Uncovering a Microbial Enigma: Isolation and Characterization of the Streamer-Generating,Iron-Oxidizing,Acidophilic Bacterium “Ferrovum myxofaciens”

A betaproteobacterium, shown by molecular techniques to have widespread global distribution in extremely acidic (pH 2 to 4) ferruginous mine waters and also to be a major component of “acid streamer” growths in mine-impacted water bodies, has proven to be recalcitrant to enrichment and isolation. A modified “overlay” solid medium was devised and used to isolate this bacterium from a number of mine water samples. The physiological and phylogenetic characteristics of a pure culture of an isolate from an abandoned copper mine (“Ferrovum myxofaciens” strain P3G) have been elucidated. “F. myxofaciens” is an extremely acidophilic, psychrotolerant obligate autotroph that appears to use only ferrous iron as an electron donor and oxygen as an electron acceptor. It appears to use the Calvin-Benson-Bassham pathway to fix CO₂ and is diazotrophic. It also produces copious amounts of extracellular polymeric materials that cause cells to attach to each other (and to form small streamer-like growth in vitro) and to different solid surfaces. “F. myxofaciens” can catalyze the oxidative dissolution of pyrite and, like many other acidophiles, is tolerant of many (cationic) transition metals. “F. myxofaciens” and related clone sequences form a monophyletic group within the Betaproteobacteria distantly related to classified orders, with genera of the family Nitrosomonadaceae (lithoautotrophic, ammonium-oxidizing neutrophiles) as the closest relatives. On the basis of the phylogenetic and phenotypic differences of “F. myxofaciens” and other Betaproteobacteria, a new family, “Ferrovaceae,” and order, “Ferrovales,” within the class Betaproteobacteria are proposed. “F. myxofaciens” is the first extreme acidophile to be described in the class Betaproteobacteria.     

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.     

Prodigiosin-like Pigments from Actinomadura (Nocardia) pelletieri and Actinomadura madurae

Thirteen red strains of Actinomadura (Nocardia) pelletieri and three of A. madurae were shown to produce prodigiosin-like pigments. Both of the two major pigments which were observed on thin-layer chromatograms had R_F values significantly greater than prodigiosin. The main pigment from A. madurae 953 was shown by mass and nuclear magnetic resonance spectroscopies to be nonylprodigiosin. The major pigment from A. pellitieri had a C₁₁H₂₂ side chain in a ring form, but it was distinctly different from metacycloprodigiosin. “Prodiginine” was proposed as a name for the invariant aromatic portion of the prodigiosin structure.     

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.     

Fluctuations, exchange processes, and water diffusion in aqueous protein systems: A study of bovine serum albumin by diverse NMR techniques

Experimental frequency, concentration, and temperature dependences of the deuteron relaxation times T₁ and T₂ of D₂O solutions of bovine serum albumin are reported and theoretically described in a closed form without formal parameters. Crucial processes of the theoretical concept are material exchange, translational diffusion of water molecules on the rugged surfaces of proteins, and tumbling of the macromolecules. It is also concluded that, apart from averaging of the relaxation rates in the diverse deuteron phases, material exchange contributes to transverse relaxation by exchange modulation of the Larmor frequency. The rate limiting factor of macromolecular tumbling is determined by the free water content. In a certain analogy to the classical free-volume theory, a “free-water-volume theory” is presented. There are two characteristic water mass fractions indicating the saturation of the hydration shells (C_s ≈ 0.3) and the onset of protein tumbling (C₀ ≈ 0.6). The existence of the translational degrees of freedom of water molecules in the hydration shells has been verified by direct measurement of the diffusion coefficient using an NMR field-gradient technique. The concentration and temperature dependences show phenomena indicating a percolation transition of clusters of free water. The threshold water content was found to be C_c^w ≈ 0.43.     

