Absence of the Outer Membrane Phospholipase A Suppresses the Temperature-Sensitive Phenotype of Escherichia coli degPMutants and Induces the Cpx and ςE Extracytoplasmic Stress Responses

ABSTRACT DegP is a periplasmic protease that is a member of both the ςE and Cpx extracytoplasmic stress regulons ofEscherichia coli and is essential for viability at temperatures above 42°C. [U-14C]acetate labeling experiments demonstrated that phospholipids were degraded indegP mutants at elevated temperatures. In addition, chloramphenicol acetyltransferase, β-lactamase, and β-galactosidase assays as well as sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis indicated that large amounts of cellular proteins are released from degP cells at the nonpermissive temperature. A mutation in pldA, which encodes outer membrane phospholipase A (OMPLA), was found to rescue degPcells from the temperature-sensitive phenotype. pldA degP mutants had a normal plating efficiency at 42°C, displayed increased viability at 44°C, showed no degradation of phospholipids, and released far lower amounts of cellular protein to culture supernatants. degP and pldA degP mutants containing chromosomal lacZ fusions to Cpx and ςE regulon promoters indicated that both regulons were activated in the pldA mutants. The overexpression of the envelope lipoprotein, NlpE, which induces the Cpx regulon, was also found to suppress the temperature-sensitive phenotype ofdegP mutants but did not prevent the degradation of phospholipids. These results suggest that the absence of OMPLA corrects the degP temperature-sensitive phenotype by inducing the Cpx and ςE regulons rather than by inactivating the phospholipase per se.

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Assembly of Colicin A in the Outer Membrane of Producing Escherichia coli Cells Requires both Phospholipase A and One Porin, but Phospholipase A Is Sufficient for Secretion

Journal of Bacteriology ◽

10.1128/jb.184.13.3723-3733.2002 ◽

2002 ◽

Vol 184 (13) ◽

pp. 3723-3733 ◽

Cited By ~ 6

Author(s):

Daniele Cavard

Keyword(s):

Escherichia Coli ◽

Monoclonal Antibodies ◽

Outer Membrane ◽

Polyclonal Antibodies ◽

Sodium Dodecyl ◽

Phospholipase A ◽

Cell Fractionation ◽

Heat Labile ◽

Molecular Masses ◽

Outer Membrane Phospholipase A

ABSTRACT Three oligomeric forms of colicin A with apparent molecular masses of about 95 to 98 kDa were detected on sodium dodecyl sulfate (SDS)-polyacrylamide gels loaded with unheated samples from colicin A-producing cells of Escherichia coli. These heat-labile forms, called colicins Au, were visualized both on immunoblots probed with monoclonal antibodies against colicin A and by radiolabeling. Cell fractionation studies show that these forms of colicin A were localized in the outer membrane whether or not the producing cells contained the cal gene, which encodes the colicin A lysis protein responsible for colicin A release in the medium. Pulse-chase experiments indicated that their assembly into the outer membrane, as measured by their heat modifiable migration in SDS gels, was an efficient process. Colicins Au were produced in various null mutant strains, each devoid of one major outer membrane protein, except in a mutant devoid of both OmpC and OmpF porins. In cells devoid of outer membrane phospholipase A (OMPLA), colicin A was not expressed. Colicins Au were detected on immunoblots of induced cells probed with either polyclonal antibodies to OmpF or monoclonal antibodies to OMPLA, indicating that they were associated with both OmpF and OMPLA. Similar heat-labile forms were obtained with various colicin A derivatives, demonstrating that the C-terminal domain of colicin A, but not the hydrophobic hairpin present in this domain, was involved in their formation.

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Kinetic characterization of Escherichia coli outer membrane phospholipase A using mixed detergent-lipid micelles

Biochemistry ◽

10.1021/bi00429a031 ◽

1989 ◽

Vol 28 (3) ◽

pp. 1139-1147 ◽

Cited By ~ 39

Author(s):

Anton J. G. Horrevoets ◽

Tilman M. Hackeng ◽

Hubertus M. Verheij ◽

Ruud Dijkman ◽

Gerard H. De Haas

Keyword(s):

Escherichia Coli ◽

Outer Membrane ◽

Phospholipase A ◽

Kinetic Characterization ◽

Outer Membrane Phospholipase A ◽

Lipid Micelles

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Bacteriocin Release Protein Triggers Dimerization of Outer Membrane Phospholipase A In Vivo

Journal of Bacteriology ◽

10.1128/jb.181.10.3281-3283.1999 ◽

1999 ◽

Vol 181 (10) ◽

pp. 3281-3283 ◽

Cited By ~ 27

Author(s):

Niek Dekker ◽

Jan Tommassen ◽

Hubertus M. Verheij

Keyword(s):

Escherichia Coli ◽

Outer Membrane ◽

Cell Envelope ◽

Lag Time ◽

Phospholipase A ◽

Cross Linking ◽

Outer Membrane Phospholipase A ◽

Bacteriocin Release Protein ◽

Chemical Cross Linking

ABSTRACT Bacteriocin release protein is known to activate outer membrane phospholipase A (OMPLA), which results in the release of colicin fromEscherichia coli. In vivo chemical cross-linking experiments revealed that the activation coincides with dimerization of OMPLA. Permeabilization of the cell envelope and dimerization were characterized by a lag time of 2 h.

