Effects of glycine hydroxamate, carbon dioxide, and oxygen on photorespiratory carbon and nitrogen metabolism in spinach mesophyll cells

The effects of added glycine hydroxamate on the photosynthetic incorporation of ¹⁴CO₂ into metabolites by isolated mesophyll cells of spinach (Spinacia oleracea L.) was investigated under conditions favorable to photorespiratory (PR) metabolism (0.04% CO₂ and 20% O₂) and under conditions leading to nonphotorespiratory (NPR) metabolism (0.2% CO₂ and 2.7% O₂). Glycine hydroxamate (GH) is a competitive inhibitor of the photorespiratory conversion of glycine to serine, CO₂ and NH₄⁺. During PR fixation, addition of the inhibitor increased glycine and decreased glutamine labeling. In contrast, labeling of glycine decreased under NPR conditions. This suggests that when the rate of glycolate synthesis is slow, the primary route of glycine synthesis is through serine rather than from glycolate. GH addition increased serine labeling under PR conditions but not under NPR conditions. This increase in serine labeling at a time when glycine to serine conversion is partially blocked by the inhibitor may be due to serine accumulation via the “reverse” flow of photorespiration from 3-P-glycerate to hydroxypyruvate when glycine levels are high. GH increased glyoxylate and decreased glycolate labeling. These observations are discussed with respect to possible glyoxylate feedback inhibition of photorespiration.     

Localization and In Situ Activities of Homoacetogenic Bacteria in the Highly Compartmentalized Hindgut of Soil-Feeding Higher Termites (Cubitermes spp.)

Methanogenesis and homoacetogenesis occur simultaneously in the hindguts of almost all termites, but the reasons for the apparent predominance of methanogenesis over homoacetogenesis in the hindgut of the humivorous species is not known. We found that in gut homogenates of soil-feeding Cubitermes spp., methanogens outcompete homoacetogens for endogenous reductant. The rates of methanogenesis were always significantly higher than those of reductive acetogenesis, whereas the stimulation of acetogenesis by the addition of exogenous H₂ or formate was more pronounced than that of methanogenesis. In a companion paper, we reported that the anterior gut regions of Cubitermes spp. accumulated hydrogen to high partial pressures, whereas H₂ was always below the detection limit (<100 Pa) in the posterior hindgut, and that all hindgut compartments turned into efficient H₂ sinks when external H₂ was provided (D. Schmitt-Wagner and A. Brune, Appl. Environ. Microbiol. 65:4490–4496, 1999). Using a microinjection technique, we found that only the posterior gut sections P3/4a and P4b, which harbored methanogenic activities, formed labeled acetate from H¹⁴CO₃⁻. Enumeration of methanogenic and homoacetogenic populations in the different gut sections confirmed the coexistence of both metabolic groups in the same compartments. However, the in situ rates of acetogenesis were strongly hydrogen limited; in the P4b section, no activity was detected unless external H₂ was added. Endogenous rates of reductive acetogenesis in isolated guts were about 10-fold lower than the in vivo rates of methanogenesis, but were almost equal when exogenous H₂ was supplied. We conclude that the homoacetogenic populations in the posterior hindgut are supported by either substrates other than H₂ or by a cross-epithelial H₂ transfer from the anterior gut regions, which may create microniches favorable for H₂-dependent acetogenesis.     

Histochemical Compartmentation of Photosynthesis in the Crassulacean Acid Metabolism Plant Crassula falcata

The succulent leaf of the obligate Crassulacean acid metabolism plant Crassula falcata comprises two distinct types of parenchyma. The peripheral tissue is dark green, whereas the central tissue is relatively colorless. We have investigated whether the conventional interpretation of Crassulacean acid metabolism—simply, temporal separation of light and dark CO₂ fixation within individual cells—is sufficient. Ribulose-1,5-bisphosphate carboxylase/oxygenase (EC 4.1.1.39) and chlorophyll, indicating the photosynthetic-carbon-reduction pathway, were concentrated in peripheral tissue. Specific activities of P-enolpyruvate carboxylase (4.1.1.31) and of NAD⁺-malic enzyme (1.1.1.39), indicating capacity for dark CO₂ fixation and release, respectively, were high in both types of parenchyma. Measured directly as malic acid decline at the beginning of the photoperiod, CO₂ “storage” occurred in both tissues. These data indicate that there is a spatial component to Crassulacean acid metabolism in C. falcata.     

