Sulfur metabolism in Beggiatoa alba

The metabolism of sulfide, sulfur, and acetate by Beggiatoa alba was investigated under oxic and anoxic conditions. B. alba oxidized acetate to carbon dioxide with the stoichiometric reduction of oxygen to water. In vivo acetate oxidation was suppressed by sulfide and by several classic respiratory inhibitors, including dibromothymoquinone, an inhibitor specific for ubiquinones. B. alba also carried out an oxygen-dependent conversion of sulfide to sulfur, a reaction that was inhibited by several electron transport inhibitors but not by dibromothymoquinone, indicating that the electrons released from sulfide oxidation were shuttled to oxygen without the involvement of ubiquinones. Intracellular sulfur stored by B. alba was not oxidized to sulfate or converted to an external soluble form under aerobic conditions. On the other hand, sulfur stored by filaments of Thiothrix nivea was oxidized to extracellular soluble oxidation products, including sulfate. Sulfur stored by filaments of B. alba, however, was reduced to sulfide under short-term anoxic conditions. This anaerobic reduction of sulfur was linked to the endogenous oxidation of stored carbon and to hydrogen oxidation.     

Methylotrophy in Freshwater Beggiatoa alba Strains

Two freshwater strains of the gammaproteobacterium Beggiatoa alba, B18LD and OH75-2a, are able to use methanol as a sole carbon and energy source under microoxic conditions. Genes encoding a methanol dehydrogenase large-subunit homolog and four enzymes of the tetrahydromethanopterin-dependent C₁ oxidation pathway were identified in B18LD. No evidence of methanotrophy was detected.     

Spectral characterization of c-type cytochromes purified from Beggiatoa alba

Three c-type cytochromes were purified from the filamentous sulfur-oxidizing bacterium, Beggiatoa alba strain B18LD, by ammonium sulfate fractionation, flat bed isoelectric focusing and gel filtration. Two of the cytochromes; flavocytochrome c-554 and cytochrome c, were similar to cytochromes found in anoxygenic photosynthetic bacteria. Flavocytochrome c-554 had an apparent molecular weight of 21,000, an isoelectric focusing point at pH 4.4, contained FMN as the flavin component and had absorption maxima at 410, 450 and 470 nm in the oxidized form and at 417, 523 and 554 nm in the dithionite-reduced from. Cytochrome c was also an acidic protein with a pI of 4.8 and an apparent molecular weight of 18,000. The absorption spectra maxima were at 400, 490 and 635 nm in the oxidized form, at 424 and 550 nm in the dithione-reduced form and at 415 and 555 nm in the dithionite-reduced plus CO form. The third cytochrome characterized, cytochrome c-553 had an apparent molecular weight of 13,000, an isoelectric point at pH 4.4 and showed absorption maxima at 411 nm in the oxidized form and at 418, 523 and 553 nm in the dithionite-reduced form. Cytochrome c-553 was also isolated as a complex with a non-heme protein with a molecular weight of 16,000. The non-heme protein altered the absorption spectra and isoelectric point of cytochrome c-553.Abbreviations IEF isoelectric focusing - M _r molecular weight - pI isoelectric point     

Properties of in vivo nitrogenase activity in Beggiatoa alba

A procedure was devised for analyzing in vivo nitrogenase activity in Beggiatoa alba B18LD which involves: (1) the induction of nitrogenase in cells pre-grown on NH₄Cl, by washing the cells free of NH₄Cl and lowering their exposure to oxygen, and (2) measuring acetylene reduction by these cells. Using this induction methodology we examined the effects of pH, temperature, and nitrogenous compounds on in vivo nitrogenase induction and activity in Beggiatoa alba B18LD. Nitrate and nitrite repressed the induction of nitrogenase activity, but glutamine did not. Induction and activity had a combined pH optimum of 6.5 to 8.0, and activity had a temperature optimum of 29°C. Ammonium and urea caused immediate inhibition of nitrogenase activity, but nitrate, nitrite, glutamine, asparagine, and other amino acids did not. Ammonium-induced inhibition was transient and incomplete, and the duration of inhibition increased in direct proportion to the amount of ammonium added. Methionine sulfoximine, a glutamine synthetase inhibitor, at a final concentration of 50 μM blocked ammonium uptake by cells, but did not prevent nitrogenase inhibition if added before ammonium. Our results imply that B. alba nitrogenase inhibition by ammonium: (1) is not directly caused by ammonium assimilation products, (2) is probably not due to an enzymatic inactivation, and (3) may be related to ammonium transport.     

