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Repression of the inner membrane lipoprotein NlpA by Rns in enterotoxigenic Escherichia coli.

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.

SUBMITTER: Bodero MD

PROVIDER: S-EPMC1855764 | biostudies-literature | 2007 Mar

REPOSITORIES: biostudies-literature

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Repression of the inner membrane lipoprotein NlpA by Rns in enterotoxigenic Escherichia coli.

Bodero Maria D MD Pilonieta M Carolina MC Munson George P GP

Journal of bacteriology 20061222 5

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 ...[more]

PMID: 17189357

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Project description:Biological membranes are essential for cell viability. Their functional characteristics strongly depend on their protein content, which consists of transmembrane (integral) and peripherally associated membrane proteins. Both integral and peripheral inner membrane proteins mediate a plethora of biological processes. Whereas transmembrane proteins have characteristic hydrophobic stretches and can be predicted using bioinformatics approaches, peripheral inner membrane proteins are hydrophilic, exist in equilibria with soluble pools, and carry no discernible membrane targeting signals. We experimentally determined the cytoplasmic peripheral inner membrane proteome of the model organism Escherichia coli using a multidisciplinary approach. Initially, we extensively re-annotated the theoretical proteome regarding subcellular localization using literature searches, manual curation, and multi-combinatorial bioinformatics searches of the available databases. Next we used sequential biochemical fractionations coupled to direct identification of individual proteins and protein complexes using high resolution mass spectrometry. We determined that the proposed cytoplasmic peripheral inner membrane proteome occupies a previously unsuspected ?19% of the basic E. coli BL21(DE3) proteome, and the detected peripheral inner membrane proteome occupies ?25% of the estimated expressed proteome of this cell grown in LB medium to mid-log phase. This value might increase when fleeting interactions, not studied here, are taken into account. Several proteins previously regarded as exclusively cytoplasmic bind membranes avidly. Many of these proteins are organized in functional or/and structural oligomeric complexes that bind to the membrane with multiple interactions. Identified proteins cover the full spectrum of biological activities, and more than half of them are essential. Our data suggest that the cytoplasmic proteome displays remarkably dynamic and extensive communication with biological membrane surfaces that we are only beginning to decipher.

Project description:Nearly a quarter of the Escherichia coli genome encodes for inner membrane proteins of which approximately a third have unassigned or poorly understood function. We had previously demonstrated that the synergy between the functional roles of the inner membrane-spanning YciB and the inner membrane lipoprotein DcrB, is essential in maintaining cell envelope integrity. In yciB dcrB cells, the abundant outer membrane lipoprotein, Lpp, mislocalizes to the inner membrane where it forms toxic linkages to peptidoglycan. Here, we report that the aberrant localization of Lpp in this double mutant is due to inefficient lipid modification at the first step in lipoprotein maturation. Both Cpx and Rcs signaling systems are upregulated in response to the envelope stress. The phosphatidylglycerol-pre-prolipoprotein diacylglyceryl transferase, Lgt, catalyzes the initial step in lipoprotein maturation. Our results suggest that the attenuation in Lgt-mediated transacylation in the double mutant is not a consequence of lowered phosphatidylglycerol levels. Instead, we posit that altered membrane fluidity, perhaps due to changes in lipid homeostasis, may lead to the impairment in Lgt function. Consistent with this idea, a dcrB null is not viable when grown at low temperatures, conditions which impact membrane fluidity. Like the yciB dcrB double mutant, dcrB null-mediated toxicity can be overcome in distinct ways - by increased expression of Lgt, deletion of lpp, or removal of Lpp-peptidoglycan linkages. The last of these events leads to elevated membrane vesiculation and lipid loss, which may, in turn, impact membrane homeostasis in the double mutant.Importance A distinguishing feature of Gram-negative bacteria is their double-membraned cell envelope which presents a formidable barrier against environmental stress. In E. coli, more than a quarter of the cellular proteins reside at the inner membrane but about a third of these proteins are functionally unassigned or their function is incompletely understood. Here, we show that the synthetic lethality underlying the inactivation of two inner membrane proteins, a small integral membrane protein YciB, and a lipoprotein, DcrB, results from the attenuation of the first step of lipoprotein maturation at the inner membrane. We propose that these two inner membrane proteins YciB and DcrB play a role in membrane homeostasis in E. coli and related bacteria.

Dataset Information

Repression of the inner membrane lipoprotein NlpA by Rns in enterotoxigenic Escherichia coli.

Publications

Repression of the inner membrane lipoprotein NlpA by Rns in enterotoxigenic Escherichia coli.

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