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Some Characteristics of the Outer Membrane Material Released by Growing Enterotoxigenic Escherichia coli

Infection and Immunity ◽

10.1128/iai.29.2.704-713.1980 ◽

1980 ◽

Vol 29 (2) ◽

pp. 704-713 ◽

Cited By ~ 1

Author(s):

Henk Gankema ◽

Jan Wensink ◽

Pieter A. M. Guinée ◽

Wim H. Jansen ◽

Bernard Witholt

Keyword(s):

Escherichia Coli ◽

Outer Membrane ◽

Polyacrylamide Gel Electrophoresis ◽

Sodium Dodecyl ◽

Freeze Fracture ◽

Sucrose Density Gradient ◽

Enterotoxigenic Escherichia Coli ◽

E Coli ◽

Freeze Fracture Electron Microscopy ◽

K 12

The high-molecular-weight material released into the medium by Escherichia coli AP1, an enterotoxigenic strain of porcine origin, has been isolated and resolved into two clearly distinct fractions, based on sucrose density gradient and differential centrifugation, chemical analysis, sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and freeze-fracture electron microscopy. These two fractions, referred to as “medium vesicles” and “medium lipopolysaccharides”, were compared with the cellular outer and cytoplasmic membranes, the periplasmic fraction, and the cytoplasmic fraction. The medium vesicles closely resembled outer membrane and accounted for 3 to 5% of the total cellular outer membrane. They contained most of the heat-labile enterotoxin (LT) activity released into the medium by E. coli AP1. The medium lipopolysaccharide consisted mostly of lipopolysaccharide and a small amount of outer membrane and contained relatively little LT activity. Based on experiments with E. coli K-12 strains, in which about 5% of the newly synthesized outer membrane is lost from areas of outer membrane synthesis, it is proposed that enterotoxigenic E. coli strains release LT as part of such newly synthesized outer membrane fragments and that released outer membrane fragments may function as physiologically significant LT carriers.

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Structural investigations of calcium binding and its role in activity and activation of outer membrane phospholipase A from Escherichia coli

Journal of Molecular Biology ◽

10.1006/jmbi.2001.4675 ◽

2001 ◽

Vol 309 (2) ◽

pp. 477-489 ◽

Cited By ~ 22

Author(s):

H.J Snijder ◽

R.L Kingma ◽

K.H Kalk ◽

N Dekker ◽

M.R Egmond ◽

...

Keyword(s):

Escherichia Coli ◽

Outer Membrane ◽

Calcium Binding ◽

Phospholipase A ◽

Structural Investigations ◽

Outer Membrane Phospholipase A

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Role of Porins in Sensitivity of Escherichia coli to Antibacterial Activity of the Lactoperoxidase Enzyme System

Applied and Environmental Microbiology ◽

10.1128/aem.71.7.3512-3518.2005 ◽

2005 ◽

Vol 71 (7) ◽

pp. 3512-3518 ◽

Cited By ~ 18

Author(s):

Philipp De Spiegeleer ◽

Jan Sermon ◽

Kristof Vanoirbeek ◽

Abram Aertsen ◽

Chris W. Michiels

Keyword(s):

Escherichia Coli ◽

Membrane Permeability ◽

Outer Membrane ◽

Enzyme System ◽

Polyacrylamide Gel Electrophoresis ◽

Sodium Dodecyl ◽

Core Oligosaccharide ◽

Nitrophenyl Phosphate ◽

Oligosaccharide Moiety ◽

Outer Membrane Permeability

ABSTRACT Lactoperoxidase is an enzyme that contributes to the antimicrobial defense in secretory fluids and that has attracted interest as a potential biopreservative for foods and other perishable products. Its antimicrobial activity is based on the formation of hypothiocyanate (OSCN−) from thiocyanate (SCN−), using H2O2 as an oxidant. To gain insight into the antibacterial mode of action of the lactoperoxidase enzyme system, we generated random transposon insertion mutations in Escherichia coli MG1655 and screened the resultant mutants for an altered tolerance of bacteriostatic concentrations of this enzyme system. Out of the ca. 5,000 mutants screened, 4 showed significantly increased tolerance, and 2 of these had an insertion, one in the waaQ gene and one in the waaO gene, whose products are involved in the synthesis of the core oligosaccharide moiety of lipopolysaccharides. Besides producing truncated lipopolysaccharides and displaying hypersensitivity to novobiocin and sodium dodecyl sulfate (SDS), these mutants were also shown by urea-SDS-polyacrylamide gel electrophoresis analysis to have reduced amounts of porins in their outer membranes. Moreover, they showed a reduced degradation of p-nitrophenyl phosphate and an increased resistance to ampicillin, two indications of a decrease in outer membrane permeability for small hydrophilic solutes. Additionally, ompC and ompF knockout mutants displayed levels of tolerance to the lactoperoxidase system similar to those displayed by the waa mutants. These results suggest that mutations which reduce the porin-mediated outer membrane permeability for small hydrophilic molecules lead to increased tolerance to the lactoperoxidase enzyme system because of a reduced uptake of OSCN−.

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