Expression of the phosphonoalanine-degradative gene cluster from Variovorax sp Pal2 is induced by growth on phosphonoalanine and phosphonopyruvate

The phosphonopyruvate hydrolase (PalA) found in Variovorax sp., Pal2, is a novel carbon-phosphorus bond cleavage enzyme, which is expressed even in the presence of high levels of phosphate, thus permitting phosphonopyruvate to be used as the sole carbon and energy source. Analysis of the regions adjacent to the palA gene revealed the presence of the five structural genes that constitute the 2-amino-3-phosphonopropionic acid (phosphonoalanine)-degradative operon. Reverse transcriptase-PCR (RT-PCR) experiments demonstrated that all five genes in the operon are transcribed as a single mRNA and that their transcription is induced by phosphonoalanine or phosphonopyruvate. Transcriptional fusions of the regulatory region of the phosphonoalanine degradative operon with the gfp gene were constructed. Expression analysis indicated that the presence of a LysR-type regulator (encoded by the palR gene) is essential for the transcription of the structural genes of the operon. Similar gene clusters were found in the sequenced genomes of six bacterial species from the Alpha-, Beta- and Gammaproteobacteria, and analysis of metagenomic libraries revealed that sequences related to palA are widely spread in the marine environment.

Cloning and characterization of a novel cis-naphthalene dihydrodiol dehydrogenase gene (narB) from Rhodococcus sp NCIMB12038

Rhodococcus sp. NCIMB112038 can utilize naphthalene as its sole carbon and energy source. The gene encoding cis-naphthalene dihydrodiol dehydrogenase (narB) of this strain has been cloned and sequenced. Expression of NCIMB12038 cis-naphthalene dihydrodiol dehydrogenase was demonstrated in Escherichia coli cells. narB encodes a putative protein of 271 amino acids and shares 39% amino acid identity with the cis-naphthalene dihydrodiol dehydrogenase from Pseudomonas putida G7. Comparison of NarB with some putative cis-dihydrodiol dehydrogenases from Rhodococcus species revealed significant differences between these proteins. NarB together with two other proteins forms a new group of cis-dihydrodiol dehydrogenases. (C) 2000 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved.

Isolation of Rhodococcus rhodochrous NCIMB13064 derivatives with new biodegradative abilities

Rhodococcus rhodochrous NCIMB13064 can dehalogenate and utilise a number of halogenated aliphatic compounds as sole carbon and energy source. Mutants of NCIMB13064 can be easily isolated with an enlarged range of 1-chloroalkane utilising ability. Dehalogenation of 1-chlorononane, 1-chlorodecane and short-chain 1-chloroalkanes (C-3-C-8) is encoded by the same plasmid pRTL1. However, a different genetic element(s) is required for the dehalogenation of 3-chloropropionic acid. Two derivatives (P200 and P400) of R. rhodochrous NCIMB13064 were isolated which had acquired the ability to utilise naphthalene as sole carbon and energy source. Both strains lost the ability to utilise short-chain 1-chloroalkanes and underwent some rearrangements associated with pRTL1 plasmid.

Plasmid pRTL1 controlling 1-chloroalkane degradation by Rhodococcus rhodochrous NCIMB 13064

Rhodococcus rhodochrous NCIMB13064 can dehalogenate and use a wide range of 1-haloalkanes as sole carbon and energy source. The 1-chloroalkane degradation phenotype may be lost by cells spontaneously or after treatment with Mitomycin C. Two laboratory derivatives of the original strain exhibited differing degrees of stability of the chloroalkane degradation marker. Plasmids of approximately 100 kbp (pRTL1) and 80 kbp (pRTL2) have been found in R. rhodochrous NCIMB13064. pRTL1 was shown to be carrying at least some genes for the dehalogenation of 1-chloroalkanes with short chain lengths (C-3 to C-9). However, no connection was found between the utilization of 1-chloroalkanes with longer chain lengths (C-12 to C-18) and the presence of pRTL1. Three separate events were observed to lead to the inability of NCIMB13064 to dehalogenate the short-chain 1-chloroalkanes; the complete loss of pRTL1, the integration of pRTL1 into the chromosome, or the deletion of a 20-kbp fragment in pRTL1. High-frequency transfer of the 1-chloroalkane degradation marker associated with pRTL1 has been demonstrated in bacterial crosses between different derivatives of R. rhodochrous NCIMB13064, (C) 1995 Academic Press, Inc.

