Is the citric acid cycle aerobic or anaerobic? I know that the oxygen is required to accept the NADH electrons so that NAD+ could be regenerated. Nevertheless, if other electron acceptors, as nitrate (NO3-), are present, could they also be used to regenerate the NAD+? I am asking this because I read a thesis claiming that “acetly-CoA enters the citric acid cycle and through anaerobic nitrate-respiration a multiple amount of ATP is generated.” If you have some literature that you can share with me I would be really grateful. Thanks.
Yes, many microbes can use electron acceptors other than oxygen.
Microbial Fuel Cells as a Platform Technology for Sustainable Wastewater Treatment
18.5.2 Electron Acceptors
The electron acceptor contributes to overcome the potential losses existing on the cathode, thus it is one of the major factors influencing power generation in MFCs. The conditions of being a good electron acceptor comprises possessing a high redox potential, presenting fast kinetics, being economically valuable, and preferably having sustainability and easy availability  . Oxygen is one of most promising electron acceptors in MFCs  . However, with the rapid progress of MFC technology as well as a better understanding of its principle, there is a broad awareness that the cathode process is far more than just an oxygen reduction reaction (ORR). Various alternative electron acceptors, such as nitrate (NO3 − ), metal ions, perchlorate, nitrobenzene, and azo dyes, have been intensively explored to achieve bioremediation in MFCs  .
In order to increase the oxygen reduction kinetics and reduce cathodic activation overpotential, different kinds of catalysts have been used in the cathode  . Platinum offers the highest catalytic performance with increased oxygen affinity and reduced activation loss, and is the most commonly used catalyst for ORR  . Logan et al. demonstrated that Pt-based MFCs could achieve a fivefold increase in power output compared to a MFC with a plain carbon cathode  . Other catalysts that are cheaper and more sustainable, such as lead dioxide  , Fe/Fe2O3  , cobalt  , manganese dioxide  , or even activated charcoal  have also been explored for oxygen reduction reactions at the cathode of MFCs. Some studies also showed the possibility of utilizing metallic oxidants (e.g., U, Cd, Cr, Cu) that can be reduced to a less toxic oxidation state. The ORR remains one of the main bottlenecks of this technology, due to its high over potential and low kinetics that are encountered  .
Biocathodes represent an innovative approach to produce sustainable cathodes using microbes as catalysts to facilitate electrochemical reduction on the cathode surface. The biocathodes eliminate the need for expensive chemical catalysts, lower construction, and operational costs, and offer flexibility in producing valuable commodities  . The biocathode requires optimal physiological conditions that promote microbial growth on the cathode surface. Unlike anode respiring bacteria in the anode, the microbes in the biocathode should have the ability to receive electrons from the cathode surface (i.e., electrotrophic). The growth of biofilms on the cathode may be achieved with special techniques such as an electrical inversion of the electrodes. Few scientific studies have confirmed that the biofilm-laden cathode can be conditioned under an oxic environment and then switched to a current generating anode. This indicates that both exoelectrogenic and electrotrophic microorganisms can be maintained in the electrode biofilms when the cathode is switched from oxic to anoxic conditions. Different approaches were taken to improve the kinetics at the cathode surface by using mediators such as ferricyanide or strong oxidants such as permanganate, catalytic electrodes with a platinum catalyst, bacteria catalyzing the oxidation of transition metals, and the bacteria catalyzing the reduction of the final oxidant (i.e., electron acceptor through either direct or indirect electron transport mechanisms including the metabolic products).
The inorganic anions nitrate (NO3 − ) and nitrite (NO2 − ) were previously thought to be inert end products of endogenous nitric oxide (NO) metabolism. However, recent studies show that these supposedly inert anions can be recycled in vivo to form NO, representing an important alternative source of NO to the classical l -arginine–NO-synthase pathway, in particular in hypoxic states. This Review discusses the emerging important biological functions of the nitrate–nitrite–NO pathway, and highlights studies that implicate the therapeutic potential of nitrate and nitrite in conditions such as myocardial infarction, stroke, systemic and pulmonary hypertension, and gastric ulceration.
