Ultrastructural changes in methicillin-resistant Staphylococcus aureus (MRSA) induced by a novel cyclic peptide ASP-1 from Bacillus subtilis: A scanning electron microscopy (SEM) study / Cambios ultraestructurales en Staphylococcusaureus resistente a meticilina (SARM) inducidos por un nuevo péptido cíclico ASP-1 de Bacillussubtilis: un estudio con microscopio electrónico de barrido (MEB)

ABSTRACT Increasing antimicrobial resistance amongStaphylococcus aureusnecessitates a new antimicrobial with a different site of action. We have isolated a novel cyclic peptide-1 (ASP-1) fromBacillussubtiliswith potent activity against methicillin-resistantS. aureus(MRSA) at a minimum inhibitory concentration (MIC) of 8-64μg/ml. Scanning electron micrographs demonstrated drastic changes in the cellular architecture of ASP-1 treated cells ofS. aureusATCC 29213 and an MRSA clinical isolate at MICs, with damages to the cell wall, membrane lysis and probable leakage of cytoplasmic contents at minimum bactericidal concentrations. The ultrastructure alterations induced by ASP-1 have also been compared with those of oxacillin-treated MRSA cells at its MIC using scanning electron microscopy.

RESUMEN El incremento de la resistencia antimicrobiana entre los tipos deS. aureusexige un nuevo agente antimicrobiano con un sitio de acción diferente. Aislamos un nuevo péptido cíclico (ASP-1) deBacillussubtiliscon potente actividad frente aS. aureusresistente a meticilina (SARM) en una concentración inhibitoria mínima (CIM) de 8-64μg/ml. Las micrografías obtenidas con microscopio electrónico de barrido mostraron cambios drásticos en la arquitectura celular de las células deS. aureusATCC 29213 tratadas con ASP-1, y un aislamiento clínico de SARM a la CIM, con daños a la pared celular, lisis de la membrana y probable fuga de contenido citoplasmático a concentraciones bactericidas mínimas. Comparamos también, las alteraciones de la ultraestructura inducidas por ASP-1 con las de células de SARM tratadas con oxacilina a su CIM, utilizando microscopio electrónico de barrido.

Extracellular expression in Bacillus subtilis of a thermostable Geobacillus stearothermophilus lipase

BACKGROUND The extracellular expression of enzymes in a secretion host such as Bacillus subtilis is a useful strategy in reducing the cost of downstream processing of industrial enzymes. Here, we present the first report of the successful extracellular expression in Bacillus subtilis WB800 of Geobacillus stearothermophilus lipase (T1.2RQ), a novel industriallydesirable thermostable lipolytic enzyme which has an excellent hydrolytic and transesterification activity. Signal peptides of a-amylase, extracellular protease, and lipase A, as well as two different promoters, were used in the secretion and expression of lipase T1.2RQ. RESULTS Lipase activity assay using p-nitrophenyl laurate showed that all three signal peptides directed the secretion of lipase T1.2RQ into the extracellular medium. The signal peptide of lipase A, resulted in the highest extracellular yield of 5.6 U/ml, which corresponds to a 6-fold increase over the parent Bacillus subtilis WB800 strain. SDS-PAGE and zymogram analysis confirmed that lipase T1.2RQ was correctly processed and secreted in its original size of 44 kDa. A comparison of the expression levels of lipase T1.2RQ in rich medium and minimal media showed that the enzyme was better expressed in rich media, with up to an 8-fold higher yield over minimal media. An attempt to further increase the lipase expression level by promoter optimization showed that, contrary to expectation, the optimized promoter exhibited similar expression levels as the original one, suggesting the need for the optimization of downstream factors. CONCLUSIONS The successful extracellular secretion of lipase T1.2RQ in Bacillus subtilis represents a remarkable feat in the industrial-scale production of this enzyme

Evaluación de los cambios en algunas propiedades físicas y químicas de un ultisolpor efecto del bacillus subtilis / Evaluation of the changes in the some physical and chemical properties of an ultisol for effect of the bacillus subtilis

El interés en estudios de biorremediación de suelos deteriorados por sobreexplotación y uso indiscriminado de agroquímicos se debe a que alteran la microflora, el sistema de autorregulación y la sustentabilidad en el largo plazo. Un Ultisol, suelo de baja fertilidad química y sometido a reducción de tamaño de agregados (<2,0 mm), solarizado para reducir la población microbiológica, fue inoculado con la bacteria Basillus subtilis en concentraciones de 106, 107, 108, 109 unidades formadoras de colonias (ufc). En 120 días se observó un incremento importante en magnitud de: estabilidad estructural, tamaño promedio de agregados, pH, fósforo disponible y una disminución del aluminio intercambiable. Estas variaciones en la respuesta estuvieron relacionadas con la actividad de los microorganismos en el suelo, y responden a la capacidad de solubilización de minerales por la bacteria, de producción de condiciones alcalinas y de biofilms, que unidos al aumento de biomasa de raíces de la planta, mucílagos y carbohidratos, coadyuvan en la formación de agregados estables y de mayor tamaño. Las propiedades físicas y químicas al final del experimento se estabilizaron en valores mayores a los encontrados en el suelo inicial, produciendo un efecto positivo general sobre el mismo, desde el punto de vista de la fertilidad global, al aumentar el fósforo disponible, disminuir la acidez intercambiable e incrementar la estabilidad y el tamaño promedio de agregados del suelo a corto plazo.

