Alkaline cyanide degradation by Pseudomonas pseudoalcaligenes CECT5344 in a batch reactor. Influence of pH.

Water containing cyanide was biologically detoxified with the bacterial strain Pseudomonas pseudoalcaligenes CECT5344 in a batch reactor. Volatilization of toxic hydrogen cyanide (HCN) was avoided by using an alkaline medium for the treatment. The operational procedure was optimized to assess cyanide biodegradation at variable pH values and dissolved oxygen concentrations. Using an initial pH of 10 without subsequent adjustment allowed total cyanide to be consumed at a mean rate of approximately 2.81 mg CN(-) L(-1) O.D.(-1) h(-1); however, these conditions posed a high risk of HCN formation. Cyanide consumption was found to be pH-dependent. Thus, no bacterial growth was observed with a controlled pH of 10; on the other hand, pH 9.5 allowed up to 2.31 mg CN(-) L(-1) O.D.(-1) h(-1) to be converted. The combination of a high pH and a low dissolved oxygen saturation (10%) minimized the release of HCN. This study contributes new basic knowledge about this biological treatment, which constitutes an effective alternative to available physico-chemical methods for the purification of wastewater containing cyanide or cyano-metal complexes.

Interactions between nitrate assimilation and 2,4-dinitrophenol cometabolism in Rhodobacter capsulatus E1F1.

The phototrophic, nitrate-photoassimilating bacterium Rhodobacter capsulatus E1F1 cometabolizes 2,4-dinitrophenol (DNP) by photoreducing it to 2-amino-4-nitrophenol under anaerobic conditions. DNP uptake and nitrate metabolism share some biochemical features, and in this article we show that both processes are influenced by each other. Thus, as was demonstrated for nitrate assimilation, DNP uptake requires a thermolabile periplasmic component. Nitrate assimilation is inhibited by DNP, which probably affects the nitrite reduction step because neither nitrate reductase activity nor the transport of nitrate or nitrite is inhibited. On the other hand, DNP uptake is competitively inhibited by nitrate, probably at the transport level, because the nitroreductase activity is not inhibited in vitro by nitrate, nitrite, or ammonium. In addition, the decrease in the intracellular DNP concentration in the presence of nitrate probably inactivates the nitroreductase. These results allow prediction of a negative environmental effect if nitrate and DNP are released together to natural habitats, because it may lead to a lower rate of DNP metabolism and to nitrite accumulation.

The assimilatory nitrate reduction system of the phototrophic bacterium Rhodobacter capsulatus E1F1.

The phototrophic bacterium Rhodobacter capsulatus E1F1 assimilates nitrate under anaerobic phototrophic growth conditions. A 17 kb DNA region encoding the nitrate assimilation (nas) system of this bacterium has been cloned and sequenced. This region includes the genes coding for a putative ABC (ATP-binding cassette)-type nitrate transporter (nasFED) and the structural genes for the enzymes nitrate reductase (nasA), nitrite reductase (nasB) and hydroxylamine reductase (hcp). Three genes code for putative regulatory proteins: a nitrite-sensitive repressor (nsrR), a transcription antiterminator (nasT) and a nitrate sensor (nasS). Other genes probably involved in nitrate assimilation are also present in this region. The sequence analysis of these genes and the biochemical properties of the purified nitrate, nitrite and hydroxylamine reductases are reviewed.

Cyanide metabolism of Pseudomonas pseudoalcaligenes CECT5344: role of siderophores.

