RESUMO
MerR metalloregulators are the central components of many biosensor platforms designed to report metal contamination. However, most MerR proteins are non-specific. This makes it difficult to apply these biosensors in the analysis of real environmental samples. On-demand implementation of molecular engineering to modify the MerR metal preferences is innovative, although it does not always yield the expected results. As the metal binding loop region (MBL) of these sensors has been proposed to be the major modulator of their specificity, we surgically switched this region for that of well-characterized specific and non-specific homologues. We found that identical modifications in different MerR proteins result in synthetic sensors displaying particular metal-detection patterns that cannot be predicted from the nature of the assembled modules. For instance, the MBL from a native Hg(II) sensor provided non-specificity or specificity toward Hg(II) or Cd(II) depending on the MerR scaffold into which it was integrated. These and other evidences reveal that residues outside the MBL are required to modulate ion recognition and transduce the input signal to the target promoter. Revealing their identity and their interactions with other residues is a critical step toward the design of more efficient biosensor devices for environmental metal monitoring.
Assuntos
Proteínas de Ligação a DNA , Mercúrio , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Proteínas de Bactérias/metabolismo , Metais/metabolismo , Mercúrio/metabolismo , Regiões Promotoras GenéticasRESUMO
Salmonella enterica sv. Typhimurium modulates the expression of factors essential for virulence, contributing to its survival against the surge of copper (Cu) in the Salmonella-containing vacuole. This bactericidal host innate immune component primarily targets the bacterial envelope, where most cuproproteins are localized. While in most enteric species periplasmic Cu homeostasis is maintained by the CusR/CusS-controlled CusCFBA efflux system encoded in the cus locus, we noticed that these genes were lost from the Salmonella-core genome. At the same time, Salmonella acquired cueP, coding for a periplasmic Cu chaperone. As cus, cueP was shown to be essential for bacterial survival in a copper-rich environment under anaerobiosis, suggesting that it can functionally substitute the CusCFBA system. In the present study, the whole Escherichia coli cus locus was reintroduced to the chromosome of the Salmonella wild-type or the ΔcueP strain. While the integrated cus locus did not affect Cu resistance under aerobic conditions, it increases Cu tolerance under anaerobiosis, irrespective of the presence or absence of cueP. In contrast to the Cus system, CueP expression is higher at high copper concentrations and persisted over time, suggesting separate functions. Finally, we observed that, regardless of the presence or absence of cus, a mutant deleted of cueP shows a deficiency in replication inside macrophages compared to the wild-type strain. Our results demonstrate that CueP and CusCFBA exert redundant functions for metal resistance, but not for intracellular survival, and therefore for the virulence of this pathogen.
RESUMO
A highly sensitive and specific Hg-whole-cell biosensor was developed from a non-selective variant of the Au sensor GolS and its regulatory pathway. The performance of this analytical tool was validated under laboratory and field-like conditions. This biosensor can be easily applied in cost-effective and portable semiquantitative devices to report Hg contamination in water.
Assuntos
Técnicas Biossensoriais/métodos , Ouro/química , Mercúrio/análise , Salmonella/metabolismo , Poluentes Químicos da Água/análise , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Água Doce/análise , Proteínas de Fluorescência Verde/química , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Ligantes , Limite de Detecção , Espectrometria de FluorescênciaRESUMO
RESUMEN Las actividades de producción piscícola en el Lago de Tota, Boyacá aumentan las concentraciones de amonio y de nitrato, nutrientes que están acelerando los procesos de eutrofización. Entre las comunidades microbianas que regulan la entrada de nitrógeno alóctono figuran las bacterias nitrificantes y la determinación de su abundancia en los ecosistemas es una medida de bioindicación de contaminación. El objetivo del estudio fue cuantificar y caracterizar bioquímicamente las bacterias nitrificantes cultivables de la zona limnética del lago. Para medir la abundancia, se usó la técnica de número más probable (NMP). Las muestras fueron tomadas en los sectores Lago Chico y Lago Grande, en zonas limnéticas, con actividad piscícola y sin intervención. Se midieron variables físicas y químicas in situ y se determinó la concentración de nutrientes en el laboratorio. Las mayores cuantificaciones de bacterias nitrificantes, se registraron en el periodo de baja precipitación y en el área de influencia de piscicultura. El grupo de bacterias oxidantes de amonio (BOA) y oxidantes de nitritos (BON) fue menor en la zona central de lago Grande respecto a zonas de piscifactorías de Hato Laguna y El Túnel. Se obtuvieron 14 aislamientos de bacterias nitrificantes, identificando los géneros Nitrosomonas spp. y Nitrobacter spp. Los cambios en la abundancia de BOA, se relacionaron con la alcalinidad, mientras que los valores de BON, se correlacionaron con la conductividad eléctrica y el nitrógeno inorgánico disuelto. Una mayor abundancia de bacterias nitrificantes, se registró en las zonas limnéticas del lago, afectadas por la actividad piscícola, asociado al aumento de nutrientes disuelto en la columna de agua.
