Sulfur Isotope Enrichment during Maintenance Metabolism in the Thermophilic Sulfate-Reducing Bacterium Desulfotomaculum putei

Values of Δ³⁴S (, where δ³⁴S_HS and indicate the differences in the isotopic compositions of the HS⁻ and SO₄²⁻ in the eluent, respectively) for many modern marine sediments are in the range of −55 to −75‰, much greater than the −2 to −46‰ ɛ³⁴S (kinetic isotope enrichment) values commonly observed for microbial sulfate reduction in laboratory batch culture and chemostat experiments. It has been proposed that at extremely low sulfate reduction rates under hypersulfidic conditions with a nonlimited supply of sulfate, isotopic enrichment in laboratory culture experiments should increase to the levels recorded in nature. We examined the effect of extremely low sulfate reduction rates and electron donor limitation on S isotope fractionation by culturing a thermophilic, sulfate-reducing bacterium, Desulfotomaculum putei, in a biomass-recycling culture vessel, or “retentostat.” The cell-specific rate of sulfate reduction and the specific growth rate decreased progressively from the exponential phase to the maintenance phase, yielding average maintenance coefficients of 10⁻¹⁶ to 10⁻¹⁸ mol of SO₄ cell⁻¹ h⁻¹ toward the end of the experiments. Overall S mass and isotopic balance were conserved during the experiment. The differences in the δ³⁴S values of the sulfate and sulfide eluting from the retentostat were significantly larger, attaining a maximum Δ³⁴S of −20.9‰, than the −9.7‰ observed during the batch culture experiment, but differences did not attain the values observed in marine sediments.Dissimilatory SO₄²⁻ reduction is a geologically ancient, anaerobic, energy-yielding metabolic process during which SO₄²⁻-reducing bacteria (SRB) reduce SO₄²⁻ to H₂S while oxidizing organic molecules or H₂. SO₄²⁻ reduction is a dominant pathway for organic degradation in marine sediments (23) and in terrestrial subsurface settings where sulfur-bearing minerals dominate over Fe³⁺-bearing minerals. For example, at depths greater than 1.5 km below land surface in the fractured sedimentary and igneous rocks of the Witwatersrand Basin of South Africa, SO₄²⁻ reduction is the dominant electron-accepting process (3, 26, 46, 48, 61).The enrichment of ³²S in biogenic sulfides, with respect to the parent SO₄²⁻, imparted by SRB, is traceable through the geologic record (10, 54). The magnitude of the Δ³⁴S (= δ³⁴S_pyrite − δ³⁴S_{barite/gypsum}, where δ³⁴S_pyrite and δ³⁴S_{barite/gypsum} are the isotopic compositions of pyrite and barite or gypsum) increases from −10‰ in the 3.47-billion-year-old North Pole deposits to −30‰ in late-Archaean deposits (55), to −75‰ in Neoproterozoic to modern sulfide-bearing marine sediments (13).The kinetic isotopic enrichment, ɛ³⁴S, deduced from trends in the δ³⁴S values of SO₄²⁻ and HS⁻ in batch culture microbial SO₄²⁻ reduction experiments using the Rayleigh relationship, ranges from −2‰ to −46‰ (6, 7, 11, 17, 22, 27, 28, 30, 31, 38, 39). The variation in ɛ³⁴S values has been attributed to the SO₄²⁻ concentration, the type of electron donor and its concentration, the SO₄²⁻ reduction rate per cell (csSRR) (22), temperature, and species-specific isotope enrichment effects. In these laboratory experiments, doubling times are on the order of hours and csSRRs range from to 0.1 to 18 fmol cell⁻¹ h⁻¹ (7, 12, 17, 22, 30, 32, 39, 40).Experiments performed during the 1960s found that the magnitude of ɛ³⁴S was inversely proportional to the csSRR for organic electron donors (16, 31, 38, 39) when SO₄²⁻ was not limiting. More-recent batch culture experiments on 3 psychrophilic (optimum growth temperature, <20°C) and mesophilic (optimum growth temperature, between 20°C and 45°C) SRB strains (7) and on 32 psychrophilic to thermophilic SRB strains (22), however, have failed to reproduce such a relationship. In 2001, Canfield (11) reported an inverse correlation between ɛ³⁴S and reduction rate using a flowthrough sediment column and demonstrated that ɛ³⁴S values of approximately −35 to −40‰ were produced when organic substrates added by way of amendment were limited with respect to SO₄²⁻. Because it was not possible to readily evaluate changes in biomass in the sediment column with changes in temperature or substrate provision rate, it was inferred that changes in ɛ³⁴S were related to changes in the csSRRs. More recently, Canfield et al. (12) observed a 6‰ variation in ɛ³⁴S values related to the temperature of the batch culture experiments relative to the optimum growth temperature. The few early experiments that were performed using H₂ as the electron donor yielded ɛ³⁴S values ranging from −3 to −19‰ (22, 38, 39), which appear to correlate with the csSRR (39). Hoek et al. (32) also found that the ɛ³⁴S values for the thermophilic SO₄²⁻ reducer Thermodesulfatator indicus increased from between −1.5‰ and −10‰ in batch cultures with high H₂ concentrations to between −24‰ and −37‰ in batch cultures grown under H₂ limitation with respect to SO₄²⁻. Detmers et al. (22) found that the average ɛ³⁴S of SRB that oxidize their organic carbon electron donor completely to CO₂ averaged −25‰, versus −9.5‰ for SRB that release acetate during their oxidation of their organic carbon electron donor. Detmers et al. (22) speculated that the greater free energy yield per mole of SO₄²⁻ from incomplete carbon oxidation relative to that for complete carbon oxidation promotes complete SO₄²⁻ reduction and hinders isotopic enrichment due to isotopic exchange of the intracellular sulfur species pools.None of these experiments, however, have yielded ɛ³⁴S factors capable of producing the Δ³⁴S values of −55 to −75‰ observed in the geological record from ∼1.0 billion years ago to today. Various schemes have been hypothesized, and observations that involve either the disproportionation of S₂O₃²⁻ (36), the disproportionation of S⁰ produced by oxidation of either H₂S or S₂O₃²⁻ (15), or the disproportionation of SO₃²⁻ (29) have been made. Attribution of the increasing Δ³⁴S values recorded for Achaean to Neoproterozoic sediments to the increasing role of H₂S oxidative pathways makes sense in the context of increasing O₂ concentrations in the atmosphere (14) but is not consistent with the lack of significant fractionation observed during oxidative reactions (29). To explain the Δ³⁴S values of −55 to −77‰ reported to occur in interstitial pore waters from 100- to 300-m-deep, hypersulfidic ocean sediments (51, 64, 67), where the presence of a S-oxidative cycle is unlikely, an alternative, elaborate model of the SO₄²⁻-reducing pathway has been proposed by Brunner and Bernasconi (9). This model attributes the large Δ³⁴S values to a multistep, reversible reduction of SO₃²⁻ to HS⁻ involving S₃O₆²⁻ and S₂O₃²⁻ (20, 25, 41, 42, 52, 66). The conditions under which the maximum ɛ³⁴S values might be expressed are a combination of elevated HS⁻ concentrations, electron donor limitations, nonlimiting SO₄²⁻ concentrations, and a very low csSRR. The csSRR for subsurface environments has been estimated from biogeochemical-flux modeling to be 10⁻⁶ to 10⁻⁷ fmol cell⁻¹ h⁻¹ (23), with a corresponding cell turnover rate greater than 1,000 years (37).Batch and chemostat culture systems, despite low growth rates, cannot completely attain a state of zero growth with constant substrate provision and therefore do not accurately reflect the in situ nutritional states of microbes in many natural settings. Retentostats, or recycling fermentor vessels, recycle 100% of biomass to the culturing vessel, allowing experimenters to culture microbial cells to a large biomass with a constant nutrient supply rate until the substrate supply rate itself becomes the growth-limiting factor and cells enter a resting state in which their specific growth rate approaches zero and they carry on maintenance metabolism (1, 47, 53, 58, 59, 62, 63). Utilizing this approach, Colwell et al. (18) were able to obtain a cell-specific respiration rate of 7 × 10⁻⁴ fmol of CH₄ cell⁻¹ h⁻¹ for a mesophilic marine methanogen, a rate that is comparable to that estimated for methanogenic communities in deep marine sediments off the coast of Peru (49).In this study, the conditions that Brunner and Bernasconi (9) hypothesized would lead to the large Δ³⁴S values seen in nature were recreated in the laboratory by limiting the electron donor supply rate with respect to the SO₄²⁻ supply rate in a retentostat vessel. The S isotopic enrichment by a resting culture of Desulfotomaculum putei at an extremely low csSRR was compared to that of a batch culture experiment to determine whether the ɛ³⁴S values produced under the former conditions approach the Δ³⁴S seen in nature.     

