Identification and modeling of a novel chloramphenicol resistance protein detected by functional metagenomics in a wetland of Lerma, Mexico

The exploration of novel antibiotic resistance determinants in a particular environment may be limited because of the presence of uncultured microorganisms. In this work, a culture independent approach based on functional metagenomics was applied to search for chloramphenicol resistance genes in agro-industrial wastewater in Lerma de Villada, Mexico. To this end, a metagenomic library was generated in Escherichia coli DH10B containing DNA isolated from environmental samples of the residual arsenic-enriched (10 mg/ml) effl uent. One resistant clone was detected in this library and further analyzed. An open reading frame similar to a multidrug resistance protein from Aeromonas salmonicida and responsible for chloramphenicol resistance was identifi ed, sequenced, and found to encode a member of the major facilitator superfamily (MFS). Our results also showed that the expression of this gene restored streptomycin sensitivity in E. coli DH10B cells. To gain further insight into the phenotype of this MFS family member, we developed a model of the membrane protein multiporter that, in addition, may serve as a template for developing new antibiotics. [Int Microbiol 2013; 16(2):103-111]Keywords: Escherichia coli; chloramphenicol; functional metagenomics; major facilitator superfamily; homology models; membrane proteins; arseni

Structure Variation And Dynamics of the Nuclear Ribosomal Intergenic Spacer Region in Key Gibberella Fujikuroi Complex Species

The intergenic spacer (IGS) region of the ribosomal DNA was cloned and sequenced in eight species within the Gibberella fujikuroi species complex with anamorphs in the genus Fusarium , a group that includes the most relevant toxigenic species. DNA sequence analyses revealed two categories of repeated elements: long repeats and short repeats of 125 and 8 bp, respectively. Long repeats were present in two copies and were conserved in all the species analyzed, whereas different numbers of short repeat elements were observed, leading to species-specific IGS sequences with different length. In Fusarium subglutinans and Fusarium nygamai , these differences seemed to be the result of duplication and deletion events. Here, we propose a model based on unequal crossing over that can explain these processes. The partial IGS sequence of 22 Fusarium proliferatum isolates was also obtained to study variation at the intraspecific level. The results revealed no differences in terms of number or pattern of repeated elements and detected frequent gene conversion events. These results suggest that the homogenization observed at the intraspecific level might not be achieved primarily by unequal crossing-over events but rather by processes associated with recombination such as gene conversion events

Salt resistance genes revealed by functional metagenomics from brines and moderate-salinity rhizosphere within a hypersaline environment

Hypersaline environments are considered one of the most extreme habitats on earth and microorganisms have developed diverse molecular mechanisms of adaptation to withstand these conditions. The present study was aimed at identifying novel genes from the microbial communities of a moderate-salinity rhizosphere and brine from the Es Trenc saltern (Mallorca, Spain), which could confer increased salt resistance to Escherichia coli. The microbial diversity assessed by pyrosequencing of 16S rRNA gene libraries revealed the presence of communities that are typical in such environments and the remarkable presence of three bacterial groups never revealed as major components of salt brines. Metagenomic libraries from brine and rhizosphere samples, were transferred to the osmosensitive strain E. coli MKH13, and screened for salt resistance. Eleven genes that conferred salt resistance were identified, some encoding for well-known proteins previously related to osmoadaptation such as a glycerol transporter and a proton pump, whereas others encoded proteins not previously related to this function in microorganisms such as DNA/RNA helicases, an endonuclease III (Nth) and hypothetical proteins of unknown function. Furthermore, four of the retrieved genes were cloned and expressed in Bacillus subtilis and they also conferred salt resistance to this bacterium, broadening the spectrum of bacterial species in which these genes can function. This is the first report of salt resistance genes recovered from metagenomes of a hypersaline environment. © 2015 Mirete, Mora-Ruiz, Lamprecht-Grandío, de Figueras, Rosselló-Móra and González-Pastor.This work was funded by the Spanish Ministry of Science and Innovation (CGL2012-39627-C03/02 and 03); the latter also supported with European Regional Development Fund (FEDER). MM-R Ph.D. is supported by fellowship CVU 265934 of the National Council of Science and Technology (CONACyT), MexicoPeer Reviewe

Design and validation of an instrument to listen the student voice in specialized open classrooms