In this study, we demonstrated that strain DHT3 converts estrogens into androgens via a cobalamin-mediated methylation and subsequently catabolizes the androgenic intermediates to HIP through the established 2,3-seco pathway. The discovery completes central pathways for bacterial steroid catabolism (Fig. 1). Briefly, anaerobic bacteria utilize a convergent catabolic pathway (the 2,3-seco pathway) to catabolize sterols, androgens, and estrogens, while aerobic bacteria adopt divergent pathways to catabolize 1) sterols and androgens (9,10-seco pathway) and 2) estrogens (4,5-seco pathway). Nevertheless, all 3 steroid catabolic pathways finally converge at HIP (Fig. 1) and HIP catabolic genes are conserved in the genomes of all characterized steroid-utilizing bacteria (SI Appendix, Fig. S11) (11, 60).
Cobamides such as cobalamin are a family of cobalt-containing tetrapyrrole biomolecules with essential biochemical functions in all 3 domains of life, serving as the prosthetic group for various methyltransferases, isomerases, and reductive dehalogenases (61). Before this study, the known methyl acceptors for cobalamin-dependent methyltransferases included tetrahydrofolate and homocysteine in most organisms (43, 48, 62) as well as CoM and tetrahydromethanopterin in methanogenic archaea (38, 53). Here, we demonstrated that estrogens are terminal methyl acceptors of a cobalamin-dependent methyltransferase in a denitrifying proteobacterium, revealing an unexpected role of cobalamin in steroid metabolism. Given that sex steroids are involved in bidirectional metabolic interactions between bacteria and their eukaryotic hosts, finding retroconversion of estrogens into androgens in bacteria portends unexplored microbe–host metabolic interdependencies via this Emt-mediated estrogen methylation reaction. Therefore, the emtABCD gene cluster can serve as a biomarker to elucidate the occurrence of retroconversion of estrogens in eukaryotic microbiota.
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We thank Z. Keresztes, V. Muntean, T. Szőke-Nagy, M. Alexe, A. Cristea and A. Baricz for their technical support during sampling and sample preparation, and E. A. Levei and M. Șenilă for contributions to chemical analyses. P.-A.B was supported by the research grant PN-III-P4-ID-PCE-2016-0303 (Romanian National Authority for Scientific Research). H.L.B. was supported by the research grants PN-III-P4-ID-PCE-2016-0303 (Romanian National Authority for Scientific Research) and STAR-UBB Advanced Fellowship-Intern (Babeș-Bolyai University). A.-Ş.A. was supported by the research grants: 17-04828 S (Grant Agency of the Czech Republic) and MSM200961801 (Academy of Sciences of the Czech Republic). M.M. was supported by the Postdoctoral Programme PPPLZ L200961651 (Academy of Sciences of the Czech Republic). R.G. was supported by the research grant 17-04828 S (Grant Agency of the Czech Republic).
The primary aim of this study was to examine the response of the ε-proteobacterium S. gotlandica GD1 T toward changes in DIC and pH. This could be achieved by assessing the impact of changes in DIC concentration and pH on growth and maximal cell numbers of S. gotlandica GD1 T in batch culture growth experiments.
Hydrogen sulfide is the major substrate in anoxic waters and S. gotlandica GD1 T seems to be primarily responsible for hydrogen sulfide oxidation in the Baltic Sea (Grote et al. 2012 ). However, at the oxic–anoxic interface and the upper sulfidic zone thiosulfate concentrations are in a similar range as hydrogen sulfide concentrations (Bruckner et al. 2013 ) and thiosulfate serves as an alternative substrate for S. gotlandica GD1 T . We used thiosulfate as electron donor in the experiments as substrate concentrations can be much better controlled compared to hydrogen sulfide. Although thiosulfate seemed to be entirely consumed at the end of the experiment (see Fig. 3), earlier experiments with this strain did not produce higher cell numbers with higher thiosulfate concentrations (Bruckner et al. 2013 Labrenz et al. 2013 ). Therefore, other potentially limiting factors, related to cell concentration, have to be considered, such as the accumulation of inhibitory metabolic products.
