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Research of the group of Thomas Brüser

Our research focuses on twin-arginine translocation (Tat) systems that transport folded proteins across prokaryotic cytoplasmic membranes, on membrane stress sensing and response mediated by the phage shock protein (Psp) system in Escherichia coli, the periplasmic maturation and modification of pyoverdines in fluorescent pseudomonads, and the formation of biofilms by plant growth-promoting rhizobacteria.

Tat transport -  For many years it was believed that only unfolded protein chains can be translocated across membranes. This view changed during the 90s when it became apparent that some proteins are translocated as oligomers, with a transport-initializing signal peptide at only one subunit. The co-translocation of protein subunits that do not possess their own signal peptide clearly indicated that the oligomer must have been formed prior to translocation. Today, this unusual transport system has been identified and named "Tat system" for "twin-arginine translocation system", as transported proteins possess N-terminal signal peptides with a characteristic sequence motif that usually includes two neighbouring "twin-arginines". Proteins that are translocated by this system have been identified, specific requirements for transport have been determined and research on the Tat system is at a stage where it focuses on mechanistic aspects.

The mechanism of Tat transport must be novel and unusual, simply because the transported proteins are much too large for any described "standard" mechanism. Some transported Tat substrates are even larger in diameter than the membrane thickness. It is known that Tat transport requires a membrane energetization and is - in contrast to the better understood Sec transport - independent from soluble factors, such as ATP. Only two components, TatA and TatC, are necessary to build up the translocation machinery. In many bacteria,  such as the model organism Escherichia coli, Tat systems include a third component, TatB, that resembles TatA but covers distinct functionalities. Plants also contain TatABC systems, as their plastids are phylogenetically derived from symbiotic cyanobacteria.

We currently analyze the assembly of the Tat translocon and try to reveal the translocation mechanism. For that purpose, we use a broad range of tools, ranging from molecular biology, protein biochemistry and cell biology to bacterial physiology.

 

Psp system -  Bacteria often experience environmental changes that harm the integrity of the membrane and therefore can be regarded as "membrane stress". It has been observed for many of these conditions that a regulon is upregulated that consists of the pspABCDE operon and the monocistronic pspG gene. The corresponding promoters depend on the alternative sigma factor sigma 54, which is activated by the bacterial enhancer binding protein PspF that is divergently encoded upstream of the pspABCDE operon. The sigma 54 activator PspF in turn is regulated by the first component encoded by the pspABCDE operon, PspA. PspA is the key regulator that somehow integrates the various stress signals to result in the desired activity of PspF and consequently in the desired response on sigma 54 dependent psp transcription level. It is believed that the PspB and PspC components sense membrane stress signals and interact with PspA to transmit these signals. We are interested in the signal sensing by the various Psp components, the signal transmission to PspA and the mode of the signal integration by PspA. To analyze the aspects, we combine a wide range of structural and molecular biological tools.

Periplasmic pyoverdine maturation and modification - Fluorescent pseudomonads produce the siderophore pyoverdine that ensures iron-supply under iron-limiting conditions, such as host environments. Therefore, pyoverdines are highly important for pathogens, such as Pseudomonas aeruginosa, as well as for beneficial pseudomonads, such as plant growth promoting Pseudomonas fluorescens strains. Pyoverdines are derived from non-ribosomally synthesized peptides that are transported into the bacterial periplasm, where a fluorophore is formed by intramolecular cyclization and oxidation reactions, and where also an N-terminal glutamic acid residue is modified to a varyety of derivatives, including succinamide, succinic acid, ketoglutaric acid, malamide, and malic acid. While fluorophore formation has been shown to depend on the action of PvdP, the role of at least three periplasmic enzymes encoded in pyoverdine biosynthesis clusters was unresolved so far and are in the focus of this project. In 2016, we could demonstrate that PvdN is an unusual enzyme that converts the glutamic acid residue in one step to succinamide. The enzyme catalyzes a novel PLP-dependent oxidative decarboxylation under retention of the amine of glutamic acid. The conversion of the fluorophore-attached glutamic acid residue to succinamide is an alternative pathway to the transamination of the glutamic acid to α-ketoglutaric acid. We recently could also identify the transaminase, PtaA, that is responsible for this conversion in fluorescent pseudomonads.

Biofilm formation by plant growth-promoting rhizobacteria - Many bacteria have a positive influence on growth or health of plants. These "plant growth-promoting rhizobacteria" (PGPRs) live close to, at the surface of, or even inside of plant roots. They often help to suppress growth of pathogenic bacteria (biocontrol), they may contribute to the availability of nutrients (biofertilization), or they can even stimulate plant root formation more directly. We are interested in the biofilms that many of these PGPRs form on root surfaces. We would like to know understand the role of protein factors required for biofilm formation and their influence on plant growth promotion.