Coupling of Methanothermobacter thermautotrophicus Methane Formation and Growth in Fed-Batch and Continuous Cultures under Different H2 Gassing Regimens

In nature, H₂- and CO₂-utilizing methanogenic archaea have to couple the processes of methanogenesis and autotrophic growth under highly variable conditions with respect to the supply and concentration of their energy source, hydrogen. To study the hydrogen-dependent coupling between methanogenesis and growth, Methanothermobacter thermautotrophicus was cultured in a fed-batch fermentor and in a chemostat under different 80% H₂-20% CO₂ gassing regimens while we continuously monitored the dissolved hydrogen partial pressures (p_H2). In the fed-batch system, in which the conditions continuously changed the uptake rates by the growing biomass, the organism displayed a complex and yet defined growth behavior, comprising the consecutive lag, exponential, and linear growth phases. It was found that the in situ hydrogen concentration affected the coupling between methanogenesis and growth in at least two respects. (i) The microorganism could adopt two distinct theoretical maximal growth yields (Y_{CH4 max}), notably approximately 3 and 7 g (dry weight) of methane formed mol⁻¹, for growth under low (p_H2 < 12 kPa)- and high-hydrogen conditions, respectively. The distinct values can be understood from a theoretical analysis of the process of methanogenesis presented in the supplemental material associated with this study. (ii) The in situ hydrogen concentration affected the “specific maintenance” requirements or, more likely, the degree of proton leakage and proton slippage processes. At low p_H2 values, the “specific maintenance” diminished and the specific growth yields approached Y_{CH4 max}, indicating that growth and methanogenesis became fully coupled.     

Candidatus Eremiobacterota,a metabolically and phylogenetically diverse terrestrial phylum with acid-tolerant adaptations

Candidatus phylum Eremiobacterota (formerly WPS-2) is an as-yet-uncultured bacterial clade that takes its name from Ca. Eremiobacter, an Antarctic soil aerobe proposed to be capable of a novel form of chemolithoautotrophy termed atmospheric chemosynthesis, that uses the energy derived from atmospheric H₂-oxidation to fix CO₂ through the Calvin-Benson-Bassham (CBB) cycle via type 1E RuBisCO. To elucidate the phylogenetic affiliation and metabolic capacities of Ca. Eremiobacterota, we analysed 63 public metagenome-assembled genomes (MAGs) and nine new MAGs generated from Antarctic soil metagenomes. These MAGs represent both recognized classes within Ca. Eremiobacterota, namely Ca. Eremiobacteria and UBP9. Ca. Eremiobacteria are inferred to be facultatively acidophilic with a preference for peptides and amino acids as nutrient sources. Epifluorescence microscopy revealed Ca. Eremiobacteria cells from Antarctica desert soil to be coccoid in shape. Two orders are recognized within class Ca. Eremiobacteria: Ca. Eremiobacterales and Ca. Baltobacterales. The latter are metabolically versatile, with individual members having genes required for trace gas driven autotrophy, anoxygenic photosynthesis, CO oxidation, and anaerobic respiration. UBP9, here renamed Ca. Xenobia class. nov., are inferred to be obligate heterotrophs with acidophilic adaptations, but individual members having highly divergent metabolic capacities compared to Ca. Eremiobacteria, especially with regard to respiration and central carbon metabolism. We conclude Ca. Eremiobacterota to be an ecologically versatile phylum with the potential to thrive under an array of “extreme” environmental conditions.Subject terms: Soil microbiology, Microbial ecology     

