Characterization of a two-gene operon epeRA involved in multidrug resistance in Streptomyces clavuligerus

[EN] Two genes, epeR and epeA, are located downstream of argH in the Streptomyces clavuligerus genome. EpeR belongs to the TetR family of transcriptional regulators. It is homologous to PqrA of Streptomyces coelicolor (74.3% identity) and to NfxB of Pseudomonas aeruginosa (30.9% identity). EpeA encodes a protein with 14 transmembrane spanning domains (TMS) of the major facilitator superfamily. It shares 68.9% identity to PqrB of S. coelicolor and 46.5% identity to LfrA, conferring resistance to fluoroquinolones in Mycobacterium smegmatis. Disruption of epeR results in a S. clavuligerus epeR::aph mutant which shows increased resistance to ethidium bromide and proflavine (16- and 32-fold higher than the wild type). Taking into consideration the sensitivity to drugs of different transformants carrying functional copies of either epeR or epeA, it might be concluded that both genes appear to be co-transcribed, with epeR encoding a regulatory protein which controls the expression of epeASIThis work was supported by CICYT grant BIO2003-3274 and GEN2003-20245. We thank Matthew Smith (University of Nottingham) and María Alvarez (University of León, Spain) for critical reading of the manuscript

CcaR is an autoregulatory protein that binds to the ccaR and cefD-cmcI promoters of the cephamycin C-clavulanic acid cluster in Streptomyces clavuligerus

[EN] The putative regulatory CcaR protein, which is encoded in the β-lactam supercluster of Streptomyces clavuligerus, has been partially purified by ammonium sulfate precipitation and heparin affinity chromatography. In addition, it was expressed in Escherichia coli, purified as a His-tagged recombinant protein (rCcaR), and used to raise anti-rCcaR antibodies. The partially purified CcaR protein from S. clavuligerus was able to bind DNA fragments containing the promoter regions of the ccaR gene itself and the bidirectional cefD-cmcI promoter region. In contrast, CcaR did not bind to DNA fragments with the promoter regions of other genes of the cephamycin-clavulanic acid supercluster including lat, blp, claR, car-cyp, and the unlinked argR gene. The DNA shifts obtained with CcaR were prevented by anti-rCcaR immunoglobulin G (IgG) antibodies but not by anti-rabbit IgG antibodies. ccaR and the bidirectional cefD-cmcI promoter region were fused to the xylE reporter gene and expressed in Streptomyces lividans and S. clavuligerus. These constructs produced low catechol dioxygenase activity in the absence of CcaR; activity was increased 1.7- to 4.6-fold in cultures expressing CcaR. Amplification of the ccaR promoter region lacking its coding sequence in a high-copy-number plasmid in S. clavuligerus ATCC 27064 resulted in a reduced production of cephamycin C and clavulanic acid, by 12 to 20% and 40 to 60%, respectively, due to titration of the CcaR regulator. These findings confirm that CcaR is a positively acting autoregulatory protein able to bind to its own promoter as well as to the cefD-cmcI bidirectional promoter regionSIThis work was supported by the Spanish Ministry of Science and Technology (FD97-1419-CO2-O2). I. Santamarta received a fellowship from the University of León. We are grateful to A. de la Fuente, F. J. Enguita, and C. de Torre for their interest and helpful discussions, to A. Jiménez for revising the manuscript, and to M. Mediavilla for technical assistance

Natural and synthetic tetracycline-inducible promoters for use in the antibiotic-producing bacteria Streptomyces

Bacteria in the genus Streptomyces are major producers of antibiotics and other pharmacologically active compounds. Genetic and physiological manipulations of these bacteria are important for new drug discovery and production development. An essential part of any ‘genetic toolkit’ is the availability of regulatable promoters. We have adapted the tetracycline (Tc) repressor/operator (TetR/tetO) regulatable system from transposon Tn10 for use in Streptomyces. The synthetic Tc controllable promoter (tcp), tcp830, was active in a wide range of Streptomyces species, and varying levels of induction were observed after the addition of 1–100 ng/ml of anhydrotetracycline (aTc). Streptomyces coelicolor contained an innate Tc-controllable promoter regulated by a TetR homologue (SCO0253). Both natural and synthetic promoters were active and inducible throughout growth. Using the luxAB genes expressing luciferase as a reporter system, we showed that induction factors of up to 270 could be obtained for tcp830. The effect of inducers on the growth of S.coelicolor was determined; addition of aTc at concentrations where induction is optimal, i.e. 0.1–1 μg/ml, ranged from no effect on growth rate to a small increase in the lag period compared with cultures with no inducer