Adaptation of Methanogenic Communities to the Cofermentation of Cattle Excreta and Olive Mill Wastes at 37°C and 55°C

The acclimatization of methanogens to two-phase olive mill wastes (TPOMW) was investigated in pilot fermenters started up with cattle excreta (37°C) and after changing their feed to excreta plus TPOMW (37°C or 55°C) or TPOMW alone (37°C) until a steady state was reached (28 days). Methanogenic diversity was screened using a phylogenetic microarray (AnaeroChip), and positive targets were quantified by real-time PCR. Results revealed high phylogenetic richness, with representatives of three out of the four taxonomic orders found in digesters. Methanosarcina dominated in the starting excreta (>96% of total 16S rRNA gene copies; over 45 times more abundant than any other methanogen) at high acetate (0.21 g liter⁻¹) and ammonia N concentrations (1.3 g liter⁻¹). Codigestion at 37°C induced a 6-fold increase of Methanosarcina numbers, correlated with CH₄ production (r_Pearson = 0.94; P = 0.02). At 55°C, the rise in temperature and H₂ partial pressure induced a burst of Methanobacterium, Methanoculleus, Methanothermobacter, and a group of uncultured archaea. The digestion of excreta alone resulted in low but constant biogas production despite certain oscillations in the methanogenic biomass. Unsuccessful digestion of TPOMW alone was attributed to high Cu levels inducing inhibition of methanogenic activity. In conclusion, the versatile Methanosarcina immediately adapted to the shift from excreta to excreta plus TPOMW and was responsible for the stimulated CH₄ production at 37°C. Higher temperatures (55°C) fostered methanogenic diversity by promoting some H₂ scavengers while yielding the highest CH₄ production. Further testing is needed to find out whether there is a link between increased methanogenic diversity and reactor productivity.Turning residues into energy is a societal and scientific priority due to climate change, fossil fuel exhaustion, and waste accumulation. In 2006, in Europe (EU27), less than 3% of electricity production came from biomass and wastes (11). Biogas plants, which anaerobically treat organic wastes to produce energy, are increasingly promoted in Europe, but their distribution is highly biased (35). While thousands of full- and farm-scale biogas plants are spread over central and northern Europe, anaerobic digestion technology in Mediterranean countries—Portugal, Spain, Italy, Greece, and Turkey—is in its early stages (35). These nations and other circum-Mediterranean countries lead in the production of olive oil and thus in olive mill wastes and wastewaters, which have a huge biogas production potential due to their lipid composition (1). Spain alone generates one-third of the world''s oil production and millions of tons of two-phase olive mill wastes (TPOMW) per year. TPOMW are mostly burned or composted (28), hence releasing methane into the atmosphere. This compels a change in strategy: methane production from TPOMW should be optimized in engineered environments and transformed into energy.TPOMW is a humid residue containing the olive pulp and stone. Its anaerobic digestibility is hampered by its low pH, low ammonia N, and high content in antimicrobial substances (1). However, it has been successfully fermented under laboratory conditions by supplementing it with nutrients and increasing the reactor organic loading rate stepwise (2) or by codigesting it with residues with a high buffering capacity, e.g., cattle excreta (17). These approaches seem to facilitate the adaptation of the methane-producing anaerobic community to the environmental conditions that TPOMW impose.Methanogenic archaea—microbes clustered within five orders of the Euryarchaeota—constitute the last step in the trophic chain of decomposers degrading organic matter in oxygen-free environments (36). Methanogenesis is often the rate-limiting step of anaerobic digestion of organic wastes (3) due to the fast duplication times of bacteria, which generate all substrates for the slow-growing methane-producing archaea. It is also the most sensitive step in processing imbalances (4), likely due to the lack of functional redundancy among methanogens (8). High concentrations of volatile fatty acids, salts, ammonia, and heavy metals can be inhibitory for methanogens (5, 22) and are the most common reasons for reactor failure (3). Our objective was to understand the adaptation of methanogenic communities to TPOMW. We investigated methanogenic diversity and abundance in pilot digesters fed with cattle excreta and after changing their feed to TPOMW or TPOMW plus excreta. We expected that mixing both residues would allow a faster adaptation and more efficient performance of the methanogenic communities in digesting TPOMW. The cofermentation was evaluated at 37°C and 55°C. During an acclimatization period of 28 days, we screened the methanogenic diversity using an in-house-devised phylogenetic microarray, the AnaeroChip (13), and quantified dominant genera by real-time quantitative PCR (qPCR). We have taken primers from the literature, and we present four new sets of genus-specific primers and SYBR green I-optimized assays for quantifying methanogens in anaerobic environments.     

Combined Gel Probe and Isotope Labeling Technique for Measuring Dissimilatory Nitrate Reduction to Ammonium in Sediments at Millimeter-Level Resolution

Dissimilatory NO₃⁻ reduction in sediments is often measured in bulk incubations that destroy in situ gradients of controlling factors such as sulfide and oxygen. Additionally, the use of unnaturally high NO₃⁻ concentrations yields potential rather than actual activities of dissimilatory NO₃⁻ reduction. We developed a technique to determine the vertical distribution of the net rates of dissimilatory nitrate reduction to ammonium (DNRA) with minimal physical disturbance in intact sediment cores at millimeter-level resolution. This allows DNRA activity to be directly linked to the microenvironmental conditions in the layer of NO₃⁻ consumption. The water column of the sediment core is amended with ¹⁵NO₃⁻ at the in situ ¹⁴NO₃⁻ concentration. A gel probe is deployed in the sediment and is retrieved after complete diffusive equilibration between the gel and the sediment pore water. The gel is then sliced and the NH₄⁺ dissolved in the gel slices is chemically converted by hypobromite to N₂ in reaction vials. The isotopic composition of N₂ is determined by mass spectrometry. We used the combined gel probe and isotopic labeling technique with freshwater and marine sediment cores and with sterile quartz sand with artificial gradients of ¹⁵NH₄⁺. The results were compared to the NH₄⁺ microsensor profiles measured in freshwater sediment and quartz sand and to the N₂O microsensor profiles measured in acetylene-amended sediments to trace denitrification.Nitrate accounts for the eutrophication of many human-affected aquatic ecosystems (19, 21). Sediment bacteria may mitigate NO₃⁻ pollution by denitrification and anaerobic ammonium oxidation (anammox), which produce N₂ (13, 18). However, inorganic nitrogen is retained in aquatic ecosystems when sediment bacteria reduce NO₃⁻ to NH₄⁺ by dissimilatory nitrate reduction to ammonium (DNRA) (5, 12, 16, 39). Hence, DNRA contributes to rather than counteracts eutrophication (23). DNRA may be the dominant pathway of dissimilatory NO₃⁻ reduction in sediments that are rich in electron donors, such as labile organic carbon and sulfide (4, 8, 17, 38, 55). High rates of DNRA are thus found in sediments affected by coastal aquaculture (8, 36) and settling algal blooms (16).DNRA, denitrification, and the chemical factors that control the partitioning between them (e.g., sulfide) should ideally be investigated in undisturbed sediments. The redox stratification of sediments involves vertical concentration gradients of pore water solutes. These gradients are often very steep, and their measurement requires high-resolution techniques, such as microsensors (26, 42) and gel probes (9, 54). If, for instance, the influence of sulfide on DNRA and denitrification is to be investigated, one wants to know exactly the sulfide concentration in the layers of DNRA and denitrification activity, as well as the flux of sulfide into these layers. This information can easily be obtained using H₂S and pH microsensors (22, 43). It is less trivial to determine the vertical distribution of DNRA and denitrification activity in undisturbed sediments. Denitrification activity can be traced using a combination of the acetylene inhibition technique (51) and N₂O microsensors (1). Acetylene inhibits the last step of denitrification, and therefore, N₂O accumulates in the layer of denitrification activity (44). This method underestimates the denitrification activity in sediments with high rates of coupled nitrification-denitrification because acetylene also inhibits nitrification (50).The vertical distribution of DNRA activity in undisturbed sediment has, to the best of our knowledge, never been determined; thus, the microenvironmental conditions in the layer of DNRA activity remain unknown. Until now, the influence of chemical factors on DNRA and denitrification in sediments has been assessed by slurry incubations (4, 12, 30), by flux measurements with sealed sediment cores (7, 47) or flowthrough sediment cores (16, 27, 37), and in one case, in reconstituted sediment cores sliced at centimeter-level resolution (39). Here, we present a new method, the combined gel probe and isotope labeling technique, to determine the vertical distribution of the net rates of DNRA in sediments. The sediments remain largely undisturbed and the NO₃⁻ amendments are within the range of in situ concentrations. The DNRA measurements can be related to the microprofiles of potential influencing factors measured in close vicinity of the gel probe. This allows DNRA activity to be directly linked with the microenvironmental conditions in the sediment.     

