Elie Dassa

The genome sequence of the entomopathogenic bacterium Photorhabdus luminescens.

Photorhabdus luminescens is a symbiont of nematodes and a broad-spectrum insect pathogen. The complete genome sequence of strain TT01 is 5,688,987 base pairs (bp) long and contains 4,839 predicted protein-coding genes. Strikingly, it encodes a large number of adhesins, toxins, hemolysins, proteases and lipases, and contains a wide array of antibiotic synthesizing genes. These proteins are likely to play a role in the elimination of competitors, host colonization, invasion and bioconversion of the insect cadaver, making P. luminescens a promising model for the study of symbiosis and host-pathogen interactions. Comparison with the genomes of related bacteria reveals the acquisition of virulence factors by extensive horizontal transfer and provides clues about the evolution of an insect pathogen. Moreover, newly identified insecticidal proteins may be effective alternatives for the control of insect pests.

Plant ABC proteins – a unified nomenclature and updated inventory

The ABC superfamily comprises both membrane-bound transporters and soluble proteins involved in a broad range of processes, many of which are of considerable agricultural, biotechnological and medical potential. Completion of the Arabidopsis and rice genome sequences has revealed a particularly large and diverse complement of plant ABC proteins in comparison with other organisms. Forward and reverse genetics, together with heterologous expression, have uncovered many novel roles for plant ABC proteins, but this progress has been accompanied by a confusing proliferation of names for plant ABC genes and their products. A consolidated nomenclature will provide much-needed clarity and a framework for future research.

The ABC of ABCs: a phylogenetic and functional classification of ABC systems in living organisms

ATP binding cassette (ABC) systems constitute one of the most abundant superfamilies of proteins. They are involved not only in the transport of a wide variety of substances, but also in many cellular processes and in their regulation. In this paper, we made a comparative analysis of the properties of ABC systems and we provide a phylogenetic and functional classification. This analysis will be helpful to accurately annotate ABC systems discovered during the sequencing of the genome of living organisms and to identify the partners of the ABC ATPases.

Getting In or Out: Early Segregation Between Importers and Exporters in the Evolution of ATP-Binding Cassette (ABC) Transporters

Abstract. ATP-binding cassette (ABC) systems, also called traffic ATPases, are found in eukaryotes and prokaryotes and almost all participate in the transport of a wide variety of molecules. ABC systems are characterized by a highly conserved ATPase module called here the ABC module, involved in coupling transport to ATP hydrolysis. We have used the sequence of one of the first representatives of bacterial ABC transporters, the MalK protein, to collect 250 closely related sequences from a nonredundant protein sequence database. The sequences collected by this objective method are all known or putative ABC transporters. After having eliminated short protein sequences and duplicates, the 197 remaining sequences were subjected to a phylogenetic analysis based on a mutational similarity matrix. An unrooted tree for these modules was found to display two major branches, one grouping all collected uptake systems and the other all collected export systems. This remarkable disposition strongly suggests that the divergence between these two functionally different types of ABC systems occurred once in the history of these systems and probably before the differentiation of prokaryotes and eukaryotes. We discuss the implications of this finding and we propose a model accounting for the generation and the diversification of ABC systems.

Subunit interactions in ABC transporters: a conserved sequence in hydrophobic membrane proteins of periplasmic permeases defines an important site of interaction with the ATPase subunits

The cytoplasmic membrane proteins of bacterial binding protein‐dependent transporters belong to the superfamily of ABC transporters. The hydrophobic proteins display a conserved, at least 20 amino acid EAA‐‐‐G‐‐‐‐‐‐‐‐‐I‐LP region exposed in the cytosol, the EAA region. We mutagenized the EAA regions of MalF and MalG proteins of the Escherichia coli maltose transport system. Substitutions at the same positions in MalF and MalG have different phenotypes, indicating that EAA regions do not act symmetrically. Mutations in malG or malF that slightly affect or do not affect transport, determine a completely defective phenotype when present together. This suggests that EAA regions of MalF and MalG may interact during transport. Maltose‐negative mutants fall into two categories with respect to the cellular localization of the MalK ATPase: in the first, MalK is membrane‐bound, as in wild‐type strains, while in the second, it is cytosolic, as in strains deleted in the malF and malG genes. From maltose‐negative mutants of the two categories, we isolated suppressor mutations within malK that restore transport. They map mainly in the putative helical domain of MalK, suggesting that EAA regions may constitute a recognition site for the ABC ATPase helical domain.