Fluorescenz- und elektronenmikroskopische Untersuchungen an Beggiatoa alba und Thiothrix nivea

Ohne Zusammenfassung     

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--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.     

Protein synthesis by Beggiatoa alba B18LD in the presence and absence of sulfide

The interaction of sulfide oxidation and protein synthesis by Beggiatoa alba B18LD was investigated using the incorporation of radiolabeled leucine to estimate protein synthesis. Leucine was assimilated into whole cells in the presence of 6.1 mM acetate at a rate of 0.6 nmol · min^-1 · mg protein^-1, 43% of which was incorporated into the protein fraction. Protein synthesis by B. alba was unaffected by 1 mM sulfide, whether or not the cells had been preincubated with sulfide. B. alba oxidized radioactive sulfide to sulfur within 30 s of addition of the label, whether or not the organism was preinduced by sulfide. Furthermore, chloramphenicol, which inhibited protein synthesis, did not significantly inhibit sulfide oxidation by sulfide-induced or uninduced B. alba. This indicates that sulfide oxidation is a constitutive process. Enrichments of sulfur inclusions from B. alba B18LD that were analyzed by polyacrylamide gel electrophoresis demonstrated two enriched peptides with M_r values of 13,000 and 15,000. The 13,000 and 15,000 M_r peptide bands were more evident in cells grown in a medium containing sulfide than in cells from a medium lacking sulfide. Although sulfide did not increase the rate of overall protein synthesis, the synthesis of a few peptides was increased by the addition of sulfide to the growth medium. Among those, the 15,000 M_r peptide was one of the most distinctive.Non-standard abbreviations SDS-PAGE Sodium dodecyl sulfate polyacrylamide gel electrophoresis - PPO 2,5-diphenyloxazole - POPOP 1,4-bis [5-phenyl-2-oxazolyl]-benzene - BSS basal salts solution - BH Beggiatoa heterotrophic (medium) - BSO Beggiatoa sulfide oxidation (medium) - CM chloramphenicol - TCA trichloroacetic acid - M_r molecular mass     

Heterotrophic Metabolism of the Chemolithotroph Thiobacillus ferrooxidans

Glucose-6-phosphate dehydrogenase and the enzymes of the Entner-Doudoroff pathway, 6-phosphogluconate dehydrase and 2-keto-3-deoxy-6-phosphogluconate aldolase (assayed together), are induced during heterotrophic growth of Thiobacillus ferrooxidans on an iron-glucose-supplemented medium or on glucose alone. By contrast, autotrophic cells (iron-grown) contain low levels of these enzymes. Fructose 1, 6-diphosphate aldolase, an enzyme of the Embden-Meyerhof pathway, is present at low levels irrespective of the growth medium, suggesting that this enzyme is not involved in energy-yielding reactions but merely provides intermediates for biosynthesis. The Entner-Doudoroff and pentose-phosphate pathways are the principle means through which glucose is dissimilated and is presumed to be concerned with energy production. Isotopic studies showed that a high rate of CO(2) formation from specifically labeled glucose came from carbon atoms 1 and 4. An unexpectedly high rate of evolution of CO(2) also came from carbon 6, suggesting that the triose phosphate formed during glucose breakdown and specifically as a result of 2-keto-3-deoxy-6-phosphogluconate aldolase activity, was metabolized via some unorthodox metabolic route. Cells grown in the iron-supplemented and glucose-salts media have a complete tricarboxylic acid cycle, whereas autotrophically grown T. ferrooxidans lacked both alpha-ketoglutarate dehydrogenase and reduced nicotinamide adenine dinucleotide oxidase. Two isocitrate dehydrogenases [nicotinamide adenine dinucleotide (NAD) and NAD phosphate (NADP) specific] were present. NAD-linked enzyme was constitutive, whereas the NADP-linked enzyme was induced upon adaptation of autotrophic cells to heterotrophic growth.     

