Coupling site-directed mutagenesis with high-level expression: large scale production of mutant porins from<i>E. coli</i>

Prilipov, Alexej; Phale, Prashant S.; Gelder, Patrick Van; Rosenbusch, Jürg P.; Koebnik, Ralf

doi:10.1111/j.1574-6968.1998.tb13027.x

Cited by 214 publications

(113 citation statements)

References 26 publications

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“…Finally, LamB protein was added in excess of the 5:1 LamB͞ ratio shown to be sufficient to open all of the phage. This ratio was established by adding LamB until we saw no further effect on the efficiency of DNA ejection (similar values for this ratio were determined elsewhere (13,17); an octylpolyoxyethylene concentration of 1% by volume was sufficient for all of the LamB to be solubilized (18). The samples were first incubated at 25°C for 15 min to allow phage binding to LamB, followed by DNA ejection (which should be complete within 1 min; ref.…”

Section: Methodsmentioning

confidence: 99%

Osmotic pressure inhibition of DNA ejection from phage

Evilevitch

Lavelle

Knobler

et al. 2003

Proc. Natl. Acad. Sci. U.S.A.

302

381

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Bacterial viral capsids in aqueous solution can be opened in vitro by addition of their specific receptor proteins, with consequent full ejection of their genomes. We demonstrate that it is possible to control the extent of this ejection by varying the external osmotic pressure. In the particular case of bacteriophage , the ejection is 50% inhibited by osmotic pressures (of polyethylene glycol) comparable to those operative in the cytoplasm of host bacteria; it is completely suppressed by a pressure of 20 atmospheres. Furthermore, our experiments monitor directly a dramatic decrease of the stress inside the unopened phage capsid upon addition of polyvalent cations to the host solution, in agreement with many recent theories of DNA interactions.V iral capsids are often highly pressurized. In the case of double-stranded DNA bacteriophages, for example, there are large forces acting to push the genome out through the tail of the viral particle. Recent experiments (1) and theory (2, 3) have established that the forces inside these capsids reach very high values (of order 50 pN) when the genomes are fully packaged. These forces are estimated to correspond to pressures of the order of 50 atm (1 atm ϭ 101.3 kPa; refs. 1-4). Such high values of stress result from the DNA being strongly bent and confined. Specifically, the inner diameter of the capsid is comparable to the DNA persistence length (50 nm), and the typical interaxial spacings are small enough (2.5 nm; ref. 5) that neighboring chains experience strong repulsions. It is precisely these forces that drive the ejection of the genome into the host cell after the tail of the capsid has been bound to its receptor protein in the outer cell membrane; equivalently, it is these forces that must be exerted by the motor protein responsible for packaging of the viral genome (1).Previous in vitro experiments have demonstrated that DNA ejection is fast (occurring on a time scale of seconds) and complete when phage capsids are opened by their receptors in aqueous solution (6). The capsids are permeable to water and salt ions, so there is no difference in hydrostatic pressure between the inside and outside of the capsid, and osmotic equilibrium is also maintained (7). The pressure difference between the capsid and the solution is therefore associated only with the confinement of the DNA. When the capsid is opened, ejection proceeds until this pressure difference falls to zero. Clearly, at this point the force driving the ejection has also dropped to zero. For the in vivo situation, however, ejection does not occur into a simple salt solution. The DNA is ejected into a highly concentrated colloidal suspension, the cell cytoplasm, which is characterized by high concentrations of proteins and other macromolecular structures. In general, then, one expects this colloidal solution to give rise to a force that resists the entry of DNA (8). More explicitly, because the ejection force associated with stress inside the capsid drops monotonically as the ejection proceeds, there will be a...

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Section: Methodsmentioning

confidence: 99%

Osmotic pressure inhibition of DNA ejection from phage

Evilevitch

Lavelle

Knobler

et al. 2003

Proc. Natl. Acad. Sci. U.S.A.

302

381

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“…Strains, Plasmids, and Growth Conditions-E. coli strains DH5␣ (24) and the multi-porin mutant BL21(DE3)Omp8 (25) were used for plasmid construction and PapC purification, respectively. Bacteria were grown in LB broth containing 100 g/ml ampicillin at 37°C with aeration.…”

Section: Methodsmentioning

confidence: 99%

Modulating Effects of the Plug, Helix, and N- and C-terminal Domains on Channel Properties of the PapC Usher