New Mode of Energy Metabolism in the Seventh Order of Methanogens as Revealed by Comparative Genome Analysis of “Candidatus Methanoplasma termitum”

The recently discovered seventh order of methanogens, the Methanomassiliicoccales (previously referred to as “Methanoplasmatales”), so far consists exclusively of obligately hydrogen-dependent methylotrophs. We sequenced the complete genome of “Candidatus Methanoplasma termitum” from a highly enriched culture obtained from the intestinal tract of termites and compared it with the previously published genomes of three other strains from the human gut, including the first isolate of the order. Like all other strains, “Ca. Methanoplasma termitum” lacks the entire pathway for CO₂ reduction to methyl coenzyme M and produces methane by hydrogen-dependent reduction of methanol or methylamines, which is consistent with additional physiological data. However, the shared absence of cytochromes and an energy-converting hydrogenase for the reoxidation of the ferredoxin produced by the soluble heterodisulfide reductase indicates that Methanomassiliicoccales employ a new mode of energy metabolism, which differs from that proposed for the obligately methylotrophic Methanosphaera stadtmanae. Instead, all strains possess a novel complex that is related to the F₄₂₀:methanophenazine oxidoreductase (Fpo) of Methanosarcinales but lacks an F₄₂₀-oxidizing module, resembling the apparently ferredoxin-dependent Fpo-like homolog in Methanosaeta thermophila. Since all Methanomassiliicoccales also lack the subunit E of the membrane-bound heterodisulfide reductase (HdrDE), we propose that the Fpo-like complex interacts directly with subunit D, forming an energy-converting ferredoxin:heterodisulfide oxidoreductase. The dual function of heterodisulfide in Methanomassiliicoccales, which serves both in electron bifurcation and as terminal acceptor in a membrane-associated redox process, may be a unique characteristic of the novel order.     

A Study of Formate Production and Oxidation in Leaf Peroxisomes during Photorespiration

When glycolate was metabolized in peroxisomes isolated from leaves of spinach beet (Beta vulgaris L., var. vulgaris) formate was produced. Although the reaction mixture contained glutamate to facilitate conversion of glycolate to glycine, the rate at which H₂O₂ became “available” during the oxidation of [1-¹⁴C]glycolate was sufficient to account for the breakdown of the intermediate [1-¹⁴C]glyoxylate to formate (C₁ unit) and ¹⁴CO₂. Under aerobic conditions formate production closely paralleled ¹⁴CO₂ release from [1-¹⁴C]glycolate which was optimal between pH 8.0 and pH 9.0 and was increased 3-fold when the temperature was raised from 25 to 35 C, or when the rate of H₂O₂ production was increased artificially by addition of an active preparation of fungal glucose oxidase.     

