[Changes in the composition of anionic membrane phospholipids influence the protein secretion and biogenesis of cell envelope in Escherichia coli].

The secretion of alkaline phosphatase (PhoA) and peculiarities of biogenesis of the cell envelope were studied in Escherichia coli strains HD30/pHD 102 and HDL11 with controlled synthesis of anionic phospholipids, phosphatidylglycerol, and cardiolipin. Inactivation of the pgsA gene encoding the synthesis of anionic phospholipids or changes in the regulation of its expression by an environmental factor caused changes in the metabolism and composition of membrane phospholipids, which resulted in a decrease in the secretion of alkaline phosphatase through the cytoplasmic membrane and an increase in PhoA secretion from the periplasm into the culture medium. A conforming increase was observed in exopolysaccharide secretion, as well as a decrease in the contents of lipopolysaccharide and lipopolyprotein of the outer membrane that determine the membrane barrier properties. The results obtained testify that anionic phospholipids play a significant role in protein secretion and are probably involved in the interrelation between the protein secretion and biogenesis of cell envelope components.

[The unbalance of membrane phospholipid composition of Escherichia coli affects the PPHO promoter activity].

Activity of phosphate PPHO or arabinose PBAD promoter of Escherichia coli has been studied depending on the content of zwitter-ionic phosphatidylethanolamine (PE) and anionic phospholipids in membranes. In the absence of PE or under significant decrease in the content of anionic phospholipids, there is a significant decline of PPHO promoter activity but not PBAD promoter. Since the PPHO promoter belongs to the Pho-regulon--a member of the family of two-component regulatory systems of signal transduction having membrane sensors, the regulation of gene expression by phospholipids is presumed to be realized through a membrane sensor.

[Interaction of effect on secretion of alkaline phosphatase from E. coli by charge of the N-terminal part of the signal peptide and proteins SecB and SecA]. / Vzaimosviazannoe vliianie na sekretsiiu shchelochnoi fosfatazy E. coli zariada N-kontsevoi oblasti signal'nogo peptida i belkov SecB i SecA.

The cytoplasmic step of posttranslational secretion in Escherichia coli is catalyzed by export-specific chaperone SecB and translocational ATPase SecA. In addition, the efficiency of secretion depends on the charge of the signal peptide (SP). Substitution of positively charged Lys(-20) with noncharged Ala or negatively charged Glu in the N-terminal region of SP of the alkaline phosphatase (PhoA) precursor (prePhoA) was shown to decrease the PhoA secretion in the periplasm. The effect on secretion increased in the absence of SecB and was especially high on SecA inactivation. A change in SP charge strengthened the SecA and SecB dependences of secretion. On evidence of immunoprecipitation, the charge of the N-terminal region of SP had no effect on prePhoA interaction with the cytoplasmic secretion factors, suggesting no direct binding between this region and SecA or SecB. Yet the charge of the N-terminal region proved to affect the functions of SP as an intramolecular chaperone and a factor of prePhoA targeting to the membrane in cooperation with SecA and SecB.

[Inactivation of pgsA gene affects biosynthesis and secretion of Escherichia coli alkaline phosphatase]. / Inaktivatsiia gena pgsA vliiaet na biosintez i sekretsiiu shchelochnoi fosfatazy Escherichia coli.

Inactivation of pgsA, which is responsible for biosynthesis of anionic phospholipid phosphatidylglycerol (PG), was shown to affect biosynthesis and secretion of alkaline phosphatase (PhoA) in Escherichia coli. A decrease in PG, but not in total anionic phospholipids, correlated with reduction of PhoA secretion, suggesting the role of PG in this process. A dramatic decrease in PG (from 18 to 3, but not 8, percent of the total phospholipids) inhibited not only secretion, but also synthesis of PhoA. In addition, pgsA inactivation expedited repression of PhoA synthesis by exogenous orthophosphate.

[Interaction of SecB and SecA with the N-terminal region of mature alkaline phosphatase on its secretion in Escherichia coli]. / Vzaimodeistvie belkov SecB i SecA s N-kontsevoi oblast'iu zreloi shchelochnoi fosfatazy Escherichia coli pri ee sekretsii.