The interest about the studies of bioremediation in deteriorated soils under and over exploitation, indiscriminate use of agrochemicals that alter the microflora, the self regulation system and the sustainability in the long term. An Ultisol, with poor chemical properties and low fertility, because the reduction of aggregates size (< 2.0 mm). Solarize to reduce the microbiological population, it was inoculated with the bacteria Basillus subtilis in concentrations of 106, 107, 108, 109 colony forming units (cfu). In 120 days important increment in magnitude of: structural stability, size average of aggregates, pH, available phosphorus. Also diminish the interchangeable aluminum. These variations in the answer were related with the activity of the microorganisms in the soil, and it responds so that solubilisation capacity of minerals because the bacteria activity, production of alkaline conditions and biofilms too. Furthermore the increase of plant roots biomass, mucilages and carbohydrates cooperate in the formation of stable aggregates and bigger size of them. The physical and chemical properties at the end of the experiment were stabilized in bigger values than found in the initial soil. Confirming a positive general effect overall the soil, since the point of view of the global fertility, when increasing the available phosphorus, reduce the interchangeable acidity and increase the soil stability and average size aggregates in the short term.

Identification and annotation of promoter regions in microbial genome sequences on the basis of DNA stability.

Analysis of various predicted structural properties of promoter regions in prokaryotic as well as eukaryotic genomes had earlier indicated that they have several common features,such as lower stability, higher curvature and less bendability, when compared with their neighboring regions. Based on the difference in stability between neighboring upstream and downstream regions in the vicinity of experimentally determined transcription start sites, a promoter prediction algorithm has been developed to identify prokaryotic promoter sequences in whole genomes. The average free energy (E) over known promoter sequences and the difference (D) between E and the average free energy over the entire genome (G)are used to search for promoters in the genomic sequences. Using these cutoff values to predict promoter regions across entire Escherichia coli genome,we achieved a reliability of 70% when the predicted promoters were cross verified against the 960 transcription start sites (TSSs) listed in the Ecocyc database. Annotation of the whole E.coli genome for promoter region could be carried out with 49% accuracy. The method is quite general and it can be used to annotate the promoter regions of other prokaryotic genomes.

ARC: automated resource classifier for agglomerative functional classification of prokaryotic proteins using annotation texts.

Functional classification of proteins is central to comparative genomics. The need for algorithms tuned to enable integrative interpretation of analytical data is felt globally. The availability of a general,automated software with built-in flexibility will significantly aid this activity. We have prepared ARC (Automated Resource Classifier), which is an open source software meeting the user requirements of flexibility. The default classification scheme based on keyword match is agglomerative and directs entries into any of the 7 basic non-overlapping functional classes: Cell wall, Cell membrane and Transporters (C), Cell division (D), Information (I), Translocation (L), Metabolism (M), Stress(R), Signal and communication (S) and 2 ancillary classes: Others (O) and Hypothetical (H).The keyword library of ARC was built serially by first drawing keywords from Bacillus subtilis and Escherichia coli K12. In subsequent steps,this library was further enriched by collecting terms from archaeal representative Archaeoglobus fulgidus, Gene Ontology, and Gene Symbols. ARC is 94.04% successful on 6,75,663 annotated proteins from 348 prokaryotes. Three examples are provided to illuminate the current perspectives on mycobacterial physiology and costs of proteins in 333 prokaryotes. ARC is available at http://arc.igib.res.in.

Ausencia de actividad antimicrobiana de un extracto acuoso liofilizado de Aloe vera (Sabila) / Absence of antimicrobial activity of lyophilized aqueous extract of Aloe vera (sabila)

Se estudio la actividad antimicrobiana de dos concentraciones (10 y 50 mg/mL) de un extracto acuoso liofilizado de hojas de Aloe vera (sabila), mediante el sistema de ensayo de difusion en agar, con una bateri aminima de cepas de microorganismos compuesta por cuatro bacterias (Staphylococcus aureus, Bacillus subtilils, Escherichia coli y Pseudomonas aeruginosa) y una levadura (Candida albicans). Los resultados indican que solo frente al Staphylococcus aureus se obteine una ligera actividad inhibitoria, al compararla con la que produce el control positivo (estreptomicina). Para el resto de los microorganismos estudiados la respuesta es negativa. Estos resultados permiten desestimar el uso del extracto acuoso liofilizado de Aloe vera como antimicrobiano, en tanto que sugiere explorar este efecto con otro tipo de extracto con el objetivo de avalar o no la utilizacion de esta planta como antimicrobiano

Actividad antimicrobiana del Schinus terebentifolius Raddi (copal) / Antimicrobial activity of Schinus terebentifolius Raddi (copal)

Se estudio la actividad antimicrobiana de diferentes concentraciones de un extracto fluido (etanol al 30 porciento) de hojas de Schinus terebenthifolius Raddi (copal) con una bateria minima de cepas de microorganismos que incluye Staphylococcus aureus y Bacillus subtilis, como grampositivo, escherichia coli y Pseudomonas aeruginosa, como gramnegativo y la levadura, Candida albicans, mediante el metdo de difusion en agar. Los resultados obtenidos indican que en la menor concentracion utilizada, del 10 porciento del extracto fluido, no se aprecia inhibicion de ninguno de los microorganismos evaluados, mientras que en las concentraciones del 50 y el 100 porciento del extracto fluido hay respuesta de inhibicion frente a las bacterias grampositiva y gramnegativa, pero no asi con la levadura. Estos resultados contribuyen a ratificar experimentalmente el uso tradicional que se hace de esta planta como antimicrobiano, ademas sugiere la elaboracion de formas farmaceuticas que permitan ampliar con mayor eficiencia su utilizacion para este objetivo