A periplasmic D-malate:cytochrome c oxidoreductase (DMCO) was identified in Ectopseudomonas oleovorans CECT5344, utilizing 2-(4-iodophenyl)-3-(4-nitrophenyl)-5-phenyl tetrazolium chloride (INT) as an artificial electron acceptor. The assay was adapted for a spectrophotometric or native polyacrylamide gel electrophoresis (PAGE) analysis. The DMCO-encoding gene (BN5_4044) was cloned and expressed in Escherichia coli, enabling a partial purification and biochemical characterization. In addition to D-malate, the enzyme oxidizes D-2-hydroxyglutarate and, to a lesser extent, D-lactate, with cytochrome c also serving as an electron acceptor. DMCO requires Zn2+ for activity and exists as a dimer, as determined by gel filtration. The in vitro reconstitution of the electron transfer from D-malate to oxygen was achieved using spheroplasts, enriched periplasmic fractions, and cytochrome c. This extracytoplasmic respiration, unique among homologs of this protein, may eliminate the need for a dedicated inner membrane transporter, thereby avoiding potential upstream respiratory bottlenecks. In the context of bioremediation, and particularly regarding the cyanide metabolism, this D-malate oxidation to oxaloacetate facilitates detoxification by forming the corresponding cyanohydrin, which can be subsequently assimilated for growth.

Summary The transcriptomic analysis (RNA‐seq) of a fur mutant of P. pseudoalcaligenes CECT 5344 has revealed that Fur regulates the expression of more than 100 genes in this bacterial strain, most of them negatively. The highest upregulated genes in response to fur deletion, with respect to the wild type, both cultivated in LB medium, corresponded to genes implicated in iron uptake. They include both TonB‐dependent siderophore transporters for the active transport across the outer membrane, and ABC‐type and MSF‐type transporters for the active transport across the cytoplasmic membrane. Therefore, the main response of this bacterium to iron limitation is expressing genes necessary for metabolism of Fe siderophores produced by other microorganisms (xenosiderophores). The number of genes whose expression decreased in the fur− mutant, as well as its normalized expression (fold change), was lower. Among them, it is remarkable the presence of one of the two cas operons of the two CRISP/Cas clusters was detected in the genome of this bacterium. The transcriptome was validated by qPCR, including the decrease in the expression of cas genes (cse1). The expression of cse1 was also decreased by limiting the amount of iron, carbon or nitrogen in the medium, or by adding menadione, a compound that causes oxidative stress. The higher decrease in cse1 expression was triggered by the addition of cyanide in minimal medium. These results suggest that this bacterium responds to stress conditions, and especially to cyanide, taking a reasonable risk with respect to both the uptake of (TonB‐dependent receptors gates) and the tolerance to (reduced immunity) foreign nucleic acids. In conjunction, this can be considered a yet unknown molecular mechanism forcing bacterial evolution.

The effect of pH on bacterial cell-growth and the evolution of extracellular pH triggered by bacterial growth has been monitored for three bacterial strains, Escherichia coli ATCC 25922 and Pseudomonas putida KT2440 as reference strains, and Pseudomonas pseudoalcaligenes CECT 5344 because of its capacity to assimilate cyanide as the sole nitrogen source under alkaline conditions. In a first instance, the influence of the initial pH in the growth curve has been texted in LB-medium adjusted to pH 6, 7 and 8, for E. coli and P. putida, and 7.5, 8.25 and 9 for P. pseudoalcaligenes. Although the initial pH were different, the pH of the extracellular medium at the end of the stationary phase converged to a certain pH that is specific for each bacterium. Similar experiments were carried out in minimal medium with glucose as the carbon source. In this case, the pHs of the culture of both Pseudomonadaceae strains were almost constant, whereas it suddenly dropped during the exponential growth phase of E. coli. When the initial pH was 6 the extracellular pH fell sharply to 4.5, which irreversibly prevented further cellular growth. Nevertheless, at higher initial pH values subsequent cellular growth of E. coli restored the medium to the initial pHs values. Finally, in all cases the evolution of the pH has been shown to depend on the carbon source used. Among the sources used, cellular growth with glucose or glycerol did not affect the extracellular pH, whereas citrate caused the alkalinization of the media. This phenotype is in concordance with computational predictions, at least in the case of the genome-scale metabolic model of Pseudomonas putida KT2440.

