Nitrogen metabolism in haloarchaea

The nitrogen cycle (N-cycle), principally supported by prokaryotes, involves different redox reactions mainly focused on assimilatory purposes or respiratory processes for energy conservation. As the N-cycle has important environmental implications, this biogeochemical cycle has become a major research topic during the last few years. However, although N-cycle metabolic pathways have been studied extensively in Bacteria or Eukarya, relatively little is known in the Archaea. Halophilic Archaea are the predominant microorganisms in hot and hypersaline environments such as salted lakes, hot springs or salted ponds. Consequently, the denitrifying haloarchaea that sustain the nitrogen cycle under these conditions have emerged as an important target for research aimed at understanding microbial life in these extreme environments. The haloarchaeon Haloferax mediterranei was isolated 20 years ago from Santa Pola salted ponds (Alicante, Spain). It was described as a denitrifier and it is also able to grow using NO₃ ^-, NO₂ ^- or NH₄ ⁺ as inorganic nitrogen sources. This review summarizes the advances that have been made in understanding the N-cycle in halophilic archaea using Hfx mediterranei as a haloarchaeal model. The results obtained show that this microorganism could be very attractive for bioremediation applications in those areas where high salt, nitrate and nitrite concentrations are found in ground waters and soils.     

Coupling of Cellular Processes and Their Coordinated Oscillations under Continuous Light in Cyanothece sp. ATCC 51142, a Diazotrophic Unicellular Cyanobacterium

Unicellular diazotrophic cyanobacteria such as Cyanothece sp. ATCC 51142 (henceforth Cyanothece), temporally separate the oxygen sensitive nitrogen fixation from oxygen evolving photosynthesis not only under diurnal cycles (LD) but also in continuous light (LL). However, recent reports demonstrate that the oscillations in LL occur with a shorter cycle time of ~11 h. We find that indeed, majority of the genes oscillate in LL with this cycle time. Genes that are upregulated at a particular time of day under diurnal cycle also get upregulated at an equivalent metabolic phase under LL suggesting tight coupling of various cellular events with each other and with the cell’s metabolic status. A number of metabolic processes get upregulated in a coordinated fashion during the respiratory phase under LL including glycogen degradation, glycolysis, oxidative pentose phosphate pathway, and tricarboxylic acid cycle. These precede nitrogen fixation apparently to ensure sufficient energy and anoxic environment needed for the nitrogenase enzyme. Photosynthetic phase sees upregulation of photosystem II, carbonate transport, carbon concentrating mechanism, RuBisCO, glycogen synthesis and light harvesting antenna pigment biosynthesis. In Synechococcus elongates PCC 7942, a non-nitrogen fixing cyanobacteria, expression of a relatively smaller fraction of genes oscillates under LL condition with the major periodicity being 24 h. In contrast, the entire cellular machinery of Cyanothece orchestrates coordinated oscillation in anticipation of the ensuing metabolic phase in both LD and LL. These results may have important implications in understanding the timing of various cellular events and in engineering cyanobacteria for biofuel production.     

Adaptive capabilities of microorganisms of salt lakes of the Altai Region under conditions of early Mars

Adaptive capacity of bacteria and archaea from salt lakes of the Altai Region are discussed. It is established that halophilic archaea (genus Halorubrum) and halotolerant bacteria (genus Halomonas) grow in a wide range of pH and mineralization (in the presence of Cl^?, SO ₄ ^2? , ClO ₄ ^? , Mg²⁺) and survive at low temperatures with a minor decrease in viability.     

The methylaspartate cycle in haloarchaea and its possible role in carbon metabolism