Differential Effects of Protein Kinase B/Akt Isoforms on Glucose Homeostasis and Islet Mass

Protein kinase B (PKB)/Akt is considered to be a key target downstream of insulin receptor substrate 2 (IRS2) in the regulation of β-cell mass. However, while deficiency of IRS2 in mice results in diabetes with insulin resistance and severe failure of β-cell mass and function, only loss of the PKBβ isoform leads to a mild metabolic phenotype with insulin resistance. Other isoforms were reported not to be required for metabolic regulation. To clarify the roles of the three PKB isoforms in the regulation of islet mass and glucose homeostasis, we assessed the metabolic and pancreatic phenotypes of Pkbα, Pkbβ, and Pkbγ-deficient mice. Our study uncovered a novel role for PKBα in the regulation of glucose homeostasis, whereas it confirmed that Pkbβ^−/− mice are insulin resistant with compensatory increase of islet mass. Pkbα^−/− mice displayed an opposite phenotype with improved insulin sensitivity, lower blood glucose, and higher serum glucagon concentrations. Pkbγ^−/− mice did not show metabolic abnormalities. Additionally, our signaling analyses revealed that PKBα, but not PKBβ or PKBγ, is specifically activated by overexpression of IRS2 in β-cells and is required for IRS2 action in the islets.Adaptation of pancreatic islet mass and function relative to metabolic demand maintains glucose homeostasis and may prevent the development of type 2 diabetes. β-Cell proliferation, apoptosis, growth, and function are tightly regulated by various extracellular factors and intracellular signaling pathways (23, 24, 34). In β-cells, insulin receptor substrate 2 (IRS2) controls maintenance and expansion of islet mass (29, 31, 42). In fact, IRS2-deficient mice are insulin resistant, show β-cell failure and hyperglycemia, and finally develop diabetes (26, 42). In contrast, deficiency of IRS1 only causes insulin resistance without the development of diabetes due to a compensatory increase in functional β-cell mass (1, 38). These observations indicated that IRS2, but not IRS1, is necessary for maintenance and compensatory increase of β-cell mass. Furthermore, experiments with isolated islets revealed that overexpression of IRS2, but not of IRS1, can increase β-cell proliferation and protect cells against high-glucose-induced apoptosis (29). Downstream of IRS2, phosphoinositide 3-kinase (PI3K)-protein kinase B (PKB) signaling is considered to be the critical pathway for the regulation of β-cell mass and function (12, 15, 16, 27). The serine-threonine kinase PKB, also known as Akt, is required for various cellular processes, from the regulation of cell cycle, survival, and growth to glucose and protein metabolism. In mammals, three PKB/Akt isoforms have been characterized and named PKBα/Akt1, PKBβ/Akt2, and PKBγ/Akt3. Although encoded by different genes on different chromosomes, the three isoforms display high homology at the protein level with 80 to 85% identical residues and the same structural organization (43). However, they differ in terms of tissue-specific expression. PKBα is expressed in most tissues and PKBβ is highly expressed in insulin-responsive tissues, whereas PKBγ expression is prominent in the brain and testes (17). All three isoforms are expressed in β-cells (30, 37). The roles of PKB in different tissues have been studied in transgenic-mouse models. While Pkbα^−/− and Pkbγ^−/− mice show impaired fetal growth and brain development, respectively, glucose homeostasis is unaffected in both models (9, 11, 14, 39, 46). In contrast, Pkbβ^−/− mice are insulin resistant and mildly glucose intolerant and have less adipose tissue. Depending on the strain and gender, these mice show either late loss of β-cells followed by the development of diabetes and mild growth deficiency or compensatory increase of β-cell mass without age-dependent progression into overt hyperglycemia (10, 17). These studies suggested that PKBβ is the only isoform playing a role in the regulation of energy homeostasis. On the other hand, constitutive activation of PKBα in β-cells is sufficient to increase growth and proliferation (5, 40), and in INS1 cells it prevents free fatty acid (FFA)-induced apoptosis (44). Furthermore, antagonizing total PKB activity in β-cells by ectopic expression of a kinase-dead mutant causes defects in insulin secretion (4), suggesting that in islets PKB is required mainly for normal function of the β-cells. Although these data support the notion that PKB must play a role in pancreatic β-cells, they are not in line with the stronger metabolic phenotype displayed by IRS2-deficient mice. In fact, PKBα and PKBγ appear not to be required to regulate glucose homeostasis (9, 11, 39), and in the case of Pkbβ^−/− mice, even though glucose homeostasis is impaired due to strong peripheral insulin resistance, the overall metabolic phenotype is far less severe than in Irs2^−/− mice (10), indicating that the capacity for β-cell compensation is retained in the absence of PKBβ.The aim of this study was to clarify the role of PKB in the regulation of islet mass and to define the relevance of PKB isoforms for IRS2 action in β-cells. Although it had been shown that PKBα is dispensable for the regulation of glucose homeostasis (9, 11), we found lower blood glucose concentrations in Pkbα^−/− mice. Based on this observation, we assessed in more detail the metabolic and the endocrine pancreatic phenotypes of Pkbα-, Pkbβ-, or Pkbγ-deficient mice. In addition, glucose uptake into fat cells, insulin secretion, and islet cell proliferation were investigated. Contrary to previous assumptions implying that PKBβ is the only (or at least the main) isoform playing a role in the regulation of glucose metabolism, we present evidence that both PKBα and PKBβ isoforms are required in the periphery for regulation of glucose homeostasis. While we confirmed that Pkbβ^−/− mice are insulin resistant and glucose intolerant with compensatory increase of β-cell mass, Pkbα^−/− mice showed lower blood glucose levels, were more insulin sensitive, and revealed higher serum glucagon concentrations accompanied by a mild increase in α-cell mass and proliferation. Moreover, our in vitro experiments showed that PKBα is specifically activated by IRS2 in β-cells and that its activation is required for IRS2-induced proliferation in islets.     

Biosynthetic Pathway for γ-Cyclic Sarcinaxanthin in Micrococcus luteus: Heterologous Expression and Evidence for Diverse and Multiple Catalytic Functions of C50 Carotenoid Cyclases

