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Llamas M.A.,Estacion Experimental del Zaidin Consejo Superior de Investigaciones Cientificas | Imperi F.,University of Rome La Sapienza | Visca P.,Third University of Rome | Lamont I.L.,University of Otago
FEMS Microbiology Reviews | Year: 2014

Membrane-spanning signaling pathways enable bacteria to alter gene expression in response to extracytoplasmic stimuli. Many such pathways are cell-surface signaling (CSS) systems, which are tripartite molecular devices that allow Gram-negative bacteria to transduce an extracellular stimulus into a coordinated transcriptional response. Typically, CSS systems are composed of the following: (1) an outer membrane receptor, which senses the extracellular stimulus; (2) a cytoplasmic membrane-spanning protein involved in signal transduction from the periplasm to the cytoplasm; and (3) an extracytoplasmic function (ECF) sigma factor that initiates expression of the stimulus-responsive gene(s). Members of genus Pseudomonas provide a paradigmatic example of how CSS systems contribute to the global control of gene expression. Most CSS systems enable self-regulated uptake of iron via endogenous (pyoverdine) or exogenous (xenosiderophores, heme, and citrate) carriers. Some are also implicated in virulence, biofilm formation, and cell-cell interactions. Incorporating insights from the well-characterized alginate regulatory circuitry, this review will illustrate common themes and variations at the level of structural and functional properties of Pseudomonas CSS systems. Control of the expression and activity of ECF sigma factors are central to gene regulation via CSS, and the variety of intrinsic and extrinsic factors influencing these processes will be discussed. © 2014 Federation of European Microbiological Societies.

Bastiaansen K.C.,Estacion Experimental del Zaidin Consejo Superior de Investigaciones Cientificas | Bastiaansen K.C.,VU University Amsterdam | Van Ulsen P.,VU University Amsterdam | Wijtmans M.,VU University Amsterdam | And 2 more authors.
Journal of Biological Chemistry | Year: 2015

The Fox system of Pseudomonas aeruginosa is a cell-surface signaling (CSS) pathway employed by the bacterium to sense and respond to the presence of the heterologous siderophore ferrioxamine in the environment. This regulatory pathway controls the transcription of the foxA ferrioxamine receptor gene through the extracytoplasmic function sigma factor σFoxI. In the absence of ferrioxamine, the activity of σFoxI is inhibited by the transmembrane anti-sigma factor FoxR. Upon binding of ferrioxamine by the FoxA receptor, FoxR is processed by a complex proteolytic cascade leading to the release and activation of σFoxI. Interestingly, we have recently shown that FoxR undergoes self-cleavage between the periplasmic Gly-191 and Thr-192 residues independent of the perception of ferrioxamine. This autoproteolytic event, which is widespread among CSS anti-sigma factors, produces two distinct domains that interact and function together to transduce the presence of the signal. In this work, we provide evidence that the selfcleavage of FoxR is not an enzyme-dependent process but is induced by an N-O acyl rearrangement. Mutation analysis showed that the nucleophilic side chain of the Thr-192 residue at +1 of the cleavage site is required for an attack on the preceding Gly-191, after which the resulting ester bond is likely hydrolyzed. Because the cleavage site is well preserved and the hydrolysis of periplasmic CSS anti-sigma factors is widely observed, we hypothesize that cleavage via an N-O acyl rearrangement is a conserved feature of these proteins. © 2015, American Society for Biochemistry and Molecular Biology Inc. All rights reserved.

Campos C.,University of Evora | Cardoso H.,University of Evora | Nogales A.,University of Evora | Svensson J.,Nordic Genetic Resource Center | And 5 more authors.
PLoS ONE | Year: 2015

Arbuscular mycorrhizal fungi (AMF) are root-inhabiting fungi that form mutualistic symbioses with their host plants. AMF symbiosis improves nutrient uptake and buffers the plant against a diversity of stresses. Rhizophagus irregularis is one of the most widespread AMF species in the world, and its application in agricultural systems for yield improvement has increased over the last years. Still, from the inoculum production perspective, a lack of consistency of inoculum quality is referred to, which partially may be due to a high genetic variability of the fungus. The alternative oxidase (AOX) is an enzyme of the alternative respiratory chain already described in different taxa, including various fungi, which decreases the damage caused by oxidative stress. Nevertheless, virtually nothing is known on the involvement of AMF AOX on symbiosis establishment, as well on the existence of AOX variability that could affect AMF effectiveness and consequently plant performance. Here, we report the isolation and characterisation of the AOX gene of R. irregularis (RiAOX), and show that it is highly expressed during early phases of the symbiosis with plant roots. Phylogenetic analysis clustered RiAOX sequence with ancient fungi, and multiple sequence alignment revealed the lack of several regulatory motifs which are present in plant AOX. The analysis of RiAOX polymorphisms in single spores of three different isolates showed a reduced variability in one spore relatively to a group of spores. A high number of polymorphisms occurred in introns; nevertheless, some putative amino acid changes resulting from non-synonymous variants were found, offering a basis for selective pressure to occur within the populations. Given the AOX relatedness with stress responses, differences in gene variants amongst R. irregularis isolates are likely to be related with its origin and environmental constraints and might have a potential impact on inoculum production. © 2015 Campos et al.This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

