Unit: Bacterial Membranes
Director: Cécile Wandersman
Our work concerns membrane transports in Gram negative bacteria with four major research themes: heme acquisition systems requiring an extracellular hemophore, protein secretion by ABC exporters across the two membranes which delimit the Gram negative cells, a two component system which is involved in iron acquisition and the characterization of ABC proteins of unknown functions.More recently, we started the study of heme transport in Bartonella birtlesii.
1) Heme acquisition systems ( Sylvie Létoffé, Laurent Debarbieux, Philippe Delepelaire, Francis Biville, Fréderic Huché, Hélène Cwerman and Najla Benevides Matos)
In Gram negative bacteria, heme which is a major iron source is uptaken by highly conserved outer membrane receptors. They belong to a protein family involved in active transport of various substrates such as vitamin B12 and siderophores. Several receptors already crystallised show an N-terminal domain closing the receptor pore and exposed to the periplasm, where it can make contact with TonB. Heme receptors either recognize free heme or exogenous hemoproteins such as hemoglobin or small heme binding proteins named hemophores, produced by the bacteria (Fig. 1).
Structural and genetical studies performed on the Serratia marcescens hemophore have revealed the 3D structure of holo and apo-hemophore allowed to identify the heme iron ligands, to compare binding properties of heme-free and heme-loaded HasA. The hemophore interacts with HasR by two independent binding sites located on two β sheets on the same side of HasA. We propose that this double binding distorts the protein allowing heme transfer to the receptor (Fig. 2).
HasR also binds free heme by two histidines conserved on other heme receptors with a lower affinity, in the micromolar range. Thus, heme is transferred from HasA a protein with a very high affinity (Kd : 10-11M) to HasR a protein with a lower affinity (Kd around 10-6M). Biochemical studies revealed that heme transfer from the hemophore to the receptor, occurs in vitro and is thus energy independent (manuscript in resubmission).
Whereas heme is uptaken as a whole through heme receptors, hemophores are not transported and have to be stripped off at the cell surface: only the heme moiety being uptaken, the apo-hemophore remains outside. We showed that empty-hemophore release from the receptor is energy driven and concomittent with heme uptake.
In Gram-negative bacteria, many outer membrane receptors involved in iron uptake are positively regulated by the availability of a specific iron source via extra cytoplasmic function (ECF) sigma factors and membrane bound anti-sigma factors. An extra cytoplasmic signal releases the ECF sigma from the anti-sigma inhibition and thereby leads to the transcription of the target operons.
The S. marcescens hasR gene is subject to this type of regulation. It is induced only by holo-HasaA. Using a collection of HasA and HasR mutants, we showed that the inducing stimulus can be brought by two molecules: heme and the 51-55 peptide belonging to one of the two HasA binding sites (manuscript in revision for J. of Bacteriology). These experiments indicate that induction and heme uptake are achieved by at least partially distinct mechanisms: uptake requires hemophore recycling whereas it appears that induction could occur without empty hemophore ejection. We are presently testing this hypothesis.
Unlike known iron starvation ECF anti-sigma factors, the transcription of hasS is directly regulated by HasI and consequently also subject to the holo-hemophore signaling cascade.
We have undertaken the molecular dissection and site directed mutagenesis of HasR using a computer modeled 3D structure (Fig. 3). We found that the HasR β barrel alone forms a specific heme channel requiring the conserved histidine residue to be functional. We have identified extracellular loops which might interact with HasA.
Heme uptake in S. marcescens requires either TonB or HasB, a TonB homolog specific for heme uptake that cannot complement the other TonB-dependent functions. To determine whether HasB is specific for HasR or for heme, we cloned another S. marcescens heme receptor present in the S. marcescens DB11 (DNA sequence data base). It allows heme uptake in E. coli only with TonB suggesting that HasB is specific for the receptor HasR and not heme.
We are actually inactivating the S. marcescens DB11 hasB, tonB, hemR and hasR genes to test the mutant virulence in the Caenorhabditis elegans model developped by J. Ewbank. A recent work has shown that C. elegans and more generally Helmithia are heme auxotrophes and can use heme as an iron source. Thus, heme restriction might contribute to S. marcescens virulence on the worm.