Export-specific chaperone SecB and translocational ATPase SecA catalyze the cytoplasmic steps of Sec-dependent secretion in Escherichia coli. Their effects on secretion of periplasmic alkaline phosphatase (PhoA) were shown to depend on the N-terminal region of the mature PhoA sequence contained in the PhoA precursor. Amino acid substitutions in the vicinity of the signal peptide (positions +2, +3) not only dramatically inhibited secretion, but also reduced its dependence on SecB and SecA. Immunoprecipitation reported their impaired binding with mutant prePhoA. The results testified that SecB and SecA interact with the mature PhoA region located close to the signal peptide in prePhoA.

[Catabolism of methylphosphonic acid and its physiological regulation in Escherichia coli]. / Razlozhenie metilfosfonovoi kisloty i ego fiziologicheskaia reguliatsiia u Escherichia coli.

It was found that methyl phosphonic acid (Pn) was degraded by different Escherichia coli strains, which utilized it as the sole phosphorus source with resulting methane formation. This ability was influenced by mutations in the regulatory genes of the pho regulon. Thus, Pn was not degraded by an E. coli mutant defective in the regulatory phoB gene, responsible for the induction of pho-regulon proteins during phosphorus starvation. The intensity of Pn degradation depended on the age and concentration of the inoculum. Preincubation of bacteria in the presence of Pn accelerated subsequent degradation of both methyl phosphonic acid and its esters. Cultures developing from a small amount of inoculum degraded Pn more efficiently than heavily inoculated cultures that underwent only one cell division. However, cultures heavily inoculated with adapted cells degraded Pn as efficiently as cultures developing from a small amount of inoculum. Aeration was an important factor regulating Pn degradation: Pn was degraded more efficiently under anaerobic conditions regardless of the amount of inoculum.

[Analysis of the effect of replacing Lys(-20) in the alkaline phosphatase signal peptide on secretion of this enzyme]. / Analiz vliianiia zamen Lys(-20) v signal'nom peptide shchelochnoi fosfatazy na sekretsiiu étogo fermenta.

The effect of substitutions for the positively charged Lys(-20) in the N-terminal domain of the E. coli alkaline phosphatase signal peptide on enzyme secretion has been studied. Mutant alkaline phosphatases were obtained by the amber-suppressor method. An amber mutation was introduced in the appropriate position of the alkaline phosphatase gene using oligonucleotide-directed mutagenesis. This was followed by mutant protein synthesis in E. coli strains producing amber-suppressor tRNAs specific for Tyr, Gly, Ala, Glu, Phe, His, Cys, and Pro. All the mutant proteins can by translocated through the cytoplasmic membrane and form in the periplasm a molecule possessing an enzymatic activity. However, some amino acid substitutions decrease the rate of protein maturation their effect depends not only on the charge of the amino acid residue but also on its nature. Thus, introduction of positively charged. His and the polar uncharged Tyr is without effect, while negatively charged Glu and hydrophobic Ala, Phe and Pro residues as well as Gly and Cys have an inhibiting action. The results obtained testify to the importance of the signal peptide terminal domain primary structure in secretion.

[Disruption of processing of alkaline phosphatase as a result of single amino acid changes affects the composition and metabolism of phospholipids from Escherichia coli, secreting mutant proteins]. / Narushenie protsessinga shchelochnoi fosfatazy kak sledstvie edinichnykh zamen aminokislot, vliiaet na sostav i obmen fosfolipidov kletok Escherichia coli, sekretiruiushchikh mutantnye belki.

Amino acid substitutions in the cleavage site of the E. coli alkaline phosphatase signal peptide Val for Ala(-1) or Pro for Arg(+1) result in the block of the enzyme processing. In cells secreting such mutant proteins the relative content and rate of turnover of anionic phospholipids (phosphatidylglycerol and cardiolipin) are increased. The rise of the transfer of the phosphoglycerol residue from phosphatidylglycerol to periplasmic membrane derived oligosaccharides or to the model substrate, arbutin performed by the activity of phosphoglycerol transferase I testifies to phosphatidylglycerol accumulation on the outer surface of the cytoplasmic membrane. The results suggest of phosphatidylglycerol interaction with the alkaline phosphatase precursor and their subsequent joint translocation through the cytoplasmic membrane of E. coli.