Furfural and 5-hydroxymethylfurfural (HMF) are degradation products of lignocellulose during pretreatment operations. Furfural compounds are a group of chemical compounds whose common thread is an aldehyde group attached to a furan ring, and they constitute a problem for the development of second-generation biofuels because they act as fermentation inhibitors of the lignocellulose hydrolysates. Up to date, very few bacteria have been described to be able to eliminate them. The objective of this work was to isolate and characterize bacterial strains able to use, as the sole carbon source, 5-(hydroxymethyl)-furfural (HMF) and furan derivatives.

Most cyanide-containing industrial effluents also contain other cyano-derivatives and high amounts of metals and metal-cyanide compounds. For this reason, the biotreatment of these wastes requires the use of microorganisms capable to degrade all these different cyano-compounds and to tolerate metals. Pseudomonas pseudoalcaligenes CECT 5344 is a cyanotrophic bacterium capable of metabolize cyanide in its free form, but it is not very efficient at degrading metal-cyanide complexes. Therefore, for the optimization of the cyanide biodegradation process it is essential to find and characterize new bacterial strains, capable of assimilating metal cyanide-complexes, to complement the capacities of P. pseudoalcaligenes CECT 5344.

Pseudomonas pseudoalcaligenes CECT5344, a Gram-negative bacterium isolated from the Guadalquir River (Córdoba, Spain), is able to utilize different cyano-derivatives. Here, the complete genome sequence of P. pseudoalcaligenes CECT5344 harboring a 4,686,340 bp circular chromosome encoding 4513 genes and featuring a GC-content of 62.34% is reported. Necessarily, remaining gaps in the genome had to be closed by assembly of few long reads obtained from PacBio single molecule real-time sequencing. Here, the first complete genome sequence for the species P. pseudoalcaligenes is presented.

The Fur protein is the main sensor of cellular iron status in bacteria. In the present study, we inactivated the fur gene of Pseudomonas pseudoalcaligenes CECT5344 and characterized the resulting mutant. Our findings provide experimental evidence that, cyanide generates an intracellular signal equivalent to that triggered by iron deprivation, as witnessed by the induction of prrF and fiuA (ferrichrome receptor) expression in the presence of cyanide. The fur mutant also displayed slow growth, especially in minimal culture medium, increased sensitivity to cyanide in LB medium and as expected, resistance to manganese ions. Moreover, the mutant exhibited enhanced iron accumulation and increased sensitivity to streptonigrin, as well as to some inducers of oxidative stress, such as paraquat and menadione, yet it remained resistant to hydrogen peroxide. Surprisingly, neither the wild type strain nor the fur mutant strain produced siderophores that could be detected using the universal CAS-agar method.

SummaryPseudomonas pseudoalcaligenesCECT5344 is a Gram‐negative bacterium able to tolerate cyanide and to use it as the sole nitrogen source. We report here the first draft of the whole genome sequence of aP. pseudoalcaligenesstrain that assimilates cyanide. Three aspects are specially emphasized in this manuscript. First, some generalities of the genome are shown and discussed in the context of otherPseudomonadaceaegenomes, including genome size, G + Ccontent, core genome and singletons among other features. Second, the genome is analysed in the context of cyanide metabolism, describing genes probably involved in cyanide assimilation, like those encoding nitrilases, and genes related to cyanide resistance, like theciogenes encoding the cyanide insensitive oxidases. Finally, the presence of genes probably involved in other processes with a great biotechnological potential like production of bioplastics and biodegradation of pollutants also is discussed.

