Nelma Regina Segnini Bossolan

Disruption of Saccharomyces cerevisiae by Plantaricin 149 and investigation of its mechanism of action with biomembrane model systems

The action of a synthetic antimicrobial peptide analog of Plantaricin 149 (Pln149a) against Saccharomyces cerevisiae and its interaction with biomembrane model systems were investigated. Pln149a was shown to inhibit S. cerevisiae growth by more than 80% in YPD medium, causing morphological changes in the yeast wall and remaining active and resistant to the yeast proteases even after 24 h of incubation. Different membrane model systems and carbohydrates were employed to better describe the Pln149a interaction with cellular components using circular dichroism and fluorescence spectroscopies, adsorption kinetics and surface elasticity in Langmuir monolayers. These assays showed that Pln149a does not interact with either mono/polysaccharides or zwitterionic LUVs, but is strongly adsorbed to and incorporated into negatively charged surfaces, causing a conformational change in its secondary structure from random-coil to helix upon adsorption. From the concurrent analysis of Pln149a adsorption kinetics and dilatational surface elasticity data, we determined that 2.5 muM is the critical concentration at which Pln149a will disrupt a negative DPPG monolayer. Furthermore, Pln149a exhibited a carpet-like mechanism of action, in which the peptide initially binds to the membrane, covering its surface and acquiring a helical structure that remains associated to the negatively charged phospholipids. After this electrostatic interaction, another peptide region causes a strain in the membrane, promoting its disruption.

Qualidade bacteriológica de águas subterrâneas em cemitérios

Groundwater samples collected by piezometers from three cemeteries in geologically distinct areas of S. Paulo and Santos, Brazil, were analysed in order to determine their hygienic and sanitary conditions. Fecal coliformes, fecal streptococci, sulfite reducer clostridia and Salmonella were searched for the purpose of evaluating sanitary conditions, and total coliforms, heterotrophic bacteria, proteolitic and lipolitic microorganisms for evaluating hygienic conditions. In some samples, nitrate levels were also determined. It was discovered that these waters do not present adequate sanitary and hygienic conditions and that, in some cases, nitrate levels were extremely high (75.7 mg/l). In most samples, higher levels of fecal streptococci and sufite reducer clostridia than fecal coliforms were detected, which seems to show that the two former indicators would be more appropriate for evaluating the sanitary conditions of this kind of water. Salmonella were detected in only one of 44 samples analysed and coliphages in none. In the statistical analysis, the correlation matrix showed significant correlations among three fecal pollution indicators, as well as among anaerobic and aerobic heterotrophs and lipolitic bacteria. A direct relationship between the deterioration of water quality and the geological and hydrogeological conditions of the environment studied was observed. When cemeteries are constructed these conditions should, therefore, be taken into consideration.Foram analisadas amostras de aguas subterrâneas de tres cemiterios localizados em areas geologicamente distintas de Sao Paulo e de Santos, Brasil, com relacao as condicoes higienicas e sanitarias. Para as primeiras foram considerados os coliformes totais, bacterias heterotroficas, microrganismos proteoliticos e lipoliticos. Para as sanitarias foram pesquisados coliformes fecais, estreptococos fecais, clostridios sulfito redutores, colifagos e salmonelas. Verificou-se que as aguas nao apresentaram condicoes higienicas satisfatorias e, em alguns casos, foram encontrados niveis altos de nitrato (75,7 mg/l). A detecao de niveis mais elevados de estreptococos fecais e de clostridios sulfito redutores em relacao aos coliformes fecais, na maior parte das amostras, parece mostrar que os dois primeiros indicadores seriam mais adequados para avaliacao das condicoes sanitarias deste tipo de agua. Foi detectada Salmonella apenas em uma amostra e nao foram detectados colifagos. Na analise estatistica, foram encontradas correlacoes significantes entre tres indicadores de poluicao fecal assim como entre as contagens em placas de bacterias heterotroficas aerobias, anaerobias e lipoliticas. Foi observada uma relacao direta entre a deterioracao da qualidade da agua e as condicoes geologicas e hidrogeologicas do ambiente estudado, devendo este fator ser considerado para o planejamento e implantacao de cemiterios.

Environmental factors modulating the stability and enzymatic activity of the Petrotoga mobilis Esterase (PmEst)

Enzymes isolated from thermophilic organisms found in oil reservoirs can find applications in many fields, including the oleochemical, pharmaceutical, bioenergy, and food/dairy industries. In this study, in silico identification and recombinant production of an esterase from the extremophile bacteria Petrotoga mobilis (designated PmEst) were performed. Then biochemical, bioinformatics and structural characterizations were undertaken using a combination of synchrotron radiation circular dichroism (SRCD) and fluorescence spectroscopies to correlate PmEst stability and hydrolytic activity on different substrates. The enzyme presented a high Michaelis-Menten constant (KM 0.16 mM) and optimum activity at ~55°C for p-nitrophenyl butyrate. The secondary structure of PmEst was preserved at acid pH, but not under alkaline conditions. PmEst was unfolded at high concentrations of urea or guanidine through apparently different mechanisms. The esterase activity of PmEst was preserved in the presence of ethanol or propanol and its melting temperature increased ~8°C in the presence of these organic solvents. PmEst is a mesophilic esterase with substrate preference towards short-to medium-length acyl chains. The SRCD data of PmEst is in agreement with the prediction of an α/β protein, which leads us to assume that it displays a typical fold of esterases from this family. The increased enzyme stability in organic solvents may enable novel applications for its use in synthetic biology. Taken together, our results demonstrate features of the PmEst enzyme that indicate it may be suitable for applications in industrial processes, particularly, when the use of polar organic solvents is required.