Natural and synthetic tetracycline-inducible promoters for use in the antibiotic-producing bacteria Streptomyces

[EN] Bacteria in the genus Streptomyces are major producers of antibiotics and other pharmacologically active compounds. Genetic and physiological manipulations of these bacteria are important for new drug discovery and production development. An essential part of any ‘genetic toolkit’ is the availability of regulatable promoters. We have adapted the tetracycline (Tc) repressor/operator (TetR/ tetO ) regulatable system from transposon Tn 10 for use in Streptomyces . The synthetic Tc controllable promoter (tcp), tcp830 , was active in a wide range of Streptomyces species, and varying levels of induction were observed after the addition of 1–100 ng/ml of anhydrotetracycline (aTc). Streptomyces coelicolor contained an innate Tc-controllable promoter regulated by a TetR homologue (SCO0253). Both natural and synthetic promoters were active and inducible throughout growth. Using the luxAB genes expressing luciferase as a reporter system, we showed that induction factors of up to 270 could be obtained for tcp830 . The effect of inducers on the growth of S.coelicolor was determined; addition of aTc at concentrations where induction is optimal, i.e. 0.1–1 μg/ml, ranged from no effect on growth rate to a small increase in the lag period compared with cultures with no inducerSIThe authors acknowledge gifts of plasmids and strains from Prof. Leadlay, Prof. Hillen, Prof. Bujard, Dr Herron and Dr Paget. The authors thank Dr Sumby, Dr Ding and Wael Hussein for the construction of several plasmids and vectors. The authors also thank Prof. Williams for the use of Lucy. This work was funded by the BBSRC. Funding to pay the Open Access publication charges for this article was provided by JIS

Characterization and expression of the arginine biosynthesis gene cluster of Streptomyces clavuligerus

Revista continuada por Microbial Physiology[EN] A cluster of genes argCJBDRGH containing most of the arginine biosynthesis genes has been found in Streptomyces clavuligerus after sequencing a 8.3 kb DNA region containing overlapping sequences of two DNA fragments known to contain arginine biosynthesis genes. Subcloning, complementation of E. coli arginine auxotrophic strains and enzymatic assays confirmed the identity of each gene. S1 nuclease mapping studies and Northern hybridization analysis revealed the formation of two large transcripts corresponding to argCJBDR and argGH. The amount of each of these mRNAs is 10 to 44 times higher in a S. clavuligerus argR-disrupted mutant than in the wild type confirming the existence of an ArgR-mediated control of arginine biosynthesis gene expression. A low level constitutive monocistronic transcript of argR was observed in S. clavuligerus cells. Most of the argGH transcript initiating at an adenine 29 nt upstream of the argG initiation codon appears to stop at a termination stem and loop structure present downstream of the argG geneSIThis work was supported by grants from the CICYT (Madrid) Bio96-0827 and by Antibióticos SA, León. We thank H. Kieser (Norwich, U.K.) for providing the S. coelicolor cosmid library. Álvaro de la Fuente and Rosario Pérez-Redondo received fellowships from the PFPI (Madrid) and the University of León, respectively