Degradation of 1,3-dichloropropene by Pseudomonas cichorii 170

The gram-negative bacterium Pseudomonas cichorii 170, isolated from soil that was repeatedly treated with the nematocide 1,3-dichloropropene, could utilize low concentrations of 1,3-dichloropropene as a sole carbon and energy source, Strain 170 was also able to grow on 3-chloroallyl alcohol, 3-chloroacrylic acid, and several 1-halo-n-alkanes. This organism produced at least three different dehalogenases: a hydrolytic haloalkane dehalogenase specific for haloalkanes and two 3-chloroacrylic acid dehalogenases, one specific for cis-3-chloroacrylic acid and the other specific for trans-3-chloroacrylic acid. The haloalkane dehalogenase and the trans-3-chloroacrylic acid dehalogenase were expressed constitutively, whereas the cis-3-chloroacrylic acid dehalogenase was inducible, The presence of these enzymes indicates that 1,3-dichloropropene is hydrolyzed to 3-chloroallyl alcohol, which is oxidized in two steps to 3-chloroacrylic acid. The latter compound is then dehalogenated, probably forming malonic acid semialdehyde. The haloalkane dehalogenase gene, which is involved in the conversion of 1,3-dichloropropene to 3-chloroallyl alcohol, was cloned and sequenced, and this gene turned out to be identical to the previously studied dhaA gene of the gram-positive bacterium Rhodococcus rhodochrous NCIMB13063, Mutants resistant to the suicide substrate 1,2-dibromoethane lacked haloalkane dehalogenase activity and therefore could not utilize haloalkanes for growth. PCR analysis showed that these mutants had lost at least part of the dhaA gene.

Utilization of organophosphonates by environmental microorganisms

A survey of the utilization by environmental micro-organisms of a range of compounds containing the carbon-phosphorus (C-P) bond was carried out. Elective culture studies indicated that 15 of 19 alkylphosphonates tested served only as a sole source of phosphorus for microbial growth. Their metabolism did not lead to the extracellular release of inorganic phosphate. However, four organophosphonates - phosphonoacetate, phosphonoalanine, 2-aminoethylphosphonate and phosphonomycin - supported microbial growth when supplied as either a phosphorus source or as a carbon and energy source, with near-quantitative inorganic phosphate release. Four of five amino alkylphosphonates tested were also utilized as a nitrogen source in the presence of 1 mmol l(-1) inorganic phosphate. In a subsequent screening programme, 99% of bacterial isolates tested were able to utilize 2-aminoethylphosphonate as a sole phosphorus source, 61% as a nitrogen source, 10% as a source of nitrogen and phosphorus, and 2% as a source of carbon, nitrogen and phosphorus; 2% of isolates used phosphonoalanine as a nitrogen source. These results suggest that the uptake and metabolism of organophosphonates by bacteria is less 'tightly' regulated by phosphorus starvation than has previously been supposed.

Nanoporous metal organic framework materials for hydrogen storage

Hydrogen is expected to play an important role in future transportation as a promising alternative clean energy source to carbon-based fuels. One of the key challenges to commercialize hydrogen energy is to develop appropriate onboard hydrogen storage systems, capable of charging and discharging large quantities of hydrogen with fast enough kinetics to meet commercial requirements. Metal organic framework (MOF) is a new type of inorganic and organic hybrid nanoporous particulate materials. Its diverse networks can enhance hydrogen storage through tuning the structure and property of MOFs. The MOF materials so far developed adsorb hydrogen through weak dispersion interactions, which allow significant quantity of hydrogen to be stored at cryogenic temperatures with fast kinetics. Novel MOFs are being developed to strengthen the interactions between hydrogen and MOFs in order to store hydrogen under ambient conditions. This review surveys the development of such candidate materials, their performance and future research needs. (C) 2009 Chinese Society of Particuology and Institute of Process Engineering, Chinese Academy of Sciences. Published by Elsevier B.V. All rights reserved.