Spatial Distribution of Photosynthesis during Drought in Field-Grown and Acclimated and Nonacclimated Growth Chamber-Grown Cotton

Inhomogeneous photosynthetic activity has been reported to occur in drought-stressed leaves. In addition, it has been suggested that these water stress-induced nonuniformities in photosynthesis are caused by “patchy” stomatal closure and that the phenomenon may have created the illusion of a nonstomatal component to the inhibition of photosynthesis. Because these earlier studies were performed with nonacclimated growth chamber-grown plants, we sought to determine whether such “patches” existed in drought-treated, field-grown plants or in chamber-grown plants that had been acclimated to low leaf water potentials (ψ_leaf). Cotton (Gossypium hirsutum L.) was grown in the field and subjected to drought by withholding irrigation and rain from 24 d after planting. The distribution of photosynthesis, which may reflect the stomatal aperture distribution in a heterobaric species such as cotton, was assayed by autoradiography after briefly exposing attached leaves of field-grown plants to ¹⁴CO₂. A homogeneous distribution of radioactive photosynthate was evident even at the lowest ψ_leaf of −1.34 MPa. “Patchiness” could, however, be induced by uprooting the plant and allowing the shoot to air dry for 6 to 8 min. In parallel studies, growth chamber-grown plants were acclimated to drought by withholding irrigation for three 5-d drought cycles interspersed with irrigation. This drought acclimation lowered the ψ_leaf value at which control rates of photosynthesis could be sustained by approximately 0.7 MPa and was accompanied by a similar decline in the ψ_leaf at which patchiness first appeared. Photosynthetic inhomogeneities in chamber-grown plants that were visible during moderate water stress and ambient levels of CO₂ could be largely removed with elevated CO₂ levels (3000 μL L⁻¹), suggesting that they were stomatal in nature. However, advanced dehydration (less than approximately 2.0 MPa) resulted in “patches” that could not be so removed and were probably caused by nonstomatal factors. The demonstration that patches do not exist in drought-treated, field-grown cotton and that the presence of patches in chamber-grown plants can be altered by treatments that cause an acclimation of photosynthesis leads us to conclude that spatial heterogeneities in photosynthesis probably do not occur frequently under natural drought conditions.     

Short-Term Regulation of Nitrogenase Activity by NH4+ in Rhodobacter capsulatus: Multiple In Vivo Nitrogenase Responses to NH4+ Addition

The photosynthetic bacterium Rhodobacter capsulatus has been shown to carry out nitrogenase “switch-off,” a rapid, reversible inhibition of in vivo activity. Here, we demonstrate that highly nitrogen-limited cultures of both the wild-type strain and a draT draG mutant are capable of nitrogenase switch-off while moderately nitrogen-limited cultures show instead a “magnitude” response, with a decrease in in vivo nitrogenase activity that is proportional to the amount of added NH₄⁺.     

Heterogenous stomatal closure in response to leaf water deficits is not a universal phenomenon

The extent and occurrence of water stress-induced “patchy” CO₂ uptake across the surface of leaves was evaluated in a number of plant species. Leaves, while still attached to a plant, were illuminated and exposed to air containing [¹⁴C]CO₂ before autoradiographs were developed. Plant water deficits that caused leaf water potential depression to −1.1 megapascals during a 4-day period did result in heterogenous CO₂ assimilation patterns in bean (Phaseolus vulgaris). However, when the same level of stress was imposed more gradually (during 17 days), no patchy stomatal closure was evident. The patchy CO₂ assimilation pattern that occurs when bean plants are subjected to a rapidly imposed stress could induce artifacts in gas exchange studies such that an effect of stress on chloroplast metabolism is incorrectly deduced. This problem was characterized by examining the relationship between photosynthesis and internal [CO₂] in stressed bean leaves. When extent of heterogenous CO₂ uptake was estimated and accounted for, there appeared to be little difference in this relationship between control and stressed leaves. Subjecting spinach (Spinacea oleracea) plants to stress (leaf water potential depression to −1.5 megapascals) did not appear to cause patchy stomatal closure. Wheat (Triticum aestivum) plants also showed homogenous CO₂ assimilation patterns when stressed to a leaf water potential of −2.6 megapascals. It was concluded that water stress-induced patchy stomatal closure can occur to an extent that could influence the analysis of gas exchange studies. However, this phenomenon was not found to be a general response. Not all stress regimens will induce patchiness; nor will all plant species demonstrate this response to water deficits.     