In general, the biodegradation of a toxic compound by a micro-organism requires the concurrence of, at least, two features in the biological system: first, the capability of the micro-organism to metabolize the toxic compound, and secondly, the capacity to resist its toxic effect. Pseudomonas pseudoalcaligenes CECT5344 is a bacterium used in the biodegradation of cyanide because it is capable to use it as a nitrogen source. The present review is mainly focused on the putative role of iron-containing enzymes of the tricarboxylic acid cycle in cyanide resistance by P. pseudoalcaligenes CECT5344.

Pseudomonas pseudoalcaligenes CECT5344 can be used in cyanide bioremediation processes because it grows at pH 9.5 using 2.0 mM cyanide at the sole nitrogen source. Cyanide strongly binds to metals creating iron-deprivation conditions. The bacterium responds to the presence of cyanide by inducing several processes such as siderophore synthesis for iron capture, cyanide-insensitive respiration system and defence mechanisms against oxidative stress. Since high concentrations of cyanide cause iron deficiency and because iron is an essential nutrient, bacterial growth in the presence of cyanide requires an efficient iron uptake. Fur is a global transcription factor that regulates a diversity of biological processes such as iron homoeostasis, TCA (tricarboxylic acid) cycle metabolism and oxidative stress response. Fur's regulation of iron uptake and storage genes should play a significant role in the lives of these bacteria. In the present review, current knowledge of Fur is summarized.

The alkaliphilic bacterium Pseudomonas pseudoalcaligenes CECT5344 is able to grow with cyanide as the sole nitrogen source. Membrane fractions from cells grown under cyanotrophic conditions catalysed the production of oxaloacetate from l -malate. Several enzymic activities of the tricarboxylic acid and glyoxylate cycles in association with the cyanide-insensitive respiratory pathway seem to be responsible for the oxaloacetate formation in vivo . Thus, in cyanide-grown cells, citrate synthase and isocitrate lyase activities were significantly higher than those observed with other nitrogen sources. Malate dehydrogenase activity was undetectable, but a malate : quinone oxidoreductase activity coupled to the cyanide-insensitive alternative oxidase was found in membrane fractions from cyanide-grown cells. Therefore, oxaloacetate production was linked to the cyanide-insensitive respiration in P. pseudoalcaligenes CECT5344. Cyanide and oxaloacetate reacted chemically inside the cells to produce a cyanohydrin (2-hydroxynitrile), which was further converted to ammonium. In addition to cyanide, strain CECT5344 was able to grow with several cyano derivatives, such as 2- and 3-hydroxynitriles. The specific system required for uptake and metabolization of cyanohydrins was induced by cyanide and by 2-hydroxynitriles, such as the cyanohydrins of oxaloacetate and 2-oxoglutarate.

The female reproductive tract adapts to allow the development of the fetus and several mechanisms are required to protect the fetus from the material immune response. Recent reports have involved ulerine NK cell activity in implantation failures and abortions. Here, we have analysed the role of prostasumes in the regulation of NK cell activity. prostasomes are small vesicles secreted byt he prostatic gland that contribute to sperm protection within the female reproductive tract because their immunosuppresive properties. The phenotypical analysis performed by flow cytometry revealed that prostasomes expressed high levels of CD48 and CD86 molecules but did not express MICAIB, CD80, CD112, or CD155. In conclusion, the expression of ligands for NK receptors in prostasomes may have functional relevance in the modulation of NK cell triggering.

In a study on gene deregulation in ovarian carcinoma we found a mRNA coding for a 350 kDa protein, Drop1, to be downregulated 20‐ to 180‐fold in the majority of ovarian and mammary carcinomas. The mRNA is encoded by a set of exons in the 5′ region of the SYNE1 gene. Immunohistochemical staining for Drop1 protein by a specific monoclonal antibody corresponds to the pattern seen for the mRNA. cDNA arrays of matched pairs of tumor and normal tissue and in situ hybridizations confirmed the drastic loss of Drop1 mRNA as a common feature in uterus, cervix, kidney, lung, thyroid and pancreas carcinomas, already at early tumor stages and in all metastases. Two‐hybrid studies suggest a role of this deficiency in the malignant progression of epithelial tumors. © 2008 Wiley‐Liss, Inc.