Application of PMUs to Coordinate Hydroelectric and Wind Power Plant Operation

It is acknowledged that wind power is a stochastic energy source compared to hydroelectric generation which is easily scheduled. In this paper a scheme for coordinating wind power plant and hydroelectric power plant is presented by using PMUs to measure and control the state of wind and hydro power plants. Hydroelectric generation is proposed as a method of energy reserve and compensation in the context of wind power fluctuation in order to avoid full or partial curtailment of wind generation to benefit wind providers. The feasibility of this proposed scheme is investigated by power flow calculation and stability analysis using the IEEE 30-bus power system model.

Heating Mechanisms for Intermittent Loops in Active Region Cores from AIA/SDO EUV Observations

We investigate intensity variations and energy deposition in five coronal loops in active region cores. These were selected for their strong variability in the AIA/SDO 94 Å intensity channel. We isolate the hot Fe XVIII and Fe XXI components of the 94 Å and 131 Å by modeling and subtracting the "warm" contributions to the emission. HMI/SDO data allow us to focus on "inter-moss" regions in the loops. The detailed evolution of the inter-moss intensity time series reveals loops that are impulsively heated in a mode compatible with a nanoflare storm, with a spike in the hot 131 Å signals leading and the other five EUV emission channels following in progressive cooling order. A sharp increase in electron temperature tends to follow closely after the hot 131 Å signal confirming the impulsive nature of the process. A cooler process of growing emission measure follows more slowly. The Fourier power spectra of the hot 131 Å signals, when averaged over the five loops, present three scaling regimes with break frequencies near 0.1 min–1 and 0.7 min–1. The low frequency regime corresponds to 1/f noise; the intermediate indicates a persistent scaling process and the high frequencies show white noise. Very similar results are found for the energy dissipation in a 2D "hybrid" shell model of loop magneto-turbulence, based on reduced magnetohydrodynamics, that is compatible with nanoflare statistics. We suggest that such turbulent dissipation is the energy source for our loops

The Type IIb SN 2011dh: Two years of observations and modelling of the lightcurves

We present optical and near-infrared (NIR) photometry and spectroscopy as well as modelling of the lightcurves of the Type IIb supernova (SN) 2011dh. Our extensive dataset, for which we present the observations obtained after day 100, spans two years, and complemented with Spitzer mid-infrared (MIR) data, we use it to build an optical-to-MIR bolometric lightcurve between days 3 and 732. To model the bolometric lightcurve before day 400 we use a grid of hydrodynamical SN models, which allows us to determine the errors in the derived quantities, and a bolometric correction determined with steady-state non-local thermodynamic equilibrium (NLTE) modelling. Using this method we find a helium core mass of 3.1^+0.7<inf>-0.4</inf> M<inf>⊙</inf> for SN 2011dh, consistent within error bars with previous results obtained using the bolometric lightcurve before day 80. We compute bolometric and broad-band lightcurves between days 100 and 500 from spectral steady-state NLTE models, presented and discussed in a companion paper. The preferred 12 M<inf>⊙</inf> (initial mass) model, previously found to agree well with the observed spectra, shows a good overall agreement with the observed lightcurves, although some discrepancies exist. Time-dependent NLTE modelling shows that after day ∼600 a steady-state assumption is no longer valid. The radioactive energy deposition in this phase is likely dominated by the positrons emitted in the decay of ⁵⁶Co, but seems insufficient to reproduce the lightcurves, and what energy source is dominating the emitted flux is unclear. We find an excess in the K and the MIR bands developing between days 100 and 250, during which an increase in the optical decline rate is also observed. A local origin of the excess is suggested by the depth of the He I 20 581 Å absorption. Steady-state NLTE models with a modest dust opacity in the core (τ = 0.44), turned on during this period, reproduce the observed behaviour, but an additional excess in the Spitzer 4.5 μm band remains. Carbon-monoxide (CO) first-overtone band emission is detected at day 206, and possibly at day 89, and assuming the additional excess to bedominated by CO fundamental band emission, we find fundamental to first-overtone band ratios considerably higher than observed in SN 1987A. The profiles of the [O i] 6300 Å and Mg i] 4571 Å lines show a remarkable similarity, suggesting that these lines originate from a common nuclear burning zone (O/Ne/Mg), and using small scale fluctuations in the line profiles we estimate a filling factor of ≲ 0.07 for the emitting material. This paper concludes our extensive observational and modelling work on SN 2011dh. The results from hydrodynamical modelling, steady-state NLTE modelling, and stellar evolutionary progenitor analysis are all consistent, and suggest an initial mass of ∼12 M<inf>⊙</inf> for the progenitor.