[Isolation and certain properties of mutant alkaline phosphatase of Escherichia coli]. / Vydelenie i nekotorye svoistva mutantnykh shchelochnykh fosfataz Escherichia coli.

Natural and mutant alkaline phosphatases with amino acid substitutions in the processing site and N-terminal domain of the mature polypeptide chain Val for Ala(-1), Gln for Glu (+4) and simultaneously Gln for Glu (+4) and Ala for Arg (+1) have been isolated from the periplasm and cultural fluid of E. coli. It has been found that these substitutions have little effect on the dependence of the enzyme activity on pH, ionic strength and temperature but influence its isoenzymic spectrum and decrease (almost twofold) the maximal rate of the enzyme-catalyzed reaction. Extracellular enzymes display, in contrast with periplasmic ones, other catalytic properties (Vmax) and binding activity (Km). After translocation through the outer membrane all the enzymes display decreased Vmax and increased Km. These changes are especially well-pronounced in case of the mutant protein PhoA46 which contains an uncleaved signal peptide due to the impossibility of processing resulting from the substitution of Val for Ala(-1). The Vmax for this protein is decreased 20 times, while the Km is increased 4-fold. The protein also shows a higher (in comparison with other proteins) sensitivity towards proteolytic enzymes and is less resistant upon storage. The experimental data suggest that the changes in the N-end of alkaline phosphatase located at a long distance from its active center influence the enzyme function.

[Changes in indicators of the antioxidant defense system in patients with ischemic heart disease treated conventionally]. / Izmenenie pokazatelei sistemy antioksidantnoi zashchity organizma u bol'nykh ishemicheskoi bolezn'iu serdtsa na fone traditsionnoi terapii.

Parameters of antioxidant defense including superoxide dismutase, catalase and spontaneous chemiluminescence were studied in 68 patients with ischemic heart disease and 20 control patients. The above parameters decreased with the disease aggravation. Conventional therapy has improved antioxidant defense, the effect being dependent on effect of therapy irrespective of its variant.

[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.

[Changes in biogenesis and secretion of periplasmic alkaline phosphatase, coded by the phoA gene included in the Escherichia coli DH1 plasmid]. / Izmeneniia v biogeneze i sekretsiia v sredu periplazmaticheskoi shchelochnoi fosfatazy, kodiruemoi genom phoA v sostave plazmidy u Escherichia coli DH1.

Augmented synthesis of periplasmic alkaline phosphatase from E. coli DH1 coded by the phoA gene within the composition of plasmid pHI-7 results in alterations in the enzyme biogenesis, such as accumulation of intermediate enzyme forms corresponding to various stages of its posttranslational modification, and alternative localization. The cell cytoplasm was found to contain a large proportion of alkaline phosphatase precursors in the form of insoluble aggregates. The mature enzyme was detected in both the periplasm and the cultural fluid in the form three methazymes with an increased (as compared with the original strain) content of methazymes I and II. The cell envelope of E. coli DHI transformed by plasmid pHI-7 with the phoA gene differed from that of the original strain by an increased acid phospholipid (cardiolipin/phosphatidylglycerol) ratio as well as by increased distribution density of intramembrane particles on the surface of the cytoplasmic membrane facing the cytoplasm. Changes in the biogenesis of the enzyme during its augmented synthesis are due to disturbances in the equilibrium between the synthesis of polypeptide chains, on the one hand, and their translocation and processing, on the other, apparently as a result of restricted secretion sites and lack of inhibition of translation of secreted proteins in bacteria.

[Features of secretion of bacteriolytic enzymes and polysaccharides in bacteria from the Pseudomonadaceae family]. / Osobennosti sekretsii bakterioliticheskikh fermentov i polisakharida u bakterii iz semeistva Pseudomonadaceae.

