Emerging role of ferrous iron in bacterial growth and host–pathogen interaction: New tools for chemical (micro)biology and antibacterial therapy

Chemical tools capable of detecting ferrous iron with oxidation-state specificity have only recently become available. Coincident with this development in chemical biology has been increased study and appreciation for the importance of ferrous iron during infection and more generally in host–pathogen interaction. Some of the recent findings are surprising and challenge long-standing assumptions about bacterial iron homeostasis and the innate immune response to infection. Here, we review these recent developments and their implications for antibacterial therapy.     

EPR Monitored Redox Titration of the Cofactors of Saccharomyces cerevisiae Nar1

Electron Paramagnetic Resonance (EPR) monitored redox titrations are a powerful method to determine the midpoint potential of cofactors in proteins and to identify and quantify the cofactors in their detectable redox state.The technique is complementary to direct electrochemistry (voltammetry) approaches, as it does not offer information on electron transfer rates, but does establish the identity and redox state of the cofactors in the protein under study. The technique is widely applicable to any protein containing an electron paramagnetic resonance (EPR) detectable cofactor.A typical titration requires 2 ml protein with a cofactor concentration in the range of 1-100 µM. The protein is titrated with a chemical reductant (sodium dithionite) or oxidant (potassium ferricyanide) in order to poise the sample at a certain potential. A platinum wire and a Ag/AgCl reference electrode are connected to a voltmeter to measure the potential of the protein solution. A set of 13 different redox mediators is used to equilibrate between the redox cofactors of the protein and the electrodes. Samples are drawn at different potentials and the Electron Paramagnetic Resonance spectra, characteristic for the different redox cofactors in the protein, are measured. The plot of the signal intensity versus the sample potential is analyzed using the Nernst equation in order to determine the midpoint potential of the cofactor.     

Computational Oral Absorption Simulation for Low‐Solubility Compounds

Bile micelles play an important role in oral absorption of low‐solubility compounds. Bile micelles can affect solubility, dissolution rate, and permeability. For the pH–solubility profile in bile micelles, the Henderson–Hasselbalch equation should be modified to take bile‐micelle partition into account. For the dissolution rate, in the Nernst–Brunner equation, the effective diffusion coefficient in bile‐micelle media should be used instead of the monomer diffusion coefficient. The diffusion coefficient of bile micelles is 8‐ to 18‐fold smaller than that of monomer molecules. For permeability, the effective diffusion coefficient in the unstirred water layer adjacent to the epithelial membrane, and the free fraction at the epithelial membrane surface should be taken into account. The importance of these aspects is demonstrated here using several in vivo and clinical oral‐absorption data of low‐solubility model compounds. Using the theoretical equations, the food effect on oral absorption is further discussed.     

Evaluation of past and potential phosphorus uptake at the Orlando Easterly Wetland

The Orlando Easterly Wetland (OEW), located near Christmas, Florida, USA, is among the longer-lived treatment wetlands in the United States. It was established in the late 1980s to reduce nitrogen and phosphorus concentrations from tertiary treated wastewater bound for the St. Johns River. A goal of 0.07 mg/l total phosphorus concentration has been set by the regulating agency (St. Johns River Water Management District). In order to understand and define the operating conditions for which this target could be met, a systematic study of historic phosphorus uptake was performed using a traditional first-order model. Phosphorus uptake performance is shown to correlate well with hydraulic performance for two parallel upstream cells. The first-order model is enhanced with predictive capabilities that acknowledge the correlation between the phosphorus uptake rate constant and the hydraulic loading rate observed in the system. Inherent limitations with the first-order modeling approach are addressed and uncertainty in model performance is used to bound predictions.     

Functional trade-off of hydration strategies in old forest epiphytic cephalolichens

Although water is essential for photosynthetic activation in lichens, rates of vapor uptake and activation in humid air, which likely influence their niche preferences and distribution ranges, are insufficiently known. This study simultaneously quantifies rehydration kinetics and PSII reactivation in sympatric, yet morphologically and functionally distinct cephalolichens (Lobaria amplissima, Lobaria pulmonaria, Lobaria virens). High-temporal resolution monitoring of rehydrating thalli by automatic weighing combined with chlorophyll fluorescence imaging of maximal PSII efficiency (F_V/F_M) was applied to determine species-specific rates of vapor uptake and photosynthetic activation. The thin and loosely attached growth form of L. pulmonaria rehydrates and reactivates faster in humid air than the thick L. amplissima, with L. virens in between. This flexible hydration strategy is consistent with L. pulmonaria’s wide geographical distribution stretching from rainforests to continental forests. By contrast, the thick and resupinate L. amplissima reactivates slowly in humid air but stores much water when provided in abundance. This prolongs active periods after rain, which could represent an advantage where abundant rain and stem flow alternates with long-lasting drying. Understanding links between morphological traits and functional responses, and their ecological implications for species at risk, is crucial to conservation planning and for modelling populations under various climate scenarios.     