2) Heme transport in Bartonella birtlesii. (Francis Biville in collaboration with Henri-Jean Boulouis group (INRA)
Bartonella are intra cellular bacteria which preferentially grow in erythrocytes suggesting that they might have special pathways to use heme and protect themselves against heme toxicity. We have cloned several genes homologous to Bartonella quintana et Bartonella henselae genes which were shown to play a role in heme uptake. We are presently studying their functions which might be involved in heme uptake.
3) Protein secretion by Gram negative ABC transporters (Sandra Cescau, Laurent Debarbieux, Annick Paquelin and Philippe Delepelaire)
The type I secretion pathway allows the secretion of proteins lacking an N terminal signal peptide. It is independent of the general secretory machinery. Four secretion pathways have been actually described in Gram-negative bacteria.
The Type I transporter is characterized by the presence of the ABC protein belonging to the ATP Binding Cassette class of proteins which are implicated in the vectorial movement of solutes across biological membranes and several play essential functions. The transporter comprizes two other membrane proteins, one in the inner membrane and one in the outer membrane (Fig. 1).
Our work has largely contributed to show that bacterial ABC protein exporters are widespread in Gram-negative organisms. We have shown that in several cases (such as the E. coli hemolysin transporter) the outer membrane component belongs to the TolC family and is a multifunctional protein involved in drug efflux and colicin import. All the proteins following this pathway, including the hemophore, have a C-terminal secretion signal which remains accessible owing to cytoplasmic chaperones which are required for secretion. Our study on HasA showed that the signal interacts with the ABC protein, modulates its ATPase activity and induces the formation of a secretion multiprotein complex. A hemophore mutant lacking its last 14 C-terminal amino acids. is not secreted, but still interacts with the ABC protein and promotes a stable complex. Strains expressing such transporter jammed by the truncated protein are sensitive to SDS. It is likely that TolC is trapped in the transporter and is not avalaible to pump out detergents. This provides us a simple SDS sensitivity test on plates to study the type I secretion protein association/dissociation independently of the secretion process itself, to localize the second interaction site and to determine the role of ATP in complexe association/dissociation.
We are presently purifying the substrate bound ABC transporter multiprotein complex to study it by crystallography and cryomicroscopy. This is done in collaboration with the laboratory of Electronic Microscopy of the Institut Pasteur.
Our future researches are directed towards the study of the mechanisms of chaperone-substrate interactions and the reconstitution of a complete secretion system in vitro.
Pentapeptide scanning mutagenesis of the ABC protein is undertaken to define its domains of interaction with the substrate and the other transporter proteins.
4) Functional and phylogenetic analysis of ABC systems (Dorothée Murat and Elie Dassa)
The function of ABC proteins lacking transmembrane domains is largely unknown in prokaryotes. The deletion of the gene encoding Uup leads to an increase in precise excision of transposons. To provide insight into the molecular mechanism of Uup in transposon excision, we purified this protein and we demonstrated that it is a cytosolic ABC protein. Purified recombinant Uup binds and hydrolyzes ATP and undergoes a large conformational change in the presence of this nucleotide. This change affects a C-terminal domain of the protein, which displays predicted structural homology with the so-called little finger (LF) domain of Y family DNA polymerases. In these enzymes, this domain is involved in DNA binding and in the processivity of replication. We show that Uup binds to DNA, and that this binding is in part dependent on its C-terminal domain. Analysis of Walker motif B mutants suggests that ATP hydrolysis at the two ABC domains is strictly coordinated and is essential for the function of Uup in vivo. Our results further suggest that Uup can directly act on DNA to minimize transposon excision, a well-known example of illegitimate recombination (D. Murat et al. Manuscript in press, JBC).
Figure 1 : Has systems
Figure 2 3D structure pf Apo and Holo HasA
Figure 3 : Computer modeled Has R structure
Keywords: Membrane transport, iron acquisition, hemophore, ABC protein