Organic Carbon Utilization by Obligately and Facultatively Autotrophic Beggiatoa Strains in Homogeneous and Gradient Cultures

Marine Beggiatoa strains MS-81-6 and MS-81-1c are filamentous gliding bacteria that use hydrogen sulfide and thiosulfate as electron donors for chemolithotrophic energy generation. They are known to be capable of chemolithoautotrophic growth in sulfide gradient media; here we report the first successful bulk cultivation of these strains in a defined liquid medium. To investigate their nutritional versatilities, strains MS-81-6 and MS-81-1c were grown in sulfide-oxygen gradient media supplemented with single organic compounds. Respiration rates and biomass production relative to those of controls grown in unsupplemented sulfide-limited media were monitored to determine whether organic compounds were utilized as sources of energy and/or cell carbon. With cells grown in sulfide gradient and liquid media, we showed that strain MS-81-6 strongly regulates two enzymes, the tricarboxylic acid cycle enzyme 2-oxoglutarate dehydrogenase and the Calvin cycle enzyme ribulose-1,5-bisphosphate carboxylase/oxygenase, in response to the presence of organic carbon (acetate) in the growth medium. In contrast, strain MS-81-1c lacked 2-oxoglutarate dehydrogenase activity and regulated ribulose-1,5-bisphosphate carboxylase/oxygenase activity only slightly in response to organic substrates. Tracer experiments with radiolabeled acetate showed that strain MS-81-1c did not oxidize acetate to CO(inf2) but could synthesize approximately 20% of its cell carbon from acetate. On the basis of these results, we conclude that Beggiatoa strain MS-81-1c is an obligate chemolithoautotroph, while strain MS-81-6 is a versatile facultative chemolithoautotroph.     

Autotrophic and Heterotrophic Metabolism of Hydrogenomonas: Regulation of Autotrophic Growth by Organic Substrates

The effects of a number of organic substrates on the autotrophic metabolism of Hydrogenomonas eutropha were examined. Dual substrate (mixotrophic) cultivation in the presence of hydrogen plus either fructose or alanine allowed autotrophic growth to begin immediately after the exhaustion of the organic substrate. On the other hand, the presence of acetate, pyruvate, or glutamate caused a lengthy lag to occur before autotrophic growth commenced. With acetate or pyruvate this lag (plateau) in the dicyclic growth curve was due to the repression of ribulose diphosphate carboxylase (RDPC) synthesis during mixotrophic growth. During heterotrophic growth with glutamate, RDPC was partially repressed; however, during mixotrophic growth, RDPC activity was high. Thus the delay of autotrophic growth was not due to a repression of RDPC by glutamate. The data suggest that glutamate interferes with autotrophic metabolism by repressing the incorporation of inorganic nitrogen. The repression of these vital autotrophic functions by acetate, pyruvate, and glutamate occurred both in the presence and absence of hydrogen, i.e., during both heterotrophic and mixotrophic cultivation. The derepression of the affected systems during the plateau phase of the dicyclic growth curves was demonstrated. Carbon dioxide assimilation by whole cells agreed well with the RDPC activity of extracts from cells grown under similar conditions.     

Alteration of Carbon Metabolism by a Base Analog

The study of carbon metabolism by cultures of the yeast C. utilis exposed to 5-fluorouracil revealed that the growth rate and synthesis of macromolecules was altered. The amino acid composition of the metabolic pool of amino acids was vastly altered, but the protein composition was unchanged. It is suggested that the analog may exert a selective action on certain amino acids, and that this action may be related to a template-like mechanism.     