Antagonistic and synergistic interactions between aluminum and manganese on growth of Vigna unguiculata at low ionic strength

Concentrations of soluble aluminum (Al) and manganese (Mn) frequently reach phytotoxic levels in acid soils. While dose response relationships for these metals are well documented, the effects of combined exposure have received less attention. We have examined the effect of combinations of Al and Mn on growth and metal accumulation in Vigna unguiculata (L.) Walp. grown in solution culture under conditions of low ionic strength (conductivities typically < 100 µS cm⁻¹). The nature of interaction between these metals varied with the specific physiological response, the part of the plant investigated, and the relative amount of stress imposed. Analysis of growth data provided evidence for amelioration of metal toxicity (antagonistic effects), although this effect was dose dependent. Analysis of metal content data provided evidence for antagonistic and synergistic (exacerbation of toxicity) effects, again depending on dose. Analysis of foliar symptoms also provided evidence for antagonisms and synergisms, with the nature of the response dependent on the specific physiological response and specific plant part investigated. In contrast with previous reports, evidence for antagonistic, synergistic, and multiplicative effects on growth, metal uptake, and expression of foliar symptoms have been obtained under physiologically and environmentally relevant conditions. These results suggest a more detailed analysis of the potential for interactions between metals in the environment is required.     

Evaluation of early generations for iron chlorosis in relation to productivity in groundnut (Arachis hypogaea L.)

Five popular but iron-inefficient cultivars were crossed with three efficient genotypes and both parents and F₁s were evaluated for iron-efficiency in potted calcareous and noncalcareous soil. The iron-efficient genotypes were dark green or green in both noncalcareous and calcareous soils whereas inefficient types were light green to yellow in calcareous soil. The chlorophyll and active iron (Fe²⁺) concentration of leaves was less in iron-efficient genotypes compared to efficient types in calcareous soil and reduction of both the parameters from noncalcareous to calcareous soil was considerably high in iron-inefficient lines. There was significant correlation between visual scores, chlorophyll and active iron content. There were no differences among F₁s for iron chlorosis and they were all iron-inefficient. The frequency of iron-inefficient plants was higher than the efficient plants in all F₂ populations. But most of the productive plants came from iron-efficient segregants indicating strong association between iron-efficiency and productivity. Based on the results selection for iron-efficiency in early generations and extensive evaluation for productivity in advanced generations is suggested for developing varieties for cultivation in calcareous soils.     

TonB protein and energy transduction between membranes

TonB protein couples cytoplasmic membrane electrochemical potential to active transport of iron-siderophore complexes and vitamin B12 through high-affinity outer membrane receptors of Gram-negative bacteria. The mechanism of energy transduction remains to be determined, but important concepts have already begun to emerge. Consistent with its function, TonB is anchored in the cytoplasmic membrane by its uncleaved amino terminus while largely occupying the periplasm. Both the connection to the cytoplasmic membrane and the amino acid sequences of the anchor are essential for activity. TonB directly associates with a number of envelope proteins, among them the outer membrane receptors and cytoplasmic membrane protein ExbB. ExbB and TonB interact through their respective transmembrane domains. ExbB is proposed to recycle TonB to an active conformation following energy transduction to the outer membrane. TonB most likely associates with the outer membrane receptors through its carboxy terminus, which is required for function. In contrast, the novel prolinerich region of TonB can be deleted without affecting function. A model that incorporates this information, as well as tempered speculation, is presented.     

JAR human placental choriocarcinoma cells actively synthesize,take up and release polyamines

Uptake of polyamines has been investigated extensively in many cells, but not in placenta, where the polyamine– polyamine oxidase system is supposed to have an immunoregulatory function in pregnancy. Due to the importance of the transfer in this tissue, we have started this study. JAR human placental choriocarcinoma cells in monolayer at confluency were used as a model for measuring the key enzymes of polyamine synthesis and interconversion, rate of uptake and efflux, and the polyamine content. Polyamines were taken up by JAR cells and released by an independent mechanism. Ornithine decarboxylase and spermidine acetyltransferase activities and the rate of transport in and out of the cell were much higher than in other cells, such as L1210 cells. However the systems used for uptake and release appear in many respects to be similar to those observed in L1210 cells, but different from others. The uptake appears to be regulated by an inhibitory protein. Moreover, protein kinase C appears to be involved in the process. The efflux also is regulated as in L1210 cells, through control of H⁺ and Ca²⁺ concentration. In conclusion, this study shows that, in JAR cells, ornithine decarboxylase and spermidine acetyltransferase activities were much higher than in other cells, and so was the rate of transport in and out of the cells. As a result, a much higher polyamine content was observed.     

Influence of Nutrient Concentrations on MPN Quantification and Enrichment of Nitrate-Reducing Fe(II)-Oxidizing and Fe(III)-Reducing Bacteria from Littoral Freshwater Lake Sediments

The application of culture-dependent studies to quantify Fe-metabolizing microorganisms from the environment is a necessity, as there are so far no universal functional marker genes for application in culture-independent studies. Media composition can vary between studies, therefore, we determined the effects of three different growth media on the quantification (MPNs) and identity (via cloning and sequencing of dominant DGGE bands) of nitrate-reducing Fe(II)-oxidizers and lactate- or acetate-oxidizing Fe(III)-reducers from a lacustrine sediment: low sulphate freshwater medium (FWM), sterile filtered bicarbonate-buffered lake water (BLW) and a mixture of both (MIX). We consistently found fewer cells in the BLW than in the FWM and the MIX. The DGGE banding patterns of the microbial communities enriched in different media types clustered together according to the e^? donor and acceptor couples and not according to the medium used. Thus, although the medium composition significantly influenced the quantification and thereby conclusions on the abundance and potential significance of the targeted group within the ecosystem, biodiversity assessments through enrichment cultures were less influenced by the medium, but instead were affected by the type and concentration of the e^? donor/acceptor.