bluuebunny/crossref_metadata_2025_split huggingface dataset:
Identification and characterization of the PhhR regulon in Pseudomonas putida
Authors: Herrera M. Carmen , Duque Estrella , Rodríguez‐Herva José J. , Fernández‐Escamilla Ana M. , Ramos Juan L. 
Published: December 2009

Summary Pseudomonas putida is a soil microorganism that utilizes aromatic amino acids present in root exudates as a nitrogen source. We have previously shown that the PhhR transcriptional regulator induces phhAB genes encoding a phenylalanine hydroxylase. In this study we show, using microarray assays and promoter fusions, that PhhR is a global regulator responsible for the activation of genes essential for phenylalanine degradation, phenylalanine homeostasis and other genes of unknown function. Recently, it has been shown that phenylalanine catabolism occurs through more than one pathway. One of these possible pathways involves the metabolism of phenylalanine via tyrosine, p‐hydroxyphenylpyruvate, and homogentisate. We identified two genes within this pathway that encode an acyl‐CoA transferase involved in the metabolism of acetoacetate. All genes in this pathway were induced in response to phenylalanine in a PhhR‐proficient background. The second potential degradative pathway involves the degradation of phenylalanine to produce phenylpyruvate, which seems to be degraded via phenylacetyl‐CoA. A number of mutants in the paa genes encoding phenylacetyl‐CoA degradation enzymes fail to grow on phenylpyruvate or phenylacetate, further supporting the existence of this second pathway. We found that the PhhR regulon also includes genes involved in the biosynthesis of aromatic amino acids that are repressed in the presence of phenylalanine, suggesting the possibility of feedback at the transcriptional level. In addition, we found that PhhR modulates the level of expression of the broad‐substrate‐specificity MexEF/OprN efflux pump. Expression from this pump is under the control of mexT gene product because phenylalanine‐dependent transcription from the mexE promoter does not occur in a mexT mutant background. These results place PhhR as an important regulator in the control of bacterial responses to aromatic amino acids.
RNA binding proteins mediate the ability of a fungus to adapt to the cold
Authors: Fang Weiguo , St. Leger Raymond J. 
Published: December 2009

SummaryLittle is known about how fungi adapt to chilling. In eubacteria, cold shock proteins (CSPs) facilitate translation by destabilizing RNA secondary structure. Animals and plants have homologous cold shock domains within proteins, and additional glycine‐rich RNA binding proteins (GRPs), but their role in stress resistance is poorly understood. In this study, we identified GRP homologues in diverse fungi. However, only Aspergillus clavatus and Metarhizium anisopliae possessed cold shock domains. Both M. anisopliae's small eubacteria‐like CSP (CRP1) and its GRP (CRP2) homologue were induced by cold. Disrupting either Crp1 or Crp2 greatly reduced metabolism and conidial germination rates at low temperatures, and decreased tolerance to freezing. However, while both Crp1 and Crp2 reduced freezing‐induced production of reactive oxygen species, only Crp1 protected cells against H2O2 and increased M. anisopliae's virulence to caterpillars. Unlike CRP2, CRP1 rescued the cold‐sensitive growth defects of an Escherichia coli CSP deletion mutant, and CRP1 also demonstrated transcription anti‐termination activity, so CRP1 can regulate transcription and translation at low temperature. Expressing either Crp1 or Crp2 in yeast increased metabolism at cold temperatures and Crp1 improved tolerance to freezing. Thus besides providing a model relevant to many biological systems, Crp1 and Crp2 have potential applications in biotechnology.
Metabolic responses of novel cellulolytic and saccharolytic agricultural soil Bacteria to oxygen
Authors: Schellenberger Stefanie , Kolb Steffen , Drake Harold L. 
Published: December 2009