Characterization and In Situ Carbon Metabolism of Phototrophic Consortia

A dense population of the phototrophic consortium “Pelochromatium roseum” was investigated in the chemocline of a temperate holomictic lake (Lake Dagow, Brandenburg, Germany). Fluorescence in situ hybridization revealed that the brown epibionts of “P. roseum” constituted up to 37% of the total bacterial cell number and up to 88% of all green sulfur bacteria present in the chemocline. Specific amplification of 16S rRNA gene fragments of green sulfur bacteria and denaturing gradient gel electrophoresis fingerprinting yielded a maximum of four different DNA bands depending on the year of study, indicating that the diversity of green sulfur bacteria was low. The 465-bp 16S rRNA gene sequence of the epibiont of “P. roseum” was obtained after sorting of individual consortia by micromanipulation, followed by a highly sensitive PCR. The sequence obtained represents a new phylotype within the radiation of green sulfur bacteria. Maximum light-dependent H¹⁴CO₃⁻ fixation in the chemocline in the presence of 3-(3,4-dichlorophenyl)-1,1-dimethylurea suggested that there was anaerobic autotrophic growth of the green sulfur bacteria. The metabolism of the epibionts was further studied by determining stable carbon isotope ratios (δ¹³C) of their specific biomarkers. Analysis of photosynthetic pigments by high-performance liquid chromatography revealed the presence of high concentrations of bacteriochlorophyll (BChl) e and smaller amounts of BChl a and d and chlorophyll a in the chemocline. Unexpectedly, isorenieratene and β-isorenieratene, carotenoids typical of other brown members of the green sulfur bacteria, were absent. Instead, four different esterifying alcohols of BChl e were isolated as biomarkers of green sulfur bacterial epibionts, and their δ¹³C values were determined. Farnesol, tetradecanol, hexadecanol, and hexadecenol all were significantly enriched in ¹³C compared to bulk dissolved and particulate organic carbon and compared to the biomarkers of purple sulfur bacteria. The difference between the δ¹³C values of farnesol, the major esterifying alcohol of BChl e, and CO₂ was −7.1%, which provides clear evidence that the mode of growth of the green sulfur bacterial epibionts of “P. roseum” in situ is photoautotrophic.     

Coupling of Carbon Dioxide Fixation to the Oxyhydrogen Reaction in the Isolated Chloroplast of Chlamydomonas reinhardtii

The oxyhydrogen reaction (the reduction of O₂ to water by H₂) in the presence of CO₂ was studied in the isolated Chlamydomonas reinhardtii chloroplast by monitoring the rate of ¹⁴CO₂ incorporation into acid-stable products in the dark. The endogenous rate of CO₂ uptake (50-125 nmol/mg chlorophyll per h) was increased about 3- to 4-fold by ATP and additionally when combined with glucose, ribose-5-phosphate, and glycerate-3-phosphate. The rate was diminished 50 to 75%, respectively, when H₂ was replaced by N₂ or by air. Decrease in CO₂ uptake by dl-glyceraldehyde was taken to indicate that the regenerative phase and complete Calvin cycle turnover were involved. Diminution of CO₂ incorporation by rotenone, antimycin A, and 2,5-dibromo-3-methyl-6-isopropanol-p-benzoquinone was attributed to an inhibition of the oxyhydrogen reaction, resulting in an elevated NADPH/NADP ratio. If so, then the diminished CO₂ uptake could have been by “product inhibition” of the carbon metabolic network. Our data are consistent with the proposal (H. Gaffron [1942] J Gen Physiol 26: 241-267) that CO₂ fixation coupled to the oxyhydrogen reaction is dependent to some extent on exchloroplastic metabolism. This support is primarily ATP provided by mitochondrial respiration.     

XoxF-Type Methanol Dehydrogenase from the Anaerobic Methanotroph “Candidatus Methylomirabilis oxyfera”

“Candidatus Methylomirabilis oxyfera” is a newly discovered anaerobic methanotroph that, surprisingly, oxidizes methane through an aerobic methane oxidation pathway. The second step in this aerobic pathway is the oxidation of methanol. In Gram-negative bacteria, the reaction is catalyzed by pyrroloquinoline quinone (PQQ)-dependent methanol dehydrogenase (MDH). The genome of “Ca. Methylomirabilis oxyfera” putatively encodes three different MDHs that are localized in one large gene cluster: one so-called MxaFI-type MDH and two XoxF-type MDHs (XoxF1 and XoxF2). MxaFI MDHs represent the canonical enzymes, which are composed of two PQQ-containing large (α) subunits (MxaF) and two small (β) subunits (MxaI). XoxF MDHs are novel, ecologically widespread, but poorly investigated types of MDHs that can be phylogenetically divided into at least five different clades. The XoxF MDHs described thus far are homodimeric proteins containing a large subunit only. Here, we purified a heterotetrameric MDH from “Ca. Methylomirabilis oxyfera” that consisted of two XoxF and two MxaI subunits. The enzyme was localized in the periplasm of “Ca. Methylomirabilis oxyfera” cells and catalyzed methanol oxidation with appreciable specific activity and affinity (V_max of 10 μmol min⁻¹ mg⁻¹ protein, K_m of 17 μM). PQQ was present as the prosthetic group, which has to be taken up from the environment since the known gene inventory required for the synthesis of this cofactor is lacking. The MDH from “Ca. Methylomirabilis oxyfera” is the first representative of type 1 XoxF proteins to be described.     