It is shown that secretion of bacteriolytic complex enzymes in a bacterium of the family Pseudomonadaceae begins in the latent and early logarithmic culture growth phases. A maximum of specific bacteriolytic activity falls in the same time interval with a maximum of specific activity of lytic proteinase L2 and muramidase. The activity of concomitant enzymes--non-specific proteinase and neutral phosphatase--peaks in the middle of the logarithmic growth phase. Secretion of exopolysaccharide into the external medium begins in the second half of the logarithmic growth phase and, thus, is not coordinated with secretion of bacteriolytic enzymes. It is shown that relative content of enzymes and polysaccharides depends upon the composition of growth medium for the producer.

[Biogenesis and secretion of alkaline phosphatase and its mutant forms in Escherichia coli. II. Effect of replacing amino acids at the processing site and N-terminal domain of the mature polypeptide chain of alkaline phosphatase on its biogenesis]. / Izuchenie biogeneza i sekretsii shchelochnoi fosfatazy i ee mutantnykh form u Escherichia coli. II. Vliianie zamen aminokislot v saite protsessinga i N-kontsevom domene zreloi polipeptidnoi tsepi shchelochnoi fosfatazy na ee biogenez.

The effect of amino acid substitutions in E. coli alkaline phosphatase on its biogenesis has been studied. The substitution of Val for Ala(-1) in the signal peptide cleavage site completely inhibits all stages of posttranslational modification: processing and formation of isozymes. The absence of processing does not prevent translocation of the precursor across the cytoplasmic membrane and formation of an active enzyme macromolecule. The precursor of the above mutant protein was found in the periplasm and in the cytoplasmic membrane. The substitution of Gln for Glu(+4), as well as the double substitution of Ala for Arg(+1) and Gln for Glu(+4), in the N-terminus of mature polypeptide chain result in the change in the isozyme spectrum. Differences in the rates of processing in vivo of both mutant proteins were not revealed. However, the double amino acid substitution significantly increases the efficiency of in vitro processing. All amino acid substitutions studied have no effect on the peculiarities of biogenesis which are conditioned by oversynthesis of the enzyme encoded by the phoA gene in the plasmid: secretion into the culture medium and accumulation of precursor as insoluble aggregates in the cytoplasm. However, extracellular activities of mutant proteins differ from that of the wild-type protein, which may result from the change either in the efficiency of their secretion or in their catalytic properties.

[Biogenesis and secretion of alkaline phosphatase and its mutants in Escherichia coli. III. Substitution of N-terminal amino acids of alkaline phosphatase affect its biogenesis]. / Izuchenie biogeneza i sekretsii shchelochnoi fosfatazy i ee mutantnykhform u Escherichia coli. III. Zameny N-kontsevoi aminokisloty shchelochnoi fosfatazy vliiaiut na ee biogenez.

The effect of the N-terminal amino acid substitution on E. coli alkaline phosphatase biogenesis has been studied. The substitutions of Ser, Gln, Tyr, Leu, Gly, Ala, Glu, Phe, His, Cys, Lys and Pro for Arg(+1) were obtained by creating amber mutation at the corresponding position within phoA gene and expressing this mutated gene in E. coli strains that produce the amber-suppressor tRNAs. All mutant proteins were shown to translocate across the cytoplasmic membrane and possess enzyme activity. The introduction of Pro in +1 position disturbs the cleavage of signal peptide whereas the insertion of the other amino acids does not change the rates of processing in comparison with wild-type protein. All amino acid substitutions affect alkaline phosphatase isoenzyme composition. Some experimental evidence were also obtained on the specificity of protease, which split off N-terminal Arg during alkaline phosphatase maturation.

[Study of the biogenesis and secretion of alkaline phosphatase and its mutant forms in Escherichia coli. I. Introduction of directed mutations into the alkaline phosphatase gene]. / Izuchenie biogeneza i sekretsii shchelochnoi fosfatazy i ee mutantnykhform u Excherichia coli. I. Vvedenie napravlennykh mutatsii v gen shchelochnoi fosfatazy.

