Mutations that alter the signal sequence of alkaline phosphatase in Escherichia coli

Michaelis, S; Inouye, Hiroshi; Oliver, Donald B.; Beckwith, Jon

doi:10.1128/jb.154.1.366-374.1983

Cited by 281 publications

(67 citation statements)

References 22 publications

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“…However, the protocol used here maintained isotonic conditions, and caused less than 5% lysis as determined by GDH levels in the periplasmic fraction. These levels of spheroplast lysis in isotonic solutions are consistent with the reports of 6 10% lysis by other workers [14,18,20]. This does not fully explain the 100% periplasmic localization described by Heimberger and Eisenstark [7], but may provide a partial explanation.…”

Section: Discussionsupporting

confidence: 86%

“…Similarly, it was necessary to have a control con¢rming that a signi¢cant proportion of the cells had been converted to protoplasts. Alkaline phosphatase is found largely in the periplasm [18] and 67% of its activity was released during the protoplasting procedure con¢rming that at least that percentage of the cells had been converted to protoplasts.…”

Section: Catalase Levels In Spheroplast Fractionsmentioning

confidence: 96%

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Intracellular location of catalase-peroxidase hydroperoxidase I of Escherichia coli

Hillar

Caeseele

Loewen

1999

FEMS Microbiology Letters

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The catalase-peroxidase hydroperoxidase I of Escherichia coli has been confirmed to be located in the cytoplasm using two independent methods. Catalase activity was found predominantly (> 95%) in the cytoplasmic fraction following spheroplast formation. The cytoplasmic enzyme glucose-6-phosphate dehydrogenase and the periplasmic enzyme alkaline phosphatase were used as controls. The second method of immunogold staining for the enzyme in situ revealed an even distribution of the enzyme across the cell.

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Section: Discussionsupporting

confidence: 86%

Section: Catalase Levels In Spheroplast Fractionsmentioning

confidence: 96%

Intracellular location of catalase-peroxidase hydroperoxidase I of Escherichia coli

Hillar

Caeseele

Loewen

1999

FEMS Microbiology Letters

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“…novicida strains were transformed with plasmids containing protein phoA fusions listed in Table S1, grown in TSAP supplemented with appropriate antibiotics at 37°C, and harvested at an optical density of 600 nm of c. 0.3-0.4. Bacterial cells were permeabilized with chloroform and sodium dodecyl sulfate (SDS) and assayed for alkaline phosphatase activity by the method described by Michaelis (Michaelis et al, 1983). Statistical significance was determined using Student's t-test.…”

Section: B-galactosidase and Alkaline Phosphatase Assaysmentioning

confidence: 99%

Lipidation of the FPI protein IglE contributes toFrancisella tularensisssp.novicidaintramacrophage replication and virulence

et al. 2014

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Francisella tularensis is a Gram-negative bacterium responsible for the human disease tularemia. The Francisella pathogenicity island (FPI) encodes a secretion system related to type VI secretion systems (T6SS) which allows F. tularensis to escape the phagosome and replicate within the cytosol of infected macrophages and ultimately cause disease. A lipoprotein is typically found encoded within T6SS gene clusters and is believed to anchor portions of the secretion apparatus to the outer membrane. We show that the FPI protein IglE is a lipoprotein that incorporates 3H-palmitate and localizes to the outer membrane. A C22G IglE mutant failed to be lipidated and failed to localize to the outer membrane, consistent with C22 being the site of lipidation. Francisella tularensis ssp. novicida expressing IglE C22G is defective for replication in macrophages and unable to cause disease in mice. Bacterial two-hybrid analysis demonstrated that IglE interacts with the C-terminal portion of the FPI inner membrane protein PdpB, and PhoA fusion analysis indicated the PdpB C-terminus is located within the periplasm. We predict this interaction facilitates channel formation to allow secretion through this system.

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“…The Prl phenotype of the suppressors was scored on McConkey plates supplemented with 1% maltose. Alkaline phosphatase assay AP activity was measured by determining the rate of p-nitro-phenylphosphate (Sigma) hydrolysis in permeabilized cells, and was normalized to the A6W of the cell suspension (Michaelis et al, 1983). The rate of chimeric PAI-2-AP protein synthesis was determined by 30 s pulselabellings with [35S]-methionine and immunoprecipitation, and it was normalized to that of OmpA or of Bla.…”

Section: Reagentsmentioning

confidence: 99%

A new genetic selection identifies essential residues in SecG, a component of the Escherichia coli protein export machinery.

Bost

Belin

1995

The EMBO Journal

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The signal sequence of the murine serine protease inhibitor PAI‐2 promotes alkaline phosphatase export to the E. coli periplasm. However, high level expression of this chimeric protein interferes with cell growth. Since most suppressors of this toxic phenotype map to secA and secY, growth arrest results from a defective interaction of the chimeric protein with the export machinery. We have characterized suppressors which map in secG, a newly defined gene of the export machinery. All single amino acid substitutions map to three adjacent codons. These secG mutants have a weak Sec phenotype, as determined by their effect on export mediated by wild‐type and mutant signal sequences. Whilst a secG disruption allele also confers a weak Sec phenotype, it does not suppress the toxicity of the chimeric protein. This difference results from a selective effect of the secG suppressors on the kinetics of export mediated by the PAI‐2 signal sequence. Using a malE signal sequence mutant, which has a Mal‐phenotype in secG mutant strains, we have isolated extragenic Mal+ suppressors. Most suppressors map to secY, and several are allele‐specific. Finally, SecG overexpression accelerates the kinetics of protein export, suggesting that there are two types of functional translocation complexes: with or without SecG.

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Mutations that alter the signal sequence of alkaline phosphatase in Escherichia coli

Cited by 281 publications

References 22 publications

Intracellular location of catalase-peroxidase hydroperoxidase I of Escherichia coli

Intracellular location of catalase-peroxidase hydroperoxidase I of Escherichia coli

Lipidation of the FPI protein IglE contributes toFrancisella tularensisssp.novicidaintramacrophage replication and virulence

A new genetic selection identifies essential residues in SecG, a component of the Escherichia coli protein export machinery.

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