Isotope Labeling and Microautoradiography of Active Heterotrophic Bacteria on the Basis of Assimilation of 14CO2

Most heterotrophic bacteria assimilate CO₂ in various carboxylation reactions during biosynthesis. In this study, assimilation of ¹⁴CO₂ by heterotrophic bacteria was used for isotope labeling of active microorganisms in pure cultures and environmental samples. Labeled cells were visualized by microautoradiography (MAR) combined with fluorescence in situ hybridization (FISH) to obtain simultaneous information about activity and identity. Cultures of Escherichia coli and Pseudomonas putida assimilated sufficient ¹⁴CO₂ during growth on various organic substrates to obtain positive MAR signals. The MAR signals were comparable with the traditional MAR approach based on uptake of ¹⁴C-labeled organic substrates. Experiments with E. coli showed that ¹⁴CO₂ was assimilated during both fermentation and aerobic and anaerobic respiration. The new MAR approach, HetCO₂-MAR, was evaluated by targeting metabolic active filamentous bacteria, including “Candidatus Microthrix parvicella” in activated sludge. “Ca. Microthrix parvicella” was able to take up oleic acid under anaerobic conditions, as shown by the traditional MAR approach with [¹⁴C]oleic acid. However, the new HetCO₂-MAR approach indicated that “Ca. Microthrix parvicella,” did not significantly grow on oleic acid under anaerobic conditions with or without addition of NO₂⁻, whereas the addition of O₂ or NO₃⁻ initiated growth, as indicated by detectable ¹⁴CO₂ assimilation. This is a metabolic feature that has not been described previously for filamentous bacteria. Such information could not have been derived by using the traditional MAR procedure, whereas the new HetCO₂-MAR approach differentiates better between substrate uptake and substrate metabolism that result in growth. The HetCO₂-MAR results were supported by stable isotope analysis of ¹³C-labeled phospholipid fatty acids from activated sludge incubated under aerobic and anaerobic conditions in the presence of ¹³CO₂. In conclusion, the novel HetCO₂-MAR approach expands the possibility for studies of the ecophysiology of uncultivated microorganisms.     

Ascorbate–glutathione pathways mediated by cytokinin regulate H2O2 levels in light-controlled rose bud burst

Rosebush (Rosa “Radrazz”) plants are an excellent model to study light control of bud outgrowth since bud outgrowth only arises in the presence of light and never occurs in darkness. Recently, we demonstrated high levels of hydrogen peroxide (H₂O₂) present in the quiescent axillary buds strongly repress the outgrowth process. In light, the outgrowing process occurred after H₂O₂ scavenging through the promotion of Ascorbic acid–Glutathione (AsA–GSH)-dependent pathways and the continuous decrease in H₂O₂ production. Here we showed Respiratory Burst Oxidase Homologs expression decreased in buds during the outgrowth process in light. In continuous darkness, the same decrease was observed although H₂O₂ remained at high levels in axillary buds, as a consequence of the strong inhibition of AsA–GSH cycle and GSH synthesis preventing the outgrowth process. Cytokinin (CK) application can evoke bud outgrowth in light as well as in continuous darkness. Furthermore, CKs are the initial targets of light in the photocontrol process. We showed CK application to cultured buds in darkness decreases bud H₂O₂ to a level that is similar to that observed in light. Furthermore, this treatment restores GSH levels and engages bud burst. We treated plants with buthionine sulfoximine, an inhibitor of GSH synthesis, to solve the sequence of events involving H₂O₂/GSH metabolisms in the photocontrol process. This treatment prevented bud burst, even in the presence of CK, suggesting the sequence of actions starts with the positive CK effect on GSH that in turn stimulates H₂O₂ scavenging, resulting in initiation of bud outgrowth.

