DNA microarray analysis of the cyanotroph Pseudomonas pseudoalcaligenes CECT5344 in response to nitrogen starvation, cyanide and a jewelry wastewater.

Pseudomonas pseudoalcaligenes CECT5344 is an alkaliphilic bacterium that can use cyanide as nitrogen source for growth, becoming a suitable candidate to be applied in biological treatment of cyanide-containing wastewaters. The assessment of the whole genome sequence of the strain CECT5344 has allowed the generation of DNA microarrays to analyze the response to different nitrogen sources. The mRNA of P. pseudoalcaligenes CECT5344 cells grown under nitrogen limiting conditions showed considerable changes when compared against the transcripts from cells grown with ammonium; up-regulated genes were, among others, the glnK gene encoding the nitrogen regulatory protein PII, the two-component ntrBC system involved in global nitrogen regulation, and the ammonium transporter-encoding amtB gene. The protein coding transcripts of P. pseudoalcaligenes CECT5344 cells grown with sodium cyanide or an industrial jewelry wastewater that contains high concentration of cyanide and metals like iron, copper and zinc, were also compared against the transcripts of cells grown with ammonium as nitrogen source. This analysis revealed the induction by cyanide and the cyanide-rich wastewater of four nitrilase-encoding genes, including the nitC gene that is essential for cyanide assimilation, the cyanase cynS gene involved in cyanate assimilation, the cioAB genes required for the cyanide-insensitive respiration, and the ahpC gene coding for an alkyl-hydroperoxide reductase that could be related with iron homeostasis and oxidative stress. The nitC and cynS genes were also induced in cells grown under nitrogen starvation conditions. In cells grown with the jewelry wastewater, a malate quinone:oxidoreductase mqoB gene and several genes coding for metal extrusion systems were specifically induced.

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.

Algunos proceso psicosociales sobre el síndrome de quemarse por el trabajo (burnout) en profesionales de enfermería / Influence of psychosocial processes on burnout in nursing

El objetivo del estudio es analizar la influencia del apoyo social en el trabajo, la falta de reciprocidad percibida en los intercambios sociales y los conflictos interpersonales sobre el síndrome de quemarse por el trabajo (SQT) (burnout). La muestra consta de 706 profesionales de enfermería. El SQT se estimó mediante el MBIHSS, el apoyo social mediante una escala de 9 ítems, la falta de reciprocidad con 5 ítems, y los conflictos interpersonales con 6 ítems. Los resultados muestran que el apoyo social, la falta de reciprocidad y los conflictos interpersonales fueron predictores significativos de agotamiento emocional. En el factor realización personal resultó significativo el apoyo social y los conflictos interpersonales. En despersonalización, fueron predictores significativos el agotamiento emocional, los conflictos interpersonales y la realización personal. Se encontró un efecto modulador del apoyo social sobre la relación entre la falta de reciprocidad y el agotamiento emocional

The purpose of this study is to analyze tbe influence of social support in tbe workplace, imbalance and interpersonal conflicts on burnout. The sample consists of 706 nursing professionals. Burnout was estimated by MBI-HSS, social support by a scale of 9 items, imbalance by 5 items and interpersonal conflicts by 6 items. Results indicate tbat social support, imbalance and interpersonal conflicts were significant predictors of emotional exhaustion. Social support and interpersonal conflicts were significant predictors of personal accomplishment. And emotional exhaustion, interpersonal conflicts and personal accomplishment showed significant influence on depersonalization. Results indicate buffering effects of social support in tbe relationship between imbalance and tbe emotional exhaustion

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.

A low-redox potential heme in the dinuclear center of bacterial nitric oxide reductase: implications for the evolution of energy-conserving heme-copper oxidases.

Bacterial nitric oxide reductase (NOR) catalyzes the two-electron reduction of nitric oxide to nitrous oxide. It is a highly diverged member of the superfamily of heme-copper oxidases. The main feature by which NOR is distinguished from the heme-copper oxidases is the elemental composition of the active site, a dinuclear center comprised of heme b(3) and non-heme iron (Fe(B)). The visible region electronic absorption spectrum of reduced NOR exhibits a maximum at 551 nm with a distinct shoulder at 560 nm; these are attributed to Fe(II) heme c (E(m) = 310 mV) and Fe(II) heme b (E(m) = 345 mV), respectively. The electronic absorption spectrum of oxidized NOR exhibits a characteristic shoulder around 595 nm that exhibits complex behavior in equilibrium redox titrations. The first phase of reduction is characterized by an apparent shift of the shoulder to 604 nm and a decrease in intensity. This is due to reduction of Fe(B) (E(m) = 320 mV), while the subsequent bleaching of the 604 nm band represents reduction of heme b(3) (E(m) = 60 mV). This separation of redox potentials (>200 mV) allows the enzyme to be poised in the three-electron reduced state for detailed spectroscopic examination of the Fe(III) heme b(3) center. The low midpoint potential of heme b(3) represents a thermodynamic barrier to the complete (two-electron) reduction of the dinuclear center. This may avoid formation of a stable Fe(II) heme b(3)-NO species during turnover, which may be an inhibited state of the enzyme. It would also appear that the evolution of significant oxygen reducing activity by heme-copper oxidases was not simply a matter of the substitution of copper for non-heme iron in the dinuclear center. Changes in the protein environment that modulate the midpoint redox potential of heme b(3) to facilitate both complete reduction of the dinuclear center (a prerequisite for oxygen binding) and rapid heme-heme electron transfer were also necessary.