Estimation of DIC saturation for growth
The growth-stimulating effects of increasing DIC concentrations for phytoplankton are well documented (e.g., Iglesias-Rodriguez et al. 2008 ) whereas only few studies were performed with chemolithoautotrophic bacteria. According to the current DIC concentrations of about 2 mmol L −1 and 3.5 mmol L −1 (Frey et al. 1991 Beldowski et al. 2010 ) in the redox zones of the Baltic and the Black Sea, our results show that these DIC concentrations are well within the range supporting maximal growth of S. gotlandica GD1 T and related epsilonproteobacteria. The results suggest that a further increase in DIC concentration in the redox zones should have no additional direct effect on these bacteria.
A higher DIC concentration in the ocean causes a shift in the DIC speciation toward carbon dioxide, resulting in a decrease of pH, that is at pH 7.1 90% of the DIC speciation is hydrogen carbonate while at pH 6.3 the balance shifts to 50% hydrogen carbonate and 50% carbon dioxide (Deffeyes 1965 ). However, this shift in speciation should not have an influence on growth of S. gotlandica GD1 T as a DIC concentration of 800 μmol L −1 was already sufficient to promote maximal cell numbers (Fig. 1). Comparable saturation curves for increasing DIC concentrations had been determined for other bacterial and phytoplankton species. Clark and Flynn ( 2000 ) described the relationship between the carbon-specific growth and the DIC concentration of several marine phytoplanktons. Most of these species reached a saturation between 500 and 1000 μmol L −1 DIC. Furthermore, Dobrinski et al. ( 2005 ) showed that for the chemolithoautotrophic γ-proteobacterium Thiomicrospira crunogena, isolated from a hydrothermal vent, the half-saturation DIC concentration was 220 μmol L −1 and saturation was reached at 1000 μmol L −1 DIC, which is in the same range as the values determined for S. gotlandica GD1 T . In contrast, Scott and Cavanaugh ( 2007 ) showed that the chemoautotrophic Solemya velum symbionts reach saturation at a CO2 concentration of 100 μmol L −1 . However, this saturation is also well within the range of the CO2 concentration in the environment, where S. velum was collected. In addition, they could prove that these symbionts rely on CO2 and not on bicarbonate.
Genomic data indicate that S. gotlandica GD1 T is capable of using both CO2 and bicarbonate by converting intracellular bicarbonate to CO2 with the carbonic anhydrase (Grote et al. 2012 ). In addition, Dobrinski et al. ( 2005 ) could prove that the chemolithoautotrophic γ-proteobacterium Thiomicrospira crunogena has the ability to use both external CO2 and bicarbonate. Therefore, it seems probable that S. gotlandica GD1 T has also the ability to use both external CO2 and bicarbonate as inorganic carbon source.
Effects of different pH values on chemolithoautotrophic growth
The pH range at which S. gotlandica GD1 T grew well (pH 6.6–8.0) was relatively narrow compared to that of other chemolithoautotrophic proteobacteria. For example several γ-proteobacterial Thiomicrospira species from hydrothermal vents grew at a wide pH range of 5.3–8.5 or 4.0–7.5 (Brinkhoff et al. 1999 ). Also for other chemoautrophic ε-proteobacteria a relatively wide tolerable pH range was found, for example for Sulfurimonas paralvinellae and Sulfurimonas autotrophica, the pH range was 5.4–8.6 (optimum 6.1) and 5.0–9.0 (optimum 6.5), respectively (Inagaki et al. 2003 Takai et al. 2006 ). Sulfurimonas denitrificans, the closest cultivated relative of S. gotlandica GD1 T (Grote et al. 2012 ), has a pH optimum of 7.0 (Timmer-ten Hoor 1975 ). Most of these investigated bacteria with a more extended pH range compared to S. gotlandica GD1 exist in habitats such as hydrothermal vents, where pH changes are frequent and rapid. Thus, the extended pH range suggests that it is an adaptation to the extremely variable conditions, whereas S. gotlandica str. GD1 was isolated from a relatively stable habitat. Scott and Cavanaugh ( 2007 ) confirm this conclusion with their studies about chemoautotrophic γ-proteobacteria, living as endosymbionts in sulfidic/oxic interfaces. These Solemya velum symbionts have also a relatively narrow range of pH optimum (between pH 7.4 and 8.5), showing the same sharp decline in growth directly below and above these levels.