Departamento de Bioquimica, Biologia Molecular y Genetica, Facultad de Veterinaria, Universidad de Extremadura, Avda de la Universidad s/n, 10071 Caceres, Spain. Cyanide is a toxic nitrogen compound for almost every living organism since it binds irreversibly to haem-proteins, (i.e. cytochromes involved in all known respiratory processes [1]). In the nature, there are bacteria, algae, fungi, and many plants, which are able to produce cyanide. However, the contamination of the environment with cyanide is mainly due to human activities. In this sense, mining, electroplating and jewellery industries generate effluents with a high concentration of cyanide. There are many possible methods for treating wastes containing cyanide. The latest cyanide disposal method is cyanide biodegradation, which is an environmentally friendly and quite inexpensive treatment method [2]. Some microorganisms have been described to be able to degrade cyanide at a neutral pH, but under this condition a high concentration of cyanide evaporates as hydrocyanic acid (HCN), a weak acid with a pK

ABSTRACT Pseudomonas pseudoalcaligenes CECT5344 grows in minimal medium containing cyanide as the sole nitrogen source. Under these conditions, an O2-dependent respiration highly resistant to cyanide was detected in cell extracts. The structural genes for the cyanide-resistant terminal oxidase, cioA and cioB, are clustered and encode the integral membrane proteins that correspond to subunits I and II of classical cytochrome bd, although the presence of heme d in the membrane could not be detected by difference spectra. The cio operon from P. pseudoalcaligenes presents a singular organization, starting upstream of cioAB by the coding sequence of a putative ferredoxin-dependent sulfite or nitrite reductase and spanning downstream two additional open reading frames that encode uncharacterized gene products. PCR amplifications of RNA (reverse transcription-PCR) indicated the cyanide-dependent up-regulation and cotranscription along the operon. The targeted disruption of cioA eliminates both the expression of the cyanide-stimulated respiratory activity and the growth with cyanide as the nitrogen source, which suggests a critical role of this cytochrome bd-related oxidase in the metabolism of cyanide by P. pseudoalcaligenes CECT5344.

The determinants of the thioredoxin (TRX)-dependent redox regulation of the chloroplastic NADP-malate dehydrogenase (NADP-MDH) from the eukaryotic green alga Chlamydomonas reinhardtii have been investigated using site-directed mutagenesis. The results indicate that a single C-terminal disulfide is responsible for this regulation. The redox midpoint potential of this disulfide is less negative than that of the higher plant enzyme. The regulation is of an all-or-nothing type, lacking the fine-tuning provided by the second N-terminal disulfide found only in NADP-MDH from higher plants. The decreased stability of specific cysteine/alanine mutants is consistent with the presence of a structural disulfide formed by two cysteine residues that are not involved in regulation of activity. Measurements of the ability of C. reinhardtii thioredoxin f (TRX f) to activate wild-type and site-directed mutants of sorghum (Sorghum vulgare) NADP-MDH suggest that the algal TRX f has a redox midpoint potential that is less negative than most those of higher plant TRXs f. These results are discussed from an evolutionary point of view.

Malate synthase (EC 2.3.3.9, formerly EC 4.1.2.2) has been investigated in the unicellular green algae Chlamydomonas reinhardtii. The molecular characteristics and the regulation of gene expression have been investigated for the enzyme. A full-length malate synthase cDNA has been isolated, containing an open reading frame of 1,641 bp encoding a polypeptide of 546 amino acids. This protein shares the conserved signature of the malate synthase family, along with the catalytic residues essential for enzymatic activity and a C-terminal motif that matches the consensus for glyoxysome import. Functionality studies have been facilitated by heterologous expression of the malate synthase cDNA in Escherichia coli. The remarkable metabolic versatility of the alga has been used to analyse the metabolic control of malate synthase gene expression. The data strongly support the role of acetate and light as the main regulatory effectors, and the existence of cross-talk between the two signalling pathways.