidases are able to grow with glucose as the carbon source but not with the aerobic substrates succinate or lactate. One of these, GV101, is a deletion mutant of cytochrome o and a point mutation of cytochrome d. The other, GK100, is a total deletion mutant of all the genes for both cytochromes. When these mutants were transformed with a plasmid containing the gene for the bacterial hemoglobin from Vitreoscilla, they were capable of growth in the presence of succinate or lactate and showed aerobic respiration in the presence of these substrates, unlike the parent strains. Cells transformed with a plasmid containing the gene for the hemoglobin but lacking the native promoter did not express the hemoglobin and did not respire. Membrane vesicles prepared from the cells consumed oxygen in the presence of succinate. This succinate-supported respiration decreased with successive washings of the vesicles but was restored by adding E. coli cytosol containing the hemoglobin or by adding the hemoglobin purified from Vitreoscilla. This respiration was inhibited by cyanide.

Aerobiosis↗
PubMed1989

Evidence for partial export of Vitreoscilla hemoglobin into the periplasmic space in Escherichia coli. Implications for protein function.

The Vitreoscilla hemoglobin protein has been implicated in earlier studies to serve a globin-like function under oxygen-limited growth conditions. Evidence is presented using fractionation as well as proteinase K accessibility techniques to prove that a considerable amount of this protein is localized in the periplasmic space of the cell. Genetic evidence points towards the existence of information within the N-terminal domain of the protein that plays a role in the process of protein export. However, this sequence is not cleaved in the process of translocation. Analysis of the primary structure of this region reveals several unusual features. Instead of positively charged residues at its amino terminus, it has a negative charge. The overall hydrophobicity of the central region of this sequence is significantly lower than in typical leader peptides due to the presence of a charged residue. In keeping with the likelihood that such an export signal may not be very efficient, a substantial fraction of the total cellular hemoglobin can also be detected in the cytoplasm. Heme is incorporated in both cytoplasmic and periplasmic globin as indicated by the ability of protein from both fractions to bind carbon monoxide. The secretion of this protein into the periplasm raises questions concerning the physiological significance of its localization. Dimensional analysis of a model based on the facilitated diffusion hypothesis, which was initially proposed to account for the effects of eukaryotic globins on oxygen transport, suggests that periplasmic globin can support an additional oxygen flux to the respiratory apparatus that may be physiologically significant.

Amino Acid Sequence↗
PubMed1990

Presence of the bacterial hemoglobin gene improves alpha-amylase production of a recombinant Escherichia coli strain.

A recombinant plasmid (pMK57) was constructed by cloning the Bacillus stearothermophilus alpha-amylase gene into pUC8; plasmid pMK79 was then derived from pMK57 by inserting the bacterial (Vitreoscilla) hemoglobin gene into the latter plasmid. Both pMK57 and pMK79 were transformed into Escherichia coli strain JM 103 to make strains MK57 and MK79, respectively. Both MK57 and MK79 produced alpha-amylase and MK79 produced hemoglobin. MK79 outgrew MK57 in shake flasks in LB medium, the advantage of the former appearing in late log phase. MK79 produced more alpha-amylase than MK57, on both per cell and per volume bases, in both mid and late log phases; the maximum advantage of MK79 (on a per volume basis) occurred in late log phase, at which time it produced 3.3 times as much alpha-amylase as MK57. The numbers of copies per cell of both pMK57 and pMK79 were significantly lower than that of pUC8.

Cloning, Molecular↗
PubMed1988

Cloning, characterization and expression of the bacterial globin gene from Vitreoscilla in Escherichia coli.

The genomic locus responsible for production of the globin portion of Vitreoscilla hemoglobin (VtHb), the only well-characterized bacterial hemoglobin (Hb), has been cloned and expressed in Escherichia coli. A 17-mer oligodeoxynucleotide, corresponding to a region of the VtHb amino acid sequence was used as a hybridization probe to screen a Vitreoscilla genomic library constructed in broad-host-range cosmid vector pVK102. E. coli, carrying recombinant pVK102:H5 which contained a 16.5-kb insert of Vitreoscilla genomic DNA, produced three to four times more Hb than Vitreoscilla. Restriction mapping and subcloning revealed that the globin-coding gene (vgb) was completely localized on a 1.4-kb HindIII-SalI fragment of the 16.5-kb insert. Production of VtHb still occurred when this 1.4-kb fragment was cloned in plasmids pUC8 and pUC9 in opposite orientations, suggesting the presence of a Vitreoscilla promoter on this fragment. A single copy of this gene on the chromosome was indicated by Southern-blot analysis, and a 450-500-nt RNA transcript specific for the globin gene was detected after Northern hybridization. A partially purified Hb preparation from E. coli harboring the recombinant plasmid had identical spectral properties and subunit molecular size as authentic VtHb. The Hb in respiring cells of E. coli was in the physiologically functional oxyHb form.