Light-induced bud outgrowth in rosebush results from cytokinin-mediated peroxide scavenging and glutathione metabolism stimulation.     

Metagenomic insights into the metabolism and evolution of a new Thermoplasmata order (Candidatus Gimiplasmatales)

Thermoplasmata is a widely distributed and ecologically important archaeal class in the phylum Euryarchaeota. Because few cultures and genomes are available, uncharacterized Thermoplasmata metabolisms remain unexplored. In this study, we obtained four medium- to high-quality archaeal metagenome-assembled genomes (MAGs) from the filamentous fragments of black-odorous aquatic sediments (Foshan, Guangdong, China). Based on their 16S rRNA gene and ribosomal protein phylogenies, the four MAGs belong to the previously unnamed Thermoplasmata UBA10834 clade. We propose that this clade (five reference genomes from the Genome Taxonomy Database (GTDB) and four MAGs from this study) be considered a new order, Candidatus Gimiplasmatales. Metabolic pathway reconstructions indicated that the Ca. Gimiplasmatales MAGs can biosynthesize isoprenoids and nucleotides de novo. Additionally, some taxa have genes for formaldehyde and acetate assimilation, and the Wood–Ljungdahl CO₂-fixation pathway, indicating a mixotrophic lifestyle. Sulfur reduction, hydrogen metabolism, and arsenic detoxification pathways were predicted, indicating sulfur-, hydrogen-, and arsenic-transformation potentials. Comparative genomics indicated that the H₄F Wood–Ljungdahl pathway of both Ca. Gimiplasmatales and Methanomassiliicoccales was likely obtained by the interdomain lateral gene transfer from the Firmicutes. Collectively, this study elucidates the taxonomic and potential metabolic diversity of the new order Ca. Gimiplasmatales and the evolution of this subgroup and its sister lineage Methanomassiliicoccales.     

Characterization and Partial Purification of Aldose-6-phosphate Reductase (Alditol-6-Phosphate:NADP 1-Oxidoreductase) from Apple Leaves

The Pereskia are morphologically primitive, leafed members of the Cactaceae. Gas exchange characteristics using a dual isotope porometer to monitor ¹⁴CO₂ and tritiated water uptake, diurnal malic acid fluctuations, phosphoenolpyruvate carboxylase, and malate dehydrogenase activities were examined in two species of the genus Pereskia, Pereskia grandifolia and Pereskia aculeata. Investigations were done on well watered (control) and water-stressed plants. Nonstressed plants showed a CO₂ uptake pattern indicating C₃ carbon metabolism. However, diurnal fluctuations in titratable acidity were observed similar to Crassulacean acid metabolism. Plants exposed to 10 days of water stress exhibited stomatal opening only during an early morning period. Titratable acidity, phosphoenolpyruvate carboxylase activity, and malate dehydrogenase activity fluctuations were magnified in the stressed plants, but showed the same diurnal pattern as controls. Water stress causes these cacti to shift to an internal CO₂ recycling (“idling”) that has all attributes of Crassulacean acid metabolism except nocturnal stomata opening and CO₂ uptake. The consequences of this shift, which has been observed in other succulents, are unknown, and some possibilities are suggested.     

