Erich Lanka

Crystal Structure of the Hexameric Traffic ATPase of the Helicobacter pylori Type IV Secretion System

Abstract The type IV secretion system of Helicobacter pylori consists of 10–15 proteins responsible for transport of the transforming protein CagA into target epithelial cells. Secretion of CagA crucially depends on the hexameric ATPase, HP0525, a member of the VirB11-PulE family. We present the crystal structure of a binary complex of HP0525 bound to ADP. Each monomer consists of two domains formed by the N- and C-terminal halves of the sequence. ADP is bound at the interface between the two domains. In the hexamer, the N- and C-terminal domains form two rings, which together form a chamber open on one side and closed on the other. A model is proposed in which HP0525 functions as an inner membrane pore, the closure and opening of which is regulated by ATP binding and ADP release.

Common mechanisms in bacterial conjugation and Ti-mediated T-DNA transfer to plant cells

Monika Lees1 and Erich Lanka Max-Planck-lnstitut fiir Molekulare Genetik Abteilung Schuster lhnestrasse 73 D-14195 Berlin-Dahlem Federal Republic of Germany Introduction Recently, the conjugative transfer system encoded by the broad host-range plasmid RP4 (IncP) has attracted broad interest. This interest stems mainly from the recognition of two characteristics: the RP4 transfer apparatus can mo- bilize DNA to a variety of phylogenetically distinct microor- ganisms, including gram-negative and gram-positive bac- teria, and even yeasts; and data have accumulated to suggest a close relationship between RPCmediated bac- terial conjugation and T-DNA transfer from Agrobacterium spp. to plant cells. Plasmid RP4 (60 kb) was originally identified in a Pseu- domonas clinical isolate. Nearly half of the genome of the plasmid is devoted to conjugative transfer functions. These are encoded by two distinct regions of the plasmid, known as Tral and Tra2. Tral encodes the gene products responsible for the initiation of conjugative DNA synthesis and contains the replication start point, the so-called origin of transfer (oriT). Functions of Tra2 together with one com- ponent of Tral are needed to establish physical contact between the donor and recipient cells. Agrobacterium tumefaciens is a plant pathogen that ge- netically transforms plant cells. Introduction of a distinct DNA element, the T-DNA, into the nuclear genome of the plant leads to tumor formation (Zambryski, 1992). The T-DNA is located on a large extrachromosomal element (-200 kb), called Ti (for tumor-inducing) plasmid. The Ti plasmid encompasses the virulence region (vir) that pro- vides most of the products that mediate actual T-DNA movement. The vir loci consist of six complementation groups (- 30 kb). Some of them are either essential for (A Less1 et al., 1993). This relationship includes amino acid sequence similarities, gene organization, and physical properties of the gene products. Most of the Tra2 and VirB proteins are hydrophobic, and some carry potential signal sequences at their N-termini, predicting a membrane- associated location. This suggests that, following attach- ment, VirB proteins in the bacterial envelope interact with the cell surface of the plant to stabilize this initial contact. Whether a pilus-like structure is involved in Agrobacteria- plant contact still remains unclear. However, it is notewor- thy that proteins encoded by virB2 of Ti and tr6C RP4 show sequence similarities to the pilus subunit (TraA) the Escherichia coli F factor (Balzer, 1993; Shirasu and Kado, 1993). Considering that the RP4 Mpf functions are responsible for pilus formation, the question arises as to whether the same machinery is involved in creation of a DNA transfer channel. This seems likely, since most of the RP4 Mpf

VirB11 ATPases are dynamic hexameric assemblies: New insights into bacterial type IV secretion

The coupling of ATP binding/hydrolysis to macromolecular secretion systems is crucial to the pathogenicity of Gram‐negative bacteria. We reported previously the structure of the ADP‐bound form of the hexameric traffic VirB11 ATPase of the Helicobacter pylori type IV secretion system (named HP0525), and proposed that it functions as a gating molecule at the inner membrane, cycling through closed and open forms regulated by ATP binding/hydrolysis. Here, we combine crystal structures with analytical ultracentrifugation experiments to show that VirB11 ATPases indeed function as dynamic hexameric assemblies. In the absence of nucleotide, the N‐terminal domains exhibit a collection of rigid‐body conformations. Nucleotide binding ‘locks’ the hexamer into a symmetric and compact structure. We propose that VirB11s use the mechanical leverage generated by such nucleotide‐dependent conformational changes to facilitate the export of substrates or the assembly of the type IV secretion apparatus. Bio chemical characterization of mutant forms of HP0525 coupled with electron microscopy and in vivo assays support such hypothesis, and establish the relevance of VirB11s ATPases as drug targets against pathogenic bacteria.

