Kinetics and distribution of alcohol oxidising activity in Acholeplasma and Mycoplasma species

Alcohol metabolism by Acholeplasma and Mycoplasma cell suspensions was determined using changes in dissolved oxygen tension to monitor oxygen uptake. All seven Acholeplasma test species oxidised ethanol and (where tested) propanol, butanol and pentanol. The rate of oxidation, at any particular substrate concentration, decreased with increasing alcohol molecular mass. Amongst 20 Mycoplasma species tested, M. agalactiae, M. bovis, M. dispar, M. gallisepticum, M. pneumoniae and M. ovipneumoniae oxidised ethanol. Propanol was also oxidised by M. dispar and isopropanol by M. agalactiae, M. bovis and M. ovipneumoniae. Isopropanol was oxidised at particularly high rates (V(max)100 nmol O(2) taken up min(-1) mg cell protein(-1)) and with a relatively high affinity (K(m) value<2 mM); oxygen uptake was consistent with oxidation to acetone. The significance of alcohol oxidation is unclear, as it would not be predicted to lead to ATP synthesis.     

Anaerobic oxidation of alkanes by newly isolated denitrifying bacteria

The capacity of denitrifying bacteria for anaerobic utilization of saturated hydrocarbons (alkanes) was investigated with n-alkanes of various chain lengths and with crude oil in enrichment cultures containing nitrate as electron acceptor. Three distinct types of denitrifying bacteria were isolated in pure culture. A strain (HxN1) with oval-shaped, nonmotile cells originated from a denitrifying enrichment culture with crude oil and was isolated with n-hexane (C₆H₁₄). Another strain (OcN1) with slender, rod-shaped, motile cells was isolated from an enrichment culture with n-octane (C₈H₁₈). A third strain (HdN1) with oval, somewhat pleomorphic, partly motile cells originated from an enrichment culture with aliphatic mineral oil and was isolated with n-hexadecane (C₁₆H₃₄). Cells of hexane-utilizing strain HxN1 grew homogeneously in the growth medium and did not adhere to the alkane phase, in contrast to the two other strains. Quantification of substrate consumption and cell growth revealed the capacity for complete oxidation of alkanes under strictly anoxic conditions, with nitrate being reduced to dinitrogen. Received: 3 August / Accepted: 6 October 1999     

Sulphide detoxification in Hediste diversicolor and Marenzelleria viridis, two dominant polychaete worms within the shallow coastal waters of the southern Baltic Sea

The polychaete worms Marenzelleria viridis (Verrill 1873) and Hediste diversicolor (O.F. Müller) form the main part of the macro-zoobenthos in soft-bottomed shallow inlets of the Baltic Sea. Due to high eutrophication within these waters the animals are exposed to low oxygen and high sulphide concentrations. Specimens of both species from a low salinity location (S 8 ‰) were compared concerning their physiological abilities in coping with this hostile environment. Sulphide detoxification occurred in both polychaetes even during severe hypoxia with the main end-product being thiosulphate. In absence of sulphide nearly no end-products of anaerobic metabolism were found in the worms during moderate hypoxia (pO₂=7 kPa). In presence of hydrogen sulphide, succinate, a sensitive indicator of anaerobic metabolism, was accumulated in higher amounts at low sulphide concentrations (0.3 mM) already. Oxygen consumption and ATP production was determined in isolated mitochondria of both species. Both polychaetes were able to perform enzymatic sulphide oxidation in the mitochondria at concentrations up to 50 μM. This process was coupled with oxidative phosphorylation. At least in M. viridis sulphide respiration was not completely inhibited by cyanide, suggesting an alternative oxidation pathway, which by-passes the cytochrome-c-oxidase. The two species did not differ in the rate of sulphide detoxification, but H. diversicolor produced about as twice as much ATP from mitochondrial sulphide oxidation. Differences in mitochondrial sulphide oxidation are probably related to the different life strategies of the worms.     