Emotional response to images of wind turbines: A psychophysiological study of their visual impact on the landscape

Social acceptance for wind turbines is variable, providing a challenge to the implementation of this energy source. Psychological research could contribute to the science of climate change. Here we focus on the emotional responses to the visual impact of wind turbines on the landscape, a factor which dominates attitudes towards this technology. Participants in the laboratory viewed images of turbines and other constructions (churches, pylons and power-plants) against rural scenes, and provided psychophysiological and self-report measures of their emotional reactions. We hypothesised that the emotional response to wind turbines would be more negative and intense than to control objects, and that this difference would be accentuated for turbine opponents. As predicted, the psychophysiological response to turbines was stronger than the response to churches, but did not differ from that of other industrial constructions. In contrast with predictions, turbines were rated as less aversive and more calming compared with other industrial constructions, and equivalent to churches. Supporters and non-supporters did not differ significantly from each other. We discuss how a methodology using photo manipulations and emotional self-assessments can help estimate the emotional reaction to the visual impact on the landscape at the planning stage for new wind turbine applications.

Discrete-Time Conserved Quantities for Damped Oscillators

Numerical sound synthesis is often carried out using the finite difference time domain method. In order to analyse the stability of the derived models, energy methods can be used for both linear and nonlinear settings. For Hamiltonian systems the existence of a conserved numerical energy-like quantity can be used to guarantee the stability of the simulations. In this paper it is shown how to derive similar discrete conservation laws in cases where energy is dissipated due to friction or in the presence of an energy source due to an external force. A damped harmonic oscillator (for which an analytic solution is available) is used to present the proposed methodology. After showing how to arrive at a conserved quantity, the simulation of a nonlinear single reed shows an example of an application in the context of musical acoustics.

The effect of polyphosphate kinase gene deletion on polyhydroxyalkanoate accumulation and carbon metabolism in Pseudomonas putida KT2440.

The primary enzyme involved in polyphosphate (polyP) synthesis, polyP kinase (ppk), has been deleted in Pseudomonas putida KT2440. This has resulted in a threefold to sixfold reduction in polyhydroxyalkanoate (PHA) accumulation compared with the wild type under conditions of nitrogen limitation, with either temperature or oxidative (H2O2) stress, when grown on glucose. The accumulation of PHA by Δppk mutant was the same as the wild type under nitrogen-limiting growth conditions. There was no difference in polyP levels between wild-type and Δppk strains under all growth conditions tested. In the Δppk mutant proteome, polyP kinase (PPK) was undetectable, but up-regulation of the polyp-associated proteins polyP adenosine triphosphate (ATP)/nicotinamide adenine dinucleotide (NAD) kinase (PpnK), a putative polyP adenosine monophosphate (AMP) phosphotransferase (PP_1752), and exopolyphosphatase was observed. Δppk strain exhibited significantly retarded growth with glycerol as carbon and energy source (42 h of lag period compared with 24 h in wild-type strain) but similar growth to the wild-type strain with glucose. Analysis of gene transcription revealed downregulation of glycerol kinase and the glycerol facilitator respectively. Glycerol kinase protein expression was also downregulated in the Δppk mutant. The deletion of ppk did not affect motility but reduced biofilm formation. Thus, the knockout of the ppk gene has resulted in a number of phenotypic changes to the mutant without affecting polyP accumulation.

