The C-terminal domain of the Uup protein is a DNA-binding coiled coil motif.

The bacterial Uup protein belongs to the REG subfamily of soluble ATP-binding cassette (ABC) ATPases, and is implicated in precise excision of transposons. In Escherichia coli, the uup gene encodes a 72 kDa polypeptide that comprises two ABC domains, separated by a linker region, and a 12kDa C-terminal domain (CTD). Uup binds double-stranded DNA with no sequence specificity, and we previously demonstrated that the CTD domain is a crucial region that participates in DNA-binding activity. We report herein the NMR structure of Uup CTD, consisting of an intramolecular antiparallel two-stranded coiled coil motif. Structural comparison with analogous coiled coil domains reveals that Uup CTD contains an atypical 3(10)-helix in the α-hairpin region that contributes to the hydrophobic core. Using NMR titration experiments, we identified residues of the CTD domain involved in the binding to double-stranded DNA. These residues are located on two opposite surfaces at the base of the coiled coil, formed by the N- and C-terminal extremities, where a strictly conserved proline residue induces an overwinding of the coiled coil. Finally, preliminary analysis of NMR spectra recorded on distinct Uup constructs precludes a fully flexible positioning of the CTD domain in full-length Uup. These structural data are the first reported for a non-ATPase domain within ABC REG subfamily.

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.

C-terminal domain of the Uup ATP-binding cassette ATPase is an essential folding domain that binds to DNA.

The Uup protein belongs to a subfamily of soluble ATP-binding cassette (ABC) ATPases that have been implicated in several processes different from transmembrane transport of molecules, such as transposon precise excision. We have demonstrated previously that Escherichia coli Uup is able to bind DNA. DNA binding capacity is lowered in a truncated Uup protein lacking its C-terminal domain (CTD), suggesting a contribution of CTD to DNA binding. In the present study, we characterize the role of CTD in the function of Uup, on its overall stability and in DNA binding. To this end, we expressed and purified isolated CTD and we investigated the structural and functional role of this domain. The results underline that CTD is essential for the function of Uup, is stable and able to fold up autonomously. We compared the DNA binding activities of three versions of the protein (Uup, UupDeltaCTD and CTD) by an electrophoretic mobility shift assay. CTD is able to bind DNA although less efficiently than intact Uup and UupDeltaCTD. These observations suggest that CTD is an essential domain that contributes directly to the DNA binding ability of Uup.

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.

Structural and functional diversity calls for a new classification of ABC transporters.

Members of the ATP-binding cassette (ABC) transporter superfamily translocate a broad spectrum of chemically diverse substrates. While their eponymous ATP-binding cassette in the nucleotide-binding domains (NBDs) is highly conserved, their transmembrane domains (TMDs) forming the translocation pathway exhibit distinct folds and topologies, suggesting that during evolution the ancient motor domains were combined with different transmembrane mechanical systems to orchestrate a variety of cellular processes. In recent years, it has become increasingly evident that the distinct TMD folds are best suited to categorize the multitude of ABC transporters. We therefore propose a new ABC transporter classification that is based on structural homology in the TMDs.

ATP-Binding Cassette Systems of Brucella.

Brucellosis is a prevalent zoonotic disease and is endemic in the Middle East, South America, and other areas of the world. In this study, complete inventories of putative functional ABC systems of five Brucella species have been compiled and compared. ABC systems of Brucella melitensis 16M, Brucella abortus 9-941, Brucella canis RM6/66, Brucella suis 1330, and Brucella ovis 63/290 were identified and aligned. High numbers of ABC systems, particularly nutrient importers, were found in all Brucella species. However, differences in the total numbers of ABC systems were identified (B. melitensis, 79; B. suis, 72; B. abortus 64; B. canis, 74; B. ovis, 59) as well as specific differences in the functional ABC systems of the Brucella species. Since B. ovis is not known to cause human brucellosis, functional ABC systems absent in the B. ovis genome may represent virulence factors in human brucellosis.

Deletion of the Escherichia coli uup gene encoding a protein of the ATP binding cassette superfamily affects bacterial competitiveness.

