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Summary ExbBD is part of a cytoplasmic membrane molecular motor driven by the proton-motive force. It belongs to the larger family of motors involved in nutriment import across the outer membrane of Gram-negative bacteria (ExbBD), flagellar rotation (MotAB) or late steps of cell division in Gram-negative bacteria (TolQR). ExbB and ExbD are integral membrane proteins with three (ExbB) or one (ExbD) transmembrane segment. Here we have solved by single-particle cryo-EM the structures of ExbB alone and of the ExbB-ExbD complex of the opportunistic pathogen Serratia marcescens . ExbB Sm alone behaves as a stable pentamer, and the complex displays the ExbB 5 -ExbD 2 stoichiometry. This is similar to what has been observed for ExbB-ExbD complexes from Escherichia coli and Pseudomonas savastanoi as well as MotAB complexes from various species. We identified residues located in the first TM of ExbB Sm and ExbB Ec that are likely involved in the interaction with TonB/HasB and that are essential for function. ExbB Sm has a ca. 40 residues long periplasmic extension absent in E. coli . Such long ExbB’s are found in some Gammaproteobacteria, and several genera of Alphaproteobacteria. We show that this extension interacts with HasB, a dedicated TonB paralog from the heme acquisition system (Has) from S. marcescens . We also show that it is involved in heme acquisition via the Has system from S. marcescens . ExbB Sm represents thus a new class of ExbB protein and our results shed light on the specificity determinants between the ExbB-ExbD complex and their associated TonB partners.

Energized nutrient import in bacteria needs the interaction between a TonB-dependent transporter (TBDT) and a TonB protein. The mechanism of energy and signal transfer between these two proteins is not well understood. They belong to two membranes separated by the periplasmic space and possess each a disordered and flexible region. Therefore, the membranes, their distance and geometrical constraints together with the protein dynamics are important factors for deciphering this trans-envelope system. Here we report the first example of the interaction of a TBDT with a TonB protein in the presence of both membranes. By combining molecular dynamics simulations in a membrane model, in vitro and in vivo phenotypic experiments we obtained the comprehensive network of interaction between HasR, a heme/hemophore receptor and its dedicated TonB protein, HasB.

International audience; Haem is involved in essential processes. It is toxic and thus is not found free in living organisms but almost entirely sequestered by haem carrier proteins. We investigated the mechanisms of haem transfer between the proteins of a bacterial haem acquisition system involving haemophores. Haemophores are secreted by several Gram-negative bacteria and are able to extract haem (assimilated as an iron source) from haemoproteins and deliver it to specific outer membrane receptors. The Serratia marcescens haemophore (HasA) is folded into a globular form and tyrosine and histidine are involved in haem ligation. Interaction with the receptor is of high affinity (5 nM) and does not involve haem. Identification and study of mutants with altered binding properties led to the description of two regions of the haemophore that bind to the receptor. They consist of residues involved in two beta strands located on the same side of HasA. Each region is sufficient for high affinity binding. The synthetic peptide corresponding to one beta strand competes with the corresponding haemophore region for binding to the receptor, suggesting that the two binding regions are independent binding sites. We propose a model for haem release and transfer to the receptor.

Two chloroplast-encoded proteins of photosystem II (PSII) have recently been the object of considerable interest. One is a rapidly synthesized 32-kD protein (Bottomley et al. 1974; Eaglesham and Ellis 1974; Hottman-Falk et al. 1982), also identified as a herbicide-binding protein (Pfister et al. 1981) and designated the QB protein (Hirschberg et al. 1984) because of its proposed role in a protein-quinone complex, which serves as the second stable electron acceptor of PSII (Arntzen et al. 1982). The other is a protein of similar size, called D2, that has several regions of homology with the QB protein (Rochaix et al. 1984). In this paper, we briefly review what is known about the primary structure of these two proteins and their genes and the mutations in the gene for the QB protein that affect herbicide resistance and cross-resistance.

Funder: LABEX DYNAMO ANR-11-LABEX-0011-01, Funder: ANR HEMESTOCKEXCHANGE ANR-12-BSV3-0022-01, Funder: ANR HEMESTOCKEXCHANGE ANR-12-BSV3-0022-01 ANR LABEX DYNAMO ANR-11-LABEX-0011-01, ExbB and ExbD are cytoplasmic membrane proteins that associate with TonB to convey the energy of the proton-motive force to outer membrane receptors in Gram-negative bacteria for iron uptake. The opportunistic pathogen Serratia marcescens (Sm) possesses both TonB and a heme-specific TonB paralog, HasB. ExbBSm has a long periplasmic extension absent in other bacteria such as E. coli (Ec). Long ExbB’s are found in several genera of Alphaproteobacteria, most often in correlation with a hasB gene. We investigated specificity determinants of ExbBSm and HasB. We determined the cryo-EM structures of ExbBSm and of the ExbB-ExbDSm complex from S. marcescens. ExbBSm alone is a stable pentamer, and its complex includes two ExbD monomers. We showed that ExbBSm extension interacts with HasB and is involved in heme acquisition and we identified key residues in the membrane domain of ExbBSm and ExbBEc, essential for function and likely involved in the interaction with TonB/HasB. Our results shed light on the class of inner membrane energy machinery formed by ExbB, ExbD and HasB.