Oxygen Responses and Mat Formation by Beggiatoa spp

The behavioral response of single Beggiatoa sp. filaments moving on a gas-permeable membrane was studied by the combined use of microscopy and oxygen microelectrodes during controlled oscillations of oxygen tension. The bacteria reacted to increasing oxygen by reversing the direction of movement. The same step-up phobic response to oxygen was observed when a filament tip or loop glided into a stable microgradient of increasing oxygen. The response was sensitive to a change in oxygen tension of <5% of air saturation min⁻¹. The response time was 20 to 50 s. Frequently, only part of the filament responded, which led to the formation of sharp bends, loops, and coils. This partial response facilitated the positioning of the long filaments within the narrow O₂-H₂S interface. The structure of whole Beggiatoa mats on sediment surfaces varied from loose to dense in relation to shallow or steep oxygen gradients in the 0.3- to 2-mm-thick, unstirred boundary layer. In an illuminated sediment Beggiatoa spp. lived together with photosynthetic organisms and migrated vertically in accordance with light/dark variations. The combined effect of phobic responses to light and oxygen can explain this migration.     

Biochemical Composition of Dissolved Organic Carbon Derived from Phytoplankton and Used by Heterotrophic Bacteria

The molecular size distribution and biochemical composition of the dissolved organic carbon released from natural communities of lake phytoplankton (photosynthetically produced dissolved organic carbon [PDOC]) and subsequently used by heterotrophic bacteria were determined in three lakes differing in trophic status and concentration of humic substances. After incubation of epilimnetic lake water samples with H¹⁴CO₃^- over one diel cycle, the phytoplankton were removed by size-selective filtration. The filtrates, still containing most of the heterotrophic bacteria, were reincubated in darkness (heterotrophic incubation). Differences in the amount and composition of PDO¹⁴C between samples collected before the heterotrophic incubation and samples collected afterwards were considered to be a result of bacterial utilization. The PDO¹⁴C collected at the start of the heterotrophic incubations always contained both high (>10,000)- and low (<1,000)-molecular-weight (MW) components and sometimes contained intermediate-MW components as well. In general, bacterial turnover rates of the low-MW components were fairly rapid, whereas the high-MW components were utilized slowly or not at all. In the humic lake, the intermediate-MW components accounted for a large proportion of the net PDO¹⁴C and were subject to rapid bacterial utilization. This fraction probably consisted almost entirely of polysaccharides of ca. 6,000 MW. Amino acids and peptides, other organic acids, and carbohydrates could all be quantitatively important parts of the low-MW PDO¹⁴C that was utilized by the heterotrophic bacteria, but the relative contributions of these fractions differed widely. It was concluded that, generally, low-MW components of PDOC are quantitatively much more important to the bacteria than are high-MW components, that PDOC released from phytoplankton does not contain substances of quantitative importance as bacterial substrates in all situations, and that high-MW components of PDOC probably contribute to the buildup of refractory, high-MW dissolved organic carbon in pelagic environments.     

Particulate and Dissolved Organic Carbon Production by the Heterotrophic Nanoflagellate Pteridomonas danica Patterson and Fenchel

Abstract We established a budget of organic carbon utilization of a starved heterotrophic nanoflagellate, Pteridomonas danica, incubated in batch cultures with Escherichia coli as model prey. The cultures were sampled periodically for biomass determinations and total organic carbon dynamics: total organic carbon, total organic carbon <1 μm, and dissolved organic carbon (DOC, <0.2 μm). During the 22 h incubation period, P. danica underwent biovolume variations of 3.2-fold. Gross growth efficiency was 22% and net growth efficiency 40%. P. danica respired 33% and egested 44% of the ingested E. coli carbon during lag and exponential growth phases. The form of the organic carbon egested varied. Of the total ingested carbon, 9% was egested in the form of DOC and occurred mainly during the exponential growth phase; 35% was egested in the form of particulate organic carbon (POC), ranging in size from 0.2 to 1 μm, and took place during the lag phase. P. danica could have reingested as much of 58% of this previously produced POC during the exponential growth phase as food scarcity increased. We concluded that POC egestion by flagellates could represent a significant source of submicrometric particles and colloidal organic matter. In addition, flagellate reingestion of egested POC could play a nonnegligible role in the microbial food web. Finally, the methodology reported in this study has proved to be a useful tool in the study of carbon metabolism in aquatic microorganisms. Received: 31 July 1998; Accepted: 2 March 1999     