Mapingire

Henderson

Duret

et al. 2009

Journal of Biological Chemistry

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The chaperone/usher system is one of the best characterized pathways for protein secretion and assembly of cell surface appendages in Gram-negative bacteria. In particular, this pathway is used for biogenesis of the P pilus, a key virulence factor used by uropathogenic Escherichia coli to adhere to the host urinary tract. The P pilus individual subunits bound to the periplasmic chaperone PapD are delivered to the outer membrane PapC usher, which serves as an assembly platform for subunit incorporation into the pilus and secretion of the pilus fiber to the cell surface. PapC forms a dimeric, twin pore complex, with each monomer composed of a 24-stranded transmembrane ␤-barrel channel, an internal plug domain that occludes the channel, and globular N-and C-terminal domains that are located in the periplasm. Here we have used planar lipid bilayer electrophysiology to characterize the pore properties of wild type PapC and domain deletion mutants for the first time. The wild type pore is closed most of the time but displays frequent short-lived transitions to various open states. In comparison, PapC mutants containing deletions of the plug domain, an ␣-helix that caps the plug domain, or the N-and C-terminal domains form channels with higher open probability but still exhibiting dynamic behavior. Removal of the plug domain results in a channel with extremely large conductance. These observations suggest that the plug gates the usher channel closed and that the periplasmic domains and ␣-helix function to modulate the gating activity of the PapC twin pore.The cell envelope of Gram-negative bacteria contains a vast array of protein machineries dedicated to the translocation of polypeptides across the cytoplasmic membrane, periplasm, and outer membrane (OM) 3 (1, 2). Some of these complexes also participate in the assembly of surface-exposed appendages, such as flagella and pili (fimbriae). One of the most thoroughly studied secretion systems is the chaperone/usher pathway, responsible for the biogenesis of a superfamily of virulenceassociated surface structures, including P and type 1 pili (3). These pili play essential roles in the pathogenesis of uropathogenic Escherichia coli by providing a tool for attachment of the bacteria to host urothelial cells (4 -6). P pili, encoded by the chromosomal pap gene cluster, are critical virulence factors for infection of the kidney by uropathogenic E. coli and the development of pyelonephritis. The P pilus is composed of multiple subunits of PapA, which form a rigid helical rod. A thin linear tip fibrillum is located at the distal end of the pilus and is made of four different subunits (PapK, PapF, PapE, and the adhesin PapG) that assemble in a precise order and stoichiometry (3). The minor pilin, PapH, anchors the pilus rod to the cell surface (7).The pilus subunits are first translocated through the cytoplasmic membrane via the Sec general secretory pathway (8). Once in the periplasm, the subunits form binary complexes with the PapD chaperone. The details of the binding interact...

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“…FhuA and FecA were individually mutated to cysteine by using the QuikChange Site-Directed Mutagenesis Kit (Stratagene, La Jolla, CA) to attach a thiol-reactive spin label. Proteins containing a single cysteine mutation were overexpressed in an E. coli BL21(DE3) strain lacking all of the outer membrane porins such as OmpA, OmpC, and LamB (40). Cells were grown in LB media containing 0.1 mg/ml ampicillin, and the transcription of FhuA and FecA was initiated by the addition of isopropyl-␤-D-thiogalactoside.…”

Section: Methodsmentioning

confidence: 99%

Substrate-dependent transmembrane signaling in TonB-dependent transporters is not conserved

Kim

Fanucci

Cafiso

2007

Proc. Natl. Acad. Sci. U.S.A.

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Site-directed spin labeling (SDSL) was used to examine and compare transmembrane signaling events in the bacterial outermembrane transport proteins BtuB, FecA, and FhuA. These proteins extract energy for transport by coupling to the transperiplasmic protein TonB, an interaction that is thought to be mediated by the Ton box, a highly conserved energy-coupling motif in these transporters. In the ferric citrate transporter, FecA, SDSL indicates that the Ton box undergoes a substrate-induced disorder transition similar to that seen for BtuB, the vitamin B12 transporter. This conformational change produces an aqueous exposed, highly disordered protein fragment, which likely regulates transporter-TonB interactions. However, in the ferrichrome transporter, FhuA, SDSL does not reveal a substrate-induced unfolding transition. In this protein, with or without substrate, the Ton box conformation is found to be highly dynamic and constitutively unfolded. In addition, SDSL indicates that structural features seen in high-resolution models are not found in membrane-associated FhuA. Taken together, these data indicate that the Ton box of FhuA may always be available for interactions with TonB, implying that transporterTonB interactions in FhuA are either constitutive or not regulated by the Ton box configuration.EPR spectroscopy ͉ membrane protein ͉ site-directed spin labeling

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Coupling site-directed mutagenesis with high-level expression: large scale production of mutant porins fromE. coli

Cited by 214 publications

References 26 publications

Osmotic pressure inhibition of DNA ejection from phage

Osmotic pressure inhibition of DNA ejection from phage

Modulating Effects of the Plug, Helix, and N- and C-terminal Domains on Channel Properties of the PapC Usher

Substrate-dependent transmembrane signaling in TonB-dependent transporters is not conserved

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