ArgR of Streptomyces coelicolor is a versatile regulator

[EN] ArgR is the regulator of arginine biosynthesis genes in Streptomyces species. Transcriptomic comparison by microarrays has been made between Streptomyces coelicolor M145 and its mutant S. coelicolor ΔargR under control, unsupplemented conditions, and in the presence of arginine. Expression of 459 genes was different in transcriptomic assays, but only 27 genes were affected by arginine supplementation. Arginine and pyrimidine biosynthesis genes were derepressed by the lack of ArgR, while no strong effect on expression resulted on arginine supplementation. Several nitrogen metabolism genes expression as glnK, glnA and glnII, were downregulated in S. coelicolor ΔargR. In addition, downregulation of genes for the yellow type I polyketide CPK antibiotic and for the antibiotic regulatory genes afsS and scbR was observed. The transcriptomic data were validated by either reverse transcription-PCR, expression of the gene-promoter coupled to the luciferase gene, proteomic or by electrophoresis mobility shift assay (EMSA) using pure Strep-tagged ArgR. Two ARG-boxes in the arginine operon genes suggest that these genes are more tightly controlled. Other genes, including genes encoding regulatory proteins, possess a DNA sequence formed by a single ARG-box which responds to ArgR, as validated by EMSASIThis work was supported by Grants from the Spanish Comisión Interministerial de Ciencia y Tecnología GEN2003-20245, BIO2009-09820, and by the European Project LSHM-CT-2004-005224. AB received a fellowship from the Ministry of Science and Innovation. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript

ArgR of Streptomyces coelicolor is a pleiotropic transcriptional regulator: effect on the transcriptome, antibiotic production, and differentiation in liquid cultures

[EN] ArgR is a well-characterized transcriptional repressor controlling the expression of arginine and pyrimidine biosynthetic genes in bacteria. In this work, the biological role of Streptomyces coelicolor ArgR was analyzed by comparing the transcriptomes of S. coelicolor ΔargR and its parental strain, S. coelicolor M145, at five different times over a 66-h period. The effect of S. coelicolor ArgR was more widespread than that of the orthologous protein of Escherichia coli, affecting the expression of 1544 genes along the microarray time series. This S. coelicolor regulator repressed the expression of arginine and pyrimidine biosynthetic genes, but it also modulated the expression of genes not previously described to be regulated by ArgR: genes involved in nitrogen metabolism and nitrate utilization; the act, red, and cpk genes for antibiotic production; genes for the synthesis of the osmotic stress protector ectoine; genes related to hydrophobic cover formation and sporulation (chaplins, rodlins, ramR, and whi genes); all the cwg genes encoding proteins for glycan cell wall biosynthesis; and genes involved in gas vesicle formation. Many of these genes contain ARG boxes for ArgR binding. ArgR binding to seven new ARG boxes, located upstream or near the ectA-ectB, afsS, afsR, glnR, and redH genes, was tested by DNA band-shift assays. These data and those of previously assayed fragments permitted the construction of an improved model of the ArgR binding site. Interestingly, the overexpression of sporulation genes observed in the ΔargR mutant in our culture conditions correlated with a sporulation-like process, an uncommon phenotypeSIGrant BIO2013-34723 from the Spanish Ministry of Science and Innovation to PL. Work in the AM's laboratory was funded by the European Research Council (ERC Starting Grant; Strp-differentiation 280304) and by the Spanish Ministry of Economy and Competitiveness (Grant BIO2015-65709-R)

Applying a positive behaviour support plan in school context

El Plan de Apoyo Conductual Positivo es una práctica basada en la evidencia que tras mostrar su eficacia para tratar conductas desafiantes en el ámbito de la discapacidad se está empezando a aplicar con éxito en los colegios de educación regular para responder a las necesidades asociadas a la conducta. El objetivo de este estudio es describir el proceso por el que se empieza a implantar este tipo de intervención proactiva en un Colegio de Educación Infantil y Primaria (CEIP) mediante un programa de formación de carácter teórico-práctico dirigido a once profesores de ese CEIP, y analizar los efectos que el programa tiene sobre la conducta y el clima social de sus aulas. Se encontraron diferencias estadísticamente significativas entre las puntuaciones obtenidas antes y después del programa en el Test de Alteración de la Conducta en la Escuela (Arias, Ayuso, Gil y González, 2006), la adaptación del Inventario de conductas en clase (Curwin y Mendler, 1983) realizada por Fernández (1998), y una adaptación de la escala para evaluar el clima social del aula (Pérez, Ramos y López, 2009). Se contrastan los resultados con los obtenidos con métodos cualitativos y se describe el proceso por el que el profesorado va transformando sus creencias y comienza a aplicar en su aula las estrategias que conducen a la creación de una escuela positiva.Positive Behavior Support Planning is an evidence based practice which, after showing its efficiency to deal with challenging behaviors in the disability world, is beginning to be applied with success in regular schools to respond to the needs associated with behaviour. The aim of this study is to describe the process through which this kind of proactive intervention is applied in an Infant and Primary Education School (IPES) with a theoretical and practical training directed to eleven teachers of that IPES, and to analyze the effects that the program has on behaviour and classroom social climate. Significant statistical differences were found between the scores obtained before and after applying the program in the Test of Challenging Behaviors in the School (Arias, Ayuso, Gil y González, 2006), the adaptation of the Inventory of behaviors in the Classroom (Curwin y Mendler, 1983) made by Fernández (1998), and an adaptation of a Scate to Evaluate Social Climate in the Classroom (Pérez, Ramos y López, 2009). Results are contrasted with those obtained with qualitative methods, and the process by which teachers keep on changing their beliefs and begin to apply in their classrooms the strategies leading to create a positive school is described