Las voces del alumnado con necesidades educativas especiales graves y permanentes, generalmente, han sido excluidas de los procesos de investigación en los que han estado implicados. De ahí, la necesidad de disponer de instrumentos que permitan recoger sus opiniones y darles visibilidad, siendo el objetivo principal de este trabajo el diseño y la validación de un cuestionario adaptado a las características del alumnado con necesidades educativas especiales graves y permanentes escolarizados en las Aulas Abiertas Especializadas (AAE). Se ha contado con la participación de 21 profesionales expertos en educación inclusiva y en metodología, empleando como estrategia metodológica principal la técnica de grupos focales. La naturaleza mixta de los datos conlleva un doble tratamiento, para lo cual se empleó el programa Atlas-Ti Versión 8, así como el paquete estadístico SPSS V.24 para analizar la concordancia inter-jueces. Los resultados muestran que se ha diseñado un cuestionario válido para el fin que se pretendía, erigiéndose los grupos focales como una vía adecuada para la construcción y validación de instrumentos.The voices of students with serious and permanent special educational needs have been excluded from the research processes in which they have been involved. Therefore, it is necessary to have instruments to collect their opinions and give them visibility. The main objective of this work is to design and validate an adapted questionnaire to the characteristics of students with serious and permanent special education needs who are enrolled in the Open Specialized Classrooms. This work has counted with the participation of 21 professionals in inclusive education and methodology, using the technique of focal groups as the main methodological strategy. The mixed nature of the data implies a double treatment, for which the Atlas-Ti Version 8 program was used and the statistical package SPSS V.24 to analyse the inter-judge concordance. The results show that a valid questionnaire has been designed for the purpose that was intended. Moreover, the focus groups have been established as an adequate way to construct and validate instruments.Este trabajo forma parte del proyecto de investigación “Medidas específicas de atención a la diversidad: evaluación de las aulas abiertas especializadas en la Región de Murcia” concedido por el Ministerio de Economía y Competitividad, España

Novel Genes Involved in Resistance to Both Ultraviolet Radiation and Perchlorate From the Metagenomes of Hypersaline Environments

Microorganisms that thrive in hypersaline environments on the surface of our planet are exposed to the harmful effects of ultraviolet radiation. Therefore, for their protection, they have sunscreen pigments and highly efficient DNA repair and protection systems. The present study aimed to identify new genes involved in UV radiation resistance from these microorganisms, many of which cannot be cultured in the laboratory. Thus, a functional metagenomic approach was used and for this, small-insert libraries were constructed with DNA isolated from microorganisms of high-altitude Andean hypersaline lakes in Argentina (Diamante and Ojo Seco lakes, 4,589 and 3,200 m, respectively) and from the Es Trenc solar saltern in Spain. The libraries were hosted in a UV radiation-sensitive strain of Escherichia coli (recA mutant) and they were exposed to UVB. The resistant colonies were analyzed and as a result, four clones were identified with environmental DNA fragments containing five genes that conferred resistance to UV radiation in E. coli. One gene encoded a RecA-like protein, complementing the mutation in recA that makes the E. coli host strain more sensitive to UV radiation. Two other genes from the same DNA fragment encoded a TATA-box binding protein and an unknown protein, both responsible for UV resistance. Interestingly, two other genes from different and remote environments, the Ojo Seco Andean lake and the Es Trenc saltern, encoded two hypothetical proteins that can be considered homologous based on their significant amino acid similarity (49%). All of these genes also conferred resistance to 4-nitroquinoline 1-oxide (4-NQO), a compound that mimics the effect of UV radiation on DNA, and also to perchlorate, a powerful oxidant that can induce DNA damage. Furthermore, the hypothetical protein from the Es Trenc salterns was localized as discrete foci possibly associated with damaged sites in the DNA in cells treated with 4-NQO, so it could be involved in the repair of damaged DNA. In summary, novel genes involved in resistance to UV radiation, 4-NQO and perchlorate have been identified in this work and two of them encoding hypothetical proteins that could be involved in DNA damage repair activities not previously described.Fil: Lamprecht Grandío, María. Consejo Superior de Investigaciones Cientificas. Centro de Astrobiologia.; EspañaFil: Cortesão, Marta. Consejo Superior de Investigaciones Cientificas. Centro de Astrobiologia.; EspañaFil: Mirete, Salvador. Consejo Superior de Investigaciones Cientificas. Centro de Astrobiologia.; EspañaFil: Benguigui de la Cámara, Macarena. Consejo Superior de Investigaciones Cientificas. Centro de Astrobiologia.; EspañaFil: de Figueras, Carolina G.. Consejo Superior de Investigaciones Cientificas. Centro de Astrobiologia.; EspañaFil: Pérez Pantoja, Danilo. Universidad Tecnológica Metropolitana; EspañaFil: White, Joseph John. Consejo Superior de Investigaciones Cientificas. Centro de Astrobiologia.; EspañaFil: Farias, Maria Eugenia. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tucumán. Planta Piloto de Procesos Industriales Microbiológicos; ArgentinaFil: Rosselló Móra, Ramon. Instituto Mediterráneo Estudios Avanzados; EspañaFil: González Pastor, José Eduardo. Consejo Superior de Investigaciones Cientificas. Centro de Astrobiologia.; Españ

