Traffic ATPases Research Articles

VirB11, a traffic ATPase, mediated flagella assembly and type IV pilus morphogenesis to control the motility and virulence of Xanthomonas albilineans.

Xanthomonas albilineans (Xal) is a gram-negative bacterial pathogen responsible for developing sugarcane leaf scald disease, which engenders significant economic losses within the sugarcane industry. In the current study, homologous recombination exchange was carried out to induce mutations within the virB/D4-like type IV secretion system (T4SS) genes of Xal. The results revealed that the virB11-deletion mutant (ΔvirB11) exhibited a loss in swimming and twitching motility. Application of transmission electron microscopy analysis further demonstrated that the ΔvirB11 failed to develop flagella formation and type IV pilus morphology and exhibited reduced swarming behaviour and virulence. However, these alterations had no discernible impact on bacterial growth. Comparative transcriptome analysis between the wild-type Xal JG43 and the deletion-mutant ΔvirB11 revealed 123 differentially expressed genes (DEGs), of which 28 and 10 DEGs were notably associated with flagellar assembly and chemotaxis, respectively. In light of these findings, we postulate that virB11 plays an indispensable role in regulating the processes related to motility and chemotaxis in Xal.

NMR resonance assignments for the GSPII-B domain of the traffic ATPase PilF from Thermus thermophilus in the apo and the c-di-GMP-bound state.

The PilF protein from the thermophilic bacterium Thermus thermophilus is a traffic ATPase powering the assembly of the DNA translocation machinery as well as of type 4 pili. Thereby PilF mediates the natural transformability of T. thermophilus. PilF contains a C-terminal ATPase domain and three N-terminal domains with partial homology to so-called general secretory pathway II (GSPII) domains. These three GSPII domains (GSPII-A, GSPII-B and GSPII-C) are essential for pilus assembly and twitching motility. They show varying degrees of sequence homology to the N-terminal domain of the ATPase MshE from Vibrio cholerae which binds the bacterial second messenger molecule c-di-GMP. NMR experiments demonstrate that the GSPII-B domain of PilF also binds c-di-GMP with high affinity and forms a 1:1 complex in slow exchange on the NMR time scale. As a prerequisite for structural studies of c-di-GMP binding to the GSPII-B domain of T. thermophilus PilF we present here the NMR resonance assignments for the apo and the c-di-GMP bound state of GSPII-B. In addition, we map the binding site for c-di-GMP on the GSPII-B domain using chemical shift perturbation data and compare the dynamics of the apo and the c-di-GMP-bound state of the GSPII-B domain based on {1H},15N-hetNOE data.

NMR resonance assignments for the GSPII-C domain of the PilF ATPase from Thermus thermophilus in complex with c-di-GMP.

The natural transformation system of the thermophilic bacterium Thermus thermophilus is one of the most efficient DNA transport systems in terms of DNA uptake rate and promiscuity. The DNA transporter of T. thermophilus plays an important role in interdomain DNA transfer in hot environments. PilF is the traffic ATPase that provides the energy for the assembly of the DNA translocation machinery and the functionally linked type IV pilus system in T. thermophilus. In contrast to other known traffic ATPases, the N-terminal region of PilF harbors three consecutive domains with homology to general secretory pathway II (GSPII) domains. These GSPII-like domains influence pilus assembly, twitching motility and transformation efficiency. A structural homolog of the PilF GSPII-like domains, the N-terminal domain of the traffic ATPase MshE from Vibrio cholerae, was recently crystallized in complex with the bacterial second messenger c-di-GMP. In order to study the consequences of c-di-GMP binding on the three-dimensional architecture of PilF, we initiated structural studies on the PilF GSPII-like domains. Here, we present the 1H, 13C and 15N chemical shift assignments for the isolated PilF GSPII-C domain from T. thermophilus in complex with c-di-GMP. In addition, the structural dynamics of the complex was investigated in an {1H},15N-hetNOE experiment.