Cyanide is one of the most potent and toxic chemicals produced by industry. The jewelry industry of Córdoba (Spain) generates a wastewater (residue) that contains free cyanide, as well as large amounts of cyano-metal complexes. Cyanide is highly toxic to living systems because it forms very stable complexes with transition metals that are essential for protein function. In spite of its extreme toxicity, some organisms have acquired mechanisms to avoid cyanide poisoning. The biological assimilation of cyanide needs the concurrence of three separate processes: (i) a cyanide-insensitive respiratory chain, (ii) a system for iron acquisition (siderophores) and (iii) a cyanide assimilation pathway. Siderophores are low-molecular-mass compounds (600-1500 Da) that scavenge iron (Fe(3+)) ions (usually with extremely high affinity) from the environment under iron-limiting conditions. There are two main classes of siderophores: catechol and hydroxamate types. The catechol-type siderophores chelate ferric ion via a hydroxy group, whereas the hydroxamate-type siderophores bind iron via a carbonyl group with the adjacent nitrogen. In the presence of cyanide, bacterial proliferation requires this specific metal uptake system because siderophores are able to break down cyano-metal complexes. Pseudomonas pseudoalcaligenes CECT5344 is able to use free cyanide or cyano-metal complexes as nitrogen source. A proteomic approach was used for the isolation and identification, in this strain, of a protein that was induced in the presence of cyanide, namely CN0, that is involved in siderophore biosynthesis in response to cyanide. An overview of bacterial cyanide degradation pathways and the involvement of siderophores in this process are presented.

Alkaline cyanide biodegradation by Pseudomonas pseudoalcaligenes CECT5344.

Pseudomonas pseudoalcaligenes CECT5344 uses cyanide, cyanate, beta-cyanoalanine, and other cyanoderivatives as nitrogen sources under alkaline conditions, which prevents volatile HCN (pK(a) 9.2) formation. The cyanide consumed by this strain is stoichiometrically converted into ammonium. In addition, this bacterium grows with the heavy metal, cyanide-containing waste water generated by the jewellery industry, and is also a cyanide-resistant strain which induces an alternative oxidase and a siderophore-based mechanism for iron acquisition in the presence of cyanide. The detection of cyanase and beta-cyanoalanine nitrilase activities in cyanide-induced cells suggests their implication in the cyanide degradation pathway.

Rhodococcus sp. RB1 grows in the presence of high nitrate and nitrite concentrations and assimilates nitrate in moderately saline environments.

Rhodococcus sp. RB1 was able to thrive in media with up to 0.9 M NaCl or KCl and in the presence of high concentrations of nitrate (up to 0.9 M) and nitrite (up to 60 mM), but only under oxic conditions. An adaptation period was not required for salt tolerance, but a rapid extrusion of K+ and intake of Na+ was observed after addition of 0.5 M NaCl. Nitrate assimilation was limited by the carbon supply, but nitrite was not accumulated in the culture medium, even at nitrate concentrations as high as 0.8 M, thus suggesting that nitrite reduction does not limit nitrate assimilation. The presence of NaCl or KCl did not affect nitrate or nitrite uptake, which were completely inhibited by ammonium or glutamine. Rhodococcus sp. RB1 nitrate reductase had an apparent molecular mass of 142 kDa and used NADH and reduced bromophenol blue or viologens as electron donors, independently of the presence of salt. The enzyme was associated with an NADH-diaphorase activity and was induced by nitrate and repressed by ammonium or glutamine, thus showing typical biochemical and regulatory properties of bacterial assimilatory NADH-nitrate reductases. The enzyme was active in vitro in the presence of 3 M NaCl or KCI, but the maximal activity was observed at 0.5 M salt. Addition of 2 M NaCl increased the optimal temperature of the enzyme from 12 to 32 degrees C, but the optimal pH (10.3) was unaffected.

Assimilation of D-malate by Rhodobacter capsulatus E1F1.

Rhodobacter capsulatus grew by using either L- or D-malate as carbon sources under light/anaerobic conditions. The cellular yields were the same with D- or L-malate. Both L-malate dehydrogenase and L-malic enzyme activities were detected in cell-free extracts from cells grown in both isomers. By contrast, a racemase activity converting D-malate into L-malate was induced only when D-malate was present in the culture medium. This racemase activity was Mn2+-dependent and was measured by coupling it either to the malate dehydrogenase or to the fumarase activities. The racemase activity was partially purified by anion-exchange chromatography.

Role for draTG and rnf genes in reduction of 2,4-dinitrophenol by Rhodobacter capsulatus.