High-resolution seismic reflection investigation of gas accumulation and seepage in the tidal channels of the Ria of Aveiro barrier lagoon (Portugal)

O metano é um gás de estufa potente e uma importante fonte de energia. A importância global e impacto em zonas costeiras de acumulações e escape de gás metano são ainda pouco conhecidas. Esta tese investiga acumulações e escape de gás em canais de maré da Ria de Aveiro com dados de cinco campanhas de reflexão sísmica de alta resolução realizadas em 1986, 1999, 2002 e 2003. Estas incluem três campanhas de Chirp (RIAV99, RIAV02 e RIAV02A) e duas campanhas de Boomer (VOUGA86 e RIAV03). O processamento dos dados de navegação incluíram filtros de erros, correcções de sincronização de relógios de sistemas de aquisição de dados, ajuste de “layback” e estimativa da posição de “midpoint”. O processamento do sinal sísmico consistiu na correcção das amplitudes, remoção de ruído do tipo “burst”, correcções estáticas, correcção do “normal move-out”, filtragem passabanda, desconvolução da assinatura e migração Stolt F-K. A análise da regularidade do trajecto de navegação, dos desfasamentos entre horizontes e dos modelos de superfícies foi utilizada para controlo de qualidade, e permitiu a revisão e melhoria dos parâmetros de processamento. A heterogeneidade da cobertura sísmica, da qualidade do sinal, da penetração e da resolução, no seu conjunto constrangeram o uso dos dados a interpretações detalhadas, mas locais, de objectos geológicos da Ria. É apresentado um procedimento para determinar a escolha de escalas adequadas para modelar os objectos geológicos, baseado na resolução sísmica, erros de posicionamento conhecidos e desfasamentos médios entre horizontes. As evidências de acumulação e escape de gás na Ria de Aveiro incluem turbidez acústica, reflexões reforçadas, cortinas acústicas, domas, “pockmarks” e alinhamentos de “pockmarks” enterradas, horizontes perturbados e plumas acústicas na coluna de água (flares). A estratigrafia e a estrutura geológica controlam a distribuição e extensão das acumulações e escape de gás. Ainda assim, nestes sistemas de baixa profundidade de água, as variações da altura de maré têm um impacto significativo na detecção de gás com métodos acústicos, através de alterações nas amplitudes originais de reflexões reforçadas, turbidez acústica e branqueamento acústico em zonas com gás. Os padrões encontrados confirmam que o escape de bolhas de gás é desencadeado pela descida da maré. Há acumulações de gás em sedimentos Holocénicos e no substrato de argilas e calcários do Mesozóico. Evidências directas de escape de gás em sondagens em zonas vizinhas, mostraram gás essencialmente biogénico. A maioria do gás na área deve ter sido gerado em sedimentos lagunares Holocénicos. No entanto, a localização e geometria de estruturas de escape de fluidos em alguns canais de maré, seguem o padrão de fracturas do substrato Mesozóico, indicando uma possível fonte mais profunda de gás e que estas fracturas funcionam como condutas preferenciais de migração dos fluidos e exercem um controlo estrutural na ocorrência de gás na Ria.