Bacteria use a variety of mechanisms for intercellular communication. Here we show that deletion of the uup gene, which encodes a soluble ATP binding cassette (ABC) ATPase, renders the mutant strain sensitive to its parent when they are grown together in the same medium. Our data suggest that the decrease in viability of the mutant is dependent on direct cell-to-cell contact with the parent strain. Furthermore, we show that the presence of intact Walker B motifs in Uup is required for immunity or resistance to the parental strain, suggesting that ATP hydrolysis is an important determinant of this phenotype.

ATP-binding cassette systems in Burkholderia pseudomallei and Burkholderia mallei.

BACKGROUND: ATP binding cassette (ABC) systems are responsible for the import and export of a wide variety of molecules across cell membranes and comprise one of largest protein superfamilies found in prokarya, eukarya and archea. ABC systems play important roles in bacterial lifestyle, virulence and survival. In this study, an inventory of the ABC systems of Burkholderia pseudomallei strain K96243 and Burkholderia mallei strain ATCC 23344 has been compiled using bioinformatic techniques. RESULTS: The ABC systems in the genomes of B. pseudomallei and B. mallei have been reannotated and subsequently compared. Differences in the number and types of encoded ABC systems in belonging to these organisms have been identified. For example, ABC systems involved in iron acquisition appear to be correlated with differences in genome size and lifestyles between these two closely related organisms. CONCLUSION: The availability of complete inventories of the ABC systems in B. pseudomallei and B. mallei has enabled a more detailed comparison of the encoded proteins in this family. This has resulted in the identification of ABC systems which may play key roles in the different lifestyles and pathogenic properties of these two bacteria. This information has the potential to be exploited for improved clinical identification of these organisms as well as in the development of new vaccines and therapeutics targeted against the diseases caused by these organisms.

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.

Manual and expert annotation of the nearly complete genome sequence of Staphylococcus sciuri strain ATCC 29059: A reference for the oxidase-positive staphylococci that supports the atypical phenotypic features of the species group.

Staphylococcus sciuri is considered to be one of the most ancestral species in the natural history of the Staphylococcus genus that consists of 48 validly described species. It belongs to the basal group of oxidase-positive and novobiocin-resistant staphylococci that diverged from macrococci approximately 250 million years ago. Contrary to other groups, the S. sciuri species group has not developed host-specific colonization strategies. Genome analysis of S. sciuri ATCC 29059 provides here the first genetic basis for atypical traits that would support the switch between the free-living style and the infective state in animals and humans. From among the most remarkable features, it was noticed in this extensive study that there were a number of phosphoenolpyruvate:carbohydrate phosphotransferase systems (PTS), almost twice as many as any other staphylococci, and the co-occurrence of mevalonate and non-mevalonate pathways for isoprenoid synthesis. The sequenced strain was devoid of the main virulence factors present in Staphylococcus aureus, although it exhibited numerous heme and iron acquisition systems, as well as crt and aldH genes necessary for gold pigment synthesis. The sensing and signaling networks, exemplified by a large and typical repertoire of two-component regulatory systems and a complete panel of master regulators, such as agr, rex, mgrA, rot, sarA and sarR genes, depict the background in which S. aureus virulence genes were later acquired. An additional sigma factor, a distinct set of electron transducer elements and many gene operons similar to those found in Bacillus spp. would constitute the most visible remnant links with Bacillaceae organisms.

The identification and evaluation of ATP binding cassette systems in the intracellular bacterium Francisella tularensis.

Francisella tularensis is a facultative intracellular bacterium responsible for the disease tularemia. Analysis of the fully sequenced genome of the virulent F. tularensis strain SCHU S4 has led to the identification of twenty ATP binding cassette (ABC) systems, of which five appear to be non-functional. The fifteen complete systems comprise three importers, five exporters, four systems involved in non-transport processes, and three systems of unknown or ill-defined function. The number and classification of the ABC systems in F. tularensis is similar to that observed in other intracellular bacteria, indicating that some of these systems may be important for the intracellular lifestyle of these organisms. Among the ABC systems identified in the genome are systems that may be involved in the virulence of F. tularensis SCHU S4. Six ABC system proteins were evaluated as candidate vaccine antigens against tularemia, although none provided significant protection against F. tularensis. However, a greater understanding of these systems may lead to the development of countermeasures against F. tularensis.

