Development of vectors and specialized Streptomyces strains for functional metagenomics

Motivation: Antimicrobial resistance (AMR) are one of the most important existing threats to public health aggravated by theoveruse of antimicrobials. It is estimated that the mortality from drug-resistance infections would increase up to 10 millions by2050. One of the major hurdles in microbial ecology is the inability to culture most of the microbial diversity present in ecosystemsunder laboratory conditions. The molecular analysis strategies used to examine the microbial community DNA, also known asthe metagenome, have been denoted as metagenomics techniques. With the use of functional metagenomics we are focus ourefforts on searching new antimicrobials. For the expression of genes from metagenomics DNA, Escherichia coli is the host mostused for function-based screening. However, the potential of Streptomyces as a surrogate host for the production of heterologousproteins has shown to be interesting for expression of proteins that are difficult to express in other bacterial host system.Methods: Basing on previous work, we are using a construction of specialized vectors with modified heterologous expressionsystem that incorporate viral components. The construction consist in the T7 RNA-polymerase gene (gene1), synthesized witha codon optimization for Streptomyces, that encodes the phage RNA-polimerase which is insensitive to many of the bacterialtermination signals. The gene1 expression is regulated by a inducible expression system based on PnitA-NitR regulatory system.To test that our system work properly, we have introduced in the cosmid for the metagenomic library construction, which isreplicative in Streptomyces, a reporter gene (xylE) whose gene product is a catechol dioxygenase which converts the colourlesssubstrate catechol to an intensely yellow oxidation product.Results: This part of the project aims to evaluate the efficience of heterologous expression measuring the catechol dioxygenaseactivity in solid and liquid media. In solid media we have seen that the Streptomyces colonies turned yellow when catechol wasapplied indicating that the expression system is working efficiently. The main objective of this project is to develop a metagenomiclibrary, with an improved heterologous expression, from DNA originating in several strains of Streptomyces in order to searchfor new antimicrobial resistance

Characterization of the antimicrobial activity of a clone with metagenomic DNA from a refinery.

Motivation: The increase in the number of antibiotic resistance mechanisms in microorganisms added to the low rate of newantimicrobial (AM) development supposes a threat for public health that should be urgently approached. Bacteria are one ofthe biggest sources of AM but only a small fraction of them can be cultured in vitro. To overcome this limitation our lab uses afunctional metagenomic approach and had previously developed a heterologous expression system that allows the study of allthe gene pool from a specific environmen1,2. Using this strategy we found different clones whose metagenomic DNA expresssion produces AM activity against Micrococcus luteus and also against the Methicillin-resistant Staphylococcus aureus (MRSA), one of the World Health Organization’s main priorities regarding resistant bacteria. In this work we study the properties and the activity of the AM produced by three different subclones with homologous genes related to phenol metabolism that were found in two different metagenomic libraries and we further characterize one of these subclones, pMPO1718, which proceeds from a refinery metagenomic library.Methods: The AM is produced in liquid cultures, the antimicrobial production is increased with arabinose for 6-7h and thenthe supernatant is filtered and lyophilized. The AM activity is tested on LB soft agar plates inoculated with the target strains.The AM is separated in different fractions by chromatography to study in which of them the activity is present.Results: We have defined a protocol to produce the AM in minimum media complemented with tryptophan and we haveobserved AM activity against M.luteus in the filtered culture and even higher activity in the total culture extracted with acetone50%. The last one was analyzed by high performance liquid chromatography and mass spectrometry by Fundación MEDINAand the comparison with databases showed that it may be an AM not previously described. Some AM properties arecharacterized in this work such as the minimum inhibitory concentration, the minimum bactericidal concentration, itsthermostability, its activity in different solvents or against different bacteria.Conclusions: The filtered supernatant of the three different subclones has AM activity against M.luteus and MRSA producedby phenol hydroxylase related genes. This AM is thermostable until more than 100ºC, has bactericidal activity and can beproduced either in LB medium or in M9 medium complemented with tryptophan

Mapa de Géneros Discursivos Escolares y Estrategias de Producción Textual para diagnosticar las demandas de escritura en las materias del currículo de un Centro de Secundaria

www.redactext.es www.didactext.netDepto. de Didáctica de las Lenguas, Artes y Educación FísicaFac. de EducaciónFALSEUniversidad Complutense de Madridsubmitte

Concepciones de escritura de estudiantes de tres especialidades del Máster de Formación de Profesorado de Secundaria y propuesta de un taller de escritura académica y profesional

El proyecto explorará las concepciones predominantes sobre enseñanza y aprendizaje de la escritura en alumnos de distintas especialidades (Lengua Castellana y Literatura, Ciencias Naturales y Ciencias Sociales) del Máster de Formación del Profesorado