Search for: All records

Absorbance measurements on intact chemolithotrophic microorganisms that respire aerobically on soluble iron are described that used a novel integrating cavity absorption meter to eliminate the effects of light scattering on the experimental results. Steady state kinetic measurements on ferric iron production by intact cells revealed that the Michaelis Menten equation described the initial rates of product formation for at least 8 different chemolithotrophic microorganisms in 6 phyla distributed equally among the archaea and the Gram negative and Gram positive eubacteria. Cell-monitored turnover measurements during aerobic respiration on soluble iron by the same 12 intact microorganisms revealed six different patterns of iron-dependent absorbance changes, suggesting that there may be at least six different sets of prosthetic groups and biomolecules that can accomplish aerobic respiration on soluble iron. Detailed kinetic studies revealed that the 3-component iron respiratory chain of Acidithiobacillus ferrooxidans functioned as an ensemble with a single macroscopic rate constant when the iron-reduced proteins were oxidized in the presence of excess molecular oxygen. The principal member of this 3-component system was a cupredoxin called rusticyanin that was present in the periplasm of At. ferrooxidans at an approximate concentration of 350 mg/mL, an observation that provides new insights into the crowded environments in the periplasms of Gram negative eubacteria that conduct electrons across their periplasm. The ability to conduct direct spectrophotometric measurements under noninvasive physiological conditions represents a new and powerful approach to examine the rates and extents of biological events in situ without disrupting the complexity of the live cellular environment. 

Proteins that oxidize extracellular substrates in Gram-positive bacteria are poorly understood. Ferrimicrobium acidiphilum is an actinobacterium that respires aerobically on extracellular ferrous ions at pH 1.5. In situ absorbance measurements were conducted on turbid suspensions of intact Fm. acidiphilum using an integrating cavity absorption meter designed for that purpose. Initial velocity kinetic studies monitored the appearance of product ferric ions in the presence of catalytic quantities of cells. Cell-catalyzed iron oxidation obeyed the Michaelis-Menten equation with values for KM and Vmax of 71 µM and 0.29 fmol/min/cell, respectively. Limited-turnover kinetic studies were conducted with higher concentrations of cells to detect and monitor changes in the absorbance properties of cellular redox proteins when the cells were exposed to limited quantities of soluble reduced iron. A single a-type cytochrome with reduced absorbance peaks at 448 and 605 nm was the only redox-active chromophore that was visible as the cells respired aerobically on iron. The reduced cytochrome 605 exhibited mathematical and correlational properties that were consistent with the hypothesis that oxidation of the cytochrome constituted the rate-limiting step in the aerobic respiratory process with a turnover number of 35 ± 2 s-1. Genomic and proteomic analyses showed that Fm. acidiphilum could and did express only two a-type heme copper terminal oxidases. Cytochrome 605 was associated with the terminal oxidase gene that is located between nucleotides 31090 and 33039, inclusive, in the annotated circular genome of this bacterium. 

The contamination in groundwater due to the presence of uranium is nowadays a subject of concern due to the severe health problems associated with renal failure, genotoxicity and cancer. The standard methods to detect uranium require time-consuming processes and expensive non-portable equipment, so these measurements are rarely performed in-field, which increases the time until water samples are analysed. Furthermore, the few portable methods available do not allow quantitative analysis and the detection limit is often not low enough to reach the recommendations for drinking water (30 ppb or 126 nM of uranium). For the first time, we propose a portable, fast, inexpensive and sensitive paper-based biosensor able to detectin situU(VI) in water samples: U(VI) selective gold nanoparticle-based lateral flow strips. Antibody-coated gold nanoparticles are used as labels in the proposed lateral flow system because of their biocompatibility; in addition, these nanoparticles provide high sensitivity due to their intense plasmonic effect. The antibody used in the assay recognizes soluble U(VI) complexed to the chelator, 2,9-dicarboxyl-1,10-phenanthroline (DCP). Because of the small size of the U(VI)-DCP complex, this assay employs a competitive format that reaches a limit of detection of 36.38 nM, lower than the action level (126 nM) established by the World Health Organization and the U.S. Environmental Protection Agency for drinking waters.