The Enterococcus hirae copper chaperone CopZ delivers copper(I) to the CopY repressor.
Expression of the cop operon which effects copper homeostasis in Enterococcus hirae is controlled by the copper responsive repressor CopY. Purified Zn(II)CopY binds to a synthetic cop promoter fragment in vitro. Here we show that the 8 kDa protein CopZ acts as a copper chaperone by specifically delivering copper(I) to Zn(II)CopY and releasing CopY from the DNA. As shown by gel filtration and luminescence spectroscopy, two copper(I) are thereby quantitatively transferred from Cu(I)CopZ to Zn(II)CopY, with displacement of the zinc(II) and transfer of copper from a non-luminescent, exposed, binding site in CopZ to a luminescent, solvent shielded, binding site in CopY. (+info)
Study of the response of a biofilm bacterial community to UV radiation.
We have developed a bioluminescent whole-cell biosensor that can be incorporated into biofilm ecosystems. RM4440 is a Pseudomonas aeruginosa FRD1 derivative that carries a plasmid-based recA-luxCDABE fusion. We immobilized RM4440 in an alginate matrix to simulate a biofilm, and we studied its response to UV radiation damage. The biofilm showed a protective property by physical shielding against UV C, UV B, and UV A. Absorption of UV light by the alginate matrix translated into a higher survival rate than observed with planktonic cells at similar input fluences. UV A was shown to be effectively blocked by the biofilm matrix and to have no detectable effects on cells contained in the biofilm. However, in the presence of photosensitizers (i.e., psoralen), UV A was effective in inducing light production and cell death. RM4440 has proved to be a useful tool to study microbial communities in a noninvasive manner. (+info)
Factors influencing expression of luxCDABE and nah genes in Pseudomonas putida RB1353(NAH7, pUTK9) in dynamic systems.
Bioluminescent reporter organisms have been successfully exploited as analytical tools for in situ determination of bioavailable levels of contaminants in static environmental samples. Continued characterization and development of such reporter systems is needed to extend the application of these bioreporters to in situ monitoring of degradation in dynamic environmental systems. In this study, the naphthalene-degrading, lux bioreporter bacterium Pseudomonas putida RB1353 was used to evaluate the relative influences of cell growth stage, cell density, substrate concentration, oxygen tension, and background carbon substrates on both the magnitude of the light response and the rate of salicylate disappearance. The effect of these variables on the lag time required to obtain maximum luminescence and degradation was also monitored. Strong correlations were observed between the first three factors and both the magnitude and induction time of luminescence and degradation rate. The maximum luminescence response to nonspecific background carbon substrates (soil extract broth or Luria broth) was 50% lower than that generated in response to 1 mg of sodium salicylate liter(-1). Oxygen tension was evaluated over the range of 0.5 to 40 mg liter(-1), with parallel inhibition to luminescence and degradation rate (20 mg of sodium salicylate liter(-1)) observed at 1.5 mg liter(-1) and below and no effect observed above 5 mg liter(-1). Oxygen tensions from 2 to 4 mg liter(-1) influenced the magnitude of luminescence but not the salicylate degradation rate. The results suggest that factors causing parallel shifts in the magnitude of both luminescence and degradation rate were influencing regulation of the nah operon promoters. For factors that cause nonparallel shifts, other regulatory mechanisms are explored. This study demonstrates that lux reporter bacteria can be used to monitor both substrate concentration and metabolic response in dynamic systems. However, each lux reporter system and application will require characterization and calibration. (+info)
A nisin bioassay based on bioluminescence.
A Lactococcus lactis subsp. lactis strain that can sense the bacteriocin nisin and transduce the signal into bioluminescence was constructed. By using this strain, a bioassay based on bioluminescence was developed for quantification of nisin, for detection of nisin in milk, and for identification of nisin-producing strains. As little as 0.0125 ng of nisin per ml was detected within 3 h by this bioluminescence assay. This detection limit was lower than in previously described methods. (+info)
Nitrogen availability to Pseudomonas fluorescens DF57 is limited during decomposition of barley straw in bulk soil and in the barley rhizosphere.