Human paraoxonases (PON1, PON2, and PON3) are lactonases with overlapping and distinct substrate specificities

The paraoxonase (PON) gene family in humans has three members, PON1, PON2, and PON3. Their physiological role(s) and natural substrates are uncertain. We developed a baculovirus-mediated expression system, suitable for all three human PONs, and optimized procedures for their purification. The recombinant PONs are glycosylated with high-mannose-type sugars, which are important for protein stability but are not essential for their enzymatic activities. Enzymatic characterization of the purified PONs has revealed them to be lactonases/lactonizing enzymes, with some overlapping substrates (e.g., aromatic lactones), but also to have distinctive substrate specificities. All three PONs metabolized very efficiently 5-hydroxy-eicosatetraenoic acid 1,5-lactone and 4-hydroxy-docosahexaenoic acid, which are products of both enzymatic and nonenzymatic oxidation of arachidonic acid and docosahexaenoic acid, respectively, and may represent the PONs' endogenous substrates. Organophosphates are hydrolyzed almost exclusively by PON1, whereas bulky drug substrates such as lovastatin and spironolactone are hydrolyzed only by PON3. Of special interest is the ability of the human PONs, especially PON2, to hydrolyze and thereby inactivate N-acyl-homoserine lactones, which are quorum-sensing signals of pathogenic bacteria. None of the recombinant PONs protected low density lipoprotein against copper-induced oxidation in vitro.     

Thioredoxin-interacting protein deficiency disrupts the fasting-feeding metabolic transition

Through a positional cloning approach, the thioredoxin-interacting protein gene (Txnip) was recently identified as causal for a form of combined hyperlipidemia in mice (Bodnar, J. S., A. Chatterjee, L. W. Castellani, D. A. Ross, J. Ohmen, J. Cavalcoli, C. Wu, K. M. Dains, J. Catanese, M. Chu, S. S. Sheth, K. Charugundla, P. Demant, D. B. West, P. de Jong, and A. J. Lusis. 2002. Positional cloning of the combined hyperlipidemia gene Hyplip1. Nat. Genet. 30: 110-116). We now show that Txnip-deficient mice in the fed state exhibit a metabolic profile similar to fasted mice, including increased levels of plasma ketone bodies and free fatty acids, decreased glucose, and increased hepatic expression of peroxisome proliferator-activated receptor-gamma coactivator-1alpha, phosphoenolpyruvate carboxykinase, glucose-6-phosphatase, and acyl-CoA oxidase. Dramatic differences in the expression of key metabolic enzymes were also observed in other tissues, and the fat-to-muscle ratio of Txnip-deficient mice was increased by approximately 40%. We demonstrate an effect of Txnip on the redox status, as the Txnip-deficient mice in the fed state had a significant increase in the ratio of NADH to NAD(+). Surprisingly, we observed that Txnip-deficient mice and wild-type mice had similar levels of thioredoxin activity, suggesting that the effects of Txnip deficiency may be mediated in part by other interactions. These results indicate a role for Txnip in the metabolic response to feeding and the maintenance of the redox status.     

The more--the better? Estimating the inhibitory activity of alpha-tocopherol towards lipid oxidation

Tocopherols are considered to be powerful antioxidants, but prooxidative effects are discussed for higher concentrations. The aim of this in vitro study was to investigate the dose-dependent inhibition of oxidation product formation caused by alpha-tocopherol, and to estimate the range of maximum antioxidant activity of alpha-tocopherol at different stages of lipid oxidation. Alpha-tocopherol was added to rapeseed oil triglycerides (ROTG, purified rapeseed oil) in concentrations ranging from 25 to 1500 micromol/kg ROTG. The inhibitory activity of alpha-tocopherol increased up to a concentration of 100 micromol/kg ROTG. A concentration of 125 micromol alpha-tocopherol/ kg ROTG did not result in an improved antioxidant effect. The formation of volatile secondary oxidation products followed the same trend, and the maximum inhibitory effect was also found for 100 micromol alpha-tocopherol/kg. Further, concentrations between 250 and 1500 micromol alpha-tocopherol/kg ROTG clearly caused increased formation of hydroperoxides during the induction period. However, compared to the control, all tested alpha-tocopherol concentrations resulted in a reduction of hydroperoxide formation and no prooxidative effects were observed.     

