Coimbra, Portugal
Coimbra, Portugal

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Santos S.S.,IMAR CMA | Pardal S.,IMAR CMA | Proenca D.N.,IMAR CMA | Ramos J.A.,IMAR CMA | And 4 more authors.
FEMS Microbiology Ecology | Year: 2012

The diversity of the cloacal microbial community in migratory shorebirds, caught at the Tagus estuary, Portugal, was assessed by cultivation (R2A and Nutrient Agar media) and denaturing gradient gel electrophoresis profiling (DGGE) to provide a better understanding of the birds' potential to harbor and disperse pathogens. Three different bird species belonging to four different populations were studied: common redshank (Tringa totanus), black-winged stilt (Himantopus himantopus) and nominate and Icelandic populations of black-tailed godwit (Limosa limosa). DGGE profiling and partial 16S RNA gene sequences of 240 isolates, and 26 DGGE bands resulting in 58 clones, were analyzed. Most isolates were members of the phylum Firmicutes and Actinobacteria and only a small portion belonged to the Proteobacteria and Deinococcus-Thermus phyla. Potentially pathogenic strains carried by the birds were found such as Helicobacter and Staphylococcus in all bird species, and Clostridium, Mycobacterium, Rhodococcus, Legionella and Corynebacterium in black-winged stilts. Unexpectedly, bacteria from the phylum Deinococcus-Thermus were isolated in shorebirds and were present in all the bird species studied. © 2012 Federation of European Microbiological Societies.


Suan G.,University Claude Bernard Lyon 1 | Mattioli E.,University Claude Bernard Lyon 1 | Pittet B.,University Claude Bernard Lyon 1 | Lecuyer C.,University Claude Bernard Lyon 1 | And 6 more authors.
Earth and Planetary Science Letters | Year: 2010

The Early Toarcian Oceanic Anoxic Event (T-OAE), about 183 myr ago, was a global event of environmental and carbon cycle perturbations, which deeply affected both marine biota and carbonate production. Nevertheless, the long-term environmental conditions prevailing prior to the main phase of marine extinction and carbonate production crisis remain poorly understood. Here we present a ∼ 8 myr-long record of Early Pliensbachian-Middle Toarcian environmental changes from the Lusitanian Basin, Portugal, in order to address the long-term paleoclimatic evolution that ultimately led to carbonate production and biotic crises during the T-OAE. Paleotemperature estimates derived from the oxygen isotope compositions of well-preserved brachiopod shells from two different sections reveal a pronounced ∼ 5 °C cooling in the Late Pliensbachian (margaritatus-spinatum ammonite Zones boundary). This cooling event is followed by a marked ∼ 7-10 °C seawater warming in the Early Toarcian that, after a second cooling event in the mid-polymorphum Zone, culminates during the T-OAE. Calcium carbonate (CaCO3) contents, the amount of nannofossil calcite and the mean size of the major pelagic carbonate producer Schizosphaerella, all largely covary with paleotemperatures, indicating a coupling between climatic conditions and both pelagic and neritic CaCO3 production. Furthermore, the cooling and warming episodes coincided with major marine regressions and transgressions, respectively, suggesting that the growth and decay of ice caps may have exerted a strong control on sea-level fluctuations throughout the studied time interval. This revised chronology of environmental changes shows important similarities with Neogene and Paleozoic episodes of deglacial black shale formation, and thus prompts the reevaluation of ice sheet dynamics as a possible agent of Mesozoic events of extinction and organic-rich sedimentation. © 2009 Elsevier B.V. All rights reserved.