The substrate utilization did not differ significantly between pHs 7.1 and 6.6. However, at a pHs below 6.6 the situation changed drastically and growth of S. gotlandica GD1 was obviously impaired, and consumption of nitrate and thiosulfate was strongly reduced. Thus, the important functional role of S. gotlandica GD1 T in the redoxcline nitrogen and sulfur cycles would probably be impacted.
While intracellular pH was not measured in this study, it is supposed that the intracellular pH varies by around 0.1 units per unit change in the external pH (Hackstadt 1983 ). According to Booth ( 1985 ) changes in pH outside the pH optimum leads to an inhibition of both enzyme activity and cell growth (Booth 1985 ). However, thus far the exact mechanism of intracellular pH regulation is not completely understood. Earlier studies have shown that the regulation is energy dependent and requires a high respiratory rate (Booth 1985 ). Hence, the high substrate utilization per bacterium at pHs 6.55 could be at least partially explained by regulation of the intracellular pH outside the optimum pH.
As substrate utilization in the batch culture experiments was significantly higher than under environmental conditions, the results rather reflect the carrying capacity of S. gotlandica GD1 T at the given conditions. Future studies should aim to more accurately simulate in situ conditions (e.g., with chemostat cultures). Due to global warming and higher CO2 concentrations in the atmosphere an increase in the DIC concentration and a decrease of pH by about 0.3 units in the ocean, known as ocean acidification, is predicted (Houghton et al. 2001 ). Our results suggest that a direct impact of acidification on S. gotlandica GD1 T and related organisms should not be very strong, as the optimum pH range for this model organism is still within the range of the predicted changes in pH. On the other hand, indirect effects on S. gotlandica GD1 T might be more important than direct ones. For example there is evidence that nitrification, the process which delivers nitrate for denitrifying bacteria, is negatively influenced by a decrease in pH and an increase in pCO2 (Huesemann et al. 2002 Denecke and Liebig 2003 Hutchins et al. 2009 ).
Increased pulmonary blood flow in shunt lambs.
All shunt lambs had a palpable thrill and an increase in the oxygen saturation between the right ventricle and pulmonary artery, demonstrating patency of the graft. In fact, the pulmonary-to-systemic blood flow ratio of shunt lambs was 2.9 ± 0.9. Compared with 2-wk-old control lambs, 2-wk-old shunt lambs had increased mean pulmonary arterial pressure (21.7 ± 3.2 vs. 15.3 ± 2.3 mmHg), left pulmonary blood flow (147.3 ± 23.4 vs. 55.1 ± 13.2), and left atrial pressure (6.8 ± 3.7 vs. 2.7 ± 1.6 mmHg) (P < 0.05). Right atrial pressure, heart rate, and mean systemic arterial pressure values were similar between the groups.
Reduced expression of the enzymes involved in carnitine homeostasis in shunt lambs.
Initially, we measured the expression of the three important mitochondrial enzymes involved in carnitine metabolism: CPT1-B, CPT2 (involved in the transport of long-chain fatty acids from cytosol to mitochondrial matrix), and CrAT (transesterfies short-chain acyl chains) in 2-wk old shunt and age-matched control lambs. There was a significant decrease in the expression of both CPT1-B (Fig. 1, A and B) and CPT2 enzymes (Fig. 1, C and D) in shunts compared with age-matched control lambs. Similarly, both CrAT expression (Fig. 2, A and B) and activity (control: 152.9 ± 46.1 units/mg vs. shunt: 7.43 ± 3.33 units/mg, P < 0.05 vs. control Fig. 2C) were significantly decreased in shunt compared with age-matched control lambs.
Fig. 1.Carnitine palmitoyltransferase (CPT) expression in peripheral lung tissue from control and shunt lambs at 2 wk of age. A: protein extracts (50 μg) prepared from peripheral lung of shunt and control lambs were analyzed by Western blot analysis using a specific antiserum raised against CPT1-B protein. CPT1-B expression was also normalized for loading using β-actin. A representative blot is shown. B: there was a significant decrease in normalized densitometric values for CPT1-B protein in peripheral lung tissue prepared from shunt compared with control lambs. Values are means ± SE n = 6 control and 6 shunt lambs. *P < 0.05 vs. control. C: protein extracts (50 μg) prepared from peripheral lung of shunt or control lambs were analyzed by Western blot analysis using a specific antiserum raised against CPT2 protein. CPT2 expression was also normalized for loading using β-actin. A representative blot is shown. D: there was a significant decrease in normalized densitometric values for CPT2 protein in peripheral lung tissue prepared from shunt compared with control lambs. Values are means ± SE n = 6 control and 6 shunt lambs. *P < 0.05 vs. control.