Gordonia polyisoprenivorans from groundwater contaminated with landfill leachate in a subtropical area: characterization of the isolate and exopolysaccharide production

Uma linahgem de Gordonia sp. (linhagem Lc), proveniente de aguas subterrâneas contaminadas por chorume, foi caracterizada por taxonomia polifasica e estudada quanto a producao de exopolissacarideos (EPS). As celulas, bastonetes agregados, isolados ou aos pares em formato de V, tipico de bacterias corineformes, apresentaram reacao Gram positiva, catalase positiva, oxidase negativa, nao apresentaram motilidade. O organismo cresceu em aerobiose e em ambiente anoxico na presenca de NaNO3. A linhagem apresentou caracteristicas morfologicas, bioquimicas e propriedades quimiotaxonomicas tipicas do genero Gordonia e perfil de acidos micolicos e acidos graxos correspondentes aos de G. polyisoprenivorans DSM44302T. A analise do sequenciamento dos primeiros 500 pares de bases do rDNA16S da linhagem mostrou 100% de similaridade com Gordonia polyisoprenivorans DSM44302T. Experimentos conduzidos em condicoes anaerobias em meio E com sacarose ou glicose como principal fonte de carbono, mostraram que a linhagem nao cresceu quando cultivada com sacarose. Entretanto, utilizando-se glicose, a velocidade especifica maxima de crescimento foi 0,17h-1, o tempo de geracao de aproximadamente 4 horas e o maximo de producao de EPS total ocorreu durante a fase exponencial (126,17 ± 15,63 g l-1). A producao de EPS livre excedeu a de capsular e a relacao EPS livre/EPS capsular aumentou de 1,9, durante a fase exponencial para 7,8 durante a fase estacionaria. Ate entao, foram isoladas seis linhagens de G. polyisoprenivorans de diferentes ambientes. A linhagem Lc e a sexta linhagem de G. polyisoprenivorans descrita, a segunda detectada nas aguas subterrâneas em questao e a primeira cuja producao de EPS esta sendo estudada.

Comparison of the presence-absence (P-A) test and conventional methods for detection of bacteriological water quality indicators

Abstract Fifty samples of water, comprising sewage-polluted river water and artificially-contaminated spring water, were analyzed in order to compare the P-A test and the conventional membrane filter and multiple tube methods for the detection of total and fecal coliforms, fecal streptococci, Pseudomonas aeruginosa, Staphylococcus aureus and sulfite reducer clostridia. The two presumptive media proposed for the P-A test, the MacConkey broth with tryptone (MacConkey-PA) and the lactose-lauryl tryptosetryptone broth (STM-PA), were also compared. The P-A test using STM-PA medium showed better results than the MacConkey-PA when water with lower levels of contamination (artificially-contaminated spring water) was analyzed. For coliform detection the P-A test with 48 h of incubation showed better results than with an incubation period of 5 days. For the detection of other indicators such as Ps. aeruginosa , sulfite reducer clostridia, and fecal streptococci in water with low levels of contamination the incubation period should be extended, as very different results after 48 and 120 h incubation were obtained with percentages of positivity being respectively, for Ps. aeruginosa , 48%, 76%; for sulfite reducer clostridia, 16%, 48%; and, for fecal streptococci 24%, 92%. Similar results were obtained for sewage polluted river water. Staphylococcus aureus was not detected by the P-A test. The P-A test using the STM-PA medium showed a good performance and is a promising tool for the evaluation of bacteriological quality of drinking water, especially in tropical climates where the coliform indicator may not be adequate. This test could overcome this problem, allowing the use of a multiple indicator approach.

Use of Starved Cells of a Klebsiella Pneumoniae Strain Isolated from a Brazilian Oil Reservoir in a Transport and Plugging Experiment in a Sandstone Core

Abstract: Microbial biomass can be used as a plugging agent in high-permeability zones in applications of microbial enhanced oil recover (MEOR) technology to oil-bearing strata. Laboratory tests have shown that bacterial cells, reduced in size by starvation, can penetrate more deeply when injected into a porous medium and also grow therein after nutrient is added. Transport and growth of Klebsiella pneumoniae starved cells through a Rio Bonito sandstone core (440 mD of permeability) were tested, to verify application to oil recovery. The injection of starved cells followed by nutrient medium at a flow rate of 1.8 mL/h, promoted cell growth in the core, which raised the inner pressure from 0.016 to 13.7 psi, resulting in a 99.9% reduction in the permeability of the core. The biobarrier formed persisted during the injection of 4 pore volumes of brine into the core at a flow rate of 1.8 mL/h, keeping the permeability reduced to 99.7%.

Using a board game to teach protein synthesis to high school students

Abstract The present work aimed to investigate the contribution of the board game ‘Synthesizing Proteins’ to the understanding of protein synthesis by high school students, based on the socio-interactionist theory of Vygotsky. Fifteen students (six from a public school and nine from a private school) participated in the research, which had three stages: diagnosis and pre-interview, game execution, and post-interview, with collection of written and audiovisual data. The data were organised and interpreted according to a qualitative content analysis, in which we evaluated the predominant concepts, the conceptual gains and type of interactions promoted by the game in the understanding of target themes. The results indicated that the game contributed to the improvement (or construction) of a protein synthesis model by the students, providing a symbolic representation of the process through interactions guided by the rules of the game (in the game, students played the roles of molecules, and simulated mechanisms and processes). These interactions, especially of cooperative and competitive nature, may promote a meaningful, prospective learning.