Blotting, Northern↗
PubMed2003

Phylogenetic analysis of Beggiatoa spp. from organic rich sediment of Tokyo Bay, Japan.

Nitrate-accumulating filamentous bacteria from organic rich sediment of Tokyo Bay, morphologically similar to Beggiatoa species, were phylogenetically analyzed. From several sites in Tokyo Bay, Beggiatoa-like bacteria were collected. Each sample contained narrower or wider type (10 and 30 microns, respectively) of trichomes. With the nested PCR using specific primers for Beggiatoa, fragments of 16S rRNA gene were amplified and then subjected to denaturing gradient gel electrophoresis (DGGE) analysis. Sequencing and the following phylogenetic analysis indicated that they are related to large Beggiatoa species. The wider type was related to uncultured Beggiatoa clones of other geographical localities and distinct from the narrower type in Tokyo Bay. Among the narrower types, a sample from a tidal flat was genetically distinct from the sample from sites of 10 and 20 m water depth. These narrower types form a new branch in Beggiatoa/Thioploca cluster. The result of phylogenetic analysis was in accordance with the previous studies that indicate possession of nitrate-accumulation capability is congruent with phylogeny based on 16S rRNA sequences.

Environmental Monitoring↗
PubMed1990

Respiratory-driven Na+ electrical potential in the bacterium Vitreoscilla.

Vitreoscilla is a Gram-negative bacterium with unique respiratory physiology in which Na+ was implicated as a coupling cation for the generation of a transmembrane electrical gradient (delta psi). Thus, cells respiring in the presence of 110 mM Na+ generated a delta psi of -142 mV compared to only -42 and -56 mV for Li+ and choline, respectively, and even the -42 and -56 mV were insensitive to the protonophore 3,5-di-tert-butyl-4-hydroxybenzaldehyde (DTHB). The kinetics of delta psi formation and collapse correlated well with the kinetics of Na+ fluxes but not with those of H+ fluxes. Cyanide inhibited respiration, Na+ extrusion, and delta psi formation 81% or more, indicating that delta psi formation and Na+ extrusion were coupled to respiration. Experiments were performed to distinguish among three possible transport systems for this coupling: (1) a Na(+)-transporting ATPase; (2) an electrogenic Na+/H+ antiport system; (3) a primary Na+ pump directly driven by the free energy of electron transport. DCCD and arsenate decreased cellular ATP up to 86% but had no effect on delta psi, evidence against a Na(+)-transporting ATPase. Low concentrations of DTHB had no effect on delta psi; high concentrations transiently collapsed delta psi, but led to a stimulation of Na+ extrusion, the opposite of that expected for a Na+/H+ antiport system. Potassium ion, which collapses delta psi, also stimulated Na+ extrusion. The experimental evidence is against Na+ extrusion by mechanisms 1 and 2 and supports the existence of a respiratory-driven primary Na+ pump for generating delta psi in Vitreoscilla.

Arsenates↗
PubMed2003

Purification and physicochemical properties of malate dehydrogenase from bacteria of the genus Beggiatoa.

Homogeneous malate dehydrogenase (MDH) with a specific activity of 20-24 units per mg protein was purified from the sulfur bacterium Beggiatoa leptomitiformis strain D-402 grown organotrophically and lithotrophically and from the organotrophic bacterium Beggiatoa alba. MDHs from the B. leptomitiformis strain D-402 grown under organotrophic conditions and from B. alba are homodimers with the subunit molecular weight of 40 kD. Tetrameric MDH is formed in B. leptomitiformis strain D-402 grown under lithotrophic conditions. The dimeric and tetrameric forms of MDH from B. leptomitiformis D-402 display some differences in kinetic properties.

Chromatography, Gel↗