Intermediate Repeat Expansion in the ATXN2 Gene as a Risk Factor in the ALS and FTD Spanish Population

Intermediate CAG expansions in the gene ataxin-2 (ATXN2) are a known risk factor for ALS, but little is known about their role in FTD risk. Moreover, their contribution to the risk and phenotype of patients might vary in populations with different genetic backgrounds. The aim of this study was to assess the relationship of intermediate CAG expansions in ATXN2 with the risk and phenotype of ALS and FTD in the Spanish population. Repeat-primed PCR was performed in 620 ALS and 137 FTD patients in three referral centers in Spain to determine the exact number of CAG repeats. In our cohort, >= 27 CAG repeats in ATXN2 were associated with a higher risk of developing ALS (odds ratio [OR] = 2.666 [1.471-4.882]; p = 0.0013) but not FTD (odds ratio [OR] = 1.446 [0.558-3.574]; p = 0.44). Moreover, ALS patients with >= 27 CAG repeats in ATXN2 showed a shorter survival rate compared to those with = 27 repeats in ATXN2 are associated with ALS risk but not with FTD in the Spanish population. ALS patients carrying an intermediate expansion in ATXN2 show more frequent limb onset but a worse prognosis than those without expansions. In patients carrying C9orf72 expansions, the intermediate ATXN2 expansion might increase the penetrance and modify the phenotype

Gestión de recursos electrónicos en el Consorcio de Bibliotecas Universitarias Andaluzas. Una experiencia de cooperación bibliotecaria en entornos digitales

En la actualidad es difícil encontrar bibliotecas digitales nacidas bajo este concepto. La denominación representa más bien la evolución de las bibliotecas tradicionales, sobre todo universitarias, hacia un entorno en que la proliferación de recursos electrónicos en lo últimos años ha cambiado no sólo el concepto de biblioteca sino el mismo concepto de investigación, de estudio y por tanto de trabajo. Bajo esta premisa, en que señalamos recursos electrónicos como origen de bibliotecas digitales, son los consorcios bibliotecarios los que han contribuido de forma extraordinaria y definitiva a su desarrollo. La vieja idea de cooperación se ha materializado en ellos no sólo en la disposición de recursos propios sino que ha encontrado su máxima expresión en el entorno consorciado en que la adquisición y gestión compartidas han sido necesarias para dar respuesta a las necesidades que impone la proliferación de recursos electrónicos en las nuevas bibliotecas digitales. Claro ejemplo de esta afirmación, de estas nuevas formas de organización lo constituye el recientemente creado Grupo de Trabajo de Recursos Electrónicos del Consorcio de Bibliotecas Universitarias Andaluzas (CBUA), cuyo proyecto de cooperación y gestión contribuye a facilitar el acceso a la colección digital en el ámbito universitario andaluz. Se presenta el trabajo realizado hasta la fecha por el Grupo, así como las perspectivas de futuro. Se exponen la metodología de trabajo empleada, las líneas de actuación, las acciones realizadas, las herramientas utilizadas para optimizar la calidad en el acceso y difusión de los recursos de los que disponemos. Palabras clave: bibliotecas universitarias, bibliotecas digitales, gestión de recursos electrónicos, consorcios de bibliotecas, cooperación bibliotecaria