Spectroscopic characterization of a novel multiheme c-type cytochrome widely implicated in bacterial electron transport.

NapC is a member of a family of bacterial membrane-anchored tetra-heme c-type cytochromes that participate in a number of respiratory electron transport pathways. They are postulated to mediate electron transfer between membrane quinols/quinones and soluble periplasmic enzymes. The water-soluble heme domain of NapC has been expressed as a periplasmic protein. Mediated redox potentiometry and characterization by UV-visible, magnetic circular dichroism, and electron paramagnetic resonance spectroscopies demonstrates that soluble NapC contains four low spin hemes, each with bis-histidine axial ligation and with midpoint reduction potentials of -56, -181, -207, and -235 mV.

Degradation of p-nitrophenol by the phototrophic bacterium Rhodobacter capsulatus.

The phototrophic bacterium Rhodobacter capsulatus detoxified p-nitrophenol and 4-nitrocatechol. The bacterium tolerated moderate concentrations of p-nitrophenol (up to 0.5 mM) and degraded it under light at an optimal O2 pressure of 20 kPa. The bacterium did not metabolize the xenobiotic in the dark or under strictly anoxic conditions or high O2 pressure. Bacterial growth with acetate in the presence of p-nitrophenol took place with the simultaneous release of nonstoichiometric amounts of 4-nitrocatechol, which can also be degraded by the bacterium. Crude extracts from R. capsulatus produced 4-nitrocatechol from p-nitrophenol upon the addition of NAD(P)H, although at a very low rate. A constitutive catechol 1, 2-dioxygenase activity yielding cis,cis-muconate was also detected in crude extracts of R. capsulatus. Further degradation of 4-nitrocatechol included both nitrite- and CO2-releasing steps since: (1) a strain of R. capsulatus (B10) unable to assimilate nitrate and nitrite released nitrite into the medium when grown with p-nitrophenol or 4-nitrocatechol, and the nitrite concentration was stoichiometric with the 4-nitrocatechol degraded, and (2) cultures of R. capsulatus growing microaerobically produced low amounts of 14CO2 from radiolabeled p-nitrophenol. The radioactivity was also incorporated into cellular compounds from cells grown with uniformly labeled 14C-p-nitrophenol. From these results we concluded that the xenobiotic is used as a carbon source by R. capsulatus, but that only the strain able to assimilate nitrite (E1F1) can use p-nitrophenol as a nitrogen source.

Isolation of periplasmic nitrate reductase genes from Rhodobacter sphaeroides DSM 158: structural and functional differences among prokaryotic nitrate reductases.

The phototrophic bacterium Rhodobacter sphaeroides DSM 158 has a periplasmic nitrate reductase which is induced by nitrate and it is not repressed by ammonium or oxygen. In a Tn5 mutant lacking nitrate reductase activity, transposon insertion is localized in a 1.2 kb EcoRI fragment. A 0.6 kb BamHI-EcoRI segment of this region was used as a probe to isolate, from the wild-type strain, a 6.8 kb PstI fragment carrying the putative genes coding for the periplasmic nitrate reductase. In vivo protein expression and DNA sequence analysis reveal the presence in this region of three genes, napABC, probably organized in an operon. These genes are required for nitrate reduction, as deduced by mutational and complementation studies. The napA gene codes for a protein with a high homology to the periplasmic nitrate reductase from Alcaligenes eutrophus and, to a lesser extent, to other prokaryotic nitrate reductases and molybdenum-containing enzymes. The napB gene product has two haem c-binding sites and shows a high homology with the cytochrome c-type subunit of the periplasmic nitrate reductase from A. eutrophus. NAPA and NAPB proteins appear to be translated with signal peptides of 29 and 24 residues, respectively, indicating that mature proteins are located in the periplasm. The napC gene codes for a 25 kDa protein with a transmembrane sequence of 17 hydrophobic residues. NAPC has four haem c-binding sites and is homologous to the membrane-bound c-type cytochromes encoded by Pseudomonas stutzeri nirT and Escherichia coli torC genes. The phenotypes of defined insertion mutants constructed for each gene also indicate that periplasmic nitrate reductase from R. sphaeroides DSM 158 is a dimeric complex of a 90 kDa catalytic subunit (NAPA) and a 15 kDa cytochrome c (NAPB), which receives electrons from a membrane-anchored tetrahaem protein (NAPC), thus allowing electron flow between membrane and periplasm. This nitrate-reducing system differs from the assimilatory and respiratory bacterial nitrate reductases at the level of cellular localization, regulatory properties, biochemical characteristics and gene organization.

[Arterial pressure during the neonatal transitional period]. / Tensión arterial en el período de transición neonatal.

A total of 288 determinations of blood pressure were practiced in newborns using an ultrasonic unit with the purpose of establishing the blood pressure in the newborn during the neonatal transitional period in a sampling cycle divided into four stages (0:30, 4:00, 6:00 and 10:00 hours). The results were analyzed in a descriptive fashion by means of variation indexes that show the changes occurred in B.P. between one and the other stages of the sampling cycle.