Osmosensitivity of Sucrose Uptake by Immature Pea Cotyledons Disappears during Development

Sucrose uptake was studied in isolated, immature pea cotyledons (Pisum sativum L. cv Marzia) in relation to their developmental stage. During the developmental period examined the water content of the cotyledons decreased from ≈80% “stage 1” to ≈55% “stage 2”. When assayed in an isotonic medium (400 osmoles per cubic meter) the influx capacity per gram fresh weight for sucrose was almost constant during this developmental period. The influx could be analyzed into a saturable component (K_m ≈ 9 moles per cubic meter; V_max ≈ 150 nanomoles per minute per gram fresh weight) and an unsaturable component (k_i ≈ 0.5 nanomoles per minute per gram fresh weight [per mole per cubic meter]). Incubation in a hypotonic medium reduced the sucrose influx in stage 1 cotyledons, up to 80% reduction at 0 milliosmole (medium without mannitol), but had no effect on sucrose uptake by stage 2 cotyledons. Reduced uptake in a hypotonic medium (100 osmoles per cubic meter) could be attributed to a lowering of the V_max from 150 to 36 nanomoles per minute per gram fresh weight. During incubation of stage 1 cotyledons and stage 2-cotyledons in a hypotonic medium (200 osmoles per cubic meter) their volume increased by 16% and 5.6%, respectively, while the calculated turgor pressure increased from 0.2 to 0.6 megapascal for cotyledons of both developmental stages. Reduced sucrose influx in hypotonic medium, therefore, seems to be related to cell swelling (membrane stretching) rather than to increased turgor pressure.     

Resolving the Negative Potential Side (n-side) Water-accessible Proton Pathway of F-type ATP Synthase by Molecular Dynamics Simulations

The rotation of F₁F_o-ATP synthase is powered by the proton motive force across the energy-transducing membrane. The protein complex functions like a turbine; the proton flow drives the rotation of the c-ring of the transmembrane F_o domain, which is coupled to the ATP-producing F₁ domain. The hairpin-structured c-protomers transport the protons by reversible protonation/deprotonation of a conserved Asp/Glu at the outer transmembrane helix (TMH). An open question is the proton transfer pathway through the membrane at atomic resolution. The protons are thought to be transferred via two half-channels to and from the conserved cAsp/Glu in the middle of the membrane. By molecular dynamics simulations of c-ring structures in a lipid bilayer, we mapped a water channel as one of the half-channels. We also analyzed the suppressor mutant cP24D/E61G in which the functional carboxylate is shifted to the inner TMH of the c-protomers. Current models concentrating on the “locked” and “open” conformations of the conserved carboxylate side chain are unable to explain the molecular function of this mutant. Our molecular dynamics simulations revealed an extended water channel with additional water molecules bridging the distance of the outer to the inner TMH. We suggest that the geometry of the water channel is an important feature for the molecular function of the membrane part of F₁F_o-ATP synthase. The inclination of the proton pathway isolates the two half-channels and may contribute to a favorable clockwise rotation in ATP synthesis mode.     

Does Marriage Make Us Healthier? Inter-Country Comparative Evidence from China,Japan, and Korea

Objectives

This study focuses on East Asian countries and investigates the difference in the marriage premium on the health-marriage protection effect (MPE) between younger and older generations and the intra-couple education concordance effect (ECE) on the health of married individuals. This study used inter-country comparative data from China, Japan, and Korea.

Methods

This study focused on individuals (n = 7,938) in China, Japan, and Korea who were sampled from the 2010 East Asian Social Survey. To investigate MPE and ECE, four health indicators were utilized: a physical and mental components summary (PCS and MCS), self-rated health status (D_self), and happiness level (D_happy). Ordinary least squares regression was conducted by country- and gender-specific subsamples.

Results

We found that the MPE on PCS, MCS, and D_self was more significant for the older generation than for the younger generation in both China and Japan, whereas the results were inconclusive in Korea. With regard to the ECE on happiness (D_happy), for both men and women, couples tend to be happier when both the husband and the wife are well educated (“higher balanced marriage”) compared to couples with a lower level of educational achievement (“lower balanced marriage”). Significant benefits from a “higher balanced marriage” on MCS and D_self were observed for women only. In contrast, no statistically significant differences in health status were observed between “higher balanced marriage” couples and couples with different levels of educational achievements (“upward marriage” or “downward marriage”).