Structural bases for F plasmid conjugation and F pilus biogenesis in Escherichia coli

Bacterial conjugation systems are members of the large type IV secretion system (T4SS) superfamily. Conjugative transfer of F plasmids residing in the Enterobacteriaceae was first reported in the 1940s, yet the architecture of F plasmid-encoded transfer channel and its physical relationship with the F pilus remain unknown. We visualized F-encoded structures in the native bacterial cell envelope by in situ cryoelectron tomography (CryoET). Remarkably, F plasmids encode four distinct structures, not just the translocation channel or channel-pilus complex predicted by prevailing models. The F1 structure is composed of distinct outer and inner membrane complexes and a connecting cylinder that together house the envelope-spanning translocation channel. The F2 structure is essentially the F1 complex with the F pilus attached at the outer membrane (OM). Remarkably, the F3 structure consists of the F pilus attached to a thin, cell envelope-spanning stalk, whereas the F4 structure consists of the pilus docked to the OM without an associated periplasmic density. The traffic ATPase TraC is configured as a hexamer of dimers at the cytoplasmic faces of the F1 and F2 structures, where it respectively regulates substrate transfer and F pilus biogenesis. Together, our findings present architectural renderings of the DNA conjugation or "mating" channel, the channel-pilus connection, and unprecedented pilus basal structures. These structural snapshots support a model for biogenesis of the F transfer system and allow for detailed comparisons with other structurally characterized T4SSs.

The traffic ATPase PilF interacts with the inner membrane platform of the DNA translocator and type IV pili from Thermusthermophilus.

A major driving force for the adaptation of bacteria to changing environments is the uptake of naked DNA from the environment by natural transformation, which allows the acquisition of new capabilities. Uptake of the high molecular weight DNA is mediated by a complex transport machinery that spans the entire cell periphery. This DNA translocator catalyzes the binding and splitting of double‐stranded DNA and translocation of single‐stranded DNA into the cytoplasm, where it is recombined with the chromosome. The thermophilic bacterium Thermus thermophilus exhibits the highest transformation frequencies reported and is a model system to analyze the structure and function of this macromolecular transport machinery. Transport activity is powered by the traffic ATPase PilF, a soluble protein that forms hexameric complexes. Here, we demonstrate that PilF physically binds to an inner membrane assembly platform of the DNA translocator, comprising PilMNO, via the ATP‐binding protein PilM. Binding to PilMNO or PilMN stimulates the ATPase activity of PilF ~ 2‐fold, whereas there is no stimulation when binding to PilM or PilN alone. A PilMK26A variant defective in ATP binding still binds PilF and, together with PilN, stimulates PilF‐mediated ATPase activity. PilF is unique in having three conserved GSPII (general secretory pathway II) domains (A–C) at its N terminus. Deletion analyses revealed that none of the GSPII domains is essential for binding PilMN, but GSPIIC is essential for PilMN‐mediated stimulation of ATP hydrolysis by PilF. Our data suggest that PilM is a coupling protein that physically and functionally connects the soluble motor ATPase PilF to the DNA translocator via the PilMNO assembly platform.

Conjugation inhibitors compete with palmitic acid for binding to the conjugative traffic ATPase TrwD, providing a mechanism to inhibit bacterial conjugation

Bacterial conjugation is a key mechanism by which bacteria acquire antibiotic resistance. Therefore, conjugation inhibitors (COINs) are promising compounds in the fight against the spread of antibiotic resistance genes among bacteria. Unsaturated fatty acids (uFAs) and alkynoic fatty acid derivatives, such as 2-hexadecanoic acid (2-HDA), have been reported previously as being effective COINs. The traffic ATPase TrwD, a VirB11 homolog in plasmid R388, is the molecular target of these compounds, which likely affect binding of TrwD to bacterial membranes. In this work, we demonstrate that COINs are abundantly incorporated into Escherichia coli membranes, replacing palmitic acid as the major component of the membrane. We also show that TrwD binds palmitic acid, thus facilitating its interaction with the membrane. Our findings also suggest that COINs bind TrwD at a site that is otherwise occupied by palmitic acid. Accordingly, molecular docking predictions with palmitic acid indicated that it shares the same binding site as uFAs and 2-HDA, although it differs in the contacts involved in this interaction. We also identified 2-bromopalmitic acid, a palmitate analog that inhibits many membrane-associated enzymes, as a compound that effectively reduces TrwD ATPase activity and bacterial conjugation. Moreover, we demonstrate that 2-bromopalmitic and palmitic acids both compete for the same binding site in TrwD. Altogether, these detailed findings open up a new avenue in the search for effective synthetic inhibitors of bacterial conjugation, which may be pivotal for combating multidrug-resistant bacteria.