Francisco R.,IMAR CMA | Moreno A.,University of Coimbra | Morais P.V.,IMAR CMA
BioMetals | Year: 2010

Studies of Cr(VI) toxicity are generally performed using chromate salts in solution, both when studying the effects on prokaryotes and eukaryotes. Some studies on human carcinogenesis and toxicology on bacteria were done using dichromate, but comparison with chromate was never reported before, and dichromate existence was never taken into consideration and usually overlooked. This paper studied comparatively the effect of dichromate and chromate on the physiology of Ochrobactrum tritici strain 5bvl1, a highly Cr(VI)-resistant and reducing microorganism. This study demonstrated that the addition of chromate or dichromate sodium salts to growth medium at neutral pH ended-up in two different solutions with a different balance of chemical species. Cr(VI) was toxic to O. tritici strain 5bvl1, as clearly shown on growth, reduction, respiration, glucose accumulation assays and by comparing cell morphology. Moreover, the addition of sodium dichromate was always more toxic to cells when compared to chromate and achieved a higher inhibition of every parameter studied. The toxicity differences between the two Cr(VI) oxyanions indicate the possibility of a different impact of Cr(VI) contamination on the environment. This may be of major importance, considering the slight acidity of most of the arable lands which favours the presence of dichromate, the more toxic species. © Springer Science+Business Media, LLC. 2010.


Sousa T.,IMAR CMA | Chung A.-P.,IMAR CMA | Pereira A.,IMAR CMA | Pereira A.,University of Coimbra | And 3 more authors.
Metallomics | Year: 2013

Severe environmental problems arise from old uranium mines, which continue to discharge uranium (U) via acid mine drainage water, resulting in soil, subsoil and groundwater contamination. Bioremediation of U contaminated environments has been attempted, but most of the conceptual models propose U removal by cell suspensions of anaerobic bacteria. In this study, strain Rhodanobacter A2-61, isolated from Urgeiriça Mine, Portugal, was shown to resist up to 2 mM of U(vi). The conditions used (low nutrient content and pH 5) potentiated the interaction of the toxic uranyl ion with the tested strain. The strain was able to remove approximately 120 μM of U(vi) when grown aerobically in the presence of 500 μM U. Under these conditions, this strain was also able to lower the phosphate concentration in the medium and increased its capacity to take up inorganic phosphate, accumulating up to 0.52 μmol phosphate per optical density unit of the medium at 600 nm, after 24 hours, corresponding approximately to the late log phase of the bacterial culture. Microscopically dense intracellular structures with nanometer size were visible. The extent of U inside the cells was quantified by LS counting. EDS analysis of heated cells showed the presence of complexes composed of phosphate and uranium, suggesting the simultaneous precipitation of U and phosphate within the cells. XRD analysis of the cells containing the U-phosphate complexes suggested the presence of a meta-autunite-like mineral structure. SEM identified, in pyrolyzed cells, crystalline nanoparticles with shape in the tetragonal system characteristic of the meta-autunite-like mineral structures. U removal has been reported previously but mainly by cell suspensions and through release of phosphate. The innovative Rhodanobacter A2-61 can actively grow aerobically, in the presence of U, and can efficiently remove U(vi) from the environment, accumulating it in a structural form consistent with that of the mineral meta-autunite inside the cell, corresponding to effective metal immobilization. This work supports previous findings that U bioremediation could be achieved via the biomineralization of U(vi) in phosphate minerals. © 2013 The Royal Society of Chemistry.


Sousa T.,IMAR CMA | Branco R.,IMAR CMA | Piedade A.P.,University of Coimbra | Morais P.V.,IMAR CMA | Morais P.V.,University of Coimbra
PLoS ONE | Year: 2015

Ochrobactrum tritici SCII24T is a highly As-resistant bacterium, with two previously described arsenic resistance operons, ars1 and ars2. Among a large number of genes, these operons contain the arsB and Acr3 genes that encode the arsenite efflux pumps responsible for arsenic resistance. Exploring the genome of O. tritici SCII24T , an additional putative operon (ars3) was identified and revealed the presence of the Acr3-2 gene that encodes for an arsenite efflux protein but which came to prove to not be required for full As resistance. The genes encoding for arsenite efflux pumps, identified in this strain, were inactivated to develop microbial accumulators of arsenic as new tools for bioremediation. Six different mutants were produced, studied and three were more useful as biotools. O. tritici wild type and the Acr3-mutants showed the highest resistance to As(III), being able to grow up to 50 mM of arsenite. On the other hand, arsB-mutants were not able to grow at concentrations higher than 1 mM As(III), and were the most As(III) sensitive mutants. In the presence of 1 mM As(III), the strain with arsB and Acr3-1 mutated showed the highest intracellular arsenic concentration (up to 17 ng(As)/mg protein), while in assays with 5 mM As(III), the single arsB-mutant was able to accumulate the highest concentration of arsenic (up to 10 ng(As)/mg protein). Therefore, arsB is the main gene responsible for arsenite resistance in O. tritici. However, both genes arsB and Acr3-1 play a crucial role in the resistance mechanism, depending on the arsenite concentration in the medium. In conclusion, at moderate arsenite concentrations, the double arsB- and Acr3-1-mutant exhibited a great ability to accumulate arsenite and can be seen as a promising bioremediation tool for environmental arsenic detoxification. © 2015 Sousa et al.