Conclusions

This study found that (1) the MPE was more significant for the older generation, and (2) the health gap, particularly the happiness gap, between higher- and lower-balanced married couples was significant. The inter-country comparative findings are useful to explain how the role of marriage (and therefore of family) on health has been diluted due to the progress of industrialization and modernization.     

On the Resistance to Transpiration of the Sites of Evaporation within the Leaf

The rates of transpiration from the upper and lower surfaces of leaves of Gossypium hirsutum, Xanthium strumarium, and Zea mays were compared with the rates at which helium diffused across those leaves. There was no evidence for effects of CO₂ concentration or rate of evaporation on the resistance to water loss from the evaporating surface (“resistance of the mesophyll wall to transpiration”) and no evidence for any significant wall resistance in turgid tissues. The possible existence of a wall resistance was also tested in leaves of Commelina communis and Tulipa gesneriana whose epidermis could be easily peeled. Only when an epidermis was removed from a leaf, evaporation from the mesophyll tissue declined. We conclude that under conditions relevant to studies of stomatal behavior, the water vapor pressure at the sites of evaporation is equal to the saturation vapor pressure.     

Temperature-dependent water and ion transport properties of barley and sorghum roots : I. Relationship to leaf growth

Root temperature strongly affects shoot growth, possibly via “nonhydraulic messengers” from root to shoot. In short-term studies with barley (Hordeum vulgare L.) and sorghum (Sorghum bicolor L.) seedlings, the optimum root temperatures for leaf expansion were 25° and 35°C, respectively. Hydraulic conductance (L_p) of both intact plants and detached exuding roots of barley increased with increasing root temperature to a high value at 25°C, remaining high with further warming. In sorghum, the L_p of intact plants and of detached roots peaked at 35°C. In both species, root temperature did not affect water potentials of the expanded leaf blade or the growing region despite marked changes in L_p. Extreme temperatures greatly decreased ion flux, particularly K⁺ and NO₃⁻, to the xylem of detached roots of both species. Removing external K⁺ did not alter short-term K⁺ flux to the xylem in sorghum but strongly inhibited flux at high temperature in barley, indicating differences in the sites of temperature effects. Leaf growth responses to root temperature, although apparently “uncoupled” from water transport properties, were correlated with ion fluxes. Studies of putative root messengers must take into account the possible role of ions.     

Kinetics of MDR Transport in Tumor-Initiating Cells

Multidrug resistance (MDR) driven by ABC (ATP binding cassette) membrane transporters is one of the major causes of treatment failure in human malignancy. MDR capacity is thought to be unevenly distributed among tumor cells, with higher capacity residing in tumor-initiating cells (TIC) (though opposite finding are occasionally reported). Functional evidence for enhanced MDR of TICs was previously provided using a “side population” assay. This assay estimates MDR capacity by a single parameter - cell’s ability to retain fluorescent MDR substrate, so that cells with high MDR capacity (“side population”) demonstrate low substrate retention. In the present work MDR in TICs was investigated in greater detail using a kinetic approach, which monitors MDR efflux from single cells. Analysis of kinetic traces obtained allowed for the estimation of both the velocity (V _max) and affinity (K _M) of MDR transport in single cells. In this way it was shown that activation of MDR in TICs occurs in two ways: through the increase of V _max in one fraction of cells, and through decrease of K _M in another fraction. In addition, kinetic data showed that heterogeneity of MDR parameters in TICs significantly exceeds that of bulk cells. Potential consequences of these findings for chemotherapy are discussed.     

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.     

