Siderophores are biosynthetically produced and secreted by many bacteria, yeasts, fungi and plants, to scavenge for ferric iron (Fe(3+)). They are selective iron-chelators that have an extremely high affinity for binding this trivalent metal ion. The ferric ion is poorly soluble but it is the form of iron that is predominantly found in oxygenated environments. Siderophore uptake in bacteria has been extensively studied and over the last decade, detailed structural information for many of the proteins that are involved in their transport has become available. Specifically, numerous crystal structures for outer membrane siderophore transporters, as well as for soluble periplasmic siderophore-binding proteins, have been reported. Moreover, unique siderophore-binding proteins have recently been serendipitously discovered in humans, and the structures of some of their siderophore-complexes have been characterized. The binding pockets for different ferric-siderophores in these proteins have been described in great molecular detail. In addition to highlighting this structural information, in this review paper we will also briefly discuss the relevant chemical properties of iron, and provide a perspective on our current understanding of the human and bacterial iron uptake pathways. Potential clinical uses of siderophores will also be discussed. The emerging overall picture is that iron metabolism plays an extremely important role during bacterial infections. Because levels of free ferric iron in biological systems are always extremely low, there is serious competition for iron and for ferric-siderophores between pathogenic bacteria and the human or animal host.
Microbial cells in bioprocesses are usually described with averaged parameters. But in fact, single cells within populations vary greatly in characteristics such as stress resistance, especially in response to carbon source gradients. Our aim was to introduce tools to quantify population heterogeneity in bioprocesses using a combination of reporter strains, flow cytometry, and easily comprehensible parameters. We calculated mean, mode, peak width, and coefficient of variance to describe distribution characteristics and temporal shifts in fluorescence intensity. The skewness and the slope of cumulative distribution function plots illustrated differences in distribution shape. These parameters are person-independent and precise. We demonstrated this by quantifying growth-related population heterogeneity of Saccharomyces cerevisiae and Escherichia coli reporter strains in steady-state of aerobic glucose-limited chemostat cultures at different dilution rates and in response to glucose pulses. Generally, slow-growing cells showed stronger responses to glucose excess than fast-growing cells. Cell robustness, measured as membrane integrity after exposure to freeze-thaw treatment, of fast-growing cells was strongly affected in subpopulations of low membrane robustness. Glucose pulses protected subpopulations of fast-growing but not slower-growing yeast cells against membrane damage. Our parameters could successfully describe population heterogeneity, thereby revealing physiological characteristics that might have been overlooked during traditional averaged analysis.
The two main metabolic pathways involved in sugar metabolism, i.e., the pentose phosphate pathway (PPP) and the glycolytic pathway (GP), were amperometrically monitored using a double-mediator system composed of menadione and ferricyanide. With the use of the Saccharomyces cerevisiae deletion mutant, EBY44, lacking the gene encoding for the branch point enzyme phosphoglucose isomerize, selective amperometric monitoring of the PPP, mainly producing NADPH, and the GP, mainly producing NADH, could be achieved. It was found that the bioelectrocatalytic current was primarily originating from NADPH. This conclusion was supported by metabolite flux analysis, confirming that, in the presence of menadione, the cells increase the rate of NADPH-producing reactions although these processes might be detrimental to cell survival. The higher rate of in vivo NADPH-dependent menadione reduction can be ascribed to the fact that the intracellular NADPH/NADP(+) ratio is much higher than NADH/NAD(+) as well as that the former ratio is more tightly controlled. This tight control over the cofactor ratios is lost upon cell disintegration as observed from spectrophotometric assays using crude cell extract, and amperometric investigations of permeabilized cells indicate a higher rate of NADH- than NADPH-dependent menadione reduction. These in vitro experiments show a higher activity of NADH-dependent than NADPH-dependent menadione-reducing dehydrogenases in S. cerevisiae cells.
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