Evidence for Escherichia coli DcuD carrier dependent FOF1-ATPase activity during fermentation of glycerol

During fermentation Escherichia coli excrete succinate mainly via Dcu family carriers. Current work reveals the total and N,N’-dicyclohexylcarbodiimide (DCCD) inhibited ATPase activity at pH 7.5 and 5.5 in E. coli wild type and dcu mutants upon glycerol fermentation. The overall ATPase activity was highest at pH 7.5 in dcuABCD mutant. In wild type cells 50% of the activity came from the FOF1-ATPase but in dcuD mutant it reached ~80%. K+ (100 mM) stimulate total but not DCCD inhibited ATPase activity 40% and 20% in wild type and dcuD mutant, respectively. 90% of overall ATPase activity was inhibited by DCCD at pH 5.5 only in dcuABC mutant. At pH 7.5 the H+ fluxes in E. coli wild type, dcuD and dcuABCD mutants was similar but in dcuABC triple mutant the H+ flux decreased 1.4 fold reaching 1.15 mM/min when glycerol was supplemented. In succinate assays the H+ flux was higher in the strains where DcuD is absent. No significant differences were determined in wild type and mutants specific growth rate except dcuD strain. Taken together it is suggested that during glycerol fermentation DcuD has impact on H+ fluxes, FOF1-ATPase activity and depends on potassium ions

The respiratory molybdo-selenoprotein formate dehydrogenases of Escherichia coli have hydrogen: benzyl viologen oxidoreductase activity

<p>Abstract</p> <p>Background</p> <p><it>Escherichia coli </it>synthesizes three membrane-bound molybdenum- and selenocysteine-containing formate dehydrogenases, as well as up to four membrane-bound [NiFe]-hydrogenases. Two of the formate dehydrogenases (Fdh-N and Fdh-O) and two of the hydrogenases (Hyd-1 and Hyd-2) have their respective catalytic subunits located in the periplasm and these enzymes have been shown previously to oxidize formate and hydrogen, respectively, and thus function in energy metabolism. Mutants unable to synthesize the [NiFe]-hydrogenases retain a H₂: benzyl viologen oxidoreductase activity. The aim of this study was to identify the enzyme or enzymes responsible for this activity.</p> <p>Results</p> <p>Here we report the identification of a new H₂: benzyl viologen oxidoreductase enzyme activity in <it>E. coli </it>that is independent of the [NiFe]-hydrogenases. This enzyme activity was originally identified after non-denaturing polyacrylamide gel electrophoresis and visualization of hydrogen-oxidizing activity by specific staining. Analysis of a crude extract derived from a variety of <it>E. coli </it>mutants unable to synthesize any [NiFe]-hydrogenase-associated enzyme activity revealed that the mutants retained this specific hydrogen-oxidizing activity. Enrichment of this enzyme activity from solubilised membrane fractions of the hydrogenase-negative mutant FTD147 by ion-exchange, hydrophobic interaction and size-exclusion chromatographies followed by mass spectrometric analysis identified the enzymes Fdh-N and Fdh-O. Analysis of defined mutants devoid of selenocysteine biosynthetic capacity or carrying deletions in the genes encoding the catalytic subunits of Fdh-N and Fdh-O demonstrated that both enzymes catalyze hydrogen activation. Fdh-N and Fdh-O can also transfer the electrons derived from oxidation of hydrogen to other redox dyes.</p> <p>Conclusions</p> <p>The related respiratory molybdo-selenoproteins Fdh-N and Fdh-O of <it>Escherichia coli </it>have hydrogen-oxidizing activity. These findings demonstrate that the energy-conserving selenium- and molybdenum-dependent formate dehydrogenases Fdh-N and Fdh-O exhibit a degree of promiscuity with respect to the electron donor they use and identify a new class of dihydrogen-oxidizing enzyme.</p

Nanosizing Cynomorium: Thumbs up for Potential Antifungal Applications

Cynomorium coccineum L., the desert thumb, is a rather exotic, parasitic plant unable to engage in photosynthesis, yet rich in a variety of unique compounds with a wide spectrum of biological applications. Whilst extraction, separation and isolation of such compounds is time consuming, the particular properties of the plant, such as dryness, hardness and lack of chlorophyll, render it a prime target for possible nanosizing. The entire plant, the external layer (coat) as well as its peel, are readily milled and high pressure homogenized to yield small, mostly uniform spherical particles with diameters in the range of 300 to 600 nm. The best quality of particles is obtained for the processed entire plant. Based on initial screens for biological activity, it seems that these particles are particularly active against the pathogenic fungus Candida albicans, whilst no activity could be observed against the model nematode Steinernema feltiae. This activity is particularly pronounced in the case of the external layer, whilst the peeled part does not seem to inhibit growth of C. albicans. Thanks to the ease of sample preparation, the good quality of the nanosuspension obtained, and the interesting activity of this natural product, nanosized coats of Cynomorium may well provide a lead for future development and applications as “green” materials in the field of medicine, but also environmentally, for instance in agriculture

The Caucasian flora: a still-to-be-discovered rich source of antioxidants

Cellular redox homeostasis is a state of balance between the formation of Usually Reactive Oxygen and / or Nitrogen Species (ROS/RNS), endogenous antioxidant defence systems, and exogenous dietary antioxidants. The disturbance of redox homeostasis, by the overproduction of endogenous ROS/RNS, may increase the risk of development of so-called civilisation diseases. The solution seems to be either the increased production of endogenous or consumption of exogenous antioxidants. Plant-borne antioxidants act via different chemical and molecular mechanisms, such as decreasing the level of oxidative damage in cells directly by reacting with ROS/ RNS or indirectly – by inhibition of the activity and expression of free radical generating enzymes or by enhancing the activity or expression of intracellular antioxidant defence enzymes. Despite the fact that the Caucasian flora is rich of health promoting edible/medicinal plants, recent studies concerning the biological activity of these plants are very scarce. This review is summarising the state-of-art on the health-promoting potential of plants representing the Caucasian flora, whose antioxidant capacity have been investigated in various in vitro models