Haloarchaea (class Halobacteria) live in extremely halophilic conditions and evolved many unique metabolic features, which help them to adapt to their environment. The methylaspartate cycle, an anaplerotic acetate assimilation pathway recently proposed for Haloarcula marismortui, is one of these special adaptations. In this cycle, acetyl-CoA is oxidized to glyoxylate via methylaspartate as a characteristic intermediate. The following glyoxylate condensation with another molecule of acetyl-CoA yields malate, a starting substrate for anabolism. The proposal of the functioning of the cycle was based mainly on in vitro data, leaving several open questions concerning the enzymology involved and the occurrence of the cycle in halophilic archaea. Using gene deletion mutants of H. hispanica, enzyme assays and metabolite analysis, we now close these gaps by unambiguous identification of the genes encoding all characteristic enzymes of the cycle. Based on these results, we were able to perform a solid study of the distribution of the methylaspartate cycle and the alternative acetate assimilation strategy, the glyoxylate cycle, among haloarchaea. We found that both of these cycles are evenly distributed in haloarchaea. Interestingly, 83% of the species using the methylaspartate cycle possess also the genes for polyhydroxyalkanoate biosynthesis, whereas only 34% of the species with the glyoxylate cycle are capable to synthesize this storage compound. This finding suggests that the methylaspartate cycle is shaped for polyhydroxyalkanoate utilization during carbon starvation, whereas the glyoxylate cycle is probably adapted for growth on substrates metabolized via acetyl-CoA.     

可培养盐碱菌多样性的研究进展

存在于高盐强碱极端环境的微生物因其独特的生命方式,引起了广泛的关注。根据盐碱环境所含的可溶性盐成分,可分为"NaCl型"和"苏打型(Na_2CO_3/NaHCO_3)"两大类,前者的碱性pH值较低而后者碱性pH值较高。本文总结了盐碱菌适宜生长条件在盐度0.5 mol/L和碱性pH 9.0之上且有效发表的标准菌株,并对这些菌株的生物多样性及生理特性进行了阐述;可培养盐碱细菌的数量及其多样性远远大于盐碱古菌,但是盐碱细菌对高盐度和强碱性p H依赖程度相对较低。盐碱细菌主要组成依次为芽孢杆菌纲(Bacilli,占总数约40%)、γ-变形菌纲(γ-Proteobacteria,30%)、梭菌纲(Clostridia,11%)、δ-变形菌纲(δ-Proteobacteria,6%)和放线菌纲(Actinobacteria,6%),而盐碱古菌主要组成为盐古菌纲(Halobacteria,92%)和甲烷微菌纲(Methanomicrobia,8%)。这些极端微生物在生物地球化学过程中或生态循环中扮演着重要的角色和功能,挖掘和利用盐碱菌具有重要意义。     

Diurnal Variations in Cycling Kinematics

Physiological and biomechanical constraints as well as their fluctuations throughout the day must be considered when studying determinant factors in the preferred pedaling rate of elite cyclists. The aim of this study was to monitor the diurnal variation of spontaneous pedaling rate and movement kinematics over the crank cycle. Twelve male competitive cyclists performed a submaximal exercise on a cycle ergometer for 15 min at 50% of their W_max. Two test sessions were performed at 06:00 and 18:00 h on two separate days to assess diurnal variation in the study variables. For each test session, the exercise bout was divided into three equivalent 5‐min periods during which subjects were requested to use different pedal rates (spontaneous cadence, 70 and 90 rev min^?1). Pedal rate and kinematics data (instantaneous pedal velocity and angle of the ankle) were collected. The results show a higher spontaneous pedal rate in the late afternoon than in the early morning (p < 0.001). For a given pedal rate condition, there was a less variation in pedal velocity during a crank cycle in the morning than in the late afternoon. Moreover, diurnal variations were observed in ankle mobility across the crank cycle, the mean plantar flexion observed throughout the crank cycle being greater in the 18:00 h test session (p < 0.001). These results suggest that muscular activation patterns during a cyclical movement could be under the influence of circadian fluctuations.     

Proteome Analyses of Strains ATCC 51142 and PCC 7822 of the Diazotrophic Cyanobacterium Cyanothece sp. under Culture Conditions Resulting in Enhanced H2 Production

