Visualizing Interactions along the Escherichia coli Twin-Arginine Translocation Pathway Using Protein Fragment Complementation

The twin-arginine translocation (Tat) pathway is well known for its ability to export fully folded substrate proteins out of the cytoplasm of Gram-negative and Gram-positive bacteria. Studies of this mechanism in Escherichia coli have identified numerous transient protein-protein interactions that guide export-competent proteins through the Tat pathway. To visualize these interactions, we have adapted bimolecular fluorescence complementation (BiFC) to detect protein-protein interactions along the Tat pathway of living cells. Fragments of the yellow fluorescent protein (YFP) were fused to soluble and transmembrane factors that participate in the translocation process including Tat substrates, Tat-specific proofreading chaperones and the integral membrane proteins TatABC that form the translocase. Fluorescence analysis of these YFP chimeras revealed a wide range of interactions such as the one between the Tat substrate dimethyl sulfoxide reductase (DmsA) and its dedicated proofreading chaperone DmsD. In addition, BiFC analysis illuminated homo- and hetero-oligomeric complexes of the TatA, TatB and TatC integral membrane proteins that were consistent with the current model of translocase assembly. In the case of TatBC assemblies, we provide the first evidence that these complexes are co-localized at the cell poles. Finally, we used this BiFC approach to capture interactions between the putative Tat receptor complex formed by TatBC and the DmsA substrate or its dedicated chaperone DmsD. Our results demonstrate that BiFC is a powerful approach for studying cytoplasmic and inner membrane interactions underlying bacterial secretory pathways

Antioxidant effects of mono- and diacylglycerols in non-stripped and stripped soybean oil-in-water emulsions

Antioxidant activity of mono- and diacylglycerols (0.01-2.50% of oil) was observed in 1.0% non-stripped and stripped soybean oil-in-water emulsions by monitoring lipid hydroperoxides and headspace hexanal formation. Addition of 1-monooleoylglycerol only had a slight impact on the oxidative stability of non-stripped soybean oil-in-water emulsions but did inhibit lipid oxidation in emulsions prepared with stripped soybean oil. A strong antioxidant activity was observed upon addition of 1,2-dioleoyl-sn-glycerol to stripped soybean oil-in-water emulsions. However, the addition of the diacylglycerol to oil-in-water emulsions made with non-stripped oils had much less of an effect on oxidation rates. To determine if the ability of mono- and diacylglycerols to impact lipid oxidation was due to their capability to alter the physical properties of the oil-in-water emulsions, zeta potential and interfacial tension were measured. Both 1-monooleoylglycerol and 1,2-dioleoyl-sn-glycerol reduced interfacial tension. Both 1-monooleoylglycerol and 1,2-dioleoyl-sn-glycerol were also able to raise the zeta potential of the emulsions, although these increases were small (< 4 mV). The ability of diacylglycerols to only impact lipid oxidation rates in oil-in-water emulsions made with stripped oil suggests that either the concentration of endogenous diacylglycerols in refined oil is high enough to provide maximum protection against oxidation, or that other minor components in non-stripped oil can interfere with the antioxidant activity of diacylglycerol

Antioxidant and prooxidant activity behavior of phospholipids in stripped soybean oil-in-water emulsions

Phospholipids have been reported to inhibit lipid oxidation in bulk oils, but very little is known about their influence on oxidation in oil-in-water emulsions. In the present study, the impact of 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) on lipid oxidation was studied in 1% stripped soybean oil-in-water (O/W) emulsions as a function of DOPC concentration and pH (3 and 7). At pH 7.0, DOPC inhibited lipid oxidation in O/W emulsions, while DOPC was prooxidative at pH 3.0. DOPC did not affect emulsion droplet charge or size at either pH 3.0 or 7.0 The antioxidant activity at pH 7.0 was observed in a series of phospholipids (PL) that varied in fatty acid unsaturation level and chain length as well as type of phosphate head group. Overall, phosphatidylcholine with either oleic or palmitic acid were the most effective at inhibiting lipid hydroperoxide and hexanal formation of all of the PLs tested. Antioxidant mechanism of PLs could not be ascribed to their ability to decompose lipid hydroperoxides. It might be possible that, at pH 7.0, the PLs antioxidant activity is related to their ability to form structures within the lipid phase of the emulsions droplets or to chelate metals