SummaryCellulose is the most abundant biopolymer in terrestrial ecosystems and is degraded by microbial communities in soils. However, relatively little is known about the diversity and function of soil prokaryotes that might participate in the overall degradation of this biopolymer. The active cellulolytic and saccharolytic Bacteria in an agricultural soil were evaluated by 16S rRNA 13C‐based stable isotope probing. Cellulose, cellobiose and glucose were mineralized under oxic conditions in soil slurries to carbon dioxide. Under anoxic conditions, these substrates were converted primarily to acetate, butyrate, carbon dioxide, hydrogen and traces of propionate and iso‐butyrate; the production of these fermentation end‐products was concomitant with the apparent reduction of iron(III). [13C]‐cellulose was mainly degraded under oxic conditions by novel family‐level taxa of the Bacteroidetes and Chloroflexi, and a known family‐level taxon of Planctomycetes, whereas degradation under anoxic conditions was facilitated by the Kineosporiaceae (Actinobacteria) and cluster III Clostridiaceae and novel clusters within Bacteroidetes. Active aerobic sub‐communities in oxic [13C]‐cellobiose and [13C]‐glucose treatments were dominated by Intrasporangiaceae and Micrococcaceae (Actinobacteria) whereas active cluster I Clostridiaceae (Firmicutes) were prevalent in anoxic treatments. A very large number (i.e. 28) of the detected taxa did not closely affiliate with known families, and active Archaea were not detected in any of the treatments. These collective findings suggest that: (i) a large uncultured diversity of soil Bacteria was involved in the utilization of cellulose and products of its hydrolysis, (ii) the active saccharolytic community differed phylogenetically from the active cellulolytic community, (iii) oxygen availability impacted differentially on the activity of taxa and (iv) different redox guilds (e.g. fermenters and iron reducers) compete or interact during cellulose degradation in aerated soils.
Dual symbiosis of the vent shrimp Rimicaris exoculata with filamentous gamma‐ and epsilonproteobacteria at four Mid‐Atlantic Ridge hydrothermal vent fields
Authors: Petersen Jillian M. , Ramette Alban , Lott Christian , Cambon‐Bonavita Marie‐Anne , Zbinden Magali , Dubilier Nicole 
Published: December 2009

SummaryThe shrimp Rimicaris exoculata from hydrothermal vents on the Mid‐Atlantic Ridge (MAR) harbours bacterial epibionts on specialized appendages and the inner surfaces of its gill chamber. Using comparative 16S rRNA sequence analysis and fluorescence in situ hybridization (FISH), we examined the R. exoculata epibiosis from four vents sites along the known distribution range of the shrimp on the MAR. Our results show that R. exoculata lives in symbiosis with two types of filamentous epibionts. One belongs to the Epsilonproteobacteria, and was previously identified as the dominant symbiont of R. exoculata. The second is a novel gammaproteobacterial symbiont that belongs to a clade consisting exclusively of sequences from epibiotic bacteria of hydrothermal vent animals, with the filamentous sulfur oxidizer Leucothrix mucor as the closest free‐living relative. Both the epsilon‐ and the gammaproteobacterial symbionts dominated the R. exoculata epibiosis at all four MAR vent sites despite striking differences between vent fluid chemistry and distances between sites of up to 8500 km, indicating that the symbiosis is highly stable and specific. Phylogenetic analyses of two mitochondrial host genes showed little to no differences between hosts from the four vent sites. In contrast, there was significant spatial structuring of both the gamma‐ and the epsilonproteobacterial symbiont populations based on their 16S rRNA gene sequences that was correlated with geographic distance along the MAR. We hypothesize that biogeography and host–symbiont selectivity play a role in structuring the epibiosis of R. exoculata.
Streptomycetes contributing to atmospheric molecular hydrogen soil uptake are widespread and encode a putative high‐affinity [NiFe]‐hydrogenase
Authors: Constant Philippe , Chowdhury Soumitra Paul , Pratscher Jennifer , Conrad Ralf 
Published: December 2009

SummaryUptake of molecular hydrogen (H2) by soil is a biological reaction responsible for ∼80% of the global loss of atmospheric H2. Indirect evidence obtained over the last decades suggests that free soil hydrogenases with an unusually high affinity for H2 are carrying out the reaction. This assumption has recently been challenged by the isolation of Streptomyces sp. PCB7, displaying the high‐affinity H2 uptake activity previously attributed to free soil enzymes. While this finding suggests that actinobacteria could be responsible for atmospheric H2 soil uptake, the ecological importance of H2‐oxidizing streptomycetes remains to be investigated. Here, we show that high‐affinity H2 uptake activity is widespread among the streptomycetes. Among 14 streptomycetes strains isolated from temperate forest and agricultural soils, six exhibited a high‐affinity H2 uptake activity. The gene encoding the large subunit of a putative high‐affinity [NiFe]‐hydrogenase (hydB‐like gene sequence) was detected exclusively in the isolates exhibiting high‐affinity H2 uptake. Catalysed reporter deposition‐fluorescence in situ hybridization (CARD‐FISH) experiments targeting hydB‐like gene transcripts and H2 uptake assays performed with strain PCB7 suggested that streptomycetes spores catalysed the H2 uptake activity. Expression of the activity in term of biomass revealed that 106–107 H2‐oxidizing bacteria per gram of soil should be sufficient to explain in situ H2 uptake by soil. We propose that specialized H2‐oxidizing actinobacteria are responsible for the most important sink term in the atmospheric H2 budget.
Biogeography of wetland rice methanotrophs
Authors: Lüke Claudia , Krause Sascha , Cavigiolo Stefano , Greppi Diego , Lupotto Elisabetta , Frenzel Peter 
Published: December 2009