Phylogenetic and Functional Classification of ATP-Binding Cassette (ABC) Systems

ATP binding cassette (ABC) systems constitute one of the most abundant superfamilies of proteins. They are involved in the transport of a wide variety of substances, but also in many cellular processes and in their regulation. In this paper, we made a comparative analysis of the properties of ABC systems and we provide a phylogenetic and functional classification. This analysis will be helpful to accurately annotate ABC systems discovered during the sequencing of the genome of living organisms and to identify the partners of the ABC ATPases.

Genome Sequence of the Plant-Pathogenic Bacterium Dickeya dadantii 3937

Dickeya dadantii is a plant-pathogenic enterobacterium responsible for the soft rot disease of many plants of economic importance. We present here the sequence of strain 3937, a strain widely used as a model system for research on the molecular biology and pathogenicity of this group of bacteria.

Membrane topology of MaIG, an inner membrane protein from the maltose transport system of Escherichia coli

In Escherichia coli, the binding protein‐dependent transport system for maltose and maltodextrins is composed of five proteins — LamB, MaIE, MaIF, MaIG and MaIK — located in the three layers of the bacterial envelope. Proteins MaIF and MaIG are hydrophobic inner membrane components mediating the energy‐dependent translocation of substrates into the cytoplasm. In this paper, we analyse the topology of the MaIG protein by using methods based on the properties of fusions between maIG and‘phoA, a truncated gene encoding alkaline phosphatase lacking its translation initiation and exportation signals. Fusions were obtained by using either phage λTnphoA or by constructing in vitro fusions located randomly within the maIG gene. The deduced topological model suggests that MaIG spans the membrane six times and has its amino‐ and carboxy‐termini in the cytoplasm. These results will be helpful for the interpretation of the phenotypes of mutants in maIG.

Phylogenetic analyses of the ATP-binding constituents of bacterial extracytoplasmic receptor-dependent ABC-type nutrient uptake permeases

Thirty-eight ATP-binding cassette (ABC) protein constituents of bacterial extracytoplasmic receptor-dependent nutrient uptake systems, including one homologous chloroplast protein were analysed for sequence conservation and phylogenetic relatedness. The proteins were generally found to cluster in accordance with the clustering patterns previously observed for the extracytoplasmic receptors and the transmembrane channel-forming constituents of these permeases. The results suggest that these transport systems evolved from a single primordial system with minimal shuffling of the three dissimilar protein constituents of the systems.

ATP-driven MalK Dimer Closure and Reopening and Conformational Changes of the “EAA” Motifs Are Crucial for Function of the Maltose ATP-binding Cassette Transporter (MalFGK2)

We have investigated conformational changes of the purified maltose ATP-binding cassette transporter (MalFGK2) upon binding of ATP. The transport complex is composed of a heterodimer of the hydrophobic subunits MalF and MalG constituting the translocation pore and of a homodimer of MalK, representing the ATP-hydrolyzing subunit. Substrate is delivered to the transporter in complex with periplasmic maltose-binding protein (MalE). Cross-linking experiments with a variant containing an A85C mutation within the Q-loop of each MalK monomer indicated an ATP-induced shortening of the distance between both monomers. Cross-linking caused a substantial inhibition of MalE-maltose-stimulated ATPase activity. We further demonstrated that a mutation affecting the “catalytic carboxylate” (E159Q) locks the MalK dimer in the closed state, whereas a transporter containing the “ABC signature” mutation Q140K permanently resides in the resting state. Cross-linking experiments with variants containing the A85C mutation combined with cysteine substitutions in the conserved EAA motifs of MalF and MalG, respectively, revealed close proximity of these residues in the resting state. The formation of a MalK-MalG heterodimer remained unchanged upon the addition of ATP, indicating that MalG-EAA moves along with MalK during dimer closure. In contrast, the yield of MalK-MalF dimers was substantially reduced. This might be taken as further evidence for asymmetric functions of both EAA motifs. Cross-linking also caused inhibition of ATPase activity, suggesting that transporter function requires conformational changes of both EAA motifs. Together, our data support ATP-driven MalK dimer closure and reopening as crucial steps in the translocation cycle of the intact maltose transporter and are discussed with respect to a current model.