We report the cloning and characterization of the biosynthetic gene cluster (crtE, crtB, crtI, crtE2, crtYg, crtYh, and crtX) of the γ-cyclic C₅₀ carotenoid sarcinaxanthin in Micrococcus luteus NCTC2665. Expression of the complete and partial gene cluster in Escherichia coli hosts revealed that sarcinaxanthin biosynthesis from the precursor molecule farnesyl pyrophosphate (FPP) proceeds via C₄₀ lycopene, C₄₅ nonaflavuxanthin, C₅₀ flavuxanthin, and C₅₀ sarcinaxanthin. Glucosylation of sarcinaxanthin was accomplished by the crtX gene product. This is the first report describing the biosynthetic pathway of a γ-cyclic C₅₀ carotenoid. Expression of the corresponding genes from the marine M. luteus isolate Otnes7 in a lycopene-producing E. coli host resulted in the production of up to 2.5 mg/g cell dry weight sarcinaxanthin in shake flasks. In an attempt to experimentally understand the specific difference between the biosynthetic pathways of sarcinaxanthin and the structurally related ɛ-cyclic decaprenoxanthin, we constructed a hybrid gene cluster with the γ-cyclic C₅₀ carotenoid cyclase genes crtYg and crtYh from M. luteus replaced with the analogous ɛ-cyclic C₅₀ carotenoid cyclase genes crtYe and crtYf from the natural decaprenoxanthin producer Corynebacterium glutamicum. Surprisingly, expression of this hybrid gene cluster in an E. coli host resulted in accumulation of not only decaprenoxanthin, but also sarcinaxanthin and the asymmetric ɛ- and γ-cyclic C₅₀ carotenoid sarprenoxanthin, described for the first time in this work. Together, these data contributed to new insight into the diverse and multiple functions of bacterial C₅₀ carotenoid cyclases as key catalysts for the synthesis of structurally different carotenoids.Carotenoids are natural pigments synthesized by bacteria, fungi, algae, and plants, and more than 750 different carotenoids have been isolated from natural sources (17). They possess important biological functions as protectants against light and oxygen excess in photosynthetic processes (32, 38), and they have been proposed to reduce the risk of certain cancers, cardiovascular disease, and Alzheimer disease due to their antioxidative properties (20, 46). The global market for carotenoids used as food colorants and nutritional supplements was estimated at approximately $935 million in 2005 (11). More than 95% of all natural carotenoids are based on a symmetric C₄₀ phytoene backbone, and only a small number of C₃₀ and even fewer C₅₀ carotenoids have been discovered (42).C₅₀ carotenoids have multiple conjugated double bonds, and they contain at least one hydroxyl group; both these features contribute to strong antioxidative properties (17, 30, 32, 38). In nature, C₅₀ carotenoids are synthesized by bacteria of the order Actinomycetales, and to date, only two different C₅₀ carotenoid biosynthetic pathways have been described in the literature. The biosynthetic pathways of the ɛ-cyclic C₅₀ carotenoid decaprenoxanthin [2,2′-bis-(4-hydroxy-3-methybut-2-enyl)-ɛ,ɛ-carotene] and the β-cyclic C₅₀ carotenoid C.p.450 [2,2′-bis-(4-hydroxy-3-methybut-2-enyl)-β,β-carotene] have been elucidated in Corynebacterium glutamicum (22, 23) and in Dietzia sp. CQ4 (41), respectively. For both pathways, the common precursor, C₄₀ lycopene, is synthesized from C₁₅ farnesyl pyrophosphate (FPP) via the methylerythritol 4-phosphate (MEP) pathway, which is present in most eubacteria (33). Effective lycopene production has been achieved in genetically engineered noncarotenogenic hosts, such as Escherichia coli and Saccharomyces cerevisiae (9). Accordingly, the potential of using such biotechnologically relevant hosts for heterologous production of any lycopene-derived carotenoids has generated high interest.The biosynthesis of cyclic C₅₀ carotenoids from lycopene is catalyzed by lycopene elongase and carotenoid cyclases. Even though most carotenoids in plants and microorganisms exhibit cyclic structures, cyclization reactions were predominantly known for C₄₀ pathways (45) catalyzed by monomeric enzymes that have been isolated from plants and bacteria (5, 16, 27, 29, 31, 36). In C. glutamicum, the genes crtYe, crtYf, and crtEb were identified as being involved in the conversion of lycopene to the ɛ-cyclic C₅₀ carotenoid decaprenoxanthin (22, 44). Sequential elongation of lycopene into the acyclic C₅₀ carotenoid flavuxanthin was catalyzed by the crtEb gene product lycopene elongase. Subsequent cyclization to decaprenoxanthin was catalyzed by a heterodimeric C₅₀ carotenoid, ɛ-cyclase, encoded by crtYe and crtYf (22). C. glutamicum can synthesize both mono- and diglucosylated decaprenoxanthin; however, the genetic and enzymatic bases for glucosylation of decaprenoxanthin are unknown. Analogous to decaprenoxanthin, biosynthesis of the β-cyclic C₅₀ carotenoid C.p.450 in Dietzia sp. CQ4 from lycopene involves lycopene elongase and C₅₀ carotenoid β-cyclase activities (41).While most cyclic carotenoids exhibit β-rings, ɛ-ring-containing pigments are common in higher plants (7), and carotenoids substituted only with γ-rings are rarely observed in plants and algae (14). To date, no biosynthetic pathway for γ-cyclic C₅₀ carotenoids has been reported in the literature.Micrococcus luteus NCTC2665 (the “Fleming strain”) is a Gram-positive bacterium belonging to the family Micrococcaceae within the order Actinomycetales. The carotenoids, including the γ-cyclic C₅₀ sarcinaxanthin [(2R,6R,2′R,6′R)-(2,2′-bis(4-hydroxy-3-methyl-2-butenyl)-γ,γ-carotene)], synthesized by this bacterium have been identified and structurally elucidated (26). We recently isolated and characterized several wild-type M. luteus strains from the sea surface microlayer of the middle part of the Norwegian coast (39). Here, we report one additional such marine M. luteus isolate, designated Otnes7, forming color-intensive colonies indicating high sarcinaxanthin production levels. Both Otnes7 and NCTC2665 were used as M. luteus model strains, and the sarcinaxanthin biosynthetic gene clusters were cloned from both strains. The complete sarcinaxanthin biosynthetic pathway from lycopene was elucidated, including glucosylation, and we also explored the potential of using Otnes7-derived genes to achieve effective heterologous production of sarcinaxanthin in E. coli. The results add important new knowledge of the biosynthesis of C₅₀ carotenoids, and in particular, they highlight the diverse functions of C₅₀ carotenoid cyclases leading to synthesis of structurally different carotenoids.     

Independent and Cooperative Roles of Adaptor Molecules in Proximal Signaling during Fc?RI-Mediated Mast Cell Activation

Activation through FcɛRI, a high-affinity IgE-binding receptor, is critical for mast cell function during allergy. The formation of a multimolecular proximal signaling complex nucleated by the adaptor molecules SLP-76 and LAT1 is required for activation through this receptor. Based on previous T-cell studies, current dogma dictates that LAT1 is required for plasma membrane recruitment and function of SLP-76. Unexpectedly, we found that the recruitment and phosphorylation of SLP-76 were preserved in LAT1^−/− mast cells and that SLP-76^−/− and LAT1^−/− mast cells harbored distinct functional and biochemical defects. The LAT1-like molecule LAT2 was responsible for the preserved membrane localization and phosphorylation of SLP-76 in LAT1^−/− mast cells. Although LAT2 supported SLP-76 phosphorylation and recruitment to the plasma membrane, LAT2 only partially compensated for LAT1-mediated cell signaling due to its decreased ability to stabilize interactions with phospholipase Cγ (PLCγ). Comparison of SLP-76^−/− LAT1^−/− and SLP-76^−/− mast cells revealed that some functions of LAT1 could occur independently of SLP-76. We propose that while SLP-76 and LAT1 depend on each other for many of their functions, LAT2/SLP-76 interactions and SLP-76-independent LAT1 functions also mediate a positive signaling pathway downstream of FcɛRI in mast cells.Mast cell activation during allergic inflammation is mediated by the high-affinity immunoglobulin E (IgE)-binding receptor FcɛRI. Cross-linking of FcɛRI on mast cells by IgE/cognate antigen complexes results in the rapid release of a wide array of inflammatory mediators, including vasoactive amines and cytokines/chemokines that give rise to allergic symptoms, ranging in severity from simple urticaria to anaphylactic shock and death (14). As allergy affects ∼30% of the population in developed countries (13), much attention has been placed on studying the signal transduction mechanisms involved in mast cell activation downstream of FcɛRI in hopes of finding novel targets for therapeutic intervention.Signal transduction downstream of FcɛRI is initiated by the phosphorylation of the immunoreceptor tyrosine-based activation motifs (ITAMs) contained in the signaling components (β and γ chains) of the FcɛRI complex (30, 37). Once phosphorylated, these chains serve as docking sites for several protein tyrosine kinases (PTKs), including Lyn and spleen tyrosine kinase (Syk) (9, 19, 34). Recruitment of Syk to the membrane by FcɛRI results in the phosphorylation of scaffold proteins known as adaptor molecules. Adaptor proteins lack enzymatic activity but instead contain protein-binding domains that are critical for the formation of a multimolecular complex, which orchestrates downstream signaling in a temporal and spatial manner. The adaptor molecules Src homology 2 (SH2) domain-containing leukocyte phosphoprotein of 76 kDa (SLP-76) and linker of activated T cells 1 (LAT1) organize the assembly of a proximal signaling complex downstream of FcɛRI. Failure to form this complex is detrimental to FcɛRI-mediated mast cell function, as demonstrated by the finding that both SLP-76-deficient (22, 29, 41) and LAT1-deficient (25, 31, 32) mast cells display severely diminished degranulation and cytokine/chemokine production following FcɛRI ligation.Similar proximal signaling complexes are formed downstream of several different ITAM-containing receptors. Much of our understanding of the role of adaptor molecules in signal transduction has come from identification of phosphoproteins during T-cell receptor (TCR)-mediated activation of the human Jurkat T-cell line (1, 33). These studies eventually led to a paradigm describing the sequence of events in the formation of the SLP-76/LAT1 signaling complex. According to this model, SLP-76 is found constitutively bound to Grb2-related adaptor downstream of Shc (GADS) (24) and resides in the cytosol. Upon TCR activation, the tyrosines of membrane-resident LAT1 are phosphorylated and become attachment sites for proteins such as phospholipase Cγ (PLCγ) and GADS (43, 45). SLP-76 is drawn to the membrane through a GADS/LAT1 interaction, which then permits Syk family PTKs to maximally phosphorylate the N-terminal tyrosines of SLP-76 (5, 10). Several lines of evidence support this model whereby a LAT1/SLP-76 module organizes TCR signaling. First, both SLP-76- and LAT1-deficient Jurkat T cells display similar biochemical defects, such as diminished PLCγ and extracellular signal-regulated kinase (ERK) activation (10, 42). Second, T cells in SLP-76^−/− and LAT1^−/− mice are blocked at the same stage of development (7, 44). Third, SLP-76 can be coimmunoprecipitated with LAT1 but not with LAT1 harboring tyrosine-to-phenylalanine mutations (45). Finally, expression of a fusion protein comprised of the membrane-localizing domain of LAT1 and SLP-76 that forces localization of SLP-76 to the plasma membrane rescues the TCR-induced functional defects of both SLP-76- and LAT1-deficient Jurkat T cells (3). This model implies a mutually dependent relationship between SLP-76 and LAT1, where SLP-76 and LAT1 rely on each other to carry out their roles.One might suspect that this model for LAT1/SLP-76 function would operate in all other cells that utilize these adaptor molecules for ITAM-containing receptor-mediated signaling. However, the published defects of LAT1-deficient mast cells in FcɛRI-mediated signaling appeared milder than those of SLP-76-deficient mast cells, although a direct comparison has never been reported. In the present study, we show that LAT1-deficient mast cells display distinct functional and biochemical defects compared to SLP-76-deficient mast cells, implying that unlike in T cells, SLP-76 may not depend entirely on LAT1 for its function in mast cells. Surprisingly, the membrane recruitment and phosphorylation of SLP-76 were also preserved in LAT1^−/− mast cells. We show that LAT2 (also known as non-T-cell activation linker [NTAL] or linker for activation of B cells [LAB]), which is not expressed in naïve T cells but is expressed in mast cells (15), is responsible for phosphorylation and plasma membrane recruitment of SLP-76 in the absence of LAT1. However, LAT2 cannot support all LAT1/SLP-76-associated functions, such as sustained Ca²⁺ flux, likely due to decreased stability of the LAT2/SLP-76/PLCγ complex. Comparison of SLP-76^−/− LAT1^−/− and SLP-76^−/− mast cells also revealed that some functions of LAT1 could occur independently of SLP-76. We propose that although SLP-76 and LAT1 are interdependent for many of their functions, LAT2/SLP-76 interactions and SLP-76-independent LAT1 functions mediate positive signaling downstream of FcɛRI in mast cells.     