A Computational workflow for the identification of the potent inhibitor of type II secretion system traffic ATPase of Pseudomonas aeruginosa

Bacterial type II secretion system has now become an attractive target for antivirulence drug development. The aim of the present study was to characterize the binding site of the type II secretion system traffic ATPase GspER of Pseudomonas aeruginosa, and identify potent inhibitors using extensive computational and virtual screening approaches. Initially, the designed platform focused on the evolutionary relationship of ATPase GspER of P. aeruginosa, followed by protein-protein interaction network analysis to characterize its function. In addition, homology modeling, virtual screening and molecular dynamics simulation have been performed to identify potent hits and understand the ligand binding properties of ATPase GspER. According to the evolutionary relationship, high level of genetic change was observed for P. aeruginosa, bearing orthology relationships with P.alcaligenes and P.otitidis. Concurrently, the binding site analysis represented residue Gly268, Ser267, Thr270, Thr271and Lys269 in Walker A motif directly formed hydrogen bonds with the ATP, which modulates the function of ATPase GspER in the secretion process, is also validated by the molecular docking analysis and molecular dynamics simulation. Furthermore, ZINC04325133 is one of the most potent hits has been identified from virtual screening approach followed by molecular dynamics simulation and MM-GBSA binding energy analysis. These results may provide new knowledge for the development of novel therapeutic strategies against P. aeruginosa, as well as inhibiting secretion system process of a wide range of gram-negative bacteria.

Functional dissection of the three N-terminal general secretory pathway domains and the Walker motifs of the traffic ATPase PilF from Thermus thermophilus.

The traffic ATPase PilF of Thermus thermophilus powers pilus assembly as well as uptake of DNA. PilF differs from other traffic ATPases by a triplicated general secretory pathway II, protein E, N-terminal domain (GSPIIABC). We investigated the in vivo and in vitro roles of the GSPII domains, the Walker A motif and a catalytic glutamate by analyzing a set of PilF deletion derivatives and pilF mutants. Here, we report that PilF variants devoid of the first two or all three GSPII domains do not form stable hexamers indicating a role of the triplicated GSPII domain in complex formation and/or stability. A pilFΔGSPIIC mutant was significantly impaired in piliation which leads to the conclusion that the GSPIIC domain plays a vital role in pilus assembly. Interestingly, the pilFΔGSPIIC mutant was hypertransformable. This suggests that GSPIIC strongly affects transformation efficiency. A pilF∆GSPIIA mutant exhibited wild-type piliation but reduced pilus-mediated twitching motility, suggesting that GSPIIA plays a role in pilus dynamics. Furthermore, we report that pilF mutants with a defect in the ATP binding Walker A motif or in the catalytic glutamate residue are defective in piliation and natural transformation. These findings show that both, ATP binding and hydrolysis, are essential for the dual function of PilF in natural transformation and pilus assembly.

Type IV traffic ATPase TrwD as molecular target to inhibit bacterial conjugation.

Bacterial conjugation is the main mechanism responsible for the dissemination of antibiotic resistance genes. Hence, the search for specific conjugation inhibitors is paramount in the fight against the spread of these genes. In this pursuit, unsaturated fatty acids have been found to specifically inhibit bacterial conjugation. Despite the growing interest on these compounds, their mode of action and their specific target remain unknown. Here, we identified TrwD, a Type IV secretion traffic ATPase, as the molecular target for fatty acid-mediated inhibition of conjugation. Moreover, 2-alkynoic fatty acids, which are also potent inhibitors of bacterial conjugation, are also powerful inhibitors of the ATPase activity of TrwD. Characterization of the kinetic parameters of ATPase inhibition has led us to identify the catalytic mechanism by which fatty acids exert their activity. These results open a new avenue for the rational design of inhibitors of bacterial conjugation in the fight against the dissemination of antibiotic resistance genes.