Utilization of Exogenous Inorganic Carbon Species in Photosynthesis by Chlorella pyrenoidosa

The nature of the inorganic carbon utilized during photosynthesis by Chlorella pyrenoidosa was investigated using three experimental techniques (open gas analysis system with “artificial leaf” or “aqueous” chambers and O₂ electrode system) to measure carbon assimilation. Photosynthesis was studied as a function of pH and CO₂ concentration. The CO₂ concentration was inadequate to meet the requirements of photosynthesis only when HCO₃⁻ was added at high pH. Under all other conditions, the low and constant K_m (CO₂), in contrast to the highly variable K_m (HCO₃⁻), suggested that CO₂ was the major species utilized.     

Ethylene Production and Respiratory Behavior of the rin Tomato Mutant

Little or no change in ethylene or CO₂ production occurred in rin tomato mutant fruits monitored for up to 120 days after harvest. Of the abnormally ripening tomatoes investigated, including “Never ripe” (Nr Y a h, Nr c l₂ r), “Evergreen” (gf r) and “Green Flesh” (gf), only rin did not show a typical climacteric and ethylene rise.     

Rare Branched Fatty Acids Characterize the Lipid Composition of the Intra-Aerobic Methane Oxidizer “Candidatus Methylomirabilis oxyfera”

The recently described bacterium “Candidatus Methylomirabilis oxyfera” couples the oxidation of the important greenhouse gas methane to the reduction of nitrite. The ecological significance of “Ca. Methylomirabilis oxyfera” is still underexplored, as our ability to identify the presence of this bacterium is thus far limited to DNA-based techniques. Here, we investigated the lipid composition of “Ca. Methylomirabilis oxyfera” to identify new, gene-independent biomarkers for the environmental detection of this bacterium. Multiple “Ca. Methylomirabilis oxyfera” enrichment cultures were investigated. In all cultures, the lipid profile was dominated up to 46% by the fatty acid (FA) 10-methylhexadecanoic acid (10MeC_16:0). Furthermore, a unique FA was identified that has not been reported elsewhere: the monounsaturated 10-methylhexadecenoic acid with a double bond at the Δ7 position (10MeC_16:1Δ7), which comprised up to 10% of the total FA profile. We propose that the typical branched fatty acids 10MeC_16:0 and 10MeC_16:1Δ7 are key and characteristic components of the lipid profile of “Ca. Methylomirabilis oxyfera.” The successful detection of these fatty acids in a peatland from which one of the enrichment cultures originated supports the potential of these unique lipids as biomarkers for the process of nitrite-dependent methane oxidation in the environment.     

Carbon Monoxide Oxidation by Clostridium thermoaceticum and Clostridium formicoaceticum

Cultures of Clostridium formicoaceticum and C. thermoaceticum growing on fructose and glucose, respectively, were shown to rapidly oxidize CO to CO₂. Rates up to 0.4 μmol min⁻¹ mg of wet cells⁻¹ were observed. Carbon monoxide oxidation by cell suspensions was found (i) to be dependent on pyruvate, (ii) to be inhibited by alkyl halides and arsenate, and (iii) to stimulate CO₂ reduction to acetate. Cell extracts catalyzed the oxidation of carbon monoxide with methyl viologen at specific rates up to 10 μmol min⁻¹ mg of protein⁻¹ (35°C, pH 7.2). Nicotinamide adenine dinucleotide, nicotinamide adenine dinucleotide phosphate and ferredoxin from C. pasteurianum were ineffective as electron acceptors. The catalytic mechanism of carbon monoxide oxidation was “ping-pong,” indicating that the enzyme catalyzing carbon monoxide oxidation can be present in an oxidized and a reduced form. The oxidized form was shown to react reversibly with cyanide, and the reduced form was shown to react reversibly with alkyl halides: cyanide inactivated the enzyme only in the absence of carbon monoxide, and alkyl halides inactivated it only in the presence of carbon monoxide. Extracts inactivated by alkyl halides were reactivated by photolysis. The findings are interpreted to indicate that carbon monoxide oxidation in the two bacteria is catalyzed by a corrinoid enzyme and that in vivo the reaction is coupled with the reduction of CO₂ to acetate. Cultures of C. acidi-urici and C. cylindrosporum growing on hypoxanthine were found not to oxidize CO, indicating that clostridia mediating a corrinoid-independent total synthesis of acetate from CO₂ do not possess a CO-oxidizing system.