Various mutations in E. coli alkaline phosphatase gene were obtained by oligonucleotide-directed mutagenesis. They result in amino acid substitutions in the signal peptide cleavage site [Val for Ala(-1)] and in the N terminus of mature polypeptide chain: Ala for Arg(+1) and Gln for Glu(+4); Gln for Glu(+4). Enzyme activity was observed in all E. coli strains transformed by plasmids with cloned mutant genes. In addition, an amber mutation was introduced into the Arg(+1) position, and the synthesis of mutant alkaline phosphatase was shown in E. coli strains containing suppressor tRNAs specific for Ser, Gln, Tyr, Leu, Ala, Glu, Phe, Gly, His, Pro, and Cys.

[Features of the biogenesis of Escherichia coli alkaline phosphatase during its supersynthesis]. / Osobennosti biogeneza shchelochnoi fosfatazy Escherichia coli pri ee sverkhsinteze.

The oversynthesis of the secreted alkaline phosphatase (PhoA) in E.coli K12802 cells due to transformation with the PhoA+ plasmid pHI-7 leads to a change in its biogenesis--alternative localization and accumulation of the enzyme intermediate forms corresponding to different stages of the its post-translational modification. Instead of the soluble PhoA available in the parent strain mostly as a completely processed mature metazyme III localized in the periplasm, five enzyme forms were discovered in the PhoA overproducer: a cytoplasmic PhoA precursor (prePhoA) as insoluble aggregates; three soluble metazymes of a mature active form localized in the periplasm as in well as in culture medium; and a soluble high-molecular form in the periplasm. PrePhoA was isolated and purified by removal of soluble cell fractions using differential centrifugation, solubilization of membrane proteins with Triton X100, dissolution of the aggregates in the buffer with 8M urea and FPLC on MonoQ. Extracellular PhoA was purified by ultrafiltration, thermal treatment, and gel chromatography on Sepharose CL-4B. It was shown that the isolated prePhoA can be transformed into a mature form in the presence of a leader peptidase in 0.8 urea and is completely cleaved with proteinase K. Three forms of the mature PhoA vary in resistance to proteinase K and trypsin. Metazyme I, the unprocessed mature PhoA, is the most resistant to proteolysis.

[Secretion of periplasmic PhoA protein during its supersynthesis into the medium using outer membrane vesicles. Features of chemical composition of the vesicles and membranes of Escherichia coli secreting cells]. / Sekretsiia periplazmaticheskogo PhoA-belka pri ego sverkhsinteze v sredu s pomoshch'iu bezikul vneshnikh membran. Osobennosti khimicheskogo sostava vezikul i membran sekretirushchikh kletok Escherichia coli.

E. coli K12802 cells transformed by multicopy plasmid with phoA gene acquire the ability to oversynthesize alkaline phosphatase, secrete it into the cultural medium, and accumulate the precursor of this enzyme. The dynamics of enzyme production and secretion as well as cytomorphological changes revealed the existence of a mechanism of selective enzyme secretion into the medium. It is characterized by a decrease of enzyme specific activity in periplasm and its increase in cultural medium, appearance of numerous local zones of adhesion of cytoplasmic and outer membranes, formation of large extracellular outer membrane vesicles containing PhoA protein on the cell poles, and their release into the medium. We isolated the vesicles and found that they contain PhoA (in dominating quantity), several other periplasmic proteins, and matrix proteins of outer membranes. By their phospholipid and protein composition, they correspond to the fraction of outer membranes which have the largest density and sedimentation rate and, apparently, contain no lipoprotein.

[Transformation of various strains of Escherichia coli by multicopy plasmids with the phoA gene leads to secretion of a periplasmatic alkaline phosphatase into the media and accumulation of its precursor]. / Transformatsiia razlichnykh shtammov Escherichia coli mul'tikopiinymi plazmidami s genom phoA pribodit k sekretsii periplazmaticheskoi shchelochnoi fosfatazy v sredu i nakopleniiu ee predshestvennika.

Various E. coli strains have been transformed by multicopy plasmids pHI-1, pHI-7 and pPHO I carrying the entire regulatory and structural phoA sequences. All the transformants with the intact pho regulatory system have been shown to be capable of alkaline phosphatase oversynthesis and secretion into the medium. They are also able to accumulate the alkaline phosphatase precursor localized in the outer membrane fraction. The transformants of the restriction or recombination mutants are the most efficient producers of the extracellular enzyme and its precursor.