The phototrophic bacterium Rhodobacter capsulatus is able to reduce 2,4-dinitrophenol (DNP) to 2-amino-4-nitrophenol enzymatically and thus can grow in the presence of this uncoupler. DNP reduction was switched off by glutamine or ammonium, but this short-term regulation did not take place in a draTG deletion mutant. Nevertheless, the target of DraTG does not seem to be the nitrophenol reductase itself since the ammonium shock did not inactivate the enzyme. In addition to this short-term regulation, ammonium or glutamine repressed the DNP reduction system. Mutants of R. capsulatus affected in ntrC or rpoN exhibited a 10-fold decrease in nitroreductase activity in vitro but almost no DNP activity in vivo. In addition, mutants affected in rnfA or rnfC, which are also under NtrC control and encode components involved in electron transfer to nitrogenase, were unable to metabolize DNP. These results indicate that NtrC regulates dinitrophenol reduction in R. capsulatus, either directly or indirectly, by controlling expression of the Rnf proteins. Therefore, the Rnf complex seems to supply electrons for both nitrogen fixation and DNP reduction.

The assimilatory nitrate reductase from the phototrophic bacterium, Rhodobacter capsulatus E1F1, is a flavoprotein.

The assimilatory nitrate reductase from the phototrophic bacterium Rhodobacter capsulatus has been purified to electrophoretic homogeneity and its molecular and kinetic parameters determined. The native nitrate reductase is a dimer of 144 kDa composed of two subunits of 46 and 95 kDa. The purified enzyme catalyzes the electron transfer from NADH, reduced bromophenol blue or reduced viologens to nitrate. The nitrate reductase contains 1 mol FAD per mole of enzyme and also reduces cytochrome c or dichlorophenol indophenol with NADH as the electron donor. The diaphorase activity is located in the small subunit.

Molecular and Regulatory Properties of the Nitrate Reducing Systems of Rhodobacter

Phototrophic bacteria of the genus Rhodobacter possess several forms of nitrate reductase including assimilatory and dissimilatory enzymes. Assimilatory nitrate reductase from Rhodobacter capsulatus E1F1 is cytoplasmic, it uses NADH as the physiological electron donor and reduced viologens as artificial electron donors, and it is coupled to an ammonium-producing nitrite reductase. Nitrate reductase induction requires a high C/N balance and the presence of nitrate, nitrite, or nitroarenes. A periplasmic 47-kDa protein facilitates nitrate uptake, thus increasing nitrate reductase activity. Two types of dissimilatory nitrate reductases have been found in strains from Rhodobacter sphaeroides. One of them is coupled to a complete denitrifying pathway, and the other is a periplasmic protein whose physiological role seems to be the dissipation of excess reducing power, thus improving photoanaerobic growth. Periplasmic nitrate reductase does not use NADH as the physiological electron donor and is a 100-kDa heterodimeric hemoprotein that receives electrons through an electron transport chain spanning the plasma membrane. This nitrate reductase is regulated neither by the intracellular C/N balance nor by O2 pressure. The enzyme also exhibits chlorate reductase activity, and both reaction products, nitrite and chlorite, are released almost stoichiometrically into the medium; this accounts for the high resistance to chlorate or nitrite exhibited by this bacterium. Nitrate reductases from both strains seem to be coded by genes located on megaplasmids.

[Acetylcholinesterase activity in blood plasma and various regions of the central nervous system of rats poisoned with lindane. Effect of sodium pentobarbital]. / Actividad acetilcolinesterasa en plasma sanguíneo y diversas regiones del SNC de ratas intoxicadas con lindano. Efecto del pentobarbital sódico.

The effect of acute intoxication by lindane on the AChE activity in blood plasma and in crude synaptosomal fractions from six CNS areas of rats has been studied. The effect of sodium pentobarbital on the convulsive syndrome caused by the above organochlorine compound is also presented. The joint administration of sodium pentobarbital together with lindane prevented the convulsive reaction which had been produced by lindane alone. In the animals intoxicated by lindane a general increase in the AChE activity both in plasma and in the CNS areas studied has been observed. The combination of sodium pentobarbital and lindane produced a decrease in the AChE activity in all the studied areas with the single exception of the spinal cord. These results are discussed bearing in mind the possible interaction between the GABAergic and cholinergic neurotransmission systems in the CNS.