Fig. 2.Carnitine acetyltransferase (CrAT) expression and activity in peripheral lung tissue from control and shunt lambs at 2 wk of age. A: protein extracts (50 μg) prepared from peripheral lung of shunt and control lambs were analyzed by Western blot analysis using a specific antiserum raised against CrAT protein. CrAT expression was also normalized for loading using β-actin. A representative blot is shown. B: there was a significant decrease in normalized densitometric values for CrAT protein in peripheral lung tissue prepared from shunt compared with control lambs. Values are means ± SE n = 6 control and 6 shunt lambs. *P < 0.05 vs. control. C: CrAT activity was determined in protein extracts (40 μg) prepared from peripheral lung tissue from control and shunt lambs. There was a significant decrease in CrAT activity in peripheral lung tissue prepared from shunt compared with control lambs. Values are expressed as units of activity per μg of protein and are means ± SE n = 4 control and 4 shunt lambs. *P < 0.05 vs. control.
Increased nitration decreases CrAT activity in shunt lambs.
Although our data indicated that CrAT expression was decreased ∼2-fold, the CrAT activity was decreased ∼20-fold. This suggested that a posttranslational modification was altering CrAT activity. Since we had previously found that there is an increase in oxidative stress in the shunt lambs, we focused on the potential role of enzyme nitration. The levels of nitrated CrAT were determined using immunoprecipitation with a specific antiserum raised against 3-nitrotyrosine residues and were found to be significantly increased in shunt compared with age-matched control lambs (Fig. 3, Aand B). Furthermore, we found that exposing purified CrAT to peroxynitrite (10 μM, 5 min) was sufficient to significantly reduce CrAT activity (vehicle: 99.9 ± 17.74 units/μg vs. peroxynitrite: 0.6481 ± 0.24 units/μg, P < 0.05 vs. vehicle Fig. 4).
Fig. 3.Increased nitration of CrAT in peripheral lung tissue of shunt lambs at 2 wk of age. A: protein extracts (1,000 μg) prepared from peripheral lung of shunt and control lambs were subjected to immunoprecipitation (IP) using an antibody specific to 3-nitrotyrosine (3-NT) and then analyzed by Western blot analysis using a specific antiserum raised against CrAT protein. A representative immunoblot (IB) is shown with CrAT expression. Minimal binding was observed in the beads alone or in IgG preclear. B: there was a significant increase in nitrated CrAT protein in peripheral lung tissue prepared from shunt compared with control lambs. Values are means ± SE n = 6 control and 6 shunt lambs. *P < 0.05 vs. control.
Fig. 4.Peroxynitrite decreases CrAT activity. Purified pigeon breast muscle CrAT was exposed to authentic peroxynitrite or vehicle, and then the activity was determined. Peroxynitrite induced a significant decrease in CrAT activity. Values are means ± SD n = 6. *P < 0.05 vs. vehicle.
Decreased CrAT activity leads to alterations in carnitine metabolism.
CrAT plays an important role in maintaining carnitine metabolism (10). Therefore, we next determined total carnitine and free carnitine levels in the peripheral lung of shunt and control lambs. Using HPLC analysis, we found that total carnitine levels were unchanged in shunt lambs (60.54 ± 8.45 vs. 72.37 ± 10.14 nmol/g wet wt Table 1). However, free carnitine levels were decreased (34.86 ± 3.35 vs. 67.57 ± 13.43 nmol/g wet wt, P < 0.05 vs. control Table 1). Furthermore, l -carnitine levels were significantly lower in shunt compared with age-matched control lambs (20.21 ± 4.29 vs. 56.41 ± 12.27 nmol/g wet wt, P < 0.05 vs. control Table 1). Since free carnitines and other acylcarnitines contribute to total carnitine levels, these data indicate that the percentage of carnitine present as acylcarnitine (calculated as total carnitine minus free carnitine) is significantly higher in shunt lambs (40.71 ± 9 vs. 6.2 ± 4.7%, P < 0.05 vs. control Table 1).