SummaryWe focused on the functional guild of methane oxidizing bacteria (MOB) as model organisms to get deeper insights into microbial biogeography. The pmoA gene was used as a functional and phylogenetic marker for MOB in two approaches: (i) a pmoA database (> 4000 sequences) was evaluated to obtain insights into MOB diversity in Italian rice paddies, and paddy fields worldwide. The results show a wide geographical distribution of pmoA genotypes that seem to be specifically adapted to paddy fields (e.g. Rice Paddy Cluster 1 and Rice Paddy Cluster 2). (ii) On the smaller geographical scale, we designed a factorial experiment including three different locations, two rice varieties and two habitats (soil and roots) within each of three rice fields. Multivariate analysis of terminal restriction fragment analysis profiles revealed different community patterns at the three field sites, located 10–20 km apart. Root samples were characterized by high abundance of type I MOB whereas the rice variety had no effect. With the agronomical practice being nearly identical, historical contingencies might be responsible for the field site differences. Considering a large reservoir of viable yet inactive MOB cells acting as a microbial seed bank, environmental conditions might have selected and activated a different subset at a time thereby shaping the community.
Transgenic maize lines with cell‐type specific expression of fluorescent proteins in plastids
Authors: Sattarzadeh Amir , Fuller Jonathan , Moguel Salvador , Wostrikoff Katia , Sato Shirley , Covshoff Sarah , Clemente Tom , Hanson Maureen , Stern David B. 
Published: December 2009

SummaryPlastid number and morphology vary dramatically between cell types and at different developmental stages. Furthermore, in C4 plants such as maize, chloroplast ultrastructure and biochemical functions are specialized in mesophyll and bundle sheath cells, which differentiate acropetally from the proplastid form in the leaf base. To develop visible markers for maize plastids, we have created a series of stable transgenics expressing fluorescent proteins fused to either the maize ubiquitin promoter, the mesophyll‐specific phosphoenolpyruvate carboxylase (PepC) promoter, or the bundle sheath‐specific Rubisco small subunit 1 (RbcS) promoter. Multiple independent events were examined and revealed that maize codon‐optimized versions of YFP and GFP were particularly well expressed, and that expression was stably inherited. Plants carrying PepC promoter constructs exhibit YFP expression in mesophyll plastids and the RbcS promoter mediated expression in bundle sheath plastids. The PepC and RbcS promoter fusions also proved useful for identifying plastids in organs such as epidermis, silks, roots and trichomes. These tools will inform future plastid‐related studies of wild‐type and mutant maize plants and provide material from which different plastid types may be isolated.
A companion protease inhibitor for the protection of cytosol‐targeted recombinant proteins in plants
Authors: Goulet Charles , Benchabane Meriem , Anguenot Raphaël , Brunelle France , Khalf Moustafa , Michaud Dominique 
Published: December 2009

SummaryWe reported earlier the potential of tomato cathepsin D inhibitor (SlCDI) as an in‐built stabilizing agent for the protection of recombinant proteins in transgenic plant leaf crude extracts (Plant Biotechnol J.4, 359–368). Here we document the potential of SlCDI for the in situ protection of proteins in potato leaves. Total protein assays with control and SlCDI‐expressing potato lines indicated a positive impact of slcdi transgene expression on leaf protein content, with a mean relative increase of 35%–40% depending on the light regime. Out of ∼700 proteins detected on 2‐D gels, only 20 exhibited a significantly altered level on a protein‐specific basis, whereas most proteins were up‐regulated on a leaf fresh weight basis, albeit at variable rates. Quantitative reverse trancriptase‐PCR assays for rubisco activase showed similar transcript levels in leaves of test and control lines despite protein levels increased by two‐ to threefold in SlCDI‐expressing lines. These observations, along with the unrelated biological functions assigned to MS‐identified proteins up‐regulated in leaves and protease assays showing slightly increased proteasome activity in protein extracts of SlCDI‐expressing lines, suggest a general, proteasome‐independent protein stabilizing effect of SlCDI in planta. Transient expression assays with human α1‐antichymotrypsin also showed a stabilizing effect for SlCDI on heterologous proteins, leading to net levels of the human protein increased by ∼2.5‐fold in SlCDI‐expressing plants. These data illustrate, overall, the potential of SlCDI as an in vivo protein‐stabilizing agent in transgenic plant systems, useful to improve protein levels and recombinant protein accumulation.
Tubers from potato lines expressing a tomato Kunitz protease inhibitor are substantially equivalent to parental and transgenic controls
Authors: Khalf Moustafa , Goulet Charles , Vorster Juan , Brunelle France , Anguenot Raphaël , Fliss Ismaïl , Michaud Dominique 
Published: December 2009