Exploring the directionality of <i>Escherichia coli </i>formate hydrogenlyase:a membrane-bound enzyme capable of fixing carbon dioxide to organic acid

During mixed‐acid fermentation Escherichia coli produces formate, which is initially excreted out the cell. Accumulation of formate, and dropping extracellular pH, leads to biosynthesis of the formate hydrogenlyase (FHL) complex. FHL consists of membrane and soluble domains anchored within the inner membrane. The soluble domain comprises a [NiFe] hydrogenase and a formate dehydrogenase that link formate oxidation directly to proton reduction with the release of CO (2) and H(2). Thus, the function of FHL is to oxidize excess formate at low pH. FHL subunits share identity with subunits of the respiratory Complex I. In particular, the FHL membrane domain contains subunits (HycC and HycD) that are homologs of NuoL/M/N and NuoH, respectively, which have been implicated in proton translocation. In this work, strain engineering and new assays demonstrate unequivocally the nonphysiological reverse activity of FHL in vivo and in vitro. Harnessing FHL to reduce CO (2) to formate is biotechnologically important. Moreover, assays for both possible FHL reactions provide opportunities to explore the bioenergetics using biochemical and genetic approaches. Comprehensive mutagenesis of hycC did not identify any single amino acid residues essential for FHL operation. However, the HycD E199, E201, and E203 residues were found to be critically important for FHL function

The Structure of Hydrogenase-2 from <i>Escherichia coli</i>:Implications for H₂ -Driven Proton Pumping

Under anaerobic conditions Escherichia coli is able to metabolize molecular hydrogen via the action of several [NiFe]-hydrogenase enzymes. Hydrogenase-2, which is typically present in cells at low levels during anaerobic respiration, is a periplasmic-facing membrane-bound complex that functions as a proton pump to convert energy from H2 oxidation into a proton gradient; consequently, its structure is of great interest. Empirically, the complex consists of a tightly-bound core catalytic module, comprising large (HybC) and small (HybO) subunits, which is attached to an Fe-S protein (HybA) and an integral membrane protein, HybB. To date, efforts to gain a more detailed picture have been thwarted by low native expression levels of hydrogenase-2 and the labile interaction between HybOC and HybA/HybB subunits. In this paper we describe a new over-expression system that has facilitated determination of high-resolution crystal structures of HybOC and, hence, a prediction of the quaternary structure of the HybOCAB complex

The Cyst-Dividing Bacterium Ramlibacter tataouinensis TTB310 Genome Reveals a Well-Stocked Toolbox for Adaptation to a Desert Environment

Ramlibacter tataouinensis TTB310T (strain TTB310), a betaproteobacterium isolated from a semi-arid region of South Tunisia (Tataouine), is characterized by the presence of both spherical and rod-shaped cells in pure culture. Cell division of strain TTB310 occurs by the binary fission of spherical “cyst-like” cells (“cyst-cyst” division). The rod-shaped cells formed at the periphery of a colony (consisting mainly of cysts) are highly motile and colonize a new environment, where they form a new colony by reversion to cyst-like cells. This unique cell cycle of strain TTB310, with desiccation tolerant cyst-like cells capable of division and desiccation sensitive motile rods capable of dissemination, appears to be a novel adaptation for life in a hot and dry desert environment. In order to gain insights into strain TTB310's underlying genetic repertoire and possible mechanisms responsible for its unusual lifestyle, the genome of strain TTB310 was completely sequenced and subsequently annotated. The complete genome consists of a single circular chromosome of 4,070,194 bp with an average G+C content of 70.0%, the highest among the Betaproteobacteria sequenced to date, with total of 3,899 predicted coding sequences covering 92% of the genome. We found that strain TTB310 has developed a highly complex network of two-component systems, which may utilize responses to light and perhaps a rudimentary circadian hourglass to anticipate water availability at the dew time in the middle/end of the desert winter nights and thus direct the growth window to cyclic water availability times. Other interesting features of the strain TTB310 genome that appear to be important for desiccation tolerance, including intermediary metabolism compounds such as trehalose or polyhydroxyalkanoate, and signal transduction pathways, are presented and discussed

Carbon Dioxide Utilisation -The Formate Route

UIDB/50006/2020 CEEC-Individual 2017 Program Contract.The relentless rise of atmospheric CO2 is causing large and unpredictable impacts on the Earth climate, due to the CO2 significant greenhouse effect, besides being responsible for the ocean acidification, with consequent huge impacts in our daily lives and in all forms of life. To stop spiral of destruction, we must actively reduce the CO2 emissions and develop new and more efficient “CO2 sinks”. We should be focused on the opportunities provided by exploiting this novel and huge carbon feedstock to produce de novo fuels and added-value compounds. The conversion of CO2 into formate offers key advantages for carbon recycling, and formate dehydrogenase (FDH) enzymes are at the centre of intense research, due to the “green” advantages the bioconversion can offer, namely substrate and product selectivity and specificity, in reactions run at ambient temperature and pressure and neutral pH. In this chapter, we describe the remarkable recent progress towards efficient and selective FDH-catalysed CO2 reduction to formate. We focus on the enzymes, discussing their structure and mechanism of action. Selected promising studies and successful proof of concepts of FDH-dependent CO2 reduction to formate and beyond are discussed, to highlight the power of FDHs and the challenges this CO2 bioconversion still faces.publishersversionpublishe