Inventory and comparative analysis of rice and Arabidopsis ATP-binding cassette (ABC) systems.

ATP-binding cassette (ABC) proteins constitute a large superfamily found in all kingdoms of living organisms. The recent completion of two draft sequences of the rice (Oryza sativa) genome allowed us to analyze and classify its ABC proteins and to compare to those in Arabidopsis thaliana. We identified a similar number of ABC proteins in rice and Arabidopsis (121 versus 120), despite the rice genome being more than three times the size of Arabidopsis. Both Arabidopsis and rice have representative members in all seven major subfamilies of ABC ATPases (A to G) commonly found in eukaryotes. This comparative analysis allowed the detection of 29 potential orthologous sequences in Arabidopsis and rice. However, plant share with prokaryotes a specific set of ABC systems that is not detected in animals. These ABC systems might be inherited from the cyanobacterial ancestor of chloroplasts. The present work provides the first complete inventory of rice ABC proteins and an updated inventory of those proteins in Arabidopsis.

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.

The metNPQ operon of Bacillus subtilis encodes an ABC permease transporting methionine sulfoxide, D- and L-methionine.

The Bacillus subtilis yusCBA operon, which encodes an ABC-type transporter, contains an S-box motif in its promoter region. We showed that the expression of these genes is repressed via the S-box system when methionine is available. The YusCB proteins are involved in the transport of both d- and l-methionine but also methionine sulfoxide. A yusCB mutant is unable to grow in the presence of 5 microM l-methionine or 100 microM methionine sulfoxide, while it grows similarly to the wild type with 100 microM l-methionine and 1 mM methionine sulfoxide. Other uptake systems are therefore present for these two compounds. In contrast, the Yus ABC transporter corresponds to the sole d-methionine uptake system. We propose to rename yusC, yusB and yusA as metN, metP and metQ, respectively.

Comparative analysis of sequences encoding ABC systems in the genome of the microsporidian Encephalitozoon cuniculi.

Microsporidia are amitochondriate eukaryotic microbes with fungal affinities and a common status of obligate intracellular parasites. A set of 13 potential genes encoding ATP-binding cassette (ABC) systems was identified in the fully sequenced genome of Encephalitozoon cuniculi. Our analyses of multiple alignments, phylogenetic trees and conserved motifs support a distribution of E. cuniculi ABC systems within only four subfamilies. Six half transporters are homologous to the yeast ATM1 mitochondrial protein, a finding which is in agreement with the hypothesis of a cryptic mitochondrion-derived compartment playing a role in the synthesis and transport of Fe-S clusters. Five half transporters are similar to the human ABCG1 and ABCG2 proteins, involved in regulation of lipid trafficking and anthracyclin resistance respectively. Two proteins with duplicated ABC domains are clearly candidate to non-transport ABC systems: the first is homologous to mammalian RNase L inhibitor and the second to the yeast translation initiation regulator GCN20. An unusual feature of ABC systems in E. cuniculi is the lack of homologs of P-glycoprotein and other ABC transporters which are involved in multiple drug resistance in a large number of eukaryotic microorganisms.

Secondary structure and NMR resonance assignments of the C-terminal DNA-binding domain of Uup protein.

ATP-binding cassette (ABC) systems belong to a large superfamily of proteins that couple the energy released from ATP hydrolysis to a wide variety of cellular processes, including not only transport of various molecules, but also gene regulation, and DNA repair. Mutations in the bacterial uup gene, which encodes a cytosolic ABC ATPase, lead to an increase in the frequency of precise excision of transposons Tn10 and Tn5, suggesting a role of the Uup protein in DNA metabolism. Uup is a 72 kDa polypeptide which comprises two ABC domains, separated by a 75-residue linker, and a C-terminal domain (CTD) of unknown function. The Uup protein from Escherichia coli has been shown to bind DNA in vitro, and the CTD domain contributes to the DNA-binding affinity. We have produced and purified uniformly labeled (15)N- and (15)N/(13)C Uup CTD domain (region 528-635), and assigned backbone and side-chains resonances using heteronuclear NMR spectroscopy. Secondary structure evaluation based on backbone chemical shifts is consistent with the presence of three α-helices, including two long ones (residues 564-590 and 601-632), suggesting that Uup CTD may fold as an intramolecular coiled coil motif. This work provides the starting point towards determining the first atomic structure of a non-ATPase domain within the vast REG subfamily of ABC soluble ATPases.

