Membrane phospholipid composition of Escherichia coli affects secretion of periplasmic alkaline phosphatase into the medium

Secretion of alkaline phosphatase (PhoA) encoded by a gene constituent of plasmids has been studied in Escherichia coli strains with controlled synthesis of anionic phospholipids (phosphatidylglycerol and cardiolipin, strain HDL11) and zwitterionic phospholipid (phosphatidylethanolamine, strain AD93). Changing the phospholipid composition of the membrane of these strains leads to an increase in secretion of PhoA, which is usually localized in the periplasm, into the culture medium. This correlates with a higher secretion of exopolysaccharides and lower content of lipopolysaccharide in the outer membrane. The results show the possibility of coupling protein secretion into the medium with biogenesis of cell envelope components in which phospholipids are involved.     

Role of membrane potential in protein folding and domain formation during secretion in Escherichia coli

The synthesis and processing of the periplasmic components of the leucine transport system of E coli have been studied to determine the role played by transmembrane potential in protein secretion. Both the leucine-isoleucine-valine binding protein and the leucine-specific binding protein are synthesized as precursors with 23 amino acid N-terminal leader sequences. The processing of these precursors is sensitive to the transmembrane potential. Since the amino acid sequence and the crystal structure have been determined for the leucine-isoleucine-valine binding protein, it and the closely related leucine-specific binding protein represent convenient models in which to examine the mechanism of protein secretion in E coli. A model for secretion has been proposed, suggesting a role for transmembrane potential. In this model, the N-terminal amino acid sequence of the precursor is assumed to form a hairpin of two helices. The membrane potential may orient this structure to make it accessible to processing. In addition, the model suggests that a negatively charged, folded domain of the secretory protein may electrophorese toward the trans-positive side of the membrane, thus providing an additional role for the transmembrane potential.     

Protein-stimulated exchange of phosphatidylcholine between intact erythrocytes and various membrane systems

Phosphatidylcholine specific exchange protein from beef liver was found to catalyze the exchange of phosphatidylcholine between intact rat and human erythrocytes and various artificial membranes. Both multilamellar liposomes and single bilayer vesicles prepared from egg lecithin, cholesterol and phosphatidic acid (46:50:4, mol/mol) appeared to be effective phospholipid donor systems. Some merits and disadvantages of the various donor systems are discussed.     

Incorporation of heterologous outer membrane and periplasmic proteins into Escherichia coli outer membrane vesicles

Gram-negative bacteria shed outer membrane vesicles composed of outer membrane and periplasmic components. Since vesicles from pathogenic bacteria contain virulence factors and have been shown to interact with eukaryotic cells, it has been proposed that vesicles behave as delivery vehicles. We wanted to determine whether heterologously expressed proteins would be incorporated into the membrane and lumen of vesicles and whether these altered vesicles would associate with host cells. Ail, an outer membrane adhesin/invasin from Yersinia enterocolitica, was detected in purified outer membrane and in vesicles from Escherichia coli strains DH5alpha, HB101, and MC4100 transformed with plasmid-encoded Ail. In vesicle-host cell co-incubation assays we found that vesicles containing Ail were internalized by eukaryotic cells, unlike vesicles without Ail. To determine whether lumenal vesicle contents could be modified and delivered to host cells, we used periplasmically expressed green fluorescent protein (GFP). GFP fused with the Tat signal sequence was secreted into the periplasm via the twin arginine transporter (Tat) in both the laboratory E. coli strain DH5alpha and the pathogenic enterotoxigenic E. coli ATCC strain 43886. Pronase-resistant fluorescence was detectable in vesicles from Tat-GFP-transformed strains, demonstrating that GFP was inside intact vesicles. Inclusion of GFP cargo increased vesicle density but did not result in morphological changes in vesicles. These studies are the first to demonstrate the incorporation of heterologously expressed outer membrane and periplasmic proteins into bacterial vesicles.     

High level secretion of TaqI restriction endonuclease by recombinant Escherichia coli

The production and high level secretion of TaqI restriction endonuclease using bacterial secretion signal within the malE gene was achieved by cloning the PCR-amplified gene from Thermus aquaticus into E. coli. The maltose binding protein (MBP) part of the MBP-TaqI fusion protein expressed by this construct did not interfere with the biological activity of the TaqI restriction endonuclease. E. coli XL1 carrying pH185 produced 332 U ml^–1 TaqI endonuclease 81% of which was secreted into the medium without apparent cell lysis. Optimization of culture conditions and selection of the host strain were found to be important for the efficient extracellular production of this protein.     

