Stable periplasmic secretion intermediate in the general secretory pathway of Escherichia coli.

Poquet, Isabelle; Faucher, Didier; Pugsley, Anthony P.

doi:10.1002/j.1460-2075.1993.tb05653.x

Cited by 46 publications

(48 citation statements)

References 41 publications

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“…Bacterial strains, plasmids and growth medium The E. coli strains used and their relevant characteristics were PAP7336 (⌬lacZ malT c FЈ(lacI q Tn10 )); PAP105 (⌬lacZ FЈ(lacI q Tn10 )); and HN594 (⌬unc ⌬malB13 ) (Davidson and Nikaido, 1990) carrying an FЈ lacI q ; all strains carried pCHAP710 (Kornacker and Pugsley, 1989) and pCHAP1106 (Poquet et al, 1993). The bacteria were grown at 30ЊC with vigorous shaking in LB broth (Miller, 1972) or in minimal medium supplemented with all amino acids except methionine (Poquet et al, 1993) and containing 0.4% glycerol.…”

Section: Methodsmentioning

confidence: 99%

“…In order to study the second step in pullulanase secretion independently of the first (Sec-dependent) step, and to facilitate experimental analysis, we used strains of E. coli carrying pCHAP1106, which encodes a soluble (non fattyacylated) form of pullulanase (Poquet et al, 1993), and pCHAP710 (Kornacker and Pugsley, 1989), which encodes all of the factors necessary for pullulanase secretion. The soluble pullulanase is efficiently exported across the cytoplasmic membrane but accumulates in the periplasm because its transport across the outer membrane is retarded compared to the wild-type protein (due to the absence of the lipids) (Poquet et al, 1993) and because the high level of production that occurs after IPTG induction saturates the secreton.…”

Section: Experimental Strategymentioning

confidence: 99%

“…The soluble pullulanase is efficiently exported across the cytoplasmic membrane but accumulates in the periplasm because its transport across the outer membrane is retarded compared to the wild-type protein (due to the absence of the lipids) (Poquet et al, 1993) and because the high level of production that occurs after IPTG induction saturates the secreton. This allows the two secretion steps to be uncoupled.…”

Section: Experimental Strategymentioning

confidence: 99%

“…This allows the two secretion steps to be uncoupled. Furthermore, the soluble pullulanase is released directly into the medium rather than being anchored to the cell surface like the wild-type pullulanase (Poquet et al, 1993), which simplifies the otherwise complicated kinetic analysis of secretion (Pugsley, 1992). The strains used either carry a malT c allele that allows constitutive expression of the malT-regulated pul secretion genes carried by pCHAP710, or were grown in medium containing maltose to induce the expression of these genes.…”

Section: Experimental Strategymentioning

confidence: 99%

“…The sensitivity of the assay does not permit the immediate effects of CCCP to be determined. We therefore used a pulse-chase assay (Poquet et al, 1993) in which IPTG-induced cells of strain PAP7336 (pCHAP710 pCHAP1106) in minimal medium were pulselabelled with […”

Section: Cccp Reversibly Inhibits Pullulanase Secretionmentioning

confidence: 99%

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Energy requirement for pullulanase secretion by the main terminal branch of the general secretory pathway

Possot

Letellìer

Pugsley

1997

Molecular Microbiology

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SummaryThe energy requirement for the second step in pullulanase secretion by the general secretory pathway was studied in Escherichia coli. In order to uncouple the two steps in the secretion pathway (across the cytoplasmic and outer membranes, respectively) and to facilitate kinetic analysis of secretion, a variant form of pullulanase lacking its N-terminal fatty acid membrane anchor was used. The transport of the periplasmic secretion intermediate form of this protein across the outer membrane was not inhibited by concentrations of sodium arsenate in excess of those required to reduce ATP levels to Յ10% of their normal value. Pullulanase secretion was inhibited by the protonophore carbonyl cyanide m-chlorophenyl hydrazone at concentrations which were similar to those reported by others to be required to prevent solute uptake or the export and processing of preproteins across the cytoplasmic membrane, but which were in excess of those required to fully dissipate the proton-motive force and to reduce lactose uptake to a significant extent.

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Section: Methodsmentioning

confidence: 99%

Section: Experimental Strategymentioning

confidence: 99%

Section: Experimental Strategymentioning

confidence: 99%

Section: Experimental Strategymentioning

confidence: 99%

Section: Cccp Reversibly Inhibits Pullulanase Secretionmentioning

confidence: 99%

See 3 more Smart Citations

Energy requirement for pullulanase secretion by the main terminal branch of the general secretory pathway

Possot

Letellìer

Pugsley

1997

Molecular Microbiology

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The secretion pathway of IgA protease‐type proteins in gram‐negative bacteria

1993

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The pathogenic, Gram-negative bacteria, Neisseria gonorrhoeae, Neisseria meningitidis and Haemophilus influenzae, secrete immunoglobulin A1 proteases into their extracellular surroundings. An extraordinary feature in the secretory pathway of these putative virulence factors is a self-directed outer membrane transport step allowing the proteins to be secreted autonomously, even from foreign Gram-negative host cells like Escherichia coli. Here we summarize recent achievements in the understanding of IgA protease outer membrane translocation.

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A specific targeting domain in mature exotoxin A is required for its extracellular secretion from Pseudomonas aeruginosa.

Lory

1996

The EMBO Journal

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A number of Gram‐negative bacteria, including Pseudomonas aeruginosa, actively secrete a subset of periplasmic proteins into their surrounding medium. The presence of a putative extracellular targeting signal within one such protein, exotoxin A, was investigated. A series of exotoxin A truncates, fused to beta‐lactamase, was constructed. Hybrid proteins, which carry at their N‐ termini 120, 255, 355 or the entire 613 residues of the mature exotoxin A, were stable and were secreted into the extracellular medium. Hybrid proteins which carry residues 1–30 and 1–60 of the mature exotoxin A were unstable; however, they could be detected entirely within the cells after a short labeling period. A hybrid with beta‐lactamase was constructed which carried only the N‐terminal residues 1–3 and region 60–120 of exotoxin A. It was also secreted into the culture medium, suggesting that a specific 60 amino acid domain contains the necessary targeting information for translocation of exotoxin A across the outer membrane. The secretion of the hybrid proteins is independent of the passenger protein, since a similar exotoxin A‐murine interleukin 4 hybrid protein was also secreted. The extracellular targeting signal between amino acids 60 and 120 is rich in anti‐parallel beta‐sheets. It has been shown previously to be involved in the interaction of the exotoxin A with the receptors of the eukaryotic cells. In the three‐ dimensional view, the targeting region is on the toxin surface where it is easily accessible to the components of the extracellular secretion machinery.

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Stable periplasmic secretion intermediate in the general secretory pathway of Escherichia coli.

Cited by 46 publications

References 41 publications

Energy requirement for pullulanase secretion by the main terminal branch of the general secretory pathway

Energy requirement for pullulanase secretion by the main terminal branch of the general secretory pathway

The secretion pathway of IgA protease‐type proteins in gram‐negative bacteria

A specific targeting domain in mature exotoxin A is required for its extracellular secretion from Pseudomonas aeruginosa.

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