Substrate Access Mechanism in a Novel Membrane-Bound Phospholipase A of Pseudomonas aeruginosa Concordant with Specificity and Regioselectivity

Soil bacteria enter the digestive tract of wireworms at ecdysis through the dorsal exuvial split and ecdysial space. Pseudomonas aeruginosa, a potential pathogen of insects, multiplies in the enteron, but many are killed within it. The mucopeptide layer of the bacterial cell wall is affected early in the degenerative process.A surface epithelial mucoid layer provides a temporary protective barrier for the midgut epithelium against the bacteria. The bacteria affect the fine structure of the host midgut epithelium in three primary ways. (1) The cells take up and retain fluids in the cytoplasm and membrane-bound vacuoles to the point of apical rupture; the selective permeability of the membranes of the cell and vacuoles appears to be affected. (2) The surface mucoid covering is degraded, the exposed plasma membrane is disrupted, and a lytic erosion of the exposed cytoplasm occurs opposite bacterial colonies. Lesions thus form in the midgut epithelium, and could lead to perforation of the wall at these points. (3) Pathological changes occur in various cell organelles, the most striking of which are an increase in abundance of rough endoplasmic reticulum (RER) and cytoplasmic ribosomes, and a transposition of membrane material from mitochondria and Golgi complexes to RER. The functional implications of these pathological changes in fine structure are discussed.

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Purification and characterization of phosphotriesterases from Pseudomonas aeruginosa F10B and Clavibacter michiganense subsp. insidiosum SBL11

Canadian Journal of Microbiology ◽

10.1139/w05-113 ◽

2006 ◽

Vol 52 (2) ◽

pp. 157-168 ◽

Cited By ~ 10

Author(s):

Subhas Das ◽

Dileep Kumar Singh

Keyword(s):

Pseudomonas Aeruginosa ◽

Molecular Mass ◽

Half Life ◽

Indoleacetic Acid ◽

Bacterial Strains ◽

Purification And Characterization ◽

Temperature Optimum ◽

Microbial Biodegradation ◽

Membrane Bound

A microbial biodegradation of monocrotophos was studied in the present investigation. The monocrotophos-degrading enzyme was purified and characterized from two soil bacterial strains. The cells were disrupted and the membrane-bound fractions were studied for purification and characterization. Solubilization of the membrane-bound fractions released nearly 80% of the bound protein. Phase separation further enriched the enzyme fraction 34–41 times. The enzyme phosphotriesterase (PTE) from both the strains was purified to more than 1000-fold with 13%–16% yield. Purified PTE from Clavibacter michiganense subsp. insidiosum SBL11 is a monomeric enzyme with a molecular mass of 43.5 kDa (pI of 7.5), while PTE from Pseudomonas aeruginosa F10B is a heterodimeric enzyme with a molecular mass of 43 and 41 kDa (pI of 7.9 and 7.35). Both purified enzymes are stable enzymes with peak activity at pH 9.0. The enzyme from strain F10B was more thermostable (half-life = 7.3 h) than that from SBL11 (half-life = 6.4 h at 50 °C), while both showed the same temperature optimum of 37 °C. Inhibitors like dithiothreitol and EDTA inhibited the purified enzyme, while p-chloromercuribenzoic acid and indoleacetic acid had a very little effect.Key words: biodegradation, monocrotophos, phosphotriesterase, Pseudomonas aeruginosa F10B, Clavibacter michiganense subsp. insidiosum SBL11.

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A membrane‐bound esterase PA2949 from Pseudomonas aeruginosa is expressed and purified from Escherichia coli

FEBS Open Bio ◽

10.1002/2211-5463.12061 ◽

2016 ◽

Vol 6 (5) ◽

pp. 484-493 ◽

Cited By ~ 5

Author(s):

Filip Kovacic ◽

Florian Bleffert ◽

Muttalip Caliskan ◽

Susanne Wilhelm ◽

Joachim Granzin ◽

...

Keyword(s):

Escherichia Coli ◽

Pseudomonas Aeruginosa ◽

Membrane Bound

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A Study on the Effect of Lysolecithin and Phospholipase A on Membrane-Bound Galactosyltransferase

Canadian Journal of Biochemistry ◽

10.1139/o74-147 ◽

1974 ◽

Vol 52 (11) ◽

pp. 1053-1066 ◽

Cited By ~ 33

Author(s):

Sailen Mookerjea ◽

James W. M. Yung

Keyword(s):