SummaryRecombinant protease inhibitors represent useful tools for the development of insect‐resistant transgenic crops, but questions have been raised in recent years about the impact of these proteins on endogenous proteases and chemical composition of derived food products. In this study, we performed a detailed compositional analysis of tubers from potato lines expressing the broad‐spectrum inhibitor of Ser and Asp proteases, tomato cathepsin D inhibitor (SlCDI), to detect possible unintended effects on tuber composition. A compositional analysis of key nutrients and toxic chemicals was carried out with tubers ofSlCDI‐expressing and control (comparator) lines, followed by a two‐dimensional gel electrophoresis (2‐DE) proteomic profiling of total and allergenic proteins to detect eventual effects at the proteome level. No significant differences were observed among control andSlCDI‐expressing lines for most chemicals assayed, in line with the very low abundance ofSlCDI in tubers. Likewise, proteins detected after 2‐DE showed no quantitative variation among the lines, except for a few proteins in some control and test lines, independent of slcditransgene expression. Components of the patatin storage protein complex and Kunitz protease inhibitors immunodetected after 2‐DE showed unaltered deposition patterns inSlCDI‐expressing lines, clearly suggesting a null impact of slcdion the intrinsic allergenic potential of potato tubers. These data suggest, overall, a null impact of slcdiexpression on tuber composition and substantial equivalence between comparator andSlCDI‐expressing tubers despite reported effects on leaf protein catabolism. They also illustrate the usefulness of proteomics as a tool to assess the authenticity of foods derived from novel‐generation transgenic plants.
Production of compact plants by overexpression of AtSHI in the ornamental Kalanchoë
Authors: Lütken Henrik , Jensen Lilli Sander , Topp Sine Hovbye , Mibus Heiko , Müller Renate , Rasmussen Søren K. 
Published: December 2009

SummaryGrowth retardation is an important breeding aim and an essential part of horticultural plant production. Here, the potential of transferring the Arabidopsis short internode (shi) mutant phenotype was explored by expressing the AtSHI gene in the popular ornamental plant Kalanchoë. A 35S‐AtSHI construct was produced and transferred into eight genetically different cultivars of Kalanchoë by Agrobacterium tumefaciens. The resulting transgenic plants showed dwarfing phenotypes like reduced plant height and diameter, and also more compact inflorescences, as a result of increased vegetative height. The shi phenotype was stable over more than five vegetative subcultivations. Compared with Arabidopsis, the ectopic expression of AtSHI in Kalanchoë showed several differences. None of the Kalanchoë SHI‐lines exhibited alterations in leaf colour or morphology, and most lines were not delayed in flowering. Moreover, continuous treatment of lines delayed in flowering with low concentrations of gibberellins completely restored the time of flowering. These features are very important as a delay in flowering would increase plant production costs significantly. The effect of expression controlled by the native Arabidopsis SHI promoter was also investigated in transgenic Kalanchoë and resulted in plants with a longer flowering period. Two AtSHI like genes were identified in Kalanchoë indicating a widespread presence of this transcription factor. These findings are important because they suggest that transformation with the AtSHI gene could be applied to several species as a tool for growth retardation, and that this approach could substitute the use of conventional chemical growth regulation in plant production.
Chloroplast‐derived vaccine antigens confer dual immunity against cholera and malaria by oral or injectable delivery
Authors: Davoodi‐Semiromi Abdoreza , Schreiber Melissa , Nalapalli Samson , Verma Dheeraj , Singh Nameirakpam D. , Banks Robert K. , Chakrabarti Debopam , Daniell Henry 
Published: December 2009

