Anne Walburger

The Tol-Pal proteins of the Escherichia coli cell envelope: an energized system required for outer membrane integrity?

The outer membrane of gram-negative bacteria acts as a barrier against harmful lipophilic compounds and larger molecules unable to diffuse freely through the porins. However, outer membrane proteins together with the Tol-Pal and TonB systems have been exploited for the entry of macromolecules such as bacteriocins and phage DNA through the Escherichia coli cell envelope. The TonB system is involved in the active transport of iron siderophores and vitamin B12, while no more precise physiological role of the Tol-Pal system has yet been defined than its requirement for cell envelope integrity. These two systems, containing an energized inner membrane protein interacting with outer membrane proteins, share similarities.

The Tol/Pal system function requires an interaction between the C‐terminal domain of TolA and the N‐terminal domain of TolB

The Tol/Pal system of Escherichia coli is composed of the YbgC, TolQ, TolA, TolR, TolB, Pal and YbgF proteins. It is involved in maintaining the integrity of the outer membrane, and is required for the uptake of group A colicins and DNA of filamentous bacteriophages. To identify new interactions between the components of the Tol/Pal system and gain insight into the mechanism of colicin import, we performed a yeast two‐hybrid screen using the different components of the Tol/Pal system and colicin A. Using this system, we confirmed the already known interactions and identified several new interactions. TolB dimerizes and the periplasmic domain of TolA interacts with YbgF and TolB. Our results indicate that the central domain of TolA (TolAII) is sufficient to interact with YbgF, that the C‐terminal domain of TolA (TolAIII) is sufficient to interact with TolB, and that the amino terminal domain of TolB (D1) is sufficient to bind TolAIII. The TolA/TolB interaction was confirmed by cross‐linking experiments on purified proteins. Moreover, we show that the interaction between TolA and TolB is required for the uptake of colicin A and for the membrane integrity. These results demonstrate that the TolA/TolB interaction allows the formation of a trans‐envelope complex that brings the inner and outer membranes in close proximity.

Analysis of the Escherichia coli Tol-Pal and TonB systems by periplasmic production of Tol, TonB, colicin, or phage capsid soluble domains.

The aim of this review is to describe an in vivo assay of the interactions taking place in the Tol-Pal or TonB-ExbB-ExbD envelope complexes in the periplasm of Escherichia coli and between them and colicins or g3p protein of filamentous bacteriophages. Domains of colicins or periplasmic soluble domains of Tol or TonB proteins can be artificially addressed to the periplasm of bacteria by fusing them to a signal sequence from an exported protein. These domains interact specifically in the periplasm with the Tol or TonB complexes and disturb their function, which can be directly detected by the appearance of specific tol or tonB phenotypes. This technique can be used to detect new interactions, to characterize them biochemically and to map them or to induce tol or tonB phenotypes to study the functions of these two complexes.

A Sulfurtransferase Is Essential for Activity of Formate Dehydrogenases in Escherichia coli

Background: The l-cysteine desulfurase IscS interacts with FdhD, a protein essential for the activity of formate dehydrogenases. Results: IscS transfers sulfur to FdhD, and this is required to yield an active enzyme. Conclusion: FdhD is a sulfurtransferase between IscS and formate dehydrogenases. Significance: This helps us to understand how sulfur transfer occurs in living cells. l-Cysteine desulfurases provide sulfur to several metabolic pathways in the form of persulfides on specific cysteine residues of an acceptor protein for the eventual incorporation of sulfur into an end product. IscS is one of the three Escherichia coli l-cysteine desulfurases. It interacts with FdhD, a protein essential for the activity of formate dehydrogenases (FDHs), which are iron/molybdenum/selenium-containing enzymes. Here, we address the role played by this interaction in the activity of FDH-H (FdhF) in E. coli. The interaction of IscS with FdhD results in a sulfur transfer between IscS and FdhD in the form of persulfides. Substitution of the strictly conserved residue Cys-121 of FdhD impairs both sulfur transfer from IscS to FdhD and FdhF activity. Furthermore, inactive FdhF produced in the absence of FdhD contains both metal centers, albeit the molybdenum cofactor is at a reduced level. Finally, FdhF activity is sulfur-dependent, as it shows reversible sensitivity to cyanide treatment. Conclusively, FdhD is a sulfurtransferase between IscS and FdhF and is thereby essential to yield FDH activity.