NADP+ Reduction with Reduced Ferredoxin and NADP+ Reduction with NADH Are Coupled via an Electron-Bifurcating Enzyme Complex in Clostridium kluyveri

It was recently found that the cytoplasmic butyryl-coenzyme A (butyryl-CoA) dehydrogenase-EtfAB complex from Clostridium kluyveri couples the exergonic reduction of crotonyl-CoA to butyryl-CoA with NADH and the endergonic reduction of ferredoxin with NADH via flavin-based electron bifurcation. We report here on a second cytoplasmic enzyme complex in C. kluyveri capable of energetic coupling via this novel mechanism. It was found that the purified iron-sulfur flavoprotein complex NfnAB couples the exergonic reduction of NADP⁺ with reduced ferredoxin (Fd_red) and the endergonic reduction of NADP⁺ with NADH in a reversible reaction: Fd_red²⁻ + NADH + 2 NADP⁺ + H⁺ = Fd_ox + NAD⁺ + 2 NADPH. The role of this energy-converting enzyme complex in the ethanol-acetate fermentation of C. kluyveri is discussed.Clostridium kluyveri is unique in fermenting ethanol and acetate to butyrate, caproate, and H₂ (reaction 1) and in deriving a large (30%) portion of its cell carbon from CO₂. Both the energy metabolism and the pathways of biosynthesis have therefore been the subject of many investigations (for relevant literature, see references 12 and 27). (1)During growth of C. kluyveri on ethanol and acetate, approximately five ethanol and four acetate molecules are converted to three butyrate molecules and one caproate molecule (reaction 1a), and one ethanol molecule is oxidized to one acetate⁻, one H⁺, and two H₂ (reaction 1b) molecules (23, 31). How exergonic reaction 1a is coupled with endergonic reaction 1b and with ATP synthesis from ADP and P_i (ΔG^o′ = +32 kJ/mol) has remained unclear for many years. (1a) (1b)We recently showed (12) that, in Clostridium kluyveri, the exergonic reduction of crotonyl-coenzyme A (crotonyl-CoA) (E_o′ = −10 mV) with NADH (E_o′ = −320 mV) involved in reaction 1a is coupled with the endergonic reduction of ferredoxin (Fd_ox) (E_o′ = −420 mV) with NADH (E_o′ = −320 mV) involved in reaction 1b via the recently proposed mechanism of flavin-based electron bifurcation (7). The coupling reaction is catalyzed by the cytoplasmic butyryl-CoA dehydrogenase-EtfAB complex (reaction 2) (12): (2)The reduced ferredoxin (Fd_red²⁻) is assumed to be used for rereduction of NAD⁺ via a membrane-associated, proton-translocating ferredoxin:NAD oxidoreductase (RnfABCDEG) (reaction 3), and the proton motive force thus generated is assumed to drive the phosphorylation of ADP via a membrane-associated F₁F₀ ATP synthetase (reaction 4): (3) (4)The novel coupling mechanism represented by reactions 2 and 3 allowed for the first time the possibility of formulating a metabolic scheme for the ethanol-acetate fermentation that could account for the observed fermentation products and growth yields and thus for the observed ATP gains (27). One issue, however, remained open, namely, why the formation of butyrate from ethanol and acetate in the fermentation involves both an NADP⁺- and an NAD⁺-specific β-hydroxybutyryl-CoA dehydrogenase (16), considering that, in the oxidative part of the fermentation (ethanol oxidation to acetyl-CoA), only NADH is generated (8, 9, 13).The presence of a reduced ferredoxin:NADP⁺ oxidoreductase was proposed based on results of enzymatic studies performed 40 years ago. Cell extracts of Clostridium kluyveri were found to catalyze the formation of H₂ from NADPH in a ferredoxin- and NAD⁺-dependent reaction (34). The results were interpreted to indicate that C. kluyveri contains a ferredoxin-dependent hydrogenase and an NADPH:ferredoxin oxidoreductase with transhydrogenase activity. H₂ formation from NADPH was strictly dependent on the presence of NAD⁺ and was inhibited by NADH, inhibition being competitive with the presence of NAD⁺, indicating that ferredoxin reduction with NADPH is under the allosteric control of the NAD⁺/NADH couple. The cell extracts also catalyzed the NADH-dependent reduction of NADP⁺ with reduced ferredoxin (21, 34). Purification of the enzyme catalyzing these reactions was not achieved, and no function in the energy metabolism of C. kluyveri was assigned.In this communication, we report on the properties of the recombinant enzyme that catalyzes the NAD⁺-dependent reduction of ferredoxin with NADPH and the NADH-dependent reduction of NADP⁺ with reduced ferredoxin and show that the cytoplasmic heterodimeric enzyme couples the exergonic reduction of NADP⁺ with reduced ferredoxin with the endergonic reduction of NADP⁺ with NADH in a fully reversible reaction. The transhydrogenation reaction is endergonic, because in vivo the NADH/NAD⁺ ratio is generally near 0.3 and the NADPH/NADP⁺ ratio is generally above 1 (2, 30). (5)NADP⁺ reduction is most probably the physiological function of the enzyme, which is why we chose the abbreviation NfnAB (for NADH-dependent reduced ferredoxin:NADP⁺ oxidoreductase).     