Effect of bilayer membrane curvature on activity of phosphatidylcholine exchange protein

Effect of bilayer membrane curvature of substrate phosphatidylcholine and inhibitor phosphatidylserine on the activity of phosphatidylcholine exchange protein has been studied by measuring transfer of spin-labeled phosphatidylcholine between vesicles, vesicles and liposomes, and between liposomes. The transfer rate between vesicles was more than 100 times larger than that between vesicles and liposomes. The transfer rate between liposomes was still smaller than that between vesicles and liposomes and nearly the same as that in the absence of exchange protein. The markedly enhanced exchange with vesicles was ascribed to the asymmetric packing of phospholipid molecules in the outer layer of the highly curved bilayer membrane. The inhibitory effect of phosphatidylserine was also greatly dependent on the membrane curvature. The vesicles with diameter of 17 nm showed more than 20 times larger inhibitory activity than those with diameter of 22 nm. The inhibitory effect of liposomes was very small. The size dependence was ascribed to stronger binding of the exchange protein to membranes with higher curvatures. The protein-mediated transfer from vesicles to spiculated erythrocyte ghosts was about four times faster than that to cup-shaped ghosts. This was ascribed to enhanced transfer to the highly curved spiculated membrane sites rather than greater mobility of phosphatidylcholine in the spiculated ghost membrane.     

大肠杆菌Ⅲ型分泌系统2毒力岛研究进展

许多革兰氏阴性菌借助Ⅲ型分泌系统黏附在宿主细胞表面,然后跨越胞膜将特异性蛋白注入宿主细胞内,破坏宿主细胞内的多种信号通路,从而有利于细菌的感染及定殖。在肠致病性大肠杆菌(Enteropathogenic Escherichia coli,EPEC)中,除了肠细胞脱落位点(Locus of entericyte effacement,LEE)毒力岛编码的Ⅲ型分泌系统(Type Ⅲ secretion system,T3SS)外,在分析肠出血性大肠杆菌O157:H7的基因组序列时发现一个新的Ⅲ型分泌系统,大肠杆菌Ⅲ型分泌系统2(Escherichia coli type Ⅲ secretion system 2,ETT2)毒力岛。研究显示,ETT2可能在大多数菌株中不具有完整的分泌系统功能,但是其对于细菌毒力的发挥具有重要作用。因此,本文简要综述了大肠杆菌ETT2的基因特征、ETT2的分布与流行、ETT2的功能与机制等方面的主要研究进展。     

Study of interaction of export initiation domain of Escherichia coli mature alkaline phosphatase with membrane phospholipids during secretion

The efficiency of secretion of alkaline phosphatase from Escherichia coli depending on the primary structure of its N-terminal region and the content of zwitterionic phospholipid phosphatidylethanolamine and anionic phospholipids in membranes has been studied in this work to establish the peculiarities of interaction of mature protein during its secretion with membrane phospholipids. It has been shown that the effect of phosphatidylethanolamine but not anionic phospholipids on the efficiency of alkaline phosphatase secretion is determined by the primary structure of its N-terminal region. The absence of phosphatidylethanolamine appreciably reduces the efficiency of secretion of wild type alkaline phosphatase and its mutant forms with amino acid substitutions in positions +5+6 and +13+14. In contrast, secretion of the protein with amino acid substitutions in positions +2+3, significantly decreased as a result of such mutation, in the presence of phosphatidylethanolamine, reaches the level of wild type protein secretion in the absence of phosphatidylethanolamine. The results suggest an interaction of the N-terminal region of the mature protein under its translocation across the membrane with phosphatidylethanolamine.     

Incorporation of diaminopimelic acid into Escherichia coli membranes

Abstract Pulse-labeled, cell division-inhibited Escherichia coli incorporate [³H]diamino pimelic acid (DAP) into membrane fractions of light (= inner membrane) and intermediate densities and transfer the DAP rapidly into heavy membrane fractions (= outer membrane). The intermediate density fraction which has been shown to contain markers for membrane adhesion sites appears to serve as intermediary in the translocation.     

The change in membrane phospholipid composition influences protein secretion and cell envelope biogenesis in Escherichia coli

Secretion of periplasmic alkaline phosphatase (PhoA) encoded by the gene constituent of plasmids and the peculiar properties of cell envelope biogenesis in Escherichia coli strains with controlled synthesis of individual membrane phospholipids have been studied. Alkaline phosphatase secretion across the cytoplasmic membrane declines, while secretion into the culture medium intensifies under changed metabolism. The composition of anionic membrane phospholipids changes due to inactivation of the pgsA gene or regulation of its expression by environmental factor, as well as in the absence of the pssA gene which is responsible for the synthesis of the precursor for zwitter-ionic phospholipid — phosphatidylethanolamine. This correlates with intensified secretion of exopolysaccharides and lower content of lipopolysaccharide and lipoprotein which are responsible for barrier properties of the outer membrane. The results suggest a possible coupling of protein secretion with biogenesis of cell envelope components at a level of phospholipid metabolism.