Environmental impact assessment due to the intake of uranium contained in surface waters in a semi-arid region in Brazil.

In Santa Quitéria City, part of the population uses surface water for potation. These waters do not undergo any treatment before consumption. As the region has a deposit of uranium, assessing water quality becomes important. In the present study, the uranium activity concentration (AC) in becquerels per liter was determined in water samples from six points. Univariate statistics showed differences between the soluble and the particulate fraction (soluble AC > particulate AC). The particulate fraction showed no variation in AC among the six points. On the other hand, the soluble fraction and the total fraction presented different ACs between them. The multivariate statistics allowed to separate the soluble from the particulate fraction of the points. The same tools applied to the total fraction made it possible to differentiate the sampling points, grouping them ((#1, #2); (#3, #4), and (#5, #6)). The maximum mean value of AC found was 0.177 Bq∙L-1, corresponding to 25% of the chemical toxicity limit (0.72 Bq∙L-1). The maximum mean dose rate, 2.25 µSv∙year-1, is lower than the considered negligible dose rate (> 10 µSv∙year-1). The excess lifetime cancer risk was 10-6, two orders of magnitude smaller than the threshold considered for taking action. The assessment parameters used in this work indicate that the risk due to the uranium intake by the local population is negligible.

Heterologous expression of proteorhodopsin enhances H2 production in Escherichia coli when endogenous Hyd-4 is overexpressed.

Proteorhodopsin (PR) is a light harvesting protein widely distributed among bacterioplankton that plays an integral energetic role in a new pathway of marine light capture. The conversion of light into chemical energy in non-chlorophyll-based bacterial systems could contribute to overcoming thermodynamic and metabolic constraints in biofuels production. In an attempt to improve biohydrogen production yields, H2 evolution catalyzed by endogenous hydrogenases, Hyd-3 and/or Hyd-4, was measured when recombinant proteorhodopsin (PR) was concomitantly expressed in Escherichia coli cells. Higher amounts of H2 were obtained with recombinant cells in a light and chromophore dependent manner. This effect was only observed when HyfR, the specific transcriptional activator of the hyf operon encoding Hyd-4 was overexpressed in E. coli, suggesting that an excess of protons generated by PR activity could increase hydrogen production by Hyd-4 but not by Hyd-3. Although many of the subunits of Hyd-3 and Hyd-4 are very similar, Hyd-4 possesses three additional proton-translocating NADH-ubiquinone oxidoreductase subunits, suggesting that it is dependent upon ΔµH(+). Altogether, these results suggest that protons generated by proteorhodopsin in the periplasm can only enhance hydrogen production by hydrogenases with associated proton translocating subunits.

Synthetic phytochelatin surface display in Cupriavidus metallidurans CH34 for enhanced metals bioremediation.

This work describes the effects of the cell surface display of a synthetic phytochelatin in the highly metal tolerant bacterium Cupriavidus metallidurans CH34. The EC20sp synthetic phytochelatin gene was fused between the coding sequences of the signal peptide (SS) and of the autotransporter ß-domain of the Neisseria gonorrhoeae IgA protease precursor (IgAß), which successfully targeted the hybrid protein toward the C. metallidurans outer membrane. The expression of the SS-EC20sp-IgAß gene fusion was driven by a modified version of the Bacillus subtilis mrgA promoter showing high level basal gene expression that is further enhanced by metal presence in C. metallidurans. The recombinant strain showed increased ability to immobilize Pb(2+), Zn(2+), Cu(2+), Cd(2+), Mn(2+), and Ni(2+) ions from the external medium when compared to the control strain. To ensure plasmid stability and biological containment, the MOB region of the plasmid was replaced by the E. coli hok/sok coding sequence.

A metal-repressed promoter from gram-positive Bacillus subtilis is highly active and metal-induced in gram-negative Cupriavidus metallidurans.

A synthetic version of the metal-regulated gene A (mrgA) promoter from Bacillus subtilis, which in this Gram-positive bacterium is negatively regulated by manganese, iron, cobalt, or copper turned out to promote high level of basal gene expression that is further enhanced by Co(II), Cd(II), Mn(II), Zn(II), Cu(II), or Ni(II), when cloned in the Gram-negative bacterium Cupriavidus metallidurans. Promoter activity was monitored by expression of the reporter gene coding for the enhanced green fluorescent protein (EGFP), and cellular intensity fluorescence was quantified by flow cytometry. Expression levels in C. metallidurans driven by the heterologous promoter, here called pan, ranged from 20- to 53-fold the expression level driven by the Escherichia coli lac promoter (which is constitutively expressed in C. metallidurans), whether in the absence or presence of metal ions, respectively. The pan promoter did also function in E. coli in a constitutive pattern, regardless of the presence of Mn(II) or Fe(II). In conclusion, the pan promoter proved to be a powerful tool to express heterologous proteins in Gram-negative bacteria, especially in C. metallidurans grown upon high levels of toxic metals, with potential applications in bioremediation.

