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

Neutralization of Botulinum Neurotoxin by a Human Monoclonal Antibody Specific for the Catalytic Light Chain

Background: Botulinum neurotoxins (BoNT) are a family of category A select bioterror agents and the most potent biological toxins known. Cloned antibody therapeutics hold considerable promise as BoNT therapeutics, but the therapeutic utility of antibodies that bind the BoNT light chain domain (LC), a metalloprotease that functions in the cytosol of cholinergic neurons, has not been thoroughly explored. Methods and Findings: We used an optimized hybridoma method to clone a fully human antibody specific for the LC of serotype A BoNT (BoNT/A). The 4LCA antibody demonstrated potent in vivo neutralization when administered alone and collaborated with an antibody specific for the HC. In Neuro-2a neuroblastoma cells, the 4LCA antibody prevented the cleavage of the BoNT/A proteolytic target, SNAP-25. Unlike an antibody specific for the HC, the 4LCA antibody did not block entry of BoNT/A into cultured cells. Instead, it was taken up into synaptic vesicles along with BoNT/A. The 4LCA antibody also directly inhibited BoNT/A catalytic activity in vitro. Conclusions: An antibody specific for the BoNT/A LC can potently inhibit BoNT/A in vivo and in vitro, using mechanisms not previously associated with BoNT-neutralizing antibodies. Antibodies specific for BoNT LC may be valuable components o

A Transcriptional “Scream” Early Response of E. coli Prey to Predatory Invasion by Bdellovibrio

We have transcriptionally profiled the genes differentially expressed in E. coli prey cells when predatorily attacked by Bdellovibrio bacteriovorus just prior to prey cell killing. This is a brief, approximately 20–25 min period when the prey cell is still alive but contains a Bdellovibrio cell in its periplasm or attached to and penetrating its outer membrane. Total RNA was harvested and labelled 15 min after initiating a semi-synchronous infection with an excess of Bdellovibrio preying upon E. coli and hybridised to a macroarray spotted with all predicted ORFs of E. coli. SAM analysis and t-tests were performed on the resulting data and 126 E. coli genes were found to be significantly differentially regulated by the prey upon attack by Bdellovibrio. The results were confirmed by QRT-PCR. Amongst the prey genes upregulated were a variety of general stress response genes, potentially “selfish” genes within or near prophages and transposable elements, and genes responding to damage in the periplasm and osmotic stress. Essentially, the presence of the invading Bdellovibrio and the resulting damage to the prey cell elicited a small “transcriptional scream”, but seemingly no specific defensive mechanism with which to counter the Bdellovibrio attack. This supports other studies which do not find Bdellovibrio resistance responses in prey, and bodes well for its use as a “living antibiotic”

Adaptively evolved Escherichia coli for improved ability of formate utilization as a carbon source in sugar???free conditions

Background: Formate converted from CO2 reduction has great potential as a sustainable feedstock for biological production of biofuels and biochemicals. Nevertheless, utilization of formate for growth and chemical production by microbial species is limited due to its toxicity or the lack of a metabolic pathway. Here, we constructed a formate assimilation pathway in Escherichia coli and applied adaptive laboratory evolution to improve formate utilization as a carbon source in sugar-free conditions. Results: The genes related to the tetrahydrofolate and serine cycles from Methylobacterium extorquens AM1 were overexpressed for formate assimilation, which was proved by the 13C-labeling experiments. The amino acids detected by GC/MS showed significant carbon labeling due to biomass production from formate. Then, 150 serial subcultures were performed to screen for evolved strains with improved ability to utilize formate. The genomes of evolved mutants were sequenced and the mutations were associated with formate dehydrogenation, folate metabolism, and biofilm formation. Last, 90 mg/L of ethanol production from formate was achieved using fed-batch cultivation without addition of sugars. Conclusion: This work demonstrates the effectiveness of the introduction of a formate assimilation pathway, combined with adaptive laboratory evolution, to achieve the utilization of formate as a carbon source. This study suggests that the constructed E. coli could serve as a strain to exploit formate and captured CO2

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

Glass-forming property of the system diethyl sulphoxide/water and its cryoprotective action on "Escherichia coli" survival.

In this work the thermal properties of diethyl sulphoxide (Et2SO), as well as its cryoprotective ability are studied and related to other well-known cryoprotectant substances, like dimethyl sulphoxide (Me2SO). We have investigated the thermal properties of Et2SO/water systems using Differential Scanning Calorimetry at a very low heating/cooling rate (2 degrees C/min). Liquid/solid or glassy/crystalline transitions have been observed only for the solutions with content of Et2SO ranging from 5 up to 40% w/w and/or greater than 85%. In the 45-75% w/w Et2SO range we have found a noticeable glass-forming tendency and a great stability of the amorphous state to the reheating. In samples with Et2SO content ranging from 80 to 85%, we observed a great stability of the glass forming by cooling, but a lesser stability to the subsequent reheating. The glass-forming tendency of these solutions is discussed in terms of existing competitive interactions between molecules of Et2SO, on the one hand, and Et2SO and water molecules, on the other hand. The results are well explainable on the basis of the model structure of water/Et2SO solutions, deduced by Raman and infrared studies [J. Mol. Struct. 665 (2003) 285-292]. The cryoprotective ability of Et2SO on Escherichia coli survival has been also investigated, and a comparison among Et2SO and other widely used cryoprotectants, like Me2SO and glycerol has been done. Survival of E. coli, determined after freezing-thawing process, was maximal at 45% w/w Et2SO (more than 85% viability). It should be noted that at the same concentration the survival is only about 35% in the presence of Me2SO and not more than 15% in the presence of glycerol. These features are well consisted with the glass-forming properties of Et2SO