EscA is a crucial component of the type III secretion system of enteropathogenic Escherichia coli.

The virulence of many Gram-negative pathogens is associated with type III secretion systems (T3SSs), which deliver virulence effector proteins into the cytoplasm of host cells. Components of enteropathogenic Escherichia coli (EPEC) T3SS are encoded within the locus of enterocyte effacement (LEE). While most LEE-encoded T3SS proteins in EPEC have assigned names and functions, a few of them remain poorly characterized.

Quantitative proteomic analysis reveals formation of an EscL-EscQ-EscN type III complex in enteropathogenic Escherichia coli.

We characterized Orf5 and SepQ, two type III secretion (T3S) system proteins in enteropathogenic Escherichia coli, and showed that they are essential for T3S, associated with the bacterial membrane, and interact with EscN. Our findings suggest that Orf5 and SepQ are homologs of YscL and YscQ from Yersinia, respectively.

Engineering of ion sensing by the cystathionine beta-synthase module of the ABC transporter OpuA.

We have previously shown that the C-terminal cystathionine beta-synthase (CBS) domains of the nucleotide-binding domains of the ABC transporter OpuA, in conjunction with an anionic membrane surface function, act as sensor of internal ionic strength (I(in)). Here, we show that a surface-exposed cationic region in the CBS module domain is critical for ion sensing. The consecutive substitution of up to five cationic residues led to a gradual decrease of the ionic strength dependence of transport.

Structural microengineers: pathogenic Escherichia coli redesigns the actin cytoskeleton in host cells.

Several virulent bacteria have the ability to manipulate the host cell actin cytoskeleton as part of their pathogenic strategy. These pathogens subvert the host cell actin polymerization machinery for various purposes including motility within host cells, cell-to-cell spread, and to prevent phagocytic engulfment by professional phagocytes. In contrast to intracellular pathogens, pathogenic Escherichia coli (including both enterohemorrhagic and enteropathogenic E.

Ion specificity and ionic strength dependence of the osmoregulatory ABC transporter OpuA.

The ATPase subunit of the osmoregulatory ATP-binding cassette transporter OpuA from Lactococcus lactis has a C-terminal extension, the tandem cystathionine beta-synthase (CBS) domain, which constitutes the sensor that allows the transporter to sense and respond to osmotic stress (Biemans-Oldehinkel, E., Mahmood, N. A.

A sensor for intracellular ionic strength.

Cystathionine-beta-synthase (CBS) domains are found in >4,000 proteins in species from all kingdoms of life, yet their functions are largely unknown. Tandem CBS domains are associated with membrane transport proteins, most notably members of the ATP-binding cassette (ABC) superfamily; voltage-gated chloride channels and transporters; cation efflux systems; and various enzymes, transcription factors, and proteins of unknown function. We now show that tandem CBS domains in the osmoregulatory ABC transporter OpuA are sensors for ionic strength that control the transport activity through an electrostatic switching mechanism.

Functional analysis of detergent-solubilized and membrane-reconstituted ATP-binding cassette transporters.

ATP-binding cassette (ABC) transporters are vital to any living system and are involved in the translocation of a wide variety of substances, from ions and nutrients to high molecular weight proteins. This chapter describes methods used to purify and membrane reconstitute ABC transporters in a fully functional state. The procedures are largely based on our experience with substrate-binding protein-dependent ABC uptake systems from bacteria, but the approaches should be applicable to multisubunit membrane complexes in general.

ABC transporter architecture and regulatory roles of accessory domains.

We present an overview of the architecture of ATP-binding cassette (ABC) transporters and dissect the systems in core and accessory domains. The ABC transporter core is formed by the transmembrane domains (TMDs) and the nucleotide binding domains (NBDs) that constitute the actual translocator. The accessory domains include the substrate-binding proteins, that function as high affinity receptors in ABC type uptake systems, and regulatory or catalytic domains that can be fused to either the TMDs or NBDs.

On the role of the two extracytoplasmic substrate-binding domains in the ABC transporter OpuA.

Members of two transporter families of the ATP-binding cassette (ABC) superfamily use two or even four extracytoplasmic substrate-binding domains (SBDs) for transport. We report on the role of the two SBDs in the translocation cycle of the ABC transporter OpuA from Lactococcus lactis. Heterooligomeric OpuA complexes with only one SBD or one functional and one non-functional SBD (inactivated by covalent linkage of a substrate mimic) have been constructed, and the substrate binding and transport kinetics of the purified transporters, reconstituted in liposomes, have been determined.

Efficient biosynthetic incorporation of tryptophan and indole analogs in an integral membrane protein.

Biosynthetic incorporation of tryptophan (Trp) analogs such as 7-azatryptophan, 5-hydroxytryptophan, and fluorotryptophan into a protein can facilitate its structural analysis by spectroscopic techniques such as fluorescence, phosphorescence, nuclear magnetic resonance, and Fourier transform infrared. Until now, the approach has dealt primarily with soluble proteins. In this article, we demonstrate that four different Trp analogs can be very efficiently incorporated into a membrane protein as demonstrated for the mannitol transporter of Escherichia coli (EII(mtl)).