Biotecnologia e desenvolvimento sustentável / Biotechnology and sustainable development

A biotecnologia pode desempenhar um papel importante para atingir as metas da sustentabilidade. No presente trabalho, são descritos diferentes exemplos bem-sucedidos de micro-organismos especialmente desenhados para otimizar a produção de etanol, a produção de plásticos biodegradáveis a partir de recursos renováveis e a biorremediação de metais tóxicos. Esses processos biotecnológicos contribuem significantemente para promover o desenvolvimento sustentável, embora possam, por enquanto, não ser ainda competitivos em relação às tecnologias convencionais.

Biotechnology can play an important role to reach the goals of sustainability. In the present work, we describe successful examples of microorganisms especially designed for optimizing ethanol production, biodegradable plastics production from renewable resources, and toxic metals bioremediation. These biotechnological processes significantly contribute to promote sustainable development, although they may, at present, not be competitive with the conventional technologies.

A conditional suicide system for Saccharomyces cerevisiae relying on the intracellular production of the Serratia marcescens nuclease.

A conditional lethal system for biological containment of genetically modified strains of Saccharomyces cerevisiae is described. This suicide system is based on the intracellular production of the Serratia marcescens nuclease in the yeast cell, aiming at the destruction of the host genetic material. The S. marcescens nuclease, encoded by the nucA gene, is normally secreted by the bacterium into the medium. In the present work, the nucA gene, devoid of its signal peptide coding sequence, was cloned in a yeast expression vector, under control of the glucose-repressed S. cerevisiae alcohol dehydrogenase 2 gene (ADH2) promoter. When transformed into S. cerevisiae, the recombinant plasmid proved to be effective in killing the host cells upon glucose depletion from the medium, and the nuclease activity was found in lysates prepared from the transformants. In addition, the nuclease degrading effect was shown to reach chromosomal DNA in the yeast host. The killing effect of the nucA plasmid was also demonstrated in soil microcosm assays, indicating that whenever the GMM escapes into the environment where glucose is scarce, the nucA gene will be expressed and the resulting nuclease will destroy the genetic material and kill the cells. In contrast to other suicide systems that target the cell envelope, the advantage of the one described here is that it disfavours horizontal gene transfer from recombinant yeast cells to other microorganisms found in the environment.

Vacinas contra a hanseniase / Anti-leprosy vaccines

A unica vacina contra a hanseniase ja aprovada para uso humano e o uso de Mycobacterium bovis BCG, utilizada primariamente contra a tuberculose. Entretanto, a eficacia desta vacina contra a hanseniase e bastante variavel na literatura cientifica. Desta forma, a pesquisa de outras vacinas com resultados mais reprodutiveis permanece. Neste artigo, revemos as vacinas ja desenvolvidas contra a hanseniase e os possiveis novos desenvolvimentos que podem aparecer. Discutimos tambem a utilização de outras micobacterias, M. leprae inativado, antigenos recombinantes, DNA e mecanismos de modificaçao da apresentaçao antigenica.

Os diferentes testes cutaneos existentes para acompanhamento de pacientes com hanseniase / Different cutaneous tests for follow-up of leprosy patients

Testes cutaneos sao utilizados para o acompanhamento de casos de hanseniase, pois constituem um dos parametros para a classificaçao de forma clinica da doença, prognostico e mesmo para indicaçao e acompanhamento do tratamento. Estes testes foram desenvolvidos no inicio do seculo e utilizam suspensoes de bacilos extraidos de lepromas humanos ou de tatus infectados. Neste artigo, revemos as caracteristicas do teste da lepromina ou de Mitsuda e dos testes alternativos que foram desenvolvidos. Discutimos tambem o potencial da utilizaçao de antigenos recombinantes como uma possibilidade futura para obtençao de testes cutaneos mais especificos e mais padronizaveis em relaçao aqueles obitidos a partir de seres humanos ou de tatus.

Expression of heretologous amylases in the yeast Saccharomyces cerevisiae

The major microbial alcoholic fermenter, the yeast Saccharomyces cerevisiae, is unable to utilize starch for ethanol production because it lacks starch degrading enzymes. Yeast genetic engineering makes it possible to obtain strains expressing amylases from a variety of different organisms. Here we present a review of the main results obtained in our group pursuing a stable recombinant S. cerevisiae strain possessing all of the enzymatic activities needed for the production of ethanol from starchy raw materials.