Novel Denitrifying Bacterium Ochrobactrum anthropi YD50.2 Tolerates High Levels of Reactive Nitrogen Oxides

Most studies of bacterial denitrification have used nitrate (NO₃⁻) as the first electron acceptor, whereas relatively less is understood about nitrite (NO₂⁻) denitrification. We isolated novel bacteria that proliferated in the presence of high levels of NO₂⁻ (72 mM). Strain YD50.2, among several isolates, was taxonomically positioned within the α subclass of Proteobacteria and identified as Ochrobactrum anthropi YD50.2. This strain denitrified NO₂⁻, as well as NO₃⁻. The gene clusters for denitrification (nar, nir, nor, and nos) were cloned from O. anthropi YD50.2, in which the nir and nor operons were linked. We confirmed that nirK in the nir-nor operon produced a functional NO₂⁻ reductase containing copper that was involved in bacterial NO₂⁻ reduction. The strain denitrified up to 40 mM NO₂⁻ to dinitrogen under anaerobic conditions in which other denitrifiers or NO₃⁻ reducers such as Pseudomonas aeruginosa and Ralstonia eutropha and nitrate-respiring Escherichia coli neither proliferated nor reduced NO₂⁻. Under nondenitrifying aerobic conditions, O. anthropi YD50.2 and its type strain ATCC 49188^T proliferated even in the presence of higher levels of NO₂⁻ (100 mM), and both were considerably more resistant to acidic NO₂⁻ than were the other strains noted above. These results indicated that O. anthropi YD50.2 is a novel denitrifier that has evolved reactive nitrogen oxide tolerance mechanisms.Environmental bacteria maintain the global nitrogen cycle by metabolizing organic and inorganic nitrogen compounds. Denitrification is critical for maintenance of the global nitrogen cycle, through which nitrate (NO₃⁻) or nitrite (NO₂⁻) is reduced to gaseous nitrogen forms such as N₂ and nitrous oxide (N₂O) (19, 47). Decades of investigations into denitrifying bacteria have revealed their ecological impact (9), their molecular mechanisms of denitrification (13, 25, 47), and the industrial importance of removing nitrogenous contaminants from wastewater (31, 36). Bacterial denitrification is considered to comprise four successive reduction steps, each of which is catalyzed by NO₃⁻ reductase (Nar), NO₂⁻ reductase (Nir), nitric oxide (NO) reductase (Nor), and N₂O reductase (Nos). The reaction of each enzyme is linked to the electron transport chain on the cellular membrane and accompanies oxidative phosphorylation, implying that bacterial denitrification is of as much physiological significance as anaerobic respiration (25, 47). Most denitrifying bacteria are facultative anaerobes and respire with oxygen under aerobic conditions. Because denitrification is induced in the absence of oxygen, it is considered an alternative mechanism of energy conservation that has evolved as an adaptation to anaerobic circumstances (13, 47).Nitrite and NO are hazardous to bacteria, since they generate highly reactive nitrogen species (RNS) under physiological conditions and damage cellular DNA, lipid, and proteins (28, 37). Denitrifying bacteria are thought to be threatened by RNS since they reduce NO₃⁻ to generate NO₂⁻ and NO as denitrifying intermediates. Furthermore, denitrifying bacteria often inhabit environments where they are exposed to NO₂⁻ and NO and hence high levels of RNS. Recent reports suggest that pathogenic bacteria invading animal tissues are attacked by NO generated by macrophages (12). Such bacteria involve denitrifiers, and some of them, for example, Neisseria meningitidis (1) and Pseudomonas aeruginosa, acquire resistance to NO by producing Nor (44). The utilization (reduction) of NO by Brucella increases the survival of infected mice (2). These examples suggest that production of a denitrifying mechanism affects bacterial survival of threats from both endogenous and extracellular RNS. However, the mechanism of RNS tolerance induced by denitrifying bacteria is not fully understood.Ubiquitous gram-negative Ochrobactrum strains are widely distributed in soils and aqueous environments, where they biodegrade aromatic compounds (11), organophosphorus pesticides (45), and other hydrocarbons (38) and remove heavy metal ions such as chromium and cadmium (24). Having been isolated from clinical specimens, Ochrobactrum anthropi is currently recognized as an emerging opportunistic pathogen, although relatively little is known about its pathogenesis and factors contributing to its virulence (7, 30). Manipulation systems have been developed to investigate these issues at the molecular genetic level (33). Some O. anthropi strains have been identified as denitrifiers (21), although the denitrifying properties of these strains have not been investigated in detail. This study was undertaken to examine the denitrifying properties of O. anthropi in more detail. O. anthropi YD50.2 was selected for this study and was isolated herein. The strain denitrified high levels of NO₂⁻ (up to 40 mM) to dinitrogen under anaerobic conditions. The strain was highly resistant to acidified NO₂⁻ under nondenitrifying aerobic conditions. These results indicate that O. anthropi YD50.2 has mechanisms that produce tolerance to RNS.     

CO2 Uptake and Fixation by a Thermoacidophilic Microbial Community Attached to Precipitated Sulfur in a Geothermal Spring

Carbon fixation at temperatures above 73°C, the upper limit for photosynthesis, is carried out by chemosynthetic thermophiles. Yellowstone National Park (YNP), Wyoming possesses many thermal features that, while too hot for photosynthesis, presumably support chemosynthetic-based carbon fixation. To our knowledge, in situ rates of chemosynthetic reactions at these high temperatures in YNP or other high-temperature terrestrial geothermal springs have not yet been reported. A microbial community attached to precipitated elemental sulfur (S^o floc) at the source of Dragon Spring (73°C, pH 3.1) in Norris Geyser Basin, YNP, exhibited a maximum rate of CO₂ uptake of 21.3 ± 11.9 μg of C 10⁷ cells⁻¹ h⁻¹. When extrapolated over the estimated total quantity of S^o floc at the spring''s source, the S^o floc-associated microbial community accounted for the uptake of 121 mg of C h⁻¹ at this site. On a per-cell basis, the rate was higher than that calculated for a photosynthetic mat microbial community dominated by Synechococcus spp. in alkaline springs at comparable temperatures. A portion of the carbon taken up as CO₂ by the S^o floc-associated biomass was recovered in the cellular nucleic acid pool, demonstrating that uptake was coupled to fixation. The most abundant sequences in a 16S rRNA clone library of the S^o floc-associated community were related to chemolithoautotrophic Hydrogenobaculum strains previously isolated from springs in the Norris Geyser Basin. These microorganisms likely contributed to the uptake and fixation of CO₂ in this geothermal habitat.The upper temperature limit for primary production via photosynthesis is ∼73°C (7, 8, 11). At this temperature, photosynthesis is restricted to cyanobacteria of the genus Synechococcus, which generally inhabit alkaline environments (11). In acidic environments (pH < 4.0), the upper temperature limit for photosynthetic-based primary production is ∼56°C. Under these conditions, phototrophic activity is restricted to the unicellular eukaryotic red algae Cyanidium, Galdieria, and Cyanidioschyzon, collectively referred to as “cyanidia” (6, 12, 31, 48). Primary production above this temperature in acidic environments occurs through chemoautotrophy, a metabolism restricted to prokaryotes.Yellowstone National Park (YNP), WY, possesses numerous high-temperature (73 to 93°C) geothermal environments that are thought to support communities of microorganisms through chemoautotrophic-based primary production. Evidence for chemosynthesis in these environments is based on the recovery of 16S rRNA gene sequences that are affiliated with cultivated representatives of the phyla Aquificae and Crenarchaeota, many of which are capable of CO₂ fixation via the oxidation of hydrogen (H₂) and/or sulfide (HS⁻) (15, 17, 21, 24, 26, 28, 41, 46). Surprisingly, CO₂ fixation has yet to be demonstrated in situ in YNP hot spring environments (acidic or alkaline) where temperatures exceed the limits of photosynthesis and where primary production is thought to be driven by chemoautotrophic metabolism (14, 15, 28, 29).Dragon Spring, an acid-sulfate-chloride (ASC) spring located in the Norris Geyser Basin of YNP, is a likely habitat for chemoautotrophic primary production. The pH of the water is ∼3.1, and the temperature of the water at the source fluctuates from 65 to 78°C, which is well above the upper temperature limit for photosynthesis under acidic conditions. Potential electron donors for chemolithoautotrophic growth in the source water include hydrogen (H₂) and sulfide (S²⁻) at concentrations of 13 nM and 65 μM, respectively (15). In addition, submerged substrata at the spring''s source are blanketed by precipitates of elemental sulfur (S°), hereafter referred to as S^o floc (23). Inventories of bacterial and archaeal 16S rRNA genes recovered from S^o floc collected from the source of Dragon Spring indicate the presence of Crenarchaeota and Aquificae (4, 15). The latter are related to chemolithoautotrophic Hydrogenobaculum spp., representatives of which have recently been isolated from the spring (15). In the present study, we demonstrate uptake and fixation of CO₂ at a temperature of 73°C by a Hydrogenobaculum-dominated microbial community associated with S^o floc collected from the source of Dragon Spring. This is the first direct evidence of CO₂ uptake in situ by a thermoacidophilic microbial community at a temperature that precludes photosynthesis in terrestrial geothermal springs.     