ABSTRACT Fish production activities in Lake Tota, Boyacá are increasing concentrations of ammonium and nitrate, nutrients that are accelerating eutrophication processes. Some of the microbial communities that regulate the entry of allochthonous nitrogen are nitrifying bacteria, and the determination of their abundance in ecosystems is a measure of pollution bioindication. The objective of the study was to quantify and biochemically characterize the cultivable nitrifying bacteria of the limnetic zone of Lake Tota. The most probable number technique (MPN) was used to measure abundance. The samples were taken in the Lago Chico and Lago Grande sectors, in limnetic areas with fish activity and without intervention. Physical and chemical variables were measured in situ and the concentration of nutrients in the laboratory was determined. The highest quantifications of nitrifying bacteria were recorded in the area of influence of fish farming. The group of ammonium oxidizing bacteria (BOA) and nitrite oxidants (BON) was smaller in the central area of Lago Grande compared to fish farms in Hato Laguna and El Túnel. 14 isolates of nitrifying bacteria were obtained, genera Nitrosomonas spp. and Nitrobacter spp. Changes in BOA abundance were related to alkalinity, while BON values correlated with electrical conductivity and dissolved inorganic nitrogen. A greater abundance of nitrifying bacteria was recorded in the lake's limnetic areas affected by fish farming activity, associated with the increase in nutrients dissolved in the water column.
RESUMO
Several regulatory systems contribute to bacterial resistance to heavy metals controlling the expression of factors required to eliminate the intoxicant and/or to repair the damage caused by it. In Salmonella, the response to Au ions is mediated by the specific metalloregulator GolS that, among other genes, controls the expression of the RND-efflux pump GesABC. In this work, we demonstrate that CpxR/CpxA, a main cell-envelope stress-responding system, promotes gesABC transcription in the presence of Au ions at neutral pH. Deletion of either cpxA or cpxR, or mutation of the CpxR-binding site identified upstream of the GolS-operator in the gesABC promoter region reduces but does not abrogate the GolS- and Au-dependent activation of gesABC. Au also triggers the activation of the CpxR/CpxA system and deletion of the cpxRA operon severely reduces survival in the presence of the toxic metal. Our results indicate that the coordinated action of GolS and CpxR/CpxA contribute to protecting the cell from severe Au damage.
Assuntos
Transportadores de Cassetes de Ligação de ATP/genética , Antibacterianos/farmacologia , Farmacorresistência Bacteriana , Regulação Bacteriana da Expressão Gênica , Ouro/farmacologia , Salmonella enterica/efeitos dos fármacos , Proteínas de Bactérias/metabolismo , Sítios de Ligação , Óperon , Proteínas Quinases/metabolismo , Salmonella enterica/genéticaRESUMO
En este trabajo se reportan las concentraciones de aluminio en sangre completa y en solución de diálisis de 27 pacientes con insuficiencia renal crónica y sometidos a hemodiálisis en los Hospitales Miguel Pérez Carreño y Universitario de la ciudad de Caracas. Asímismo, se monitoriaron los niveles de aluminio del agua empleada en la preparación de los dializados y se estudió la movilización de este metal durante el tratamiento dialítico. Las concentraciones medias de aluminio en sangre (ca. 34g AL/L) fueron inferiores a las concentradas en Maracaibo (ca. 58g AL/L), situación que se asoció a los bajos contenidos metálicos presentes en el agua utilizada en las soluciones de hemodiálisis (ca.14g AL/L). Por otra parte, el agua de consumo humano de Caracas reveló concentraciones muy elevadas de aluminio, superiores a 475g/L. los niveles sanguíneos de aluminio estuvieron en relación directa con el contenido de este metal en la solución de diálisis y con la ingestión de antiácidos a base de hidróxido de aluminio. En ambas Unidades de Diálisis se observaron procesos de transferencia-incorporación del aluminio hacia la sangre de los pacientes. Esta situación fué promovida por los gradientes favorables de concentración (concentración de aluminio de sangre/concentracion de aluminio en el dializado) establecidos al inicio de las hemodiálisis