The availability of nitrogen to Pseudomonas fluorescens DF57 during straw degradation in bulk soil and in barley rhizosphere was studied by introducing a bioluminescent reporter strain (DF57-N3), responding to nitrogen limitation, to model systems of varying complexity. DF57-N3 was apparently not nitrogen limited in the natural and sterilized bulk soil used for these experiments. The soil was subsequently amended with barley straw, representing a plant residue with a high carbon-to-nitrogen ratio (between 60 and 100). In these systems the DF57-N3 population gradually developed a nitrogen limitation response during the first week of straw decomposition, but exclusively in the presence of the indigenous microbial population. This probably reflects the restricted ability of DF57 to degrade plant polymers by hydrolytic enzymes. The impact of the indigenous population on nitrogen availability to DF57-N3 was mimicked by the cellulolytic organism Trichoderma harzianum Rifai strain T3 when coinoculated with DF57-N3 in sterilized, straw-amended soil. Limitation occurred concomitantly with fungal cellulase production, pointing to the significance of hydrolytic activity for the mobilization of straw carbon sources, thereby increasing the nitrogen demand. Enhanced survival of DF57-N3 in natural soil after straw amendment further indicated that DF57 was cross-fed with carbon/energy sources. The natural barley rhizosphere was experienced by DF57-N3 as an environment with restricted nitrogen availability regardless of straw amendment. In the rhizosphere of plants grown in sterilized soil, nitrogen limitation was less severe, pointing to competition with indigenous microorganisms as an important determinant of the nitrogen status for DF57-N3 in this environment. Hence, these studies have demonstrated that nitrogen availability and gene expression in Pseudomonas is intimately linked to the structure and function of the microbial community. Further, it was demonstrated that the activities of cellulolytic microorganisms may affect the availability of energy and specific nutrients to a group of organisms deficient in hydrolytic enzyme activities. (+info)
Increased cortical oxidative metabolism due to sensory stimulation: implications for functional brain imaging.
Modern functional brain mapping relies on interactions of neuronal electrical activity with the cortical microcirculation. The existence of a highly localized, stimulus-evoked initial deoxygenation has remained a controversy. Here, the activity-dependent oxygen tension changes in the microcirculation were measured directly, using oxygen-dependent phosphorescence quenching of an exogenous indicator. The first event after sensory stimulation was an increase in oxygen consumption, followed by an increase in blood flow. Because oxygen consumption and neuronal activity are colocalized but the delayed blood flow is not, functional magnetic resonance imaging focused on this initial phase will yield much higher spatial resolution, ultimately enabling the noninvasive visualization of fundamental processing modules in the human brain. (+info)
Involvement of CDSP 32, a drought-induced thioredoxin, in the response to oxidative stress in potato plants.
In animal cells, yeast and bacteria, thioredoxins are known to participate in the response to oxidative stress. We recently identified a novel type of plant thioredoxin named CDSP 32 for chloroplastic drought-induced stress protein of 32 kDa. In the present work, we measured comparable increases in the glutathione oxidation ratio and in the level of chlorophyll thermoluminescence, a specific marker for thylakoid lipid peroxidation in Solanum tuberosum plants subjected to drought or oxidative treatments (photooxidative stress, gamma irradiation and methyl viologen spraying). Further, substantial accumulations of CDSP 32 mRNA and protein were revealed upon oxidative treatments. These data show for the first time in plants the induction of a thioredoxin by oxidative stress. We conclude that CDSP 32 may preserve chloroplastic structures against oxidative injury upon drought. (+info)
Design and application of a biosensor for monitoring toxicity of compounds to eukaryotes.
Here we describe an alternative approach to currently used cytotoxicity analyses through applying eukaryotic microbial biosensors. The yeast Saccharomyces cerevisiae was genetically modified to express firefly luciferase, generating a bioluminescent yeast strain. The presence of any toxic chemical that interfered with the cells' metabolism resulted in a quantitative decrease in bioluminescence. In this study, it was demonstrated that the luminescent yeast strain senses chemicals known to be toxic to eukaryotes in samples assessed as nontoxic by prokaryotic biosensors. As the cell wall and adaptive mechanisms of S. cerevisiae cells enhance stability and protect from extremes of pH, solvent exposure, and osmotic shock, these inherent properties were exploited to generate a biosensor that should detect a wide range of both organic and inorganic toxins under extreme conditions. (+info)