Novel nickel resistance genes from the rhizosphere metagenome of plants adapted to acid mine drainage

11 pages, 5 figures, 3 tables.-- PMID: 17675438 [PubMed].-- PMCID: PMC2075024.-- ISI Article Identifier: 000249969000004.-- Printed version published Oct 2007.Supporting information (Suppl. figs S1-S2, table S1) available at: http://aem.asm.org/cgi/content/full/73/19/6001/DC1Full-text available Open Access at journal siteMetal resistance determinants have traditionally been found in cultivated bacteria. To search for genes involved in nickel resistance, we analyzed the bacterial community of the rhizosphere of Erica andevalensis, an endemic heather which grows at the banks of the Tinto River, a naturally metal-enriched and extremely acidic environment in southwestern Spain. 16S rRNA gene sequence analysis of rhizosphere DNA revealed the presence of members of five phylogenetic groups of Bacteria and the two main groups of Archaea mostly associated with sites impacted by acid mine drainage (AMD). The diversity observed and the presence of heavy metals in the rhizosphere led us to construct and screen five different metagenomic libraries hosted in Escherichia coli for searching novel nickel resistance determinants. A total of 13 positive clones were detected and analyzed. Insights about their possible mechanisms of resistance were obtained from cellular nickel content and sequence similarities. Two clones encoded putative ABC transporter components, and a novel mechanism of metal efflux is suggested. In addition, a nickel hyperaccumulation mechanism is proposed for a clone encoding a serine O-acetyltransferase. Five clones encoded proteins similar to well-characterized proteins but not previously reported to be related to nickel resistance, and the remaining six clones encoded hypothetical or conserved hypothetical proteins of uncertain functions. This is the first report documenting nickel resistance genes recovered from the metagenome of an AMD environment.S.M. and C.G.D.F. are supported by Instituto Nacional de Técnica Aeroespacial (INTA) postdoctoral and technician fellowships, respectively. J.E.G.-P. benefits from a Ramón y Cajal contract from the Ministerio de Educación y Ciencia (MEC-Spain) at INTA. This work was supported by the Centro de Astrobiología (CSIC/INTA), associated with the NASA Astrobiology Institute, and a grant from the MEC-Spain (CGL2006-14238).Peer reviewe

Ansiedad social subclínica en adultos jóvenes sanos: cortisol y ansiedad subjetiva en respuesta a estrés agudo

No existe consenso sobre el patrón de liberación de cortisol y su relación con la ansiedad subjetiva en situaciones de estrés en población con ansiedad social. Nuestro objetivo fue determinar la respuesta de cortisol y ansiedad subjetiva en individuos con ansiedad social sometidos a un estresor psicosocial agudo. 26 universitarios (58.6% hombres), edad media = 21.62 ± 0.43, fueron expuestos a la versión estrés o control del Maastricht Acute Stress Test. El cortisol salival y la ansiedad subjetiva fueron medidos antes, durante y post-estrés. Los participantes mostraron un incremento en los niveles de cortisol durante las fases de estrés y post-estrés, con una respuesta significativamente mayor en aquellos con ansiedad social. Los participantes con alta ansiedad social mostraron, a nivel muestral, mayores niveles de ansiedad subjetiva, especialmente post-estrés. Sólo en la fase de estrés, el cortisol y la ansiedad subjetiva correlacionaron significativamente en los participantes socialmente ansiosos. Los hallazgos apoyan una hiperresponsividad de cortisol en población no clínica y joven con alta ansiedad social. Futuras investigaciones deberían estudiar los factores involucrados y efectos de esta respuesta fisiológica en la salud. Asimismo, se resalta la necesidad de controlar la ansiedad social en experimentos que utilicen un estresor psicosocial de laboratorio.There is no consensus about the pattern of cortisol release and its relationship with subjective anxiety in situations of stress in the population with social anxiety. Our aim was to determine the cortisol and subjective anxiety response in individuals with social anxiety subjected to an acute psychosocial stressor. 26 college students (58.6% males), mean age = 21.62 ± 0.43, were exposed to the stress or control adaptation of the Maastricht Acute Stress Test. Salivary cortisol and subjective anxiety were measured before, during, and after stress. Participants showed an increase in cortisol levels during the stress and post-stress phases, with a significantly higher response in those with high social anxiety. Participants with high social anxiety also showed, as a tendency, higher levels of subjective anxiety, especially in the post-stress phase. Only in the stress phase, cortisol and subjective anxiety correlated significantly in socially anxious participants. The findings support a cortisol hyperresponsiveness in a young, non-clinical population with high social anxiety. Future research should focus on the factors involved and the effects of this physiological response on health. Furthermore, the need to control social anxiety in experiments using a laboratory psychosocial stressor is highlighted