Gaining electricity from in situ oxidation of hydrogen produced by fermentative cellulose degradation

AIM: To exploit the fermentative hydrogen generation and direct hydrogen oxidation for the generation of electric current from the degradation of cellulose. METHODS AND RESULTS: Utilizing the metabolic activity of the mesophilic anaerobe Clostridium cellulolyticum and the thermophilic Clostridium thermocellum we show that electricity generation is possible from cellulose fermentation. The current generation is based on an in situ oxidation of microbially synthesized hydrogen at platinum-poly(tetrafluoroaniline) (Pt-PTFA) composite electrodes. Current densities of 130 mA l(-1) (with 3 g cellulose per litre medium) were achieved in poised potential experiments under batch and semi-batch conditions. Conclusions: The presented results show that electricity generation is possible by the in situ oxidation of hydrogen, product of the anaerobic degradation of cellulose by cellulolytic bacteria. SIGNIFICANCE AND IMPACT OF THE STUDY: For the first time, it is shown that an insoluble complex carbohydrate like cellulose can be used for electricity generation in a microbial fuel cell. The concept represents a first step to the utilization of macromolecular biomass components for microbial electricity generation.     

Tendency for oxidation of annelid hemoglobin at alkaline pH and dissociated states probed by redox titration

The redox titration of extracellular hemoglobin of Glossoscolex paulistus (Annelidea) was investigated in different pH conditions and after dissociation induced by pressure. Oxidation increased with increasing pH, as shown by the reduced amount of ferricyanide necessary for the oxidation of hemoglobin. This behavior was the opposite of that of vertebrate hemoglobins. The potential of half oxidation (E_{1 / 2}) changed from − 65.3 to + 146.8 mV when the pH increased from 4.50 to 8.75. The functional properties indicated a reduction in the log P₅₀ from 1.28 to 0.28 in this pH range. The dissociation at alkaline pH or induced by high pressure, confirmed by HPLC gel filtration, suggested that disassembly of the hemoglobin could be involved in the increased potential for oxidation. These results suggest that the high stability and prolonged lifetime common to invertebrate hemoglobins is related to their low tendency to oxidize at acidic pH, in contrast to vertebrate hemoglobins.     

Bacterial biodegradation of aliphatic sulfides under aerobic carbon- or sulfur-limited growth conditions

AIMS: To isolate bacteria capable of cleaving aliphatic carbon-sulfur bonds as potential biological upgrading catalysts for the reduction of molecular weight and viscosity in heavy crude oil. METHODS AND RESULTS: Thirty-one bacterial strains isolated from enrichment cultures were able to biotransform model compounds representing the aliphatic sulfide bridges found in asphaltenes. Using gas chromatography and mass spectrometry, three types of attack were identified: alkyl chain degradation, allowing use as a carbon source; nonspecific sulfur oxidation; and sulfur-specific oxidation and carbon-sulfur bond cleavage, allowing use as a sulfur source. Di-n-octyl sulfide degradation produced octylthio- and octylsulfonyl-alkanoic acids, consistent with terminal oxidation followed by beta-oxidation reactions. Utilization of dibenzyl sulfide or 1,4-dithiane as a sulfur source was regulated by sulfate, indicating a sulfur-specific activity rather than nonspecific oxidation. Finally, several isolates were also able to use dibenzothiophene as a sulfur source, and this was the preferred organic sulfur substrate for one isolate. CONCLUSIONS: The use of commercially available alkyl sulfides in enrichment cultures gave isolates that followed a range of metabolic pathways, not just sulfur-specific attack. SIGNIFICANCE AND IMPACT OF THE STUDY: These results give new insight into biodegradation of organosulfur compounds from petroleum and for biotreatment of such compounds in chemical munitions.     

Novel intermediate of Rac GTPase activation by guanine nucleotide exchange factor

The biochemical role of guanine nucleotide exchange factors (GEFs) in catalyzing small GTPase GDP-GTP exchange is thought to be twofold: stimulation of GDP dissociation and stabilization of a nucleotide-free GTPase intermediate. Here we report that TrioN, a Dbl family GEF, activates Rac1 by facilitating GTP binding to, as well as stimulating GDP dissociation from, Rac1. The TrioN-catalyzed GDP dissociation is dependent upon the structural nature and the concentration of free nucleotide, and nucleotide binding serves as the rate-limiting step of the GEF reaction. The TrioN-stimulated nucleotide exchange may undergo a novel two nucleotide-one G-protein intermediate involving two cryptic subsites on Rac1 induced by the GEF, with one subsite contributing to the recognition of the beta/gamma phosphates of the incoming GTP and another to the binding of the guanine base of the leaving GDP. We propose that the Rac GEF reaction may proceed by competitive displacement of bound GDP by GTP through a transient intermediate of GEF-[GTP-Rac-GDP].