Source of Nitrous Oxide Emissions during the Cow Manure Composting Process as Revealed by Isotopomer Analysis of and amoA Abundance in Betaproteobacterial Ammonia-Oxidizing Bacteria

A molecular analysis of betaproteobacterial ammonia oxidizers and a N₂O isotopomer analysis were conducted to study the sources of N₂O emissions during the cow manure composting process. Much NO₂⁻-N and NO₃⁻-N and the Nitrosomonas europaea-like amoA gene were detected at the surface, especially at the top of the composting pile, suggesting that these ammonia-oxidizing bacteria (AOB) significantly contribute to the nitrification which occurs at the surface layer of compost piles. However, the ¹⁵N site preference within the asymmetric N₂O molecule (SP = δ¹⁵N^α − δ¹⁵N^β, where ¹⁵N^α and ¹⁵N^β represent the ¹⁵N/¹⁴N ratios at the center and end sites of the nitrogen atoms, respectively) indicated that the source of N₂O emissions just after the compost was turned originated mainly from the denitrification process. Based on these results, the reduction of accumulated NO₂⁻-N or NO₃⁻-N after turning was identified as the main source of N₂O emissions. The site preference and bulk δ¹⁵N results also indicate that the rate of N₂O reduction was relatively low, and an increased value for the site preference indicates that the nitrification which occurred mainly in the surface layer of the pile partially contributed to N₂O emissions between the turnings.The very sensitive greenhouse gas nitrous oxide (N₂O) has a 296 times higher impact than CO₂ (39) and is also responsible for ozone depletion (10). Agricultural activities such as the use of nitrate fertilizers, livestock production, and manure management, including composting, are known to be important sources of N₂O emissions (18). To devise a strategy to mitigate N₂O emissions, it is essential to understand its sources in detail. However, the sources of N₂O emissions during the composting process are still largely unclear.In the composting process, a part of NH₄⁺-N is known to be processed through nitrification-denitrification and emitted as N₂ and N₂O. Nitrous oxide is known to be generated through both the nitrification and denitrification processes as intermediate products or by-products. Nitrous oxide emission is a very complex process because denitrifying bacteria are phylogenetically diverse (60), and nitrifiers are also known to utilize the denitrification process even under aerobic conditions (42). It is thus very difficult to estimate the relative contributions of nitrification and denitrification in actual N₂O emissions from the environment. Until now, there has been insufficient knowledge about the relative contributions of these processes to N₂O emissions during the animal manure composting process. Measurement of the actual contributions of N₂O emissions from compost piles in the field is therefore critical to establishing a strategy of mitigating N₂O emissions.Recently, a high-precision analytical technique for determining intramolecular ¹⁵N site preference in asymmetric molecules of N₂O was developed (47). Since N₂O has two N atoms within the molecule (central and outer N), distribution of a stable isotope, ¹⁵N, results in the distribution of three isotopomers, such as ¹⁵N¹⁵NO, ¹⁵N¹⁴NO, and ¹⁴N¹⁵NO. By using this newly developed innovative technique, the latter two types of molecules, which exist abundantly in the environment, can be individually measured. The difference in δ¹⁵N between δ¹⁵N^α and δ¹⁵N^β is the so-called site preference (SP = δ¹⁵N^α − δ¹⁵N^β, where ¹⁵N^α and ¹⁵N^β represent the ¹⁵N/¹⁴N ratios at the center and end sites of the nitrogen atoms, respectively). The site preference enabled us to identify the source and sinks of N₂O in the environment (48, 49, 50, 56). Using this technique, Sutka et al. (44) found that the site preference for N₂O from hydroxylamine oxidation (∼33‰) and nitrite reduction (∼0‰) differs in a pure culture study and noted that this difference can be used to distinguish the relative contributions of nitrification and denitrification sources to N₂O emissions. There have still been only several reported studies which applied this measurement technique to field N₂O samples (48, 53) or referred to the relative contributions of nitrification and denitrification. To our knowledge, the present study is the first to apply this isotopomer analysis technique to the determination of N₂O sources in the composting process. We specifically used this technique to understand the actual contributions of nitrification and denitrification to N₂O emissions during the cow manure composting process.Ammonia oxidation, the conversion of ammonium to nitrite via hydroxylamine, is an initial step of the nitrification-denitrification process and is critical to the nitrogen cycle in the terrestrial environment (4, 24). In the nitrification process, N₂O is generated as a by-product when ammonia oxidizers convert hydroxylamine to nitrite (35). Since NO₂⁻-N and NO₃⁻-N accumulate in the latter stages of the composting process (29, 30), it is obvious that nitrifiers are active in compost piles. Therefore, it is important to clarify the role and significance of ammonia oxidizers in N₂O emissions during the composting process. However, since the pure culture isolation method is so difficult and time-consuming, little is known about these ammonia oxidizers. A molecular approach based on PCR has been recently developed and has to date been used to target the ammonia monooxygenase gene (amoA) or 16S rRNA gene of betaproteobacterial ammonia oxidizers in soil, wetlands, and marine sediments (2, 3, 6, 7, 13, 32, 52). Using these techniques, substantial information about uncultured ammonia-oxidizing bacteria (AOB) that are partially or wholly responsible for nitrification in the environment will become available. Since the microbial community drastically changes through the composting process (19, 29), and a high accumulation of nitrite or nitrate will occur, especially in the latter half of the process (30), we continuously sampled and analyzed the diversity and abundance of AOB throughout the process. Our objectives in this study were to elucidate the sources of N₂O emissions during the cow manure composting process by combining the isotopomer analysis and molecular analysis of betaproteobacterial AOB.     

Potential Application of N-Carbamoyl-β-Alanine Amidohydrolase from Agrobacterium tumefaciens C58 for β-Amino Acid Production

An N-carbamoyl-β-alanine amidohydrolase of industrial interest from Agrobacterium tumefaciens C58 (βcar_At) has been characterized. βcar_At is most active at 30°C and pH 8.0 with N-carbamoyl-β-alanine as a substrate. The purified enzyme is completely inactivated by the metal-chelating agent 8-hydroxyquinoline-5-sulfonic acid (HQSA), and activity is restored by the addition of divalent metal ions, such as Mn²⁺, Ni²⁺, and Co²⁺. The native enzyme is a homodimer with a molecular mass of 90 kDa from pH 5.5 to 9.0. The enzyme has a broad substrate spectrum and hydrolyzes nonsubstituted N-carbamoyl-α-, -β-, -γ-, and -δ-amino acids, with the greatest catalytic efficiency for N-carbamoyl-β-alanine. βcar_At also recognizes substrate analogues substituted with sulfonic and phosphonic acid groups to produce the β-amino acids taurine and ciliatine, respectively. βcar_At is able to produce monosubstituted β²- and β³-amino acids, showing better catalytic efficiency (k_cat/K_m) for the production of the former. For both types of monosubstituted substrates, the enzyme hydrolyzes N-carbamoyl-β-amino acids with a short aliphatic side chain better than those with aromatic rings. These properties make βcar_At an outstanding candidate for application in the biotechnology industry.N-Carbamoyl-β-alanine amidohydrolase (NCβAA) (EC 3.5.1.6), also known as β-alanine synthase or β-ureidopropionase, catalyzes the third and final step of reductive pyrimidine degradation. In this reaction, N-carbamoyl-β-alanine or N-carbamoyl-β-aminoisobutyric acid is irreversibly hydrolyzed to CO₂, NH₃, and β-alanine or β-aminoisobutyric acid, respectively (43). Eukaryotic NCβAAs have been purified from several sources (10, 25, 33, 39, 42, 44). Nevertheless, only two prokaryotic NCβAAs, belonging to the Clostridium and Pseudomonas genera (4, 29), have been purified to date, although this activity has been inferred for several microorganisms due to the appearance of the reductive pathway of pyrimidine degradation (38, 45). Pseudomonas NCβAA is also able to hydrolyze l-N-carbamoyl-α-amino acids, and indeed, this activity is widespread in the bacterial kingdom (3, 23, 26, 46).β-Amino acids have unique pharmacological properties, and their utility as building blocks of β-peptides, pharmaceutical compounds, and natural products is of growing interest (14). β-Alanine, a natural β-amino acid, is a precursor of coenzyme A and pantothenic acid in bacteria and fungi (vitamin B₅) (7). β-Alanine is widely distributed in the central nervous systems of vertebrates and is a structural analogue of γ-amino-n-butyric acid and glycine, major inhibitory neurotransmitters, suggesting that it may be involved in synaptic transmissions (20). Another important natural β-amino acid is taurine (2-aminoethanesulfonic acid), which plays an important role in several essential processes, such as membrane stabilization, osmoregulation, glucose metabolism, antioxidation, and development of the central nervous system and the retina (9, 28, 33). 2-Aminoethylphosphonate, the most common naturally occurring phosphonate, also known as ciliatine, is an important precursor used in the biosynthesis of phosphonolipids, phosphonoproteins, and phosphonoglycans (5). β-Homoalanine (β-aminobutyric acid) has been used successfully for the design of nonnatural ligands for therapeutic application against autoimmune diseases such as rheumatoid arthritis, multiple sclerosis, or autoimmune uveitis (30). Substituted β-amino acids can be denominated β², β³, and β^2,3, depending on the position of the side chain(s) (R) on the amino acid skeleton (18). β²-Amino acids are not yet as readily available as their β³-counterparts, as they must be prepared using multistep procedures (17).We decided to characterize NCβAA (β-carbamoylase) from Agrobacterium tumefaciens C58 (βcar_At) after showing that some dihydropyrimidinases belonging to the Arthrobacter and Sinorhizobium genera are able to hydrolyze different 5- or 6-substituted dihydrouracils to the corresponding N-carbamoyl-β-amino acids (18, 22). If βcar_At could decarbamoylate the reaction products of dihydrouracils, different β-amino acids would be obtained enzymatically in the same way that α-amino acids are produced via the hydantoinase process (6, 21). We therefore describe the physical, biochemical, kinetic, and substrate specificity properties of recombinant βcar_At.     