Liver Microsomes ◽

Fatty Acyl ◽

Phospholipase A ◽

Synthetase Activity ◽

Rat Liver Microsomes ◽

Physiological Concentration ◽

Activation Effect ◽

Membrane Bound ◽

Hydrolysis Of

Addition of lysolecithin caused very marked activation of UDP-galactose:glycoprotein galactosyltransferase in rat liver microsomes and in Golgi-rich membranes. Lysolecithin activated galactosyltransferase when the enzyme was assayed both with endogenous acceptor and with exogenous proteins or monosaccharides as acceptors. Lactose synthetase activity in presence of α-lactalbumin was also stimulated by lysolecithin. Lecithin, lysophosphatidylethanolamine, lysophosphatidic acid, and glycerophosphorylcholine did not activate the enzyme, suggesting that both fatty acyl and phosphorylcholine groups of the lysolecithin molecule are required for the observed activation. The degree of activation was about the same when myristoyl-, palmitoyl-, oleoyl-, or stearoyllysolecithin were tested. The activation by lysolecithin was observed well within the physiological concentration of the lipid in the liver cell. Saturating amounts of Triton masked the effect of lysolecithin.Brief preincubation with phospholipase A activated the enzyme and generated lysolecithin in the membranes. Triton and lysolecithin activated the enzyme without any lag time, whereas phospholipase A activation was dependent on preincubation and also on an alkaline pH favorable for the hydrolysis of phospholipid. EDTA blocked the activation effect of phospholipase A but had no effect on activation by lysolecithin. Albumin and cholesterol opposed the effects of lysolecithin and phospholipase A on the enzyme. Two successive incubations of the microsomes with lysolecithin caused considerable release of the enzyme into the soluble fraction. The role of lysolecithin in the activation of the enzyme is probably related to the solubilization of the membrane and consequent enhanced interaction of the enzyme with substrate. Lysolecithin also activated N-acetylglucosaminyl- and sialyltransferase activities in microsomes. A possible role of lysolecithin is indicated in the regulation of glycosylation reactions in mammalian system.

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Free and membrane-bound forms of bacterial cytochrome c4

Biochemical Journal ◽

10.1042/bj2520427 ◽

1988 ◽

Vol 252 (2) ◽

pp. 427-435 ◽

Cited By ~ 28

Author(s):

G W Pettigrew ◽

K R Brown

Keyword(s):

Pseudomonas Aeruginosa ◽

Azotobacter Vinelandii ◽

Pseudomonas Stutzeri ◽

Growth Conditions ◽

Free Form ◽

Aerobic Growth ◽

Cytochromes C ◽

Membrane Bound ◽

Solubilized Form ◽

Attached Form

Cytochrome c4 was isolated from cells of Pseudomonas aeruginosa, Pseudomonas stutzeri and Azotobacter vinelandii. The dihaem nature, Mr of approx. 20,000 and ferrohaem spectra in the region of the alpha- and beta-peaks define this family of cytochromes c. The behaviour of the holocytochromes in SDS was atypical, but removal of the haem groups resulted in a normal migration. In all three organisms most of the cytochrome c4 was tightly bound to the membrane, but some free cytochrome was detected. The membrane-attached cytochrome could be extracted with butanol, and this solubilized form was then indistinguishable in properties from the free form. Denitrifying rather than aerobic growth conditions hardly affected the total cytochrome c4 in the two pseudomonads, but there was slightly more free form and less membrane-attached form in denitrifying growth. The nature of the attachment of cytochrome c4 to the membrane is discussed and a model is proposed for the process of solubilization.

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Phosphatidylinositol 4,5-Bisphosphate Is a Novel Coactivator of the Pseudomonas aeruginosa Cytotoxin ExoU

Infection and Immunity ◽

10.1128/iai.00414-13 ◽

2013 ◽

Vol 81 (8) ◽

pp. 2873-2881 ◽

Cited By ~ 23

Author(s):

Gregory H. Tyson ◽

Alan R. Hauser

Keyword(s):

Pseudomonas Aeruginosa ◽

Type Iii Secretion ◽

Eukaryotic Cell ◽

Secretion System ◽

Phospholipase A ◽

Effector Protein ◽

Yeast Mutant ◽

Membrane Phospholipids ◽

Content Type ◽

Ubiquitinated Proteins

ABSTRACTExoU is a potent phospholipase A2effector protein secreted by the type III secretion system ofPseudomonas aeruginosa. By cleaving plasma membrane phospholipids, it causes rapid lysis of eukaryotic cells. However, ExoU does not exhibit activity on its own but instead requires eukaryotic cell cofactors for activation. Ubiquitin and ubiquitinated proteins have been shown to activate ExoU, but previous work suggested that other cofactors are also involved. In this study, we demonstrate that phosphatidylinositol 4,5-bisphosphate [PI(4,5)P2] is another important coactivator of ExoU. PI(4,5)P2 works synergistically with ubiquitin to greatly enhance the phospholipase A2activity of ExoU. Distinct residues of ExoU were critical for activation by PI(4,5)P2 and by ubiquitin, indicating that these factors activate ExoU by discrete mechanisms. In support of the biological relevance of PI(4,5)P2 coactivation, a yeast mutant with reduced PI(4,5)P2 levels was less susceptible to the cytotoxic activity of ExoU. Together, these findings further elaborate the molecular mechanism of ExoU.

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Phospholipase A in Gram-negative bacteria and its role in pathogenesis

Microbiology ◽

10.1099/mic.0.28609-0 ◽

2006 ◽

Vol 152 (5) ◽

pp. 1263-1274 ◽

Cited By ~ 88

Author(s):

Taghrid S. Istivan ◽

Peter J. Coloe

Keyword(s):

Host Cell ◽

Virulence Factor ◽

Cell Invasion ◽

Phospholipase A ◽

Membrane Disruption ◽

Bacterial Invasion ◽

Host Cell Invasion ◽

Gram Negative Bacteria ◽

Gram Negative ◽

Membrane Bound

Phospholipase A (PLA) is one of the few enzymes present in the outer membrane of Gram-negative bacteria, and is likely to be involved in the membrane disruption processes that occur during host cell invasion. Both secreted and membrane-bound phospholipase A2 activities have been described in bacteria, fungi and protozoa. Recently there have been increasing reports on the involvement of PLA in bacterial invasion and pathogenesis. This review highlights the latest findings on PLA as a virulence factor in Gram-negative bacteria.

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