Table 1. Carnitine levels in peripheral lungs of shunt and control lambs
Data are means ± SE n = 5.
Altered carnitine metabolism is associated with mitochondrial dysfunction in shunt lambs.
Since the carnitine acyltransferase pathway has been shown to be of critical importance for maintaining normal mitochondrial function, we next determined whether shunt lambs present with markers of mitochondrial dysfunction. Because loss of SOD2 (8, 13, 18, 26, 35, 44, 48) and increases in UCP-2 (2, 27, 42, 46, 62) have been shown to be markers of mitochondrial dysfunction in other systems, we examined these markers. Our data indicate that SOD2 expression was significantly decreased (Fig. 5, A and B) in shunt lambs, whereas UCP-2 expression was significantly increased (Fig. 5, C and D). In addition, we determined the lung levels of lactate and pyruvate in shunt and control lambs to quantify mitochondrial activity. Under normal mitochondrial function, pyruvate levels are higher than lactate, and thus any increases in lactate levels decreases the pyruvate-to-lactate ratio and can be extrapolated to suggest a reduction in ATP generation by the mitochondria (57). As shown in Table 2, shunt lambs had significantly decreased pyruvate levels (0.059 ± 0.007 vs. 0.132 ± 0.028 μmol/g wet wt, P < 0.05) and increased lactate levels (0.568 ± 0.838 vs. 1.348 ± 0.214 μmol/g wet wt, P < 0.05) as well as a significant reduction in the pyruvate-to-lactate ratio.
Fig. 5.Markers of mitochondrial dysfunction are increased in shunt lambs at 2 wk of age. A: protein extracts (25 μg) prepared from peripheral lung of shunt and control lambs were analyzed by Western blot analysis using a specific antiserum raised against SOD2 protein. SOD2 expression was also normalized for loading using β-actin. A representative blot is shown. B: there was a significant decrease in normalized densitometric values for SOD2 protein in peripheral lung tissue prepared from shunt compared with control lambs. Values are means ± SE n = 6 control and 6 shunt lambs. *P < 0.05 vs. control. C: protein extracts (25 μg) prepared from peripheral lung of shunt and control lambs were analyzed by Western blot analysis using a specific antiserum raised against uncoupling protein-2 (UCP-2). UCP-2 expression was also normalized for loading using β-actin. A representative blot is shown. D: there was a significant increase in normalized densitometric values for UCP-2 protein in peripheral lung tissue prepared from shunt compared with control lambs. Values are means ± SE n = 6 control and n = 6 shunt lambs. *P < 0.05 vs. control.
Table 2. Lactate and pyruvate levels in peripheral lungs of shunt and control lambs
Data are means ± SE n = 5.
Mitochondrial dysfunction attenuates HSP90-eNOS interaction and shear-induced NO production in PAEC.
To further investigate the effect of mitochondrial dysfunction on NO signaling, we utilized cultured PAEC isolated from juvenile lambs. Mitochondrial dysfunction was induced in PAEC by using 2,4-DNP (50 μM, 0–6 h). Our data indicate that 2,4-DNP significantly decreased ATP levels in PAEC (Fig. 6A) associated with an increase in oxidative stress within the mitochondria (Fig. 6B). Furthermore, we found that this mitochondrial dysfunction was associated with a decrease in the association of HSP90 with eNOS (Fig. 7, Aand B) and shear-induced NO production (Fig. 7C) and an increase in eNOS-derived superoxide (Fig. 7D), indicating that impaired mitochondrial function reduces NO production.
Fig. 6.2,4-Dinitrophenol (2,4-DNP) decreases ATP levels and increases mitochondrial oxidative stress in pulmonary arterial endothelial cells (PAEC). A: PAEC were treated with 2,4-DNP (25 μM, 0–8 h), and the cellular ATP levels were then determined. 2,4-DNP significantly decreased cellular ATP levels. Values are means ± SE n = 6. T, time. *P < 0.05 vs. control. B: PAEC were treated with 2,4-DNP (25 μM, 4 h). Cells were then exposed to MitoSox (10 μM, 15 min) to measure mitochondrial superoxide levels (as a marker for mitochondrial dysfunction). 2,4-DNP induced a significant increase in MitoSox fluorescence (representative images are shown as an inset). Values are means ± SE n = 4. *P < 0.05 vs. untreated.