Responses of succulents to plant water stress

Experiments were performed to test the hypothesis that succulents “shift” their method of photosynthetic metabolism in response to environmental change. Our data showed that there were at least three different responses of succulents to plant water status. When plant water status of Portulacaria afra (L.) Jacq. was lowered either by withholding water or by irrigating with 2% NaCl, a change from C₃-photosynthesis to Crassulacean acid metabolism (CAM) occurred. Fluctuation of titratable acidity and nocturnal CO₂ uptake was induced in the stressed plants. Stressed Peperomia obtusifolia A. Dietr. plants showed a change from C₃-photosynthesis to internal cycling of CO₂. Acid fluctuation commenced in response to stress but exogenous CO₂ uptake did not occur. Zygocactus truncatus Haworth plants showed a pattern of acid fluctuation and nocturnal CO₂ uptake typical of CAM even when well irrigated. The cacti converted from CAM to an internal CO₂ cycle similar to Peperomia when plants were water-stressed. Reverse phase gas exchange in succulents results in low water loss to carbon gain. Water is conserved and low levels of metabolic activity are maintained during drought periods by complete stomatal closure and continual fluctuation of organic acids.     

Enhancement of CO(2) Uptake in Avena Coleoptiles by Fusicoccin

When Avena coleoptile segments are immersed in a solution containing H¹⁴CO₃⁻, the appearance of label in the tissue is stimulated approximately 3-fold by fusicoccin application. This effect is rapid (1-2 minutes lag time), dependent upon respiratory energy, inhibited by carbonyl cyanide m-chlorophenylhydrazone, but not appreciably altered by cycloheximide treatment. A large percentage of the cellular radioactivity is found in the form of malate. Preliminary experiments indicate that CO₂, as opposed to HCO₃⁻, is the favored species of “CO₂” taken up by the segments. These results are consistent with the notion that CO₂, presumably by virtue of its fixation and conversion to malic acid, participates in the early events associated with fusicoccin-enhanced acidification of the cell wall region leading to the stimulation of cell extension growth.     

Intracellular Carbonic Anhydrase Activity Sensitizes Cancer Cell pH Signaling to Dynamic Changes in CO2 Partial Pressure

Carbonic anhydrase (CA) enzymes catalyze the chemical equilibration among CO₂, HCO₃⁻ and H⁺. Intracellular CA (CA_i) isoforms are present in certain types of cancer, and growing evidence suggests that low levels correlate with disease severity. However, their physiological role remains unclear. Cancer cell CA_i activity, measured as cytoplasmic CO₂ hydration rate (k_f), ranged from high in colorectal HCT116 (∼2 s⁻¹), bladder RT112 and colorectal HT29, moderate in fibrosarcoma HT1080 to negligible (i.e. spontaneous k_f = 0.18 s⁻¹) in cervical HeLa and breast MDA-MB-468 cells. CA_i activity in cells correlated with CAII immunoreactivity and enzymatic activity in membrane-free lysates, suggesting that soluble CAII is an important intracellular isoform. CA_i catalysis was not obligatory for supporting acid extrusion by H⁺ efflux or HCO₃⁻ influx, nor for maintaining intracellular pH (pH_i) uniformity. However, in the absence of CA_i activity, acid loading from a highly alkaline pH_i was rate-limited by HCO₃⁻ supply from spontaneous CO₂ hydration. In solid tumors, time-dependence of blood flow can result in fluctuations of CO₂ partial pressure (pCO₂) that disturb cytoplasmic CO₂-HCO₃⁻-H⁺ equilibrium. In cancer cells with high CA_i activity, extracellular pCO₂ fluctuations evoked faster and larger pH_i oscillations. Functionally, these resulted in larger pH-dependent intracellular [Ca²⁺] oscillations and stronger inhibition of the mTORC1 pathway reported by S6 kinase phosphorylation. In contrast, the pH_i of cells with low CA_i activity was less responsive to pCO₂ fluctuations. Such low pass filtering would “buffer” cancer cell pH_i from non-steady-state extracellular pCO₂. Thus, CA_i activity determines the coupling between pCO₂ (a function of tumor perfusion) and pH_i (a potent modulator of cancer cell physiology).     