Tributyltin (TBT) resistance in Aeromonas molluscorum Av27

O tributilestanho (TBT) é considerado um dos xenobióticos mais tóxicos, produzidos e deliberadamente introduzidos no meio ambiente pelo Homem. Tem sido usado numa variedade de processos industriais e subsequentemente descarregado no meio ambiente. O tempo de meia-vida do TBT em águas marinhas é de várias semanas, mas em condições de anóxia nos sedimentos, pode ser de vários anos, devido à sua degradação mais lenta. Embora o TBT tenha sido descrito como sendo tóxico para eucariotas e procariotas, muitas bactérias podem ser resistentes a este composto. O presente trabalho teve como objetivo principal elucidar o mecanismo de resistência ao TBT em bactérias. Para além disso, pretendeu-se desenvolver um biorepórter para detectar TBT no ambiente. Para atingir estes objetivos foram delineadas várias tarefas cujos principais resultados obtidos se apresentam a seguir. Várias bactérias resistentes ao TBT foram isoladas de sedimento e água do Porto de Pesca Longínqua (PPL) na Ria de Aveiro, Portugal. Entre estas, Aeromonas molluscorum Av27 foi selecionada devido à sua elevada resistência a este composto (concentrações até 3 mM), à sua capacidade de degradar o TBT em compostos menos tóxicos (dibutilestanho, DBT e monobutilestanho, MBT) e também por usar o TBT como fonte de carbono. A. molluscorum Av27 foi caracterizada genotipica e fenotipicamente. Os fatores de virulência estudados mostraram que esta estirpe i) possui atividade lipolítica; ii) não é citotóxica para células de mamíferos, nomeadamente para células Vero; iii) não possui integrões de classe I e II e iv) possui cinco plasmídeos com aproximadamente 4 kb, 7 kb, 10 kb, 100 kb e mais de 100 kb. Estes resultados mostraram que a estirpe Av27 não é tóxica, aumentando assim o interesse nesta bactéria para futuras aplicações, nomeadamente na bioremediação. Os testes de toxicidade ao TBT mostraram que este composto tem um impacto negativo no crescimento desta estirpe, bem como, na densidade, no tamanho e na atividade metabólica das células e é responsável pela formação de agregados celulares. Assim, o TBT mostrou ser bastante tóxico para as bactérias interferindo com a atividade celular geral. O gene Av27-sugE, que codifica a proteína SugE pertencente à família das “small multidrug resistance proteins” (SMR), foi identificado como estando envolvido na resistência ao TBT nesta estirpe. Este gene mostrou ser sobreexpresso quando as células crescem na presença de TBT. O promotor do gene Av27-sugE foi utilizado para construir um bioreporter para detetar TBT, contendo o gene da luciferase do pirilampo como gene repórter. O biorepórter obtido reúne as características mais importantes de um bom biorepórter: sensibilidade (intervalo de limite de detecção de 1-1000 nM), rapidez (3 h são suficientes para a deteção de sinal) e, possivelmente, não é invasivo (pois foi construído numa bactéria ambiental). Usando sedimento recolhido no Porto de Pesca Longínqua da Ria de Aveiro, foi preparada uma experiência de microcosmos com o intuito de avaliar a capacidade de Av27 para bioremediar o TBT, isoladamente ou em associação com a comunidade bacteriana indígena. A análise das amostras de microcosmos por PCR-DGGE e de bibliotecas de 16S rDNA revelaram que a comunidade bacteriana é relativamente estável ao longo do tempo, mesmo quando Av27 é inoculada no sedimento. Para além disso, o sedimento estuarino demonstrou ser dominado por bactérias pertencentes ao filo Proteobacteria (sendo mais abundante as Delta e Gammaproteobacteria) e Bacteroidetes. Ainda, cerca de 13% dos clones bacterianos não revelaram nenhuma semelhança com qualquer dos filos já definidos e quase 100% afiliou com bactérias não cultiváveis do sedimento. No momento da conclusão desta tese, os resultados da análise química de compostos organoestânicos não estavam disponíveis, e por essa razão não foi possível tirar quaisquer conclusões sobre a capacidade desta bactéria remediar o TBT em sedimentos. Esses resultados irão ajudar a esclarecer o papel de A. molluscorum Av27 na remediação de TBT. Recentemente, a capacidade da estirpe Av27 remediar solo contaminado com TBT foi confirmada em bioensaios realizados com plantas, Brassica rapa e Triticum aestivum (Silva 2011a), e também com invertebrados Porcellionides pruinosus (Silva 2011B). Assim, poder-se-á esperar que a bioremediação do sedimento na experiência de microcosmos também tenha ocorrido. No entanto, só a análise química dos compostos organostânicos deverá ser conclusiva. Devido à dificuldade em realizar a análise analítica de organoestânicos, um método de bioensaio fácil, rápido e barato foi adaptado para avaliar a toxicidade do TBT em laboratório, antes de se proceder à análise química das amostras. O método provou a sua utilidade, embora tenha mostrado pouca sensibilidade quando se usam concentrações de TBT baixas. Em geral, os resultados obtidos contribuíram para um melhor entendimento do mecanismo de resistência ao TBT em bactérias e mostraram o potencial biotecnológico de A. molluscorum Av27, nomeadamente, no que refere à sua possível aplicação na descontaminação de TBT no ambiente e também no desenvolvimento de biorepórteres.