Functional Interactions of VirB11 Traffic ATPases with VirB4 and VirD4 Molecular Motors in Type IV Secretion Systems

Pilus biogenesis and substrate transport by type IV secretion systems require energy, which is provided by three molecular motors localized at the base of the secretion channel. One of these motors, VirB11, belongs to the superfamily of traffic ATPases, which includes members of the type II secretion system and the type IV pilus and archaeal flagellar assembly apparatus. Here, we report the functional interactions between TrwD, the VirB11 homolog of the conjugative plasmid R388, and TrwK and TrwB, the motors involved in pilus biogenesis and DNA transport, respectively. Although these interactions remained standing upon replacement of the traffic ATPase by a homolog from a phylogenetically related conjugative system, namely, TraG of plasmid pKM101, this homolog could not replace the TrwD function for DNA transfer. This result suggests that VirB11 works as a switch between pilus biogenesis and DNA transport and reinforces a mechanistic model in which VirB11 proteins act as traffic ATPases by regulating both events in type IV secretion systems.

Purification, crystallization and preliminary X-ray diffraction studies of the ATP-binding subunit of an ABC transporter fromGeobacillus kaustophilus

ATP-binding cassette (ABC) transporters, also known as traffic ATPases, form a large family of integral membrane proteins responsible for the translocation of a variety of chemically diverse substrates across the lipid bilayers of cellular membranes of both prokaryotes and eukaryotes by the hydrolysis of ATP. The ATP-binding subunit of an ABC transporter from Geobacillus kaustophilus, a homodimeric enzyme, was overexpressed in Escherichia coli and purified. Crystals were obtained using the microbatch-under-oil method at 291 K. X-ray diffraction data to 1.6 Å resolution were collected on SPring-8 beamline BL26B1. The crystals belonged to the orthorhombic space group I222, with unit-cell parameters a=54.94, b=78.63, c=112.96 Å. Assuming the presence of a dimer in the asymmetric unit gave a crystal volume per protein weight (VM) of 2.32 Å3 Da(-1) and a solvent content of 47%; this was consistent with the results of a dynamic light-scattering experiment, which showed a dimeric state of the protein in solution. Molecular-replacement trials using the crystal structure of HisP from the Salmonella typhimurium ATP-binding subunit of an ABC transporter as a search model did not provide a satisfactory solution, indicating that the two ATP-binding subunits of ABC transporters have substantially different structures.

Retracted: Maf acts downstream of ComGA to arrest cell division in competent cells of B. subtilis

Transformable (competent) cells of Bacillus subtilis are blocked in cell division because the traffic ATPase ComGA prevents the formation of FtsZ rings. Although ComGA-deficient cells elongate and form FtsZ rings, cell division remains blocked at a later stage and the cells become mildly filamented. Here we show that the highly conserved protein Maf is synthesized predominantly in competent cells under the direct control of the transcription factor ComK and is solely responsible for the later block in cell division. In vivo and in vitro data show that Maf binds to both ComGA and DivIVA. A point mutation in maf that interferes with Maf binding to DivIVA also interferes with the ability of Maf to inhibit cell division. Based on these findings, we propose that Maf and ComGA mediate mechanisms for the inhibition of cell division in competent cells with Maf acting downstream of ComGA. We further suggest that Maf must interact with DivIVA to inhibit cell division.

Inorganic carbon transporters of the cyanobacterial CO2 concentrating mechanism

Cyanobacteria possess an environmental adaptation known as a CO(2) concentrating mechanism (CCM) that evolved to improve photosynthetic performance, particularly under CO(2)-limiting conditions. The CCM functions to actively transport dissolved inorganic carbon species (Ci; HCO(3)(-) and CO(2)) resulting in accumulation of a pool of HCO(3)(-) within the cell that is then utilised to provide an elevated CO(2) concentration around the primary CO(2) fixing enzyme, ribulose bisphosphate carboxylase-oxygenase (Rubisco). Rubisco is encapsulated in unique micro-compartments known as carboxysomes and also provides the location for elevated CO(2) levels in the cell. Five distinct transport systems for active Ci uptake are known, including two types of Na(+)-dependent HCO(3)(-) transporters (BicA and SbtA), one traffic ATPase (BCT1) for HCO(3)(-) uptake and two CO(2) uptake systems based on modified NADPH dehydrogenase complexes (NDH-I(3) and NDH-I(4)). The genes for a number of these transporters are genetically induced under Ci limitation via transcriptional regulatory processes. The in-membrane topology structures of the BicA and SbtA HCO(3)(-) transporters are now known and this may aid in determining processes related to transporter activation during dark to light transitions or under severe Ci limitation.