TraG-Like Proteins of DNA Transfer Systems and of the Helicobacter pylori Type IV Secretion System: Inner Membrane Gate for Exported Substrates?

TraG-like proteins are potential NTP hydrolases (NTPases) that are essential for DNA transfer in bacterial conjugation. They are thought to mediate interactions between the DNA-processing (Dtr) and the mating pair formation (Mpf) systems. TraG-like proteins also function as essential components of type IV secretion systems of several bacterial pathogens such as Helicobacter pylori. Here we present the biochemical characterization of three members of the family of TraG-like proteins, TraG (RP4), TraD (F), and HP0524 (H. pylori). These proteins were found to have a pronounced tendency to form oligomers and were shown to bind DNA without sequence specificity. Standard NTPase assays indicated that these TraG-like proteins do not possess postulated NTP-hydrolyzing activity. Surface plasmon resonance was used to demonstrate an interaction between TraG and relaxase TraI of RP4. Topology analysis of TraG revealed that TraG is a transmembrane protein with cytosolic N and C termini and a short periplasmic domain close to the N terminus. We predict that multimeric inner membrane protein TraG forms a pore. A model suggesting that the relaxosome binds to the TraG pore via TraG-DNA and TraG-TraI interactions is presented.

Enzymology of DNA Transfer by Conjugative Mechanisms

Publisher Summary Bacterial conjugation is one of the major routes of genetic exchange in prokaryotes. Bacterial conjugation is still used as a tool for introducing genetic information into organisms for which transformation procedures do not exist. By using shuttle-vectors with alternative origins of vegetative replication, genetic information can be transferred and stably established across species boundaries among organisms as phylogenetically remote as Escherichia coli and Saccharomyces cerevisiae . Recently, tumorigenic DNA (T-DNA) transfer from Agrobacterium tumefaciens to plant cells has been recognized as a special form of bacterial conjugation, adapted to the requirements of transkingdom gene transfer. The T-DNA transfer system is extensively used for the genetic manipulation of plants. Shortly after antibiotics were introduced for the treatment of infectious diseases and as a supplement in animal food, bacterial strains with multiple antibiotic resistances appeared. These strains contained extrachromosomal elements, conjugative plasmids, and resistance factors that carried the genetic information for the antibiotic-resistance phenotype.

TraG from RP4 and TraG and VirD4 from Ti Plasmids Confer Relaxosome Specificity to the Conjugal Transfer System of pTiC58

Plasmid conjugation systems are composed of two components, the DNA transfer and replication system, or Dtr, and the mating pair formation system, or Mpf. During conjugal transfer an essential factor, called the coupling protein, is thought to interface the Dtr, in the form of the relaxosome, with the Mpf, in the form of the mating bridge. These proteins, such as TraG from the IncP1 plasmid RP4 (TraG(RP4)) and TraG and VirD4 from the conjugal transfer and T-DNA transfer systems of Ti plasmids, are believed to dictate specificity of the interactions that can occur between different Dtr and Mpf components. The Ti plasmids of Agrobacterium tumefaciens do not mobilize vectors containing the oriT of RP4, but these IncP1 plasmid derivatives lack the trans-acting Dtr functions and TraG(RP4). A. tumefaciens donors transferred a chimeric plasmid that contains the oriT and Dtr genes of RP4 and the Mpf genes of pTiC58, indicating that the Ti plasmid mating bridge can interact with the RP4 relaxosome. However, the Ti plasmid did not mobilize transfer from an IncQ relaxosome. The Ti plasmid did mobilize such plasmids if TraG(RP4) was expressed in the donors. Mutations in traG(RP4) with defined effects on the RP4 transfer system exhibited similar phenotypes for Ti plasmid-mediated mobilization of the IncQ vector. When provided with VirD4, the tra system of pTiC58 mobilized plasmids from the IncQ relaxosome. However, neither TraG(RP4) nor VirD4 restored transfer to a traG mutant of the Ti plasmid. VirD4 also failed to complement a traG(RP4) mutant for transfer from the RP4 relaxosome or for RP4-mediated mobilization from the IncQ relaxosome. TraG(RP4)-mediated mobilization of the IncQ plasmid by pTiC58 did not inhibit Ti plasmid transfer, suggesting that the relaxosomes of the two plasmids do not compete for the same mating bridge. We conclude that TraG(RP4) and VirD4 couples the IncQ but not the Ti plasmid relaxosome to the Ti plasmid mating