Maleita C.M.,University of Coimbra | Simoes M.J.,Technology Transfer Center | Egas C.,Technology Transfer Center | Curtis R.H.C.C.,Rothamsted Research | de O Abrantes I.M.,IMAR CMA
Plant Disease | Year: 2012

Meloidogyne hispanica infects many economically important crops worldwide. The accurate identification of this pathogen is essential for the establishment of efficient and sustainable integrated pest management programs. Portuguese M. hispanica isolates were studied by biometrical, biochemical, and molecular characteristics. Biometrical characteristics of M. hispanica females, males, and second-stage juveniles were similar to the original description. Biochemical studies revealed a unique enzyme pattern (Hi4) for M. hispanica esterases that allowed for species differentiation. Molecular analysis of the mtDNA region from COII and 16S rRNA genes resulted in amplification products (1,800 bp) similar to M. hispanica, M. ethiopica, and M. javanica, and the described HinfI was unable to discriminate M. hispanica from the other two species. Analysis of the mtDNA sequences revealed altered nucleotides among the isolates that created new restriction sites for AluI and DraIII. The resulting restriction patterns successfully discriminated between the three species, providing a new tool for Meloidogyne identification. Finally, the phylogenetic relationship between M. hispanica and several Meloidogyne spp. sequences was analyzed using mtDNA, confirming the divergence between meiotic and mitotic species and revealing the proximity of M. hispanica to closely related species. Based on the studies conducted, the application of isozyme or polymerase chain reaction restriction fragment length polymorphism analysis would be a useful and efficient methodology for M. hispanica identification. © 2012 The American Phytopathological Society.


Coelho C.,IMAR CMA | Branco R.,IMAR CMA | Natal-da-Luz T.,IMAR CMA | Sousa J.P.,IMAR CMA | And 3 more authors.
Chemosphere | Year: 2015

Chromate can be considered a potent environmental contaminant and consequently, an understanding of chromate availability and toxicity to soil biology is essential for effective ecological assessment of metal impact in soils. This study shows the response of two bacterial bioreporters, pCHRGFP1 Escherichia coli and pCHRGFP2 Ochrobactrum tritici, to increasing concentrations of chromate in two different soils. The bioreporters, carrying the regulatory gene chrB transcriptionally fused to the gfp reporter system, exhibited different features. In both, the fluorescence signal and the chromate concentration could be linearly correlated but E. coli biosensor functioned within the range of 0.5-2μM and O. tritici biosensor within 2-10μM chromate. The bioreporters were validated through comparative measurements using the chemical chromate methods of diphenylcarbazide and ionic chromatography. The bacterial sensors were used for the estimation of bioavailable fraction of chromate in a natural soil and OECD artificial soil, both spiked with chromate in increasing concentrations of 0-120mgCr(VI)kg-1 of soil. OECD soil showed a faster chromate decrease comparing to the natural soil. The toxicity of soils amended with chromate was also evaluated by ecotoxicological tests through collembolan reproduction tests using Folsomia candida as test organism. Significant correlations were found between collembolans reproduction and chromate concentration in soil (lower at high chromate concentrations) measured by biosensors. Data obtained showed that the biosensors tested are sensitive to chromate presence in soil and may constitute a rapid and efficient method to measure chromate availability in soils. © 2014 Elsevier Ltd.