Natural history of ABC systems: not only transporters.

In recent years, our understanding of the functioning of ABC (ATP-binding cassette) systems has been boosted by the combination of biochemical and structural approaches. However, the origin and the distribution of ABC proteins among living organisms are difficult to understand in a phylogenetic perspective, because it is hard to discriminate orthology and paralogy, due to the existence of horizontal gene transfer. In this chapter, I present an update of the classification of ABC systems and discuss a hypothetical scenario of their evolution. The hypothetical presence of ABC ATPases in the last common ancestor of modern organisms is discussed, as well as the additional possibility that ABC systems might have been transmitted to eukaryotes, after the two endosymbiosis events that led to the constitution of eukaryotic organelles. I update the functional information of selected ABC systems and introduce new families of ABC proteins that have been included recently into this vast superfamily, thanks to the availability of high-resolution three-dimensional structures.

Structure, function, and evolution of bacterial ATP-binding cassette systems.

SUMMARY: ATP-binding cassette (ABC) systems are universally distributed among living organisms and function in many different aspects of bacterial physiology. ABC transporters are best known for their role in the import of essential nutrients and the export of toxic molecules, but they can also mediate the transport of many other physiological substrates. In a classical transport reaction, two highly conserved ATP-binding domains or subunits couple the binding/hydrolysis of ATP to the translocation of particular substrates across the membrane, through interactions with membrane-spanning domains of the transporter. Variations on this basic theme involve soluble ABC ATP-binding proteins that couple ATP hydrolysis to nontransport processes, such as DNA repair and gene expression regulation. Insights into the structure, function, and mechanism of action of bacterial ABC proteins are reported, based on phylogenetic comparisons as well as classic biochemical and genetic approaches. The availability of an increasing number of high-resolution structures has provided a valuable framework for interpretation of recent studies, and realistic models have been proposed to explain how these fascinating molecular machines use complex dynamic processes to fulfill their numerous biological functions. These advances are also important for elucidating the mechanism of action of eukaryotic ABC proteins, because functional defects in many of them are responsible for severe human inherited diseases.

ATP hydrolysis and pristinamycin IIA inhibition of the Staphylococcus aureus Vga(A), a dual ABC protein involved in streptogramin A resistance.

In Gram-positive bacteria, a large subfamily of dual ATP-binding cassette proteins confers acquired or intrinsic resistance to macrolide, lincosamide, and streptogramin antibiotics by a far from well understood mechanism. Here, we report the first biochemical characterization of one such protein, Vga(A), which is involved in streptogramin A (SgA) resistance among staphylococci. Vga(A) is composed of two nucleotide-binding domains (NBDs), separated by a charged linker, with a C-terminal extension and without identified transmembrane domains. Highly purified Vga(A) displays a strong ATPase activity (K(m) = 78 mum, V(m) = 6.8 min(-1)) that was hardly inhibited by orthovanadate. Using mutants of the conserved catalytic glutamate residues, the two NBDs of Vga(A) were shown to contribute unequally to the total ATPase activity, the mutation at NBD2 being more detrimental than the other. ATPase activity of both catalytic sites was essential for Vga(A) biological function because each single Glu mutant was unable to confer SgA resistance in the staphylococcal host. Of great interest, Vga(A) ATPase was specifically inhibited in a non-competitive manner by the SgA substrate, pristinamycin IIA (PIIA). A deletion of the last 18 amino acids of Vga(A) slightly affected the ATPase activity without modifying the PIIA inhibition values. In contrast, this deletion reduced 4-fold the levels of SgA resistance. Altogether, our results suggest a role for the C terminus in regulation of the SgA antibiotic resistance mechanism conferred by Vga(A) and demonstrate that this dual ATP-binding cassette protein interacts directly and specifically with PIIA, its cognate substrate.