SummaryCholera and malaria are major diseases causing high mortality. The only licensed cholera vaccine is expensive; immunity is lost in children within 3 years and adults are not fully protected. No vaccine is yet available for malaria. Therefore, in this study, the cholera toxin‐B subunit (CTB) of Vibrio cholerae fused to malarial vaccine antigens apical membrane antigen‐1 (AMA1) and merozoite surface protein‐1 (MSP1) was expressed in lettuce and tobacco chloroplasts. Southern blot analysis confirmed homoplasmy and stable integration of transgenes. CTB‐AMA1 and CTB‐MSP1 fusion proteins accumulated up to 13.17% and 10.11% (total soluble protein, TSP) in tobacco and up to 7.3% and 6.1% (TSP) in lettuce, respectively. Nine groups of mice (n = 10/group) were immunized subcutaneously (SQV) or orally (ORV) with purified antigens or transplastomic tobacco leaves. Significant levels of antigen‐specific antibody titres of immunized mice completely inhibited proliferation of the malarial parasite and cross‐reacted with the native parasite proteins in immunoblots and immunofluorescence studies. Protection against cholera toxin challenge in both ORV (100%) and SQV (89%) mice correlated with CTB‐specific titres of intestinal, serum IgA and IgG1 in ORV and only IgG1 in SQV mice, but no other immunoglobulin. Increasing numbers of interleukin‐10+ T cell but not Foxp3+ regulatory T cells, suppression of interferon‐γ and absence of interleukin‐17 were observed in protected mice, suggesting that immunity is conferred via the Tr1/Th2 immune response. Dual immunity against two major infectious diseases provided by chloroplast‐derived vaccine antigens for long‐term (>300 days, 50% of mouse life span) offers a realistic platform for low cost vaccines and insight into mucosal and systemic immunity.
Twelve‐ to 14‐month‐old infants can predict single‐event probability with large set sizes
Authors: Denison Stephanie , Xu Fei 
Published: December 2009

AbstractPrevious research has revealed that infants can reason correctly about single‐event probabilities with small but not large set sizes (Bonatti, 2008; Teglas et al., 2007). The current study asks whether infants can make predictions regarding single‐event probability with large set sizes using a novel procedure. Infants completed two trials: A preference trial to determine whether they preferred pink or black lollipops and a test trial where infants saw two jars, one containing mostly pink lollipops and another containing mostly black lollipops. The experimenter removed one occluded lollipop from each jar and placed them in two separate opaque cups. Seventy‐eight percent of infants searched in the cup that contained a lollipop from the jar with a higher proportion of their preferred color object, significantly better than chance. Thus infants can reason about single‐event probabilities with large set sizes in a choice paradigm, and contrary to most findings in the infant literature, the prediction task used here appears a more sensitive measure than the standard looking‐time task.
Opportunistic infections and organ‐specific diseases in HIV‐1‐infected children: a cohort study (1990–2006)
Authors: Guillén S , García San Miguel L , Resino S , Bellón JM , González I , Jiménez de Ory S , Muñoz‐Fernández MA , Navarro ML , Gurbindo MD , De José MI , Mellado MJ , Martín‐Fontelos P , Gonzalez‐Tomé MI , Martinez J , Beceiro J , Roa MA , Ramos JT ,   on behalf of the Madrid Group for Research on Pediatric HIV Infection
Published: December 2009

ObjectivesHighly active antiretroviral therapy (HAART) has dramatically changed the natural history of HIV infection in children, but there are few studies in the literature about the incidence of clinical manifestations after HAART in this population, compared with adults. The aim of this study was to describe the influence of the widespread use of HAART on the development of opportunistic infections and organ‐specific diseases in HIV‐infected children.MethodsAn observational study of a cohort of 366 vertically HIV‐infected children followed from 1990 to 2006 was carried out. According to the main antiretroviral protocol used, three calendar periods (CPs) were defined and compared: CP1 (1990–1996: no patients on HAART), CP2 (1997–1999: <60% on HAART) and CP3 (2000–2006: >60% on HAART).ResultsChildren experienced a progressive increase in CD4 T cell count (P<0.05) and a decrease in HIV viral load from 1996 onwards (P<0.05). Similarly, rates of death, AIDS, opportunistic infections (bacteraemia, candidosis, cryptosporidiosis and bacterial pneumonia) and organ‐specific diseases (wasting syndrome, thrombocytopenia, cardiomyopathy, lymphoid interstitial pneumonia and HIV‐associated encephalopathy) were lower in CP2 and CP3 than in CP1.ConclusionsThis study provides evidence of improved clinical outcomes in HIV‐infected children over time and shows that mortality, AIDS, opportunistic infections and organ‐specific diseases declined as HAART was progressively instituted in this population.