PubMed | IMAR CMA
Type: | Journal: Journal of hazardous materials | Year: 2011

Hexavalent chromium (Cr(VI)) is a toxic environmental contaminant which detoxification consists in reduction to Cr(III). In this work, the Cr(VI)-resistant and reducing Ochrobactrum tritici 5bvl1 produced phosphate nanoparticles upon exposure to Cr(VI) and Fe(III), effectively removing chromium from solution. Under Cr(VI) stress, higher siderophore production by strain 5bvl1 was observed. Cr(VI) toxicity was decreased in presence of Fe(III), increasing the growth and Cr(VI)-reduction rates in cell cultures, lowering the amount of morphologically compromised cells and promoting chromium immobilization as insoluble extracellular phosphate complexes. The formation of phosphate nanoparticles increased with Cr(VI) and Fe(III) concentrations and was also stimulated by Ni(II). Under these experimental conditions, nanoparticle formation occurred together with enhanced inorganic phosphate consumption by cells and increased polyphosphate kinase (PPK) activity. NMR analysis of the particles showed the presence of both polyphosphate and phosphonate together with orthophosphate, and FT-IR supported these results, also showing evidences of Cr(III) coordination. This work demonstrated that O. tritici 5bvl1 possesses protection mechanisms against chromium toxicity other than the presence of the Cr(VI) pump and SOD related enzymes previously described. Future assessment of the molecular regulation of production of these nanoparticles will open new perspectives for remediation of metal contaminated environments.


The diversity of the cloacal microbial community in migratory shorebirds, caught at the Tagus estuary, Portugal, was assessed by cultivation (R2A and Nutrient Agar media) and denaturing gradient gel electrophoresis profiling (DGGE) to provide a better understanding of the birds potential to harbor and disperse pathogens. Three different bird species belonging to four different populations were studied: common redshank (Tringa totanus), black-winged stilt (Himantopus himantopus) and nominate and Icelandic populations of black-tailed godwit (Limosa limosa). DGGE profiling and partial 16S RNA gene sequences of 240 isolates, and 26 DGGE bands resulting in 58 clones, were analyzed. Most isolates were members of the phylum Firmicutes and Actinobacteria and only a small portion belonged to the Proteobacteria and Deinococcus-Thermus phyla. Potentially pathogenic strains carried by the birds were found such as Helicobacter and Staphylococcus in all bird species, and Clostridium, Mycobacterium, Rhodococcus, Legionella and Corynebacterium in black-winged stilts. Unexpectedly, bacteria from the phylum Deinococcus-Thermus were isolated in shorebirds and were present in all the bird species studied.


PubMed | IMAR CMA and University of Coimbra
Type: | Journal: Chemosphere | Year: 2015

Chromate can be considered a potent environmental contaminant and consequently, an understanding of chromate availability and toxicity to soil biology is essential for effective ecological assessment of metal impact in soils. This study shows the response of two bacterial bioreporters, pCHRGFP1 Escherichiacoli and pCHRGFP2 Ochrobactrumtritici, to increasing concentrations of chromate in two different soils. The bioreporters, carrying the regulatory gene chrB transcriptionally fused to the gfp reporter system, exhibited different features. In both, the fluorescence signal and the chromate concentration could be linearly correlated but E. coli biosensor functioned within the range of 0.5-2 M and O. tritici biosensor within 2-10 M chromate. The bioreporters were validated through comparative measurements using the chemical chromate methods of diphenylcarbazide and ionic chromatography. The bacterial sensors were used for the estimation of bioavailable fraction of chromate in a natural soil and OECD artificial soil, both spiked with chromate in increasing concentrations of 0-120 mg Cr(VI) kg(-1) of soil. OECD soil showed a faster chromate decrease comparing to the natural soil. The toxicity of soils amended with chromate was also evaluated by ecotoxicological tests through collembolan reproduction tests using Folsomia candida as test organism. Significant correlations were found between collembolans reproduction and chromate concentration in soil (lower at high chromate concentrations) measured by biosensors. Data obtained showed that the biosensors tested are sensitive to chromate presence in soil and may constitute a rapid and efficient method to measure chromate availability in soils.

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