Sulfonylurea receptors and mechanism of sulfonylurea action.

Binding of hypoglycemic sulfonylureas and their analogues to the sulfonylurea receptor in the beta-cell plasma membrane mediates closure of the ATP-sensitive K+-channel (KATP-channel) and thereby stimulation of insulin release. The sulfonylurea receptor is a member of the traffic ATPase family with two intracellular nucleotide binding folds. The receptor binding site for hypoglycemic drugs is located at the cytoplasmic face of the plasma membrane. Mutations in the sulfonylurea receptor gene have been detected which cause familial hyper-insulinism. Non-beta-cell sulfonylurea receptors do not contribute to the therapeutic benefit of sulfonylureas, but might be involved in presumed adverse effects of sulfonylureas in the cardiovascular and the central nervous system.

A novel ATP-binding cassette transporter, ABCG6 is involved in chemoresistance of Leishmania

ATP-binding cassette (ABC) transporters constitute the biggest family of membrane proteins involved in drug resistance and other biological activities. Resistance of leishmanial parasites to therapeutic drugs continues to escalate in developing countries and in many instances it is due to overexpressed ABC efflux pumps. Progressively adapted camptothecin (CPT)-resistant parasites show overexpression of a novel ABC transporter, which was classified as ABCG6. Transfection and overexpression of LdABCG6 in wild type parasites, shows its localization primarily in the plasma membrane and flagellar pocket region. Overexpressed LdABCG6 confers substantial CPT resistance to the parasites by rapid drug efflux. Various inhibitors have been tested for their ability to revert the CPT-resistant phenotype to specifically understand the inhibition of LdABCG6 transporter. Transport experiments using everted membrane vesicles were carried out to gain an insight into the kinetics of drug transport. This study provides further knowledge of specific membrane traffic ATPase and its involvement in the chemoresistance of Leishmania.

FhuD1, a Ferric Hydroxamate-binding Lipoprotein in Staphylococcus aureus: A CASE OF GENE DUPLICATION AND LATERAL TRANSFER

Staphylococcus aureus can utilize ferric hydroxamates as a source of iron under iron-restricted growth conditions. Proteins involved in this transport process are: FhuCBG, which encodes a traffic ATPase; FhuD2, a post-translationally modified lipoprotein that acts as a high affinity receptor at the cytoplasmic membrane for the efficient capture of ferric hydroxamates; and FhuD1, a protein with similarity to FhuD2. Gene duplication likely gave rise to fhuD1 and fhuD2. While the genomic locations of fhuCBG and fhuD2 in S. aureus strains are conserved, both the presence and the location of fhuD1 are variable. The apparent redundancy of FhuD1 led us to examine the role of this protein. We demonstrate that FhuD1 is expressed only under conditions of iron limitation through the regulatory activity of Fur. FhuD1 fractions with the cell membrane and binds hydroxamate siderophores but with lower affinity than FhuD2. Using small angle x-ray scattering, the solution structure of FhuD1 resembles that of FhuD2, and only a small conformational change is associated with ferrichrome binding. FhuD1, therefore, appears to be a receptor for ferric hydroxamates, like FhuD2. Our data to date suggest, however, that FhuD1 is redundant to FhuD2 and plays a minor role in hydroxamate transport. However, given the very real possibility that we have not yet identified the proper conditions where FhuD1 does provide an advantage over FhuD2, we anticipate that FhuD1 serves an enhanced role in the transport of untested hydroxamate siderophores and that it may play a prominent role during the growth of S. aureus in its natural environments.