Rivero J.,Estacion Experimental del Zaidin Consejo Superior de Investigaciones Cientificas | Gamir J.,Jaume I University | Gamir J.,University of Fribourg | Aroca R.,Estacion Experimental del Zaidin Consejo Superior de Investigaciones Cientificas | And 2 more authors.
Frontiers in Microbiology | Year: 2015

Beneficial plant-microorganism interactions are widespread in nature. Among them, the symbiosis between plant roots and arbuscular mycorrhizal fungi (AMF) is of major importance, commonly improving host nutrition and tolerance against environmental and biotic challenges. Metabolic changes were observed in a well-established symbiosis between tomato and two common AMF: Rhizophagus irregularis and Funneliformis mosseae. Principal component analysis of metabolites, determined by non-targeted liquid chromatography-mass spectrometry, showed a strong metabolic rearrangement in mycorrhizal roots. There was generally a negative impact of mycorrhizal symbiosis on amino acid content, mainly on those involved in the biosynthesis of phenylpropanoids. On the other hand, many intermediaries in amino acid and sugar metabolism and the oxylipin pathway were among the compounds accumulating more in mycorrhizal roots. The metabolic reprogramming also affected other pathways in the secondary metabolism, mainly phenyl alcohols (lignins and lignans) and vitamins. The results showed that source metabolites of these pathways decreased in mycorrhizal roots, whilst the products derived from a-linolenic and amino acids presented higher concentrations in AMF-colonized roots. Mycorrhization therefore increased the flux into those pathways. Venn-diagram analysis showed that there are many induced signals shared by both mycorrhizal interactions, pointing to general mycorrhiza-associated changes in the tomato metabolome. Moreover, fungus-specific fingerprints were also found, suggesting that specific molecular alterations may underlie the reported functional diversity of the symbiosis. Since most positively regulated pathways were related to stress response mechanisms, their potential contribution to improved host stress tolerance is discussed. © 2015 Rivero, Gamir, Aroca, Pozo and Flors.

Martinez-Fernandez G.,Estacion Experimental del Zaidin Consejo Superior de Investigaciones Cientificas | Abecia L.,Estacion Experimental del Zaidin Consejo Superior de Investigaciones Cientificas | Arco A.,Estacion Experimental del Zaidin Consejo Superior de Investigaciones Cientificas | Cantalapiedra-Hijar G.,Estacion Experimental del Zaidin Consejo Superior de Investigaciones Cientificas | And 6 more authors.
Journal of Dairy Science | Year: 2014

The aim of this work was to investigate the effect of feeding ethyl-3-nitrooxy propionate (E3NP) and 3-nitrooxypropanol (3NP), 2 recently developed compounds with potential antimethanogenic activity, in vitro and in vivo in nonlactating sheep on ruminal methane production, fermentation pattern, the abundance of major microbial groups, and feed degradability. Three experiments were conducted, 1 in vitro and 2 in vivo. The in vitro batch culture trial (experiment 1) tested 2 doses of E3NP and 3NP (40 and 80. μL/L), which showed a substantial reduction of methane production (up to 95%) without affecting concentration of volatile fatty acids (VFA). The 2 in vivo trials were conducted over 16 d (experiment 2) and 30 d (experiment 3) to study their effects in sheep. In experiment 2, 6 adult nonpregnant sheep, with permanent rumen cannula and fed alfalfa hay and oats (60:40), were treated with E3NP at 2 doses (50 and 500. mg/animal per day). After 7, 14, and 15 d of treatment, methane emissions were recorded in respiration chambers and rumen fluid samples were collected for VFA analysis and quantification of bacterial, protozoal, and archaeal numbers by real-time PCR. Methane production decreased by 29% compared with the control with the higher dose of E3NP on d 14 to 15. A decrease in the acetate:propionate ratio was observed without detrimental effects on dry matter intake. In experiment 3, 9 adult nonpregnant sheep, with permanent rumen cannula and fed with alfalfa hay and oats (60:40), were treated with E3NP or 3NP at one dose (100. mg/animal per day) over 30 d. On d 14 and d 29 to 30, methane emissions were recorded in respiration chambers. Rumen fluid samples were collected on d 29 and 30 for VFA analysis and quantification of bacterial, protozoal, and archaeal numbers by real-time PCR. In addition, on d 22 and 23, samples of oats and alfalfa hay were incubated in the rumen of sheep to determine dry matter ruminal degradation over 24 and 48. h, respectively; no effect was observed (78.6, 78.3, and 78.8% of alfalfa and 74.2, 74.0, and 70.6% of oats in control, E3NP, and 3NP groups, respectively). A reduction in methane production was observed for both additives at d 14 and d 29 to 30. In both treatments, the acetate:propionate ratio was significantly decreased. Likewise, total concentrations of the analyzed microbial groups in the rumen showed no difference among treatments and doses for both experiments. Both tested compounds showed promise as methane inhibitors in the rumen, with no detrimental effects on fermentation or intake, which would need to be confirmed in lactating animals. © 2014 American Dairy Science Association.

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