Topic
OmpT
About: OmpT is a research topic. Over the lifetime, 257 publications have been published within this topic receiving 13374 citations.
Papers published on a yearly basis
Papers
TL;DR: Certain environmental signals (i.e., osmolarity and the presence of amino acids) are tightly coupled to the expression of toxR-regulated proteins and therefore may be signals that are directly sensed by the ToxR protein.
Abstract: The toxR gene of Vibrio cholerae encodes a transmembrane, DNA-binding protein that activates transcription of the cholera toxin operon and a gene (tcpA) for the major subunit of a pilus colonization factor. We constructed site-directed insertion mutations in the toxR gene by a novel method employing the chromosomal integration of a mobilizable suicide plasmid containing a portion of the toxR coding sequence. Mutants containing these new toxR alleles had an altered outer membrane protein profile, suggesting that two major outer membrane proteins (OmpT and OmpU) might be under the control of toxR. Physiological studies indicated that varying the concentration of the amino acids asparagine, arginine, glutamate, and serine caused coordinate changes in the expression of cholera toxin, TcpA, OmpT, and OmpU. Changes in the osmolarity of a tryptone-based medium also produced coordinate changes in the expression of these proteins. Other environmental signals (temperature and pH) had a more pronounced effect on the expression of cholera toxin and TcpA than they did on the outer membrane proteins. These results suggest that certain environmental signals (i.e., osmolarity and the presence of amino acids) are tightly coupled to the expression of toxR-regulated proteins and therefore may be signals that are directly sensed by the ToxR protein.
2,156 citations
TL;DR: The crystal structure of OmpT from Escherichia coli is presented, which shows a 10‐stranded antiparallel β‐barrel that protrudes far from the lipid bilayer into the extracellular space and a novel proteolytic mechanism, involving a His-Asp dyad and an Asp—Asp couple that activate a putative nucleophilic water molecule.
Abstract: OmpT from Escherichia coli belongs to a family of highly homologous outer membrane proteases, known as omptins, which are implicated in the virulence of several pathogenic Gram-negative bacteria. Here we present the crystal structure of OmpT, which shows a 10-stranded antiparallel β-barrel that protrudes far from the lipid bilayer into the extracellular space. We identified a putative binding site for lipopolysaccharide, a molecule that is essential for OmpT activity. The proteolytic site is located in a groove at the extracellular top of the vase-shaped β-barrel. Based on the constellation of active site residues, we propose a novel proteolytic mechanism, involving a His–Asp dyad and an Asp–Asp couple that activate a putative nucleophilic water molecule. The active site is fully conserved within the omptin family. Therefore, the structure described here provides a sound basis for the design of drugs against omptin-mediated bacterial pathogenesis. Coordinates are in the Protein Data Bank (accession No. 1I78)
300 citations
TL;DR: First direct in vivo evidence that TonB can span the periplasmic space to interact physically with outer membrane receptors is presented, indicating that ExbB plays a direct role in energy transduction and probably secondarily protects TonB protein from proteolysis.
228 citations
TL;DR: Screening a library of 6 × 105 random OmpT variants by FACS using a FRET peptide substrate with a nonpreferred Arg-Val cleavage sequence resulted in the isolation of variant proteases with catalytic activities enhanced by as much as 60-fold.
Abstract: Here we describe a high-throughput, quantitative method for the isolation of enzymes with novel substrate specificities from large libraries of protein variants. Protein variants are displayed on the surface of microorganisms and incubated with a synthetic substrate consisting of (1) a fluorescent dye (2) a positively charged moiety (3) the target scissile bond, and (4) a fluorescence resonance energy transfer (FRET) quenching partner. Enzymatic cleavage of the scissile bond results in release of the FRET quenching partner while the fluorescent product is retained on the cell surface, allowing isolation of catalytically active clones by fluorescence-activated cell sorting (FACS). Using a synthetic substrate with these characteristics, we enriched Escherichia coli expressing the serine protease OmpT from cells expressing an inactive OmpT variant by over 5,000-fold in a single round. Screening a library of 6 × 105 random OmpT variants by FACS using a FRET peptide substrate with a nonpreferred Arg-Val cleavage sequence resulted in the isolation of variant proteases with catalytic activities enhanced by as much as 60-fold. This approach represents a potentially widely applicable method for high-throughput screening of large libraries on the basis of catalytic turnover.
219 citations
TL;DR: It is demonstrated that OmpT participates in the degradation of a fusion protein secreted into the periplasmic space and should be useful for the expression of proteolytically sensitive secreted proteins.
Abstract: The Escherichia coli outer membrane protease OmpT (protease VII) has been shown to degrade several proteins in vitro, but its function in vivo is uncertain. We demonstrate that OmpT participates in the degradation of a fusion protein secreted into the periplasmic space. A strain with mutations in degP (K.L. Strauch and J. Beckwith, Proc. Natl. Acad. Sci. USA 85:1576-1580, 1988) and ompT exhibits a cumulative decrease in protein degradation and should be useful for the expression of proteolytically sensitive secreted proteins. Images
191 citations
Related Topics (5)
Performance Metrics
| Year | Papers |
|---|---|
| 2021 | 4 |
| 2020 | 11 |
| 2019 | 9 |
| 2018 | 4 |
| 2017 | 7 |
| 2016 | 1 |