Fig. 7.2, 4-DNP decreases NO signaling in PAEC. A: PAEC were treated with 2,4-DNP (25 μM, 4 h) and washed with PBS, and lysates were prepared with modified RIPA buffer. IP was performed using an antibody to endothelial nitric oxide synthase (eNOS) and then Western blot analysis was done using a specific antiserum raised against heat shock protein 90 (HSP90). Blots were also stripped and reprobed for eNOS to normalize the IP. A representative blot is shown. B: 2,4-DNP treatment caused a significant reduction in eNOS-HSP90 interaction. Values are means ± SE n = 3. *P < 0.05 vs. untreated. C: cells were treated with 2,4-DNP (25 μM, 4 h) and exposed to laminar shear stress (20 dyn/cm 2 , 15 min), and then the medium was assayed for nitrate/nitrite (NOx) as an indirect determination of NO production. 2,4-DNP significantly decreased the shear-mediated increase in NOx. Values are means ± SE n = 6. *P < 0.05 vs. no shear. †P < 0.05 vs. control. D: cells were treated with 2,4-DNP (25 μM, 4 h) in the presence and absence of the NOS inhibitor 3-ethylisothiourea (ETU 100 μM) and then exposed to laminar shear stress (20 dyn/cm 2 , 15 min). Superoxide levels were then determined using the spin trap 1-hydroxy-3-methoxycarbonyl-2,2,5,5-tetramethylpyrrolidine·HCl. 2,4-DNP significantly increased NOS-dependent superoxide generation in response to shear. Values are means ± SE n = 3. *P < 0.05 vs. no shear. †P < 0.05 vs. shear + 2,4-DNP.
Decreased HSP90-eNOS interaction and altered NO signaling in shunt lambs.
To determine the effect of decreased mitochondrial function in shunt lambs on NO signaling, we carried out immunoprecipitation studies to determine eNOS-HSP90 interactions in both 2- and 4-wk-old shunt and control lambs. Our data indicate that there was a progressive decrease in eNOS-HSP90 interactions between 2 and 4 wk of age in shunt compared with control lambs (Fig. 8, A and B) and that this was associated with a progressive decrease in relative eNOS activity (as determined by tissue NOx levels as a fraction of calcium-dependent [ 3 H]arginine metabolism, Fig. 8C) and increased NOS-dependent superoxide levels indicative of eNOS uncoupling (Fig. 8D). We also confirmed the specificity of ETU for NOS-derived superoxide by demonstrating that ETU did not quench the superoxide generated by a xanthine/xanthine oxidase superoxide-generating system (Fig. 8E).
