|Bacterial Membranes - CNRS URA 2172|
|HEAD||Pr. WANDERSMAN Cécile / firstname.lastname@example.org|
|MEMBERS||BENEVIDES MATOS Najla/Dr BIVILLE Francis/BURGOS Monica/CESCAU Sandra/Dr CHESNEAU Olivier/CWERMAN Hélène/Dr DASSA Elie/Dr DELEPELAIRE Philippe/DIAGNE Marième/FOURNIER Clémence
Dr LETOFFE Sylvie/Dr NUNEZ SAMUDIO Virginia/PAQUELIN Annick/RAJARATNAM Thomas/THEPAUT Sylvana/WISZNIOWSKI Karine
The increasing number of available genome sequences brings mountains of data that are not easy to interprete. Even in bacteria, half of the genes encoding polypeptides have unknown functions. Moreover, several polypeptides have more than one function.
Bacterial genetics is a powerful approach to discover new functions. Our laboratory has identified several new bacterial functions including those of TolC, hemophores, dipeptide permease etc.. Most of these achievements were start points of biochemical and biophysical studies performed with various collaborators.
Heme acquisition across the outer membrane
Most organisms have a complete heme biosynthetic pathway. Nevertheless, exogenous heme is taken up by bacteria as an iron source. Exogenous free heme, or heme extracted from various hemoproteins, is internalized whole and degraded in the cytosol to retrieve iron. At the same time, free heme is highly toxic owing to the generation of reactive oxygen species. Thus, heme uptake and breakdown are usually highly regulated to maintain heme homeostasy.
In several Gram-negative bacteria, heme is captured from host hemoproteins by extracellular hemophores (HasA) and then transferred to outer membrane hemophore receptors (HasR).
Genetical, biochemical and structural studies performed on the Serratia marcescens hemophore dependent heme acquisition system have revealed the heme iron ligands on HasA and on HasR, the interaction regions between the hemophore and its receptor. They have shed light on the mechanism of heme transfer from the hemophore to the receptor which does not consume energy. On the other hand, hemophore mediated transcription induction and heme uptake require energy driven by one inner membrane protein HasB. HasB belongs to the TonB family which is in most cases multifunctional, providing energy for unrelated receptors. On the contrary, HasB forms a specific pair with HasR (J. Bacteriol. 2008).
Apo and holo HasA structures (J.Mol. Biol.2007), apo and holo-HasA-HasR complex structures have been solved by X ray analysis and NMR studies.
HasR is the first 3D structure of a bacterial heme receptor.
Heme transport through the inner membrane
Once in the periplasm, heme is transported through the inner membrane by permeases consisting of one periplasmic binding protein and an inner membrane ABC transporter.
Two types of heme permeases have been identified. One type HemTUV is specific for heme whereas, in E. coliK12, there is another type of heme permease constituted by the dipeptide ABC transporter DppBCDF that functions with two optional periplasmic binding proteins DppA and MppA. Heme binding to DppA and MppA, but not to HemT is inhibited by peptides (J. Bact. 2008).
The contribution of each type of permease in heme acquisition by bacterial pathogens is actually under study. Blast searches for putative heme permeases did not reveal any hemTUV orthologs in several species such as Neisseria and Haemophilus which have functional heme outer membrane receptors and which are able to use heme as an iron source.
Many proteins including hemophores are secreted by a transporter comprizing one ABC protein and two helper envelope proteins. The secretion signal usually located at the C-terminus interacts with the ABC protein, modulates its ATPase activity and induces the formation of a secretion multiprotein complex. A hemophore mutant lacking its secretion signal is not secreted, but still interacts with the ABC protein and promotes a stable complex (J.Bacteriol.2007). We are studying the dynamics of the complex dissociation induced by the secretion signal.
ABC proteins lacking transmembrane domains
Studies on Upp (an ABC protein of unknown function) show that it has an ATP-dependent helicase activity. Uup could directly act on DNA to minimize transposon excision, a well-known example of illegitimate recombination.
Vga(A), a Staphylococcus aureus ABC protein of this family exhibits an ATPase activity inhibited by streptogramin but not by other antibiotics. Vga(A) could be involved in streptogramin efflux.
Keywords: Membrane transport, iron acquisition, hemophore, ABC protein
Biville F, Cwerman H, Létoffé S, Rossi MS, Drouet V, Ghigo JM, Wandersman C. 2004. Haemophore-mediated signalling in Serratia marcescens: a new mode of regulation for an extra cytoplasmic function (ECF) sigma factor involved in haem acquisition. Mol. Microbiol. 53:1267-1277.
Murat D, Bance P, Callebaut I, Dassa E. 2006. ATP Hydrolysis Is Essential for the Function of the Uup ATP-binding Cassette ATPase in Precise Excision of Transposons. J. Biol. Chem. 281: 6850-6859.
Izadi-Pruneyre N, Huché F, Lukat-Rodgers GS, Lecroisey A, Gilli R, Rodgers KR, Wandersman C, Delepelaire P. 2006. The Heme Transfer from the Soluble HasA Hemophore to Its Membrane-bound Receptor HasR Is Driven by Protein-Protein Interaction from a High to a Lower Affinity Binding Site*Formula. J. Biol. Chem. 281:25541-25550.
Létoffé S, Delepelaire P, Wandersman C. 2006. The housekeeping dipeptide permease is the Escherichia coli heme transporter and functions with two optional peptide binding proteins. Proc Natl Acad Sci U S A., 103:12891-12896.
Cescau S, Debarbieux L, Wandersman C. 2007. Probing the in vivo dynamics of type I protein secretion complex association through sensitivity to detergents. J. Bacteriol. 189: 1496-1504.
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