Crystal structure of the hexameric traffic ATPase of theHelicobacter pyloritype IV secretion system: insights into function and mode of action

Crystal structure of the hexameric traffic ATPase of the<i>Helicobacter pylori</i>type IV secretion system: insights into function and mode of action

Overexpression, purification, and characterization of the periplasmic space thiamin-binding protein of the thiamin traffic ATPase in Escherichia coli

Thiamin (Vitamin B 1) transport in Escherichia coli occurs by the superfamily of traffic ATPases in which the initial receptor is the periplasmic binding protein. We have cloned the periplasmic thiamin-binding protein (TBP) of the E. coli periplasmic thiamin transport system and purified the overexpressed protein to apparent homogeneity. A subsequent biochemical characterization demonstrates that TBP is a 34.205 kDa monomer. TBP also contains one tightly bound thiamin species [thiamin, thiamin monophosphate (TMP), or thiamin diphosphate (TDP)] per monomer ( K D =0.8 μM ) when isolated under conditions that would remove any loosely bound ligands. We also demonstrate that thiamin is readily exchangeable in the presence of exogenous thiamin with a k off =0.12 s −1 . The biochemical characteristics of the overexpressed, plasmid-derived TBP are indistinguishable from those determined for endogenous TBP purified from E. coli. The overexpression and purification of TBP that we present here allows the rapid isolation of large amounts of pure protein that are required for further mechanistic and structural studies and demonstrates a vast improvement over previously reported purifications.

Modification of type IV pilus-associated epithelial cell adherence and multicellular behavior by the PilU protein of Neisseria gonorrhoeae.

Expression of type IV pili (Tfp) correlates with the ability of Neisseria gonorrhoeae to colonize the human host, as well as with adherence to human epithelial tissue, twitching motility, competence for natural transformation, and autoagglutination. N. gonorrhoeae PilF (required for Tfp biogenesis) and PilT (required for twitching motility and transformation) share significant identities with members of a family of putative ATPases involved in membrane trafficking of macromolecules. An open reading frame downstream of the pilT locus encoding a 408-amino-acid protein with 33% identity with the gonococcal PilT protein and 45% identity with the PilU protein in Pseudomonas aeruginosa was characterized, and the corresponding gene was designated pilU. Unlike N. gonorrhoeae pilT mutants, pilU mutants express twitching motility and are competent for DNA transformation. However, loss-of-function mutations in pilU increased bacterial adherence to ME-180 human epithelial cells eightfold and disrupted in vitro Tfp-associated autoagglutination. Comparative alignment of N. gonorrhoeae PilU with other members of the TrbB-like family of traffic ATPases revealed a conserved carboxy-terminal domain unique to family members which are not essential for Tfp biogenesis but which specifically modify Tfp-associated phenotypes. Studies of the pilT-pilU locus by using Northern blotting, transcriptional fusions, and reverse transcription-PCR showed that the two genes encoding closely related proteins with dissimilar effects on Tfp phenotypes are transcribed from a single promoter.

Two-hybrid-based analysis of protein-protein interactions of the yeast multidrug resistance protein, Pdr5p.

The ATP-binding cassette (ABC) transporters are a large family of proteins responsible for the translocation of a variety of compounds across the membranes of both prokaryotes and eukaryotes. The inter-protein and intra-protein interactions in these traffic ATPases are still only poorly understood. In the present study we describe, for the first time, an extensive yeast two-hybrid (Y2H)-based analysis of the interactions of the cytoplasmic loops of the yeast pleiotropic drug resistance (Pdr) protein, Pdr5p, an ABC transporter of Saccharomyces cerevisiae. Four of the major cytosolic loops that have been predicted for this protein [including the two nucleotide-binding domain (NBD)-containing loops and the cytosolic C-terminal region] were subjected to an extensive inter-domain interaction study in addition to being used as baits to identify potential interacting proteins within the cell using the Y2H system. Results of these studies have revealed that the first cytosolic loop (CL1)--containing the first NBD domain--and also the C-terminal region of Pdr5p, interact with several candidate proteins. The possibility of an interaction between the CL1 loops of two neighboring Pdr5p molecules was also indicated, which could possibly have implications for dimerization of this protein.