Enzymatic and Genetic Characterization of Carbon and Energy Metabolisms by Deep-Sea Hydrothermal Chemolithoautotrophic Isolates of Epsilonproteobacteria

The carbon and energy metabolisms of a variety of cultured chemolithoautotrophic Epsilonproteobacteria from deep-sea hydrothermal environments were characterized by both enzymatic and genetic analyses. All the Epsilonproteobacteria tested had all three key reductive tricarboxylic acid (rTCA) cycle enzymatic activities—ATP-dependent citrate lyase, pyruvate:ferredoxin oxidoreductase, and 2-oxoglutarate:ferredoxin oxidoreductase—while they had no ribulose 1,5-bisphosphate carboxylase (RubisCO) activity, the key enzyme in the Calvin-Benson cycle. These results paralleled the successful amplification of the key rTCA cycle genes aclB, porAB, and oorAB and the lack of success at amplifying the form I and II RubisCO genes, cbbL and cbbM. The combination of enzymatic and genetic analyses demonstrates that the Epsilonproteobacteria tested use the rTCA cycle for carbon assimilation. The energy metabolisms of deep-sea Epsilonproteobacteria were also well specified by the enzymatic and genetic characterization: hydrogen-oxidizing strains had evident soluble acceptor:methyl viologen hydrogenase activity and hydrogen uptake hydrogenase genes (hyn operon), while sulfur-oxidizing strains lacked both the enzyme activity and the genes. Although the energy metabolism of reduced sulfur compounds was not genetically analyzed and was not fully clarified, sulfur-oxidizing Epsilonproteobacteria showed enzyme activity of a potential sulfite:acceptor oxidoreductase for a direct oxidation pathway to sulfate but no activity of AMP-dependent adenosine 5′-phosphate sulfate reductase for a indirect oxidation pathway. No activity of thiosulfate-oxidizing enzymes was detected. The enzymatic and genetic characteristics described here were consistent with cellular carbon and energy metabolisms and suggest that molecular tools may have great potential for in situ elucidation of the ecophysiological roles of deep-sea Epsilonproteobacteria.     

Role of Carbon Dioxide in Catabolism of Propane by “Nocardia paraffinicum” (Rhodococcus rhodochrous)

The catabolism of propane by “Nocardia paraffinicum” (Rhodococcus rhodochrous) has been shown to involve CO₂ fixation after its oxidation to propionic acid. “N. paraffinicum” failed to grow on either propane or 1-propanol in the absence of CO₂. The rate of propane utilization was directly related to the initial CO₂ concentration, and Warburg respirometry suggested that CO₂ was required for the catabolism of 1-propanol, propionaldehyde, and propionate but not for 2-propanol. These data also suggested that the predominant pathway for the utilization of propane by “N. paraffinicum” was through 1-propanol. The use of [2-¹⁴C]propane and ¹⁴CO₂ confirmed the catabolism of propane and the fixation of CO₂. Through the use of these isotopes and the pyruvate carboxylase inhibitor sodium arsenite, the labeled 2,4-dinitrophenylhydrazine derivative of pyruvate was trapped and isolated via thin-layer chromatography. The trapping of [¹⁴C]pyruvate in this manner was considered to be indicative of the presence of the methylmalonyl coenzyme A pathway for CO₂ fixation.     

Stable Carbon Isotopic Fractionations Associated with Inorganic Carbon Fixation by Anaerobic Ammonium-Oxidizing Bacteria

Isotopic analyses of Candidatus “Brocadia anammoxidans,” a chemolithoautotrophic bacterium that anaerobically oxidizes ammonium (anammox), show that it strongly fractionates against ¹³C; i.e., lipids are depleted by up to 47‰ versus CO₂. Similar results were obtained for the anammox bacterium Candidatus “Scalindua sorokinii,” which thrives in the anoxic water column of the Black Sea, suggesting that different anammox bacteria use identical carbon fixation pathways, which may be either the Calvin cycle or the acetyl coenzyme A pathway.