Dependence of prePhoA-phospholipid interaction in vivo and in vitro on charge of signal peptide N-terminus and content of anionic phospholipids in membranes.

Replacement of the positively charged signal peptide with neutral or negatively charged peptides due to substitution of Lys(-20) in the N-terminal region of the signal peptide leads to decreases in the rate of prePhoA membrane translocation in vivo and in the efficiency of prePhoA insertion into liposomes in vitro. The effect of anionic phospholipids on prePhoA insertion into model membranes is determined by the signal peptide N-terminus charge, while the dependence of prePhoA translocation across the cytoplasmic membrane in vivo is not, under the studied variations in the content of anionic phospholipids. This is evidence of the possibility of direct electrostatic interaction between the signal peptide N-terminus and anionic phospholipids, which in vivo, however, seems to involve some proteins of the Sec machinery.

Depletion of phosphatidylethanolamine--the major membrane phospholipid of Escherichia coli--depresses posttranslocational modification of alkaline phosphatase in the periplasm.

Unbalanced phospholipid composition due to depletion of the major phospholipid of Escherichia coli, phosphatidylethanolamine, was shown previously to significantly decrease the secretion and transcriptional expression of periplasmic alkaline phosphatase of this bacterium. The current work studies the effect of this unbalance on posttranslocational proteolytic modification of the enzyme, proceeding in the periplasm with the formation of its isoforms. It has been revealed that under phosphatidylethanolamine depletion the content of incompletely modified (intermediate) isoforms I and II increases. This increase does not depend on the level of enzyme synthesis and the mechanism of its regulation (expression of the chromosomal gene or the gene cloned in plasmid under the control of the endogenous promoter P(PHO) or exogenous promoter P(BAD)). Depression of posttranslocational modification of alkaline phosphatase in phosphatidylethanolamine-depleted cells is supposed to be a result of the lower efficiency of secretion of modifying proteinase (product of the iap gene) localized, like alkaline phosphatase, in the periplasm of Escherichia coli.

Depletion of phosphatidylethanolamine affects secretion of Escherichia coli alkaline phosphatase and its transcriptional expression.

In this report we demonstrate that depletion of the major phospholipid phosphatidylethanolamine, a single non-bilayer forming phospholipid of Escherichia coli, significantly reduces the secretion efficiency of alkaline phosphatase in vivo. Secretion, however, is correlated with the content in membranes of cardiolipin, which in combination with selected divalent cations has a strong tendency to adopt a non-bilayer state indicating the possible involvement of lipid polymorphism in efficient protein secretion. Depletion of this zwitterionic phospholipid also inhibits expression of the protein controlled by the endogenous P(PHO) promoter but not the P(BAD) promoter, which is suggested to be due to the effect of unbalanced phospholipid composition on the orthophosphate signal transduction system (Pho regulon) through an effect on its membrane bound sensor.

The major phospholipid of Escherichia coli, phosphatidylethanolamine, is required for efficient production and secretion of alkaline phosphatase.

The major phospholipid of the Escherichia coli membranes--the zwitterion phosphatidylethanolamine (PE)--is the only phospholipid involved in the formation of non-bilayer structure of membrane lipids, which is supposed to be necessary for efficient translocation of secreted proteins across the cytoplasmic membrane. The effect of PE on the production and secretion of alkaline phosphatase has been studied in this work using the mutant strain E. coli AD93, which is unable to synthesize PE. It was shown that this phospholipid is required for the efficient production and secretion of alkaline phosphatase. The anionic phospholipid cardiolipin in combination with divalent cations Mg2+ functionally replaces PE in these processes, participating in the regulation of lipid polymorphism.

Requirement for phospholipids of the translocation of the trimethylamine N-oxide reductase through the Tat pathway in Escherichia coli.

Trimethylamine N-oxide reductase (TorA) is an anaerobically synthesized molybdoenzyme. It is translocated across the cytoplasmic membrane in a folded conformation via the Tat pathway of Escherichia coli. The requirement for phospholipids for the export of this enzyme was analyzed in the pgsA and pss mutants lacking anionic phospholipids and phosphatidylethanolamine, respectively. Anaerobic growth did not influence phospholipid composition of the pgsA and pss mutants. Interestingly, both pgsA and pss mutations severely retarded the translocation of TorA into the periplasm. Therefore, translocation of proteins through the Tat pathway is dependent on the anionic phospholipids and on lipid polymorphism.

Interaction of mutant alkaline phosphatase precursors with membrane phospholipids in vivo and in vitro.

Positively charged amino acid residues in the N-terminal domain of the signal peptides of secreted proteins are thought to interact with negatively charged anionic phospholipids during the initiation of secretion. To test this hypothesis, substitutions of the uncharged Ala or the negatively charged Glu residue for the positively charged Lys-20 of the N-terminus of the signal peptide of Escherichia coli alkaline phosphatase were introduced using a modified method of oligonucleotide-directed mutagenesis. We found that Lys-20 is involved in the interaction of the signal peptide with anionic phospholipids in vivo and effects the efficiency of insertion of the signal peptide of isolated precursor into model phospholipid membranes in vitro. We also show that the efficiency of signal peptide insertion into the lipid bilayer depends on the fluidity of the bilayer.