Cultures of the cyanobacterial genus Cyanothece have been shown to produce high levels of biohydrogen. These strains are diazotrophic and undergo pronounced diurnal cycles when grown under N₂-fixing conditions in light-dark cycles. We seek to better understand the way in which proteins respond to these diurnal changes, and we performed quantitative proteome analysis of Cyanothece sp. strains ATCC 51142 and PCC 7822 grown under 8 different nutritional conditions. Nitrogenase expression was limited to N₂-fixing conditions, and in the absence of glycerol, nitrogenase gene expression was linked to the dark period. However, glycerol induced expression of nitrogenase during part of the light period, together with cytochrome c oxidase (Cox), glycogen phosphorylase (Glp), and glycolytic and pentose phosphate pathway (PPP) enzymes. This indicated that nitrogenase expression in the light was facilitated via higher levels of respiration and glycogen breakdown. Key enzymes of the Calvin cycle were inhibited in Cyanothece ATCC 51142 in the presence of glycerol under H₂-producing conditions, suggesting a competition between these sources of carbon. However, in Cyanothece PCC 7822, the Calvin cycle still played a role in cofactor recycling during H₂ production. Our data comprise the first comprehensive profiling of proteome changes in Cyanothece PCC 7822 and allow an in-depth comparative analysis of major physiological and biochemical processes that influence H₂ production in both strains. Our results revealed many previously uncharacterized proteins that may play a role in nitrogenase activity and in other metabolic pathways and may provide suitable targets for genetic manipulation that would lead to improvement of large-scale H₂ production.     

Relationship between a metabolic rhythm and emergence rhythm in Drosophila melanogaster

Oxygen consumption and lactic acid dehydrogenase (LDH) activity were determined for Drosophila melanogaster pupae and pharate adults exposed to 12 : 12 or 1 : 23 light-dark (LD) regime. Bimodal circadian fluctuations of oxygen consumption were found in pupae and pharate adults exposed to either LD regime and organisms appeared to demonstrate an anticipatory change in oxygen consumption associated with change in illumination. The oxygen-consumption trend for the entire period spent in the puparium showed a high at the time of emergence, but the diurnal rhythm showed a low at the time of emergence suggesting that emergence occurs at a low in the diurnal cycle. Emergence maximum showed a 3 hr lead over the oxygen-consumption maximum. Changing the LD regime produced similar changes in the phasing of both oxygen consumption and emergence rhythms. LDH activity did not demonstrate a detectable circadian rhythm but did show a steady decrease during pupal and pharate adult development.     

Isolation of extremely halophilic <Emphasis Type="Italic">Archaea</Emphasis> from a saline river in the Lut Desert of Iran,moderately resistant to desiccation and gamma radiation

Admittedly, the Lut Desert of Iran has been remained as an unexplored region from a microbiological standpoint. Domain Archaea contains extremophiles that can live in harsh habitats. Extremely halophilic archaea are exposed to different environmental stresses in the hypersaline environments such as high solar irradiance and periodic desiccation. Haloarchaeal diversity in Shoor River, a saline river in the Lut Desert (a salinity of 134.3 g L^–1 of dissolved salts), was investigated by a culture-dependent method. A large number of extremely halophilic isolates were obtained and a subset of 59 isolates was considered distinct. Firstly, the isolates were screened for their resistance under desiccation stress in 35 days. Eleven of these strains remained viable during the period in a desiccator containing silica gel. Then, three of them were randomly selected and their resistance against desiccation and ionizing radiation were determined. The isolates MS2, MS17, and MS50 were still recovered after 8 weeks in a desiccator and were moderately resistant to gamma radiation with D₁₀ value between 2 and 3 kGy. Strains MS2, MS17, and MS50 were affiliated with three species in the family Halobacteriaceae using 16S rRNA gene sequence analysis as well as morphological and biochemical characteristics—Haloterrigena jeotgali A29^T (99.6% similarity), Natrialba aegyptia 40^T (99.4% similarity) and Natrinema pallidum NCIMB 777^T (99.3% similarity), respectively. Although resistance to desiccation did not follow the sigmoid survival curve pattern of Deinococcus radiodurans, apparently haloarchaea can show a more resistance to desiccation in more long-term periods of time. This is the first report on isolation of extremely halophilic archaea belonged to the family Halobacteriaceae and their radioresistance and desiccation tolerance properties isolated from the Shoor River.     

