Ferrichrome transport in inner membrane vesicles of Escherichia coli K12.

Recent evidence suggests that in Escherichia coli, SecA/SecB and signal recognition particle (SRP) are constituents of two different pathways targeting secretory and inner membrane proteins to the SecYEG translocon of the plasma membrane. We now show that a secY mutation, which compromises a functional SecY–SecA interaction, does not impair the SRP-mediated integration of polytopic inner membrane proteins. Furthermore, under conditions in which the translocation of secretory proteins is strictly dependent on SecG for assisting SecA, the absence of SecG still allows polytopic membrane proteins to integrate at the wild-type level. These results indicate that SRP-dependent integration and SecA/SecB-mediated translocation do not only represent two independent protein delivery systems, but also remain mechanistically distinct processes even at the level of the membrane where they engage different domains of SecY and different components of the translocon. In addition, the experimental setup used here enabled us to demonstrate that SRP-dependent integration of a multispanning protein into membrane vesicles leads to a biologically active enzyme.

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Repression of the Inner Membrane Lipoprotein NlpA by Rns in Enterotoxigenic Escherichia coli

Journal of Bacteriology ◽

10.1128/jb.01714-06 ◽

2006 ◽

Vol 189 (5) ◽

pp. 1627-1632 ◽

Cited By ~ 23

Author(s):

Maria D. Bodero ◽

M. Carolina Pilonieta ◽

George P. Munson

Keyword(s):

Escherichia Coli ◽

Outer Membrane ◽

Membrane Vesicles ◽

Outer Membrane Vesicles ◽

Enterotoxigenic Escherichia Coli ◽

Start Codon ◽

Inner Membrane ◽

E Coli ◽

Inner Membrane Protein

ABSTRACT The expression of the inner membrane protein NlpA is repressed by the enterotoxigenic Escherichia coli (ETEC) virulence regulator Rns, a member of the AraC/XylS family. The Rns homologs CfaD from ETEC and AggR from enteroaggregative E. coli also repress expression of nlpA. In vitro DNase I and potassium permanganate footprinting revealed that Rns binds to a site overlapping the start codon of nlpA, preventing RNA polymerase from forming an open complex at nlpAp. A second Rns binding site between positions −152 and −195 relative to the nlpA transcription start site is not required for repression. NlpA is not essential for growth of E. coli under laboratory conditions, but it does contribute to the biogenesis of outer membrane vesicles. As outer membrane vesicles have been shown to contain ETEC heat-labile toxin, the repression of nlpA may be an indirect mechanism through which the virulence regulators Rns and CfaD limit the release of toxin.

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A Lysine Substitute for K+

Journal of Biological Chemistry ◽

10.1074/jbc.m210341200 ◽

2002 ◽

Vol 277 (51) ◽

pp. 49651-49654 ◽

Cited By ~ 67

Author(s):

Georgiy A. Belogurov ◽

Reijo Lahti

Keyword(s):

Escherichia Coli ◽

Heterologous Expression ◽

Phylogenetic Analyses ◽

Functional Characterization ◽

Membrane Vesicles ◽

Thermophilic Bacterium ◽

Inorganic Pyrophosphatase ◽

Inner Membrane ◽

Carboxydothermus Hydrogenoformans

The H+proton-translocating inorganic pyrophosphatase (H+-PPase) family is composed of two phylogenetically distinct types of enzymes: K+-dependent and K+-independent. However, to date, the sequence criteria governing this dichotomy have remained unknown. In this study, we describe the heterologous expression and functional characterization of H+-PPase from the thermophilic bacteriumCarboxydothermus hydrogenoformans. Both PPi-hydrolyzing and PPi-energized H+translocation activities of the recombinant enzyme inEscherichia coliinner membrane vesicles are strictly K+-dependent. Here we deduce the K+requirement of all available H+-PPase sequences based on the K+dependence ofC. hydrogenoformansH+-PPase in conjunction with phylogenetic analyses. Our data reveal that K+-independent H+-PPases possess conserved Lys and Thr that are absent in K+-dependent H+-PPases. We further demonstrate that a A460K substitution inC. hydrogenoformansH+-PPase is sufficient to confer K+independence to both PPihydrolysis and PPi-energized H+translocation. In contrast, a A463T mutation does not affect the K+dependence of H+-PPase.

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