Interaction of the E. coli alkaline phosphatase precursor with model phospholipid membranes.

An interaction of a precursor of the E. coli secreted alkaline phosphatase (prePhoA) containing a signal peptide and model bilayer membranes prepared from the lipids of E. coli has been studied. The interaction was evaluated by monitoring of the state of the lipid phase by fluorescence spectroscopy. The role of the signal peptide in this process was evaluated by a comparative study of the interaction of the mature alkaline phosphatase which does not contain the signal peptide with the model membranes. The precursor was shown to be inserted into the lipid bilayer since the fluorescence anisotropy of a hydrophobic probe, diphenylhexatriene, was enhanced. The intensity of the process is determined by the presence of the signal peptide and depends on the pH of the medium. The data indicate that the mature polypeptide chain of the enzyme also has affinity for the membrane.

[Permeability of the Escherichia coli outer membrane for ethidium ions and periplasmic alkaline phosphatase during increased synthesis of it]. / Pronitsaemost' vneshnei membrany Escherichia coli dlia ionov étidiia i periplazmennoi shchelochnoi fosfatazy pri ee povyshenoom sinteze.

During augmented synthesis of periplasmic alkaline phosphatase by various strains of Escherichia coli, the outer membrane of bacterial cells becomes permeable for both the enzyme and ethidium ions which do not generally penetrate inside the cells of gram-negative bacteria. In the absence of the lipoprotein in the outer membrane, its permeability for these compounds as well as its sensitivity to membranotropic agents increases, thus testifying to the influence of the lipoprotein upon certain properties of the outer membrane. A competitive interaction was found between the lipoprotein and lipopolysaccharide content in the outer membrane and their content and alkaline phosphatase secretion into the external medium. It is suggested that increased permeability of the E. coli outer membrane during augmented synthesis of the secreted protein is due to impaired biogenesis of membrane components.

[Effect on the orientation of the membrane vesicles of Escherichia coli of various methods of cell disintegration]. / Vliianie na orientatsiiu membrannykh vezikul u Escherichia coli razlichnykh sposobov dezintegratsii kletok.

As was demonstrated using the Con-A polymer, membranous fractions prepared by various cell disintegration procedures are a heterogeneous population. The population includes right side out vesicles and inside out vesicles whose proportion depends on the procedure of disintegration. The orientation of these vesicles was studied by electron microscopy, their ATPase activity was assayed by cytochemical techniques, and the morphology of the vesicles was also investigated. The authors discuss the possible effect of Con-A on the reorganisation of membranes and the activity of ATPase.

[Dependence of the efficiency of the fractionation of summary E. coli cell membranes on the method of their disintegration]. / Zavisimost' éffektivnosti fraktsionirovaniia summarnykh membran kletok E. coli ot sposoba ikh dezintegratsii.

The efficiency of fractionation in the sucrose density gradient of E. coli cell membranes obtained after cell disintegration by ultrasonic, ballistic and extrusion methods was measured. The purity of individual membrane fractions was estimated by electron microscopy and polyacrylamide gel electrophoresis. The application of ballistic disintegration and solid state extrusion did not separate inner and outer membranes. Cell disintegration by means of ultrasonic treatment and liquid state extrusion allowed reproducible separation of membranes of the two types with a sufficiently high degree of purity.

[Structural and biochemical characteristics of Escherichia coli cells with various levels of secretable alkaline phosphatase]. / Strukturno-biokhimicheskie osobennosti kletok Escherichia coli s raznym urovnem sinteza sekretiruemoi shchelochnoi fosfatazy.

The biochemical and structural peculiarities of Escherichia coli cells with different rates of synthesis of secreted alkaline phosphatase were studied under the conditions of repression, derepression and constitutive synthesis. The enzyme synthesis was shown to correlate with the redistribution of free and membrane-bound ribosomes in the cells, with an increase in their proportion, and with a reconstruction of the cell ultrastructural organization.

[Comparative characteristics of membrane preparations of Escherichia coli obtained by mechanical disruption]. / Sravnitel'naia kharakteristika preparatov membran Escherichia coli, poluchennykh mekhanicheskim razrusheniem.

Membrane preparations were obtained from Escherichia coli cells by the liquid and solid extrusion, ultrasonic treatment and ballistic disintegration. The preparations were subjected to biochemical analysis and studied by electron microscopy. The methods employed preserved, though to a different degree, the enzymic activity and integrity of the ribosome-membrane complex of the membrane preparations. The highest values were observed with the preparations obtained by ballistic and modified French-press disintegration. The membrane fractions obtained consisted of the cell wall and cytoplasmic membrane fragments heterogeneous in size and shape, and depending on the technique of disintegration were characterized by the presence or absence of a contact between them.