Roles of Siderophores,Oxalate, and Ascorbate in Mobilization of Iron from Hematite by the Aerobic Bacterium Pseudomonas mendocina

In aerobic, circumneutral environments, the essential element Fe occurs primarily in scarcely soluble mineral forms. We examined the independent and combined effects of a siderophore, a reductant (ascorbate), and a low-molecular-weight carboxylic acid (oxalate) on acquisition of Fe from the mineral hematite (α-Fe₂O₃) by the obligate aerobe Pseudomonas mendocina ymp. A site-directed ΔpmhA mutant that was not capable of producing functional siderophores (i.e., siderophore⁻ phenotype) did not grow on hematite as the only Fe source. The concentration of an added exogenous siderophore (1 μM desferrioxamine B [DFO-B]) needed to restore wild-type (WT)-like growth kinetics to the siderophore⁻ strain was ∼50-fold less than the concentration of the siderophore secreted by the WT organism grown under the same conditions. The roles of a reductant (ascorbate) and a simple carboxylic acid (oxalate) in the Fe acquisition process were examined in the presence and absence of the siderophore. Addition of ascorbate (50 μM) alone restored the growth of the siderophore⁻ culture to the WT levels. A higher concentration of oxalate (100 μM) had little effect on the growth of a siderophore⁻ culture; however, addition of 0.1 μM DFO-B and 100 μM oxalate restored the growth of the mutant to WT levels when the oxalate was prereacted with the hematite, demonstrating that a metabolizing culture benefits from a synergistic effect of DFO-B and oxalate.Iron (Fe) is essential for almost all life. However, in aerobic, circumneutral environments, Fe is bound primarily in scarcely soluble minerals and amorphous solids [e.g., the solubility product (K_SP) for amorphous Fe(OH)₃ is 10⁻³⁸] (53) and is therefore poorly bioavailable. Aerobic microorganisms directly transform mineral-bound Fe(III) into soluble, highly bioavailable forms (1), overcoming significant kinetic and thermodynamic barriers to mineral dissolution and serving as primary transporters of Fe from the geosphere into global biogeochemical cycles.A primary means by which aerobic microorganisms enhance Fe mobility and bioavailability is by secreting siderophores, which are structurally diverse, low-molecular-weight chelating agents with extremely high affinities for Fe(III) (12, 27, 37, 40). Fe(III)-siderophore stability constants can be as high as 10⁵² (1, 40), which is many orders of magnitude higher than the stability constants for low-molecular-weight organic acids, such as oxalic acid [for Fe(III) + 3 oxalate ⇆ Fe(oxalate)₃, K = 10^18.6] (45). While their high affinity for Fe(III) is clearly important for helping siderophores mobilize Fe from Fe(III) (hydr)oxides in the aqueous phase, the mechanisms of Fe mobilization appear to be complex and are the subject of much recent study (14, 17, 18, 26, 28, 49). In particular, the role of siderophores in ligand-promoted dissolution mechanisms has undergone careful evaluation in vitro. The model is described simply here as follows for amorphous Fe(OH)₃ and has been described in detail by Kraemer (26): Fe(OH)₃ + 3H⁺ ⇆ Fe(III) + 3H₂O (K_SP) (equation 1); Fe(III) + H₃L ⇆ FeL + 3H⁺ (K_FeL) (equation 2); and Fe(OH)₃ + H₃L ⇆ FeL + 3H₂O (K_eq = K_SPK_FeL = [FeL]/[H₃L]) (equation 3). The concentration of the solubilized FeL complex, according to equation 3, is determined as follows: [FeL] = [H₃L]K_{SP ×} K_FeL. The estimated concentration of siderophores in carbonic soil (∼10⁻⁸ to 10⁻⁷ M), combined with their strong affinities for Fe(III) (39), suggests that [FeL] could in principle easily be micromolar or higher and could support vigorous bacterial growth. However, the trishydroxamate siderophores that have been studied most to date adsorb only weakly to Fe(III) (hydr)oxide minerals, likely due to steric constraints, although charge repulsion may also play a role for positively charged siderophores, such as desferrioxamine B (DFO-B) (6, 26, 41, 42). Therefore, it has been proposed that siderophores act primarily in conjunction with other molecules, such as simple plant-derived carboxylic acids or reductants, which interact more strongly with mineral surfaces and release Fe directly through ligand-promoted and/or reductive mechanisms (52). This proposed “synergistic effect,” in which the combined effect of various elements is greater than the sum of the individual effects, suggests that an interaction of biogenic molecules may overcome kinetic and thermodynamic barriers to the release of Fe from minerals in the presence of siderophores. The role of the siderophore in such a synergistic system is not a direct role in surface processes; rather, the siderophore maintains a low concentration of aqueous Fe in equilibrium with the mineral (an Fe sink), thus driving the reaction toward more dissolution (26, 41). Only a low concentration of a siderophore relative to the concentrations of surface-reacting organic species is required to promote efficient dissolution (26).The synergistic effect has been observed directly in in vitro, abiotic experiments using combinations of microbe-derived siderophores and simple organic acids. A combination of environmentally relevant concentrations of oxalate (1 to 80 μM) and DFO-B (40 μM), for example, doubled the rate of Fe(III) hydroxide mineral (goethite) dissolution compared with the rate when only oxalate or DFO-B was present in a recent in vitro study (6). Actively metabolizing aerobic bacteria, which can move Fe from solution into cells and recycle or release new siderophores back into the medium, might be expected to promote the synergistic siderophore-carboxylic acid interaction even further in a batch system. Likewise, it has been suggested that organic reductants may work synergistically with siderophores. In particular, a recent study showed that exogenously added reductants significantly enhance the bioavailability of Fe to an aerobic siderophore-producing bacterium, Pseudomonas mendocina ymp (15), isolated from the Nevada Test Site and used in the work described here.As an obligate aerobe, P. mendocina ymp does not have dissimilatory reduction pathways, so that its use of iron (hydr)oxide minerals is only for acquisition of nutritional Fe and not for cellular respiration. In contrast to dissimilatory Fe-reducing bacteria, which require millimolar concentrations of Fe (2, 29-31, 36, 43, 50), P. mendocina ymp requires micromolar concentrations (19, 20, 24, 32-34). Previously, this strain''s ability to dissolve and use various mineral forms of Fe was quantified in a series of microbial growth studies (23, 24, 32-34). P. mendocina ymp is known to produce hydroxamate-containing siderophores that increase the rate of dissolution of the Fe oxide mineral hematite. A recent study demonstrated that reductants significantly enhanced the bioavailability of Fe-(hydr)oxide minerals to P. mendocina (15). The ymp strain was also shown to have endogenous Fe(III)-reducing activity, which Hersman et al. suggested could be involved in solubilizing ferric minerals (24). Likewise, closely related strains of Pseudomonas stutzeri have been shown to produce pyridine-2,6-bis(thiocarboxylic acid) (PDTC), which they can use in the reduction, transport, and detoxification of metals and metalloids (11, 16, 51). However, control experiments showed that P. mendocina did not secrete molecules that exhibited a significant amount of reducing activity under the conditions used in this study (see the supplemental material). Notably, this species does not appear to contain a set of PDTC biosynthesis genes.In this work we used the wild-type (WT) strain P. mendocina ymp along with a mutant with a site-directed markerless mutation that was not capable of producing siderophores (ΔpmhA mutant with the siderophore⁻ phenotype) (3) in a series of experiments examining siderophore use and potential synergistic effects with either a simple carboxylic acid (oxalate) or an exogenous reductant (ascorbate). Both ascorbate and oxalate are plant products that are frequently found in the shallow subsurface; their effects on in vitro Fe (hydr)oxide dissolution have been well described (6).