Crystallization and preliminary crystallographic study of the peptidoglycan-associated lipoprotein from Escherichia coli.

The peptidoglycan-associated lipoprotein (Pal) from Escherichia coli is part of the Tol--Pal multiprotein complex used by group A colicins to penetrate and kill cells. Pal homologues are found in many Gram-negative bacteria and the Tol--Pal system is thought to play a role in bacterial envelope integrity. The Pal protein comprises 152 amino acids. Crystals of the C-terminal 109-amino-acid fragment of the Pal protein have been produced. The crystals belong to the tetragonal space group I4(1), with unit-cell parameters a = b = 89.3, c = 67.2 A. There are two molecules in the asymmetric unit. Frozen crystals diffract to at least 2.8 A resolution using synchrotron radiation. Selenomethionine-substituted truncated Pal protein is currently being produced in order to use multiwavelength anomalous dispersion (MAD) for phasing.

Dynamic subcellular localization of a respiratory complex controls bacterial respiration

Respiration, an essential process for most organisms, has to optimally respond to changes in the metabolic demand or the environmental conditions. The branched character of their respiratory chains allows bacteria to do so by providing a great metabolic and regulatory flexibility. Here, we show that the native localization of the nitrate reductase, a major respiratory complex under anaerobiosis in Escherichia coli, is submitted to tight spatiotemporal regulation in response to metabolic conditions via a mechanism using the transmembrane proton gradient as a cue for polar localization. These dynamics are critical for controlling the activity of nitrate reductase, as the formation of polar assemblies potentiates the electron flux through the complex. Thus, dynamic subcellular localization emerges as a critical factor in the control of respiration in bacteria. DOI: http://dx.doi.org/10.7554/eLife.05357.001

Sulphur shuttling across a chaperone during molybdenum cofactor maturation

Formate dehydrogenases (FDHs) are of interest as they are natural catalysts that sequester atmospheric CO2, generating reduced carbon compounds with possible uses as fuel. FDHs activity in Escherichia coli strictly requires the sulphurtransferase EcFdhD, which likely transfers sulphur from IscS to the molybdenum cofactor (Mo-bisPGD) of FDHs. Here we show that EcFdhD binds Mo-bisPGD in vivo and has submicromolar affinity for GDP-used as a surrogate of the molybdenum cofactors nucleotide moieties. The crystal structure of EcFdhD in complex with GDP shows two symmetrical binding sites located on the same face of the dimer. These binding sites are connected via a tunnel-like cavity to the opposite face of the dimer where two dynamic loops, each harbouring two functionally important cysteine residues, are present. On the basis of structure-guided mutagenesis, we propose a model for the sulphuration mechanism of Mo-bisPGD where the sulphur atom shuttles across the chaperone dimer.

Redox cofactors insertion in prokaryotic molybdoenzymes occurs via a conserved folding mechanism.

A major gap of knowledge in metalloproteins is the identity of the prefolded state of the protein before cofactor insertion. This holds for molybdoenzymes serving multiple purposes for life, especially in energy harvesting. This large group of prokaryotic enzymes allows for coordination of molybdenum or tungsten cofactors (Mo/W-bisPGD) and Fe/S clusters. Here we report the structural data on a cofactor-less enzyme, the nitrate reductase respiratory complex and characterize the conformational changes accompanying Mo/W-bisPGD and Fe/S cofactors insertion. Identified conformational changes are shown to be essential for recognition of the dedicated chaperone involved in cofactors insertion. A solvent-exposed salt bridge is shown to play a key role in enzyme folding after cofactors insertion. Furthermore, this salt bridge is shown to be strictly conserved within this prokaryotic molybdoenzyme family as deduced from a phylogenetic analysis issued from 3D structure-guided multiple sequence alignment. A biochemical analysis with a distantly-related member of the family, respiratory complex I, confirmed the critical importance of the salt bridge for folding. Overall, our results point to a conserved cofactors insertion mechanism within the Mo/W-bisPGD family.