Die Mixotrophie von Beggiatoa

Zusammenfassung Axenische Kulturen von Beggiatoa wurden zur Prüfung der Frage verwendet, ob autotrophe Ernährung möglich ist. Dazu wurde eine neue Technik ausgearbeitet, bei der FeS als H₂S-Quelle dient.Es bestätigte sich, daß entweder heterotrophe Ernährung oder neben H₂S kleinste Konzentrationen organischer Energiesubstrate nötig sind. Es liegt dann eine Sonderform von Mixotrophie vor, anders als bei chemoautotrophen Bakterien.Der aus H₂S entstehende S ist ein Reservestoff, der aber nicht nur, etwa wie Stärke, der Überschuß-Speicherung dient, sondern eine Zwischenstufe in der Verwertung der Oxydationsenergie darstellt, denn es wird sogleich H₂SO₄ gebildet.Unter Bedingungen, die in Reinkulturen die stärkste Vermehrung bewirken, kann Beggiatoa wegen des Wettbewerbes in der Natur nicht leben. Die ökologisch besten Lebensumstände sind von besonderer Art und von den physiologisch optimalen verschieden.

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The mixotrophy of Beggiatoa

Summary Axenic cultures of Beggiatoa were used to answer the question whether permanent autotrophic growth is feasible, so that oxidation of sulfur compounds would supply energy for all living processes, and organic substances were not needed.For that purpose a new technique was devised with FeS as a source of H₂S. Results confirmed the assumption that for Beggiatoa either heterotrophic nutrition or in addition to H₂S minute quantities of an organic substrate are required.The colorless Cyanophycean Beggiatoa represents therefore nutritionally a special mode of mixotrophy differing from the nutrition of chemo-autotrophic bacteria.Sulfur as a reserve material does not only serve as a surplus deposit but also represents a step in the utilization of oxidation energy from H₂S to H₂SO₄.Ecologically its living conditions are characterized by low tensions of O₂ and H₂S, a supply of CO₂ and low concentrations of organic nutrients. Under optimum conditions of nutrition. Beggiatoa would not stand competition by other organisms.

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Herrn Professor C. B. van Niel in Freundschaft und Verehrung zum 70. Geburtstag gewidmet.     

Heterotrophic nitrification by Pseudomonas aeruginosa

Summary Several soil bacteria and fungi produce nitrite when provided with acetaldoxime. Nitrite formation by one isolate, identified as a strain of Pseudomonas aeruginosa, is not directly linked to growth but rather proceeds mainly after the active growth period. The added oxime-nitrogen is converted completely to nitrite, and nitrate is not formed. Extracts of the bacterium generate nitrite, but not nitrate, more rapidly from nitroethane than from the added oxime. The enzyme system catalyzing the formation of nitrite in oxime solutions is soluble and inducible, whereas the enzyme catalyzing the release of equimolar quantities of nitrite and acetaldehyde from nitroethane is constitutive. The slow rate of nitrite production when the enzyme preparation is provided with acetaldoxime is not markedly increased by added cofactors. The soluble enzymes also generate nitrite when incubated with several aliphatic and alicyclic oximes and nitro compounds. Nitroethane is not formed from acetaldoxime. The possible mechanism of this nitrification reaction catalyzed by a heterotrophic microorganism is discussed.