Fig. 8.Progressive decreases in the interaction of eNOS with HSP90 in shunt compared with control lambs. A: the interaction of eNOS with HSP90 was determined by IP using specific antiserum raised against eNOS in tissue extracts prepared from peripheral lung of shunt and control lambs at 2 and 4 wk of age. IP extracts were analyzed using antisera against either eNOS or HSP90. A representative image is shown. No specific protein bands were observed in the beads alone or in IgG preclear. B: the levels of eNOS protein associated with HSP90 relative to total eNOS protein were calculated. The data obtained indicate that there was a progressive decrease in the association of eNOS with HSP90 in shunt compared with control lambs between 2 (bars at left) and 4 wk of age (bars at right). Values are means ± SE n = 5 shunt and 5 control lambs at each age. *P < 0.05 compared with age-matched control. †P < 0.05 vs. 2-wk shunt. C: relative eNOS activity was estimated in shunt and age-matched control lambs at 2 and 4 wk of age by dividing peripheral lung tissue NOx levels by total lung eNOS activity (determined by calcium-dependent [ 3 H]arginine to [ 3 H]citrulline conversion). Relative eNOS activity was significantly lower in the shunt lambs at both 2 and 4 wk of age. Values are means ± SE n = 4 shunt and 4 control lambs at each age. *P < 0.05 compared with age-matched control. †P < 0.05 vs. 2-wk shunt. D: superoxide anion levels determined by electron paramagnetic resonance (EPR) in snap-frozen lung tissue from shunt and age-matched control lambs at 4 wk of age in the presence and absence of the NOS inhibitor ETU (100 μM). A bar graph representing the cumulative data is shown. ETU-inhibitable superoxide levels were significantly higher in the shunt lambs. Values are means ± SD n = 6 shunt and 6 control lambs at each age. *P < 0.05 compared with age-matched control. †P < 0.05 vs. 2-wk shunt. E: superoxide was generated in vitro by cross-reacting xanthine oxidase (XO 1 U/ml) with xanthine (X 1 mM) in the presence or absence of ETU (100 μM). Two control reactions, one lacking X and the other lacking XO, were included to ensure the absence of nonspecific reactions of either reagent with the EPR spin trap. The significant increase in superoxide generated by the X/XO reaction was not significantly quenched by the presence of ETU. Values are means ± SE n = 3 for each condition. *P < 0.05 compared with X alone. †P < 0.05 vs. XO alone.
Global transcriptome analysis of the tetrachloroethene-dechlorinating bacterium Desulfitobacterium hafniense Y51 in the presence of various electron donors and terminal electron acceptors
Desulfitobacterium hafniense Y51 is a dechlorinating bacterium that encodes an unusually large set of O-demethylase paralogs and specialized respiratory systems including specialized electron donors and acceptors. To use this organism in bioremediation of tetrachloroethene (PCE) or trichloroethene (TCE) pollution, expression patterns of its 5,060 genes were determined under different conditions using 60-mer probes in DNA microarrays. PCE, TCE, fumarate, nitrate, and dimethyl sulfoxide (DMSO) respiration all sustain the growth of strain Y51. Global transcriptome analyses were thus performed using various electron donor and acceptor couples (respectively, pyruvate and either fumarate, TCE, nitrate, or DMSO, and vanillate/fumarate). When TCE is used as terminal electron acceptor, resulting in its detoxification, a series of electron carriers comprising a cytochrome bd-type quinol oxidase (DSY4055-4056), a ferredoxin (DSY1451), and four Fe&ndashS proteins (DSY1626, DSY1629, DSY0733, DSY3309) are upregulated, suggesting that the products of these genes are involved in PCE oxidoreduction. Interestingly, the PCE dehalogenase cluster (pceABCT) is constitutively expressed in the media tested, with pceT being upregulated and pceC downregulated in pyruvate/TCE-containing medium. In addition, another dehalogenation enzyme (DSY1155 coding for a putative chlorophenol reductive dehalogenase), is induced 225-fold in that medium, despite not being involved in PCE respiration. Remarkably since the reducing equivalents formed during pyruvate conversion to acetyl-CoA are channeled to electron acceptors including halogenated compounds, pyruvate induces expression of a pyruvate:ferredoxin oxidoreductase. This study paves the way to understanding the physiology of D. hafniense, optimizing this microbe as a bioremediation agent, and designing bioarray sensors to monitor the presence of dechlorinating organisms in the environment.
Journal of Industrial Microbiology Biotechnology &ndash Springer Journals
Release Notes for EcoCyc Version 4.0
The most significant changes are marked with a "*".
Changes to the EcoCyc Data
- EcoCyc describes all published pathways of E. coli metabolism. The following pathways were added since version 3.7:
- carnitine metabolism, CoA-linked
- enterobactin synthesis
- phenylethylamine degradation
All genes defined in this Genbank entry (which we term the "Blattner genes") were loaded into EcoCyc by merging the Blattner genes with gene objects that existed previously in EcoCyc. The initial merging was performed programmatically by matching the gene names assigned to the Blattner genes against the gene names and synonyms maintained in EcoCyc. These matches were confirmed by checking the Swiss-Prot links provided for many genes by the Blattner group with the Swiss-Prot links that exist for many EcoCyc genes. 2497 matches were found of which 2265 were confirmed using the Swiss-Prot links. A number of additional gene correspondences were determined through manual inspection.