Nickel-resistance determinants in acidiphilium sp. Pm identified by genome-wide functional screening

11 p.-1 tab.-5 fig.Acidiphilium spp. are conspicuous dwellers of acidic, metal-rich environments. Indeed, they are among the most metalresistant organisms; yet little is known about the mechanisms behind the metal tolerance in this genus. Acidiphilium sp. PM is an environmental isolate from Rio Tinto, an acidic, metal-laden river located in southwestern Spain. The characterization of its metal resistance revealed a remarkable ability to tolerate high Ni concentrations. Here we report the screening of a genomic library of Acidiphilium sp. PM to identify genes involved in Ni resistance. This approach revealed seven different genes conferring Ni resistance to E. coli, two of which form an operon encoding the ATP-dependent protease HslVU (ClpQY). This protease was found to enhance resistance to both Ni and Co in E. coli, a function not previously reported. Other Niresistance determinants include genes involved in lipopolysaccharide biosynthesis and the synthesis of branched amino acids. The diversity of molecular functions of the genes recovered in the screening suggests that Ni resistance in Acidiphilium sp. PM probably relies on different molecular mechanisms. © 2014 San Martin-Uriz et al.This work was funded by European Research Council advanced grant ERC 250350 and Spanish Ministry of Science and Innovation grants CGL2009-11059, CGL-2009-10756/BOS and CONSOLIDER Ingenio 2010 (‘‘The Metagenome of the Iberian Peninsula’’, CE-CSD2007-00005). PSMU is supported by a ‘‘Calvo Rode´ s’’ fellowship from the National Institute for Aerospace Technology (INTA).Peer Reviewe

Salt resistance genes revealed by functional metagenomics from brines and moderate-salinity rhizosphere within a hypersaline environment

Hypersaline environments are considered one of the most extreme habitats on earth and microorganisms have developed diverse molecular mechanisms of adaptation to withstand these conditions. The present study was aimed at identifying novel genes from the microbial communities of a moderate-salinity rhizosphere and brine from the Es Trenc saltern (Mallorca, Spain), which could confer increased salt resistance to Escherichia coli. The microbial diversity assessed by pyrosequencing of 16S rRNA gene libraries revealed the presence of communities that are typical in such environments and the remarkable presence of three bacterial groups never revealed as major components of salt brines. Metagenomic libraries from brine and rhizosphere samples, were transferred to the osmosensitive strain E. coli MKH13, and screened for salt resistance. Eleven genes that conferred salt resistance were identified, some encoding for well-known proteins previously related to osmoadaptation such as a glycerol transporter and a proton pump, whereas others encoded proteins not previously related to this function in microorganisms such as DNA/RNA helicases, an endonuclease III (Nth) and hypothetical proteins of unknown function. Furthermore, four of the retrieved genes were cloned and expressed in Bacillus subtilis and they also conferred salt resistance to this bacterium, broadening the spectrum of bacterial species in which these genes can function. This is the first report of salt resistance genes recovered from metagenomes of a hypersaline environment. © 2015 Mirete, Mora-Ruiz, Lamprecht-Grandío, de Figueras, Rosselló-Móra and González-Pastor.This work was funded by the Spanish Ministry of Science and Innovation (CGL2012-39627-C03/02 and 03); the latter also supported with European Regional Development Fund (FEDER). MM-R Ph.D. is supported by fellowship CVU 265934 of the National Council of Science and Technology (CONACyT), MexicoPeer Reviewe