Reduction State of Q and Nonradiative Energy Dissipation during Photosynthesis in Leaves of a Crassulacean Acid Metabolism Plant, Kalanchoë daigremontiana Hamet et Perr

Fluorescence was measured in leaves of the CAM plant Kalanchoë daigremontiana using a pulse modulation technique at room temperature. During a 12-h light period at 500 micromole photons per square meter per second (400-700 nanometers) in air containing 350 microbar CO₂, the component of fluorescence quenching related to the reduction state of Q, the primary electron transport acceptor of PSII, remained fairly constant and showed that only 20% of Q were in the reduced form. The reduction state was slightly increased at the onset and at the end of the light period. By contrast, the nonphotochemical component of fluorescence quenching which is a measure of the fraction of nonradiative deexcitation underwent marked diurnal changes. Nonradiative energy conversion was low during the phase of most active malic acid decarboxylation in the middle of the light period when uptake of atmospheric CO₂ was negligible, and when internal CO₂ partial pressures were higher than in air; this allowed for high rates of CO₂ reduction in the chloroplasts. Nonradiative energy conversion was high during the early and the late light period when atmospheric CO₂ was taken up and internal CO₂ partial pressures were below air level. Manipulation of the internal CO₂ partial pressure during the late light period by increasing or decreasing the external CO₂ partial pressure to 1710 and 105 microbar, respectively, led to changes in the magnitude of energy dependent fluorescence quenching which were consistent with the relationship between nonradiative energy dissipation and internal CO₂ partial pressure observed during the diurnal cycle. Again, the reduction state of Q was hardly affected by these treatments. Thus, changes in electron transport rate during the diurnal CAM cycle at a given photon flux density lead primarily to alterations in the rate of nonradiative energy dissipation, with the reduction state of Q being maintained at a relatively low and constant level. Conditions are described under which nonphotochemical dissipation of excitation energy reaches a maximum value and the reduction state of Q is increased.     

Expression in Haloferax volcanii of 3-Hydroxy-3-Methylglutaryl Coenzyme A Synthase Facilitates Isolation and Characterization of the Active Form of a Key Enzyme Required for Polyisoprenoid Cell Membrane Biosynthesis in Halophilic Archaea

Enzymes of the isoprenoid biosynthetic pathway in halophilic archaea remain poorly characterized, and parts of the pathway remain cryptic. This situation may be explained, in part, by the difficulty of expressing active, functional recombinant forms of these enzymes. The use of newly available expression plasmids and hosts has allowed the expression and isolation of catalytically active Haloferax volcanii 3-hydroxy-3-methylglutaryl coenzyme A (CoA) synthase (EC 2.3.310). This accomplishment has permitted studies that represent, to the best of our knowledge, the first characterization of an archaeal hydroxymethylglutaryl CoA synthase. Kinetic characterization indicates that, under optimal assay conditions, which include 4 M KCl, the enzyme exhibits catalytic efficiency and substrate saturation at metabolite levels comparable to those reported for the enzyme from nonhalophilic organisms. This enzyme is unique in that it is the first hydroxymethylglutaryl CoA synthase that is insensitive to feedback substrate inhibition by acetoacetyl-CoA. The enzyme supports reaction catalysis in the presence of various organic solvents. Haloferax 3-hydroxy-3-methylglutaryl CoA synthase is sensitive to inactivation by hymeglusin, a specific inhibitor known to affect prokaryotic and eukaryotic forms of the enzyme, with experimentally determined K_i and k_inact values of 570 ± 120 nM and 17 ± 3 min⁻¹, respectively. In in vivo experiments, hymeglusin blocks the propagation of H. volcanii cells, indicating the critical role that the mevalonate pathway plays in isoprenoid biosynthesis by these archaea     

Photosystem II Cyclic Heterogeneity and Photoactivation in the Diazotrophic, Unicellular Cyanobacterium Cyanothece Species ATCC 51142

The unicellular, diazotrophic cyanobacterium Cyanothece sp. ATCC 51142 demonstrated important modifications to photosystem II (PSII) centers when grown under light/dark N₂-fixing conditions. The properties of PSII were studied throughout the diurnal cycle using O₂-flash-yield and pulse-amplitude-modulated fluorescence techniques. Nonphotochemical quenching (q_N) of PSII increased during N₂ fixation and persisted after treatments known to induce transitions to state 1. The q_N was high in cells grown in the dark, and then disappeared progressively during the first 4 h of light growth. The photoactivation probability, ε, demonstrated interesting oscillations, with peaks near 3 h of darkness and 4 and 10 h of light. Experiments and calculations of the S-state distribution indicated that PSII displays a high level of heterogeneity, especially as the cells prepare for N₂ fixation. We conclude that the oxidizing side of PSII is strongly affected during the period before and after the peak of nitrogenase activity; changes include a lowered capacity for O₂ evolution, altered dark stability of PSII centers, and substantial changes in q_N.     

Diurnal changes in maize leaf photosynthesis : I. Carbon exchange rate, assimilate export rate, and enzyme activities

Diurnal changes in photosynthetic parameters and enzyme activities were characterized in greenhouse grown maize plants (Zea mays L. cv Pioneer 3184). Rates of net photosynthesis and assimilate export were highest at midday, coincident with maximum irradiance. During the day, assimilate export accounted for about 80% of net carbon fixation, and the maximum export rate (35 milligrams CH₂O per square decimeter per hour) was substantially higher than the relatively constant rate maintained through the night (5 milligrams CH₂O per square decimeter per hour). Activities of sucrose phosphate synthase and NADP-malate dehydrogenase showed pronounced diurnal fluctuations; maximum enzyme activities were generally coincident with highest light intensity. Reciprocal light/dark transfers of plants throughout the diurnal cycle revealed that both enzymes were deactivated by 30 minutes of darkness during the day, and they could both be substantially activated by 30 minutes of illumination at night. During 24 hours of extended darkness, sucrose phosphate synthase activity declined progressively to an almost undetectable level, but was activated after 1.5 hours of illumination. Thus, the diurnal fluctuation in maize sucrose phosphate synthase can be explained by some form of light modulation of enzyme activity and is not due to an endogenous rhythm in activity. No diurnal fluctuations were observed in the activities of NADP-malic enzyme or fructose 6-phosphate-2-kinase. Phosphoenolpyruvate carboxylase was activated by light to some extent (about 50%) when activity was measured under suboptimal conditions in vitro. The results suggested that the rates of sucrose formation and assimilate export were closely aligned with the rate of carbon fixation and the activation state of sucrose phosphate synthase.     

Presence of Acetyl Coenzyme A (CoA) Carboxylase and Propionyl-CoA Carboxylase in Autotrophic Crenarchaeota and Indication for Operation of a 3-Hydroxypropionate Cycle in Autotrophic Carbon Fixation

The pathway of autotrophic CO₂ fixation was studied in the phototrophic bacterium Chloroflexus aurantiacus and in the aerobic thermoacidophilic archaeon Metallosphaera sedula. In both organisms, none of the key enzymes of the reductive pentose phosphate cycle, the reductive citric acid cycle, and the reductive acetyl coenzyme A (acetyl-CoA) pathway were detectable. However, cells contained the biotin-dependent acetyl-CoA carboxylase and propionyl-CoA carboxylase as well as phosphoenolpyruvate carboxylase. The specific enzyme activities of the carboxylases were high enough to explain the autotrophic growth rate via the 3-hydroxypropionate cycle. Extracts catalyzed the CO₂-, MgATP-, and NADPH-dependent conversion of acetyl-CoA to 3-hydroxypropionate via malonyl-CoA and the conversion of this intermediate to succinate via propionyl-CoA. The labelled intermediates were detected in vitro with either ¹⁴CO₂ or [¹⁴C]acetyl-CoA as precursor. These reactions are part of the 3-hydroxypropionate cycle, the autotrophic pathway proposed for C. aurantiacus. The investigation was extended to the autotrophic archaea Sulfolobus metallicus and Acidianus infernus, which showed acetyl-CoA and propionyl-CoA carboxylase activities in extracts of autotrophically grown cells. Acetyl-CoA carboxylase activity is unexpected in archaea since they do not contain fatty acids in their membranes. These aerobic archaea, as well as C. aurantiacus, were screened for biotin-containing proteins by the avidin-peroxidase test. They contained large amounts of a small biotin-carrying protein, which is most likely part of the acetyl-CoA and propionyl-CoA carboxylases. Other archaea reported to use one of the other known autotrophic pathways lacked such small biotin-containing proteins. These findings suggest that the aerobic autotrophic archaea M. sedula, S. metallicus, and A. infernus use a yet-to-be-defined 3-hydroxypropionate cycle for their autotrophic growth. Acetyl-CoA carboxylase and propionyl-CoA carboxylase are proposed to be the main CO₂ fixation enzymes, and phosphoenolpyruvate carboxylase may have an anaplerotic function. The results also provide further support for the occurrence of the 3-hydroxypropionate cycle in C. aurantiacus.