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Energy-coupled import across the bacterial outer membrane

Group Leader:	Prof. Dr. rer. nat. Dr. med. h. c. Volkmar Braun
Phone:	+49 7071 - 601 343
Fax:	+49 7071 - 601 349

Secretary:	Karin Lehmann
Phone:	+49 7071 - 601 340

Research Interest

Chaperone-dependent protein import

We have recently discovered that a periplasmic chaperone, FkpA, of E. coli is essential for the toxicity of imported colicin M. This is a novel finding since chaperones are otherwise involved in correct folding of newly synthesized proteins. The chaperone is a peptidyl-prolyl-cis-trans-isomerase (PPIase). Colicin M unfolds during translocation across the outer membrane and must refold in the periplasm to inhibit murein (peptidoglycan) biosynthesis by cleaving the polyisoprenyl carrier lipid from the murein precursor. FkpA cannot be replaced by other periplasmic chaperones. FkpA mutants resistant to colicin M carry mutations in the PPIase domain. Energy-coupled protein translocation across the outer membrane, unfolding and chaperone assisted refolding is being studied. The crystal structure of colicin M has been determined in cooperation with Kornelius Zeth and unraveled a novel structure that consists of a receptor binding domain, a translocation domain and an activity domain that forms a new type of phosphatase. Structure-function relationships are studied with randomly generated mutants in colicin M subdomains. (Stephanie Helbig, Silke Patzer, Christin Römer)

Energy transfer between membranes

Energy dependent transport across the outer membrane is powered by the proton motive force of the adjacent cytoplasmic membrane. The energy transfer device between the two membranes is formed by three proteins, TonB, ExbB, and ExbD. It is not known how the energy is harvested and how it is transferred from the cytoplasmic membrane into the outer membrane and how outer membrane transport proteins react to the energy input. There is structural information that TonB interacts with outer membrane transport proteins. The structure of the complex must be known to understand how energy coupling is achieved. We unraveled the essential functions of ExbB and ExbD and determined their transmembrane topology. We attempt to isolate the protein complex to determine its size, shape, stoichiometry, quaternary, tertiary and secondary structures. (Avijit Pramanik)

Signal transduction from the cell surface to the cytoplasm

We have discovered a transmembrane transcription device that initiates at the cell surface. A signal, no substance, is transferred across the outer membrane, the periplasm, and the cytoplasmic membrane into the cytoplasm where a specific sigma factor, FecI, is activated that directs the RNA polymerase to the promoter of an operon that encodes five genes for the uptake of ferric citrate. Cells first recognize iron deficiency and then the presence of ferric citrate in the medium. Iron deficiency leads to synthesis of a protein in the cytoplasmic membrane, FecR, and of FecI. Both proteins are required but not sufficient to initiate transcription of the ferric citrate transport genes. Ferric citrate must bind to a specific outer membrane protein that changes its structure and interacts with FecR and this in turn reacts with FecI. The nature of the signal transfer across the cytoplasmic membrane is presently investigated. It seems to involve a biochemical reaction in which the signal activates the RseP protease in the cytoplasmic membrane which releases the cytoplasmic domain of FecR that binds to and activates FecI. (S. Patzer)

Biosynthesis and transport of iron carriers (siderophores) in streptomycetes

Bacteria and fungi transport ferric iron as siderophore complexes. Siderophores serve to solubilize the extremely insoluble ferric ions at pH 7. Ferric siderophores are taken up by an energy-coupled transport across the outer membrane and the cytoplasmic membrane. Some antibiotics consist of a siderophore to which an antibiotic moiety is covalently bound, called sideromycins. In contrast to most antibiotics, the outer membrane does not form a permeability barrier for sideromycins but actively transports them by the ferric siderophore transport systems into the periplasm and from there into the cytoplasm. Active transport in contrast to diffusion lowers the minimal inhibitory concentration of the antibiotics about 100-fold, thereby lowering the amount to be applied and the side effects. The most extensively studied sideromycin is albomycin that is synthesized by certain streptomycetes. For use as an antibiotic biosynthesis of albomycin must be increased. Therefore, we identified and sequenced the biosynthetic gene cluster on a 40 kb DNA region that specifies albomycin synthesis. Moreover, since albomycin has a unique structure its biosynthesis is of interest. A novel gene cluster for a siderophore of the catecholate type was found and is being characterized since no genes for siderophores of this type have been studied in streptomycetes. Expression of genes that specify biosynthesis and transport of ferric siderophores are regulated by iron through regulatory proteins of which we identified and characterized the first one. The iron regulation of the genes in the two operons will be analyzed.(S. Patzer)

FhuA TonB ExbB ExbD

The figure illustrates the receptor and transport functions of the FhuA protein and its coupling to
the inner membrane protein complex TonB, ExbB and ExbD. FhuA consist of a beta-barrel which is
tightly closed by a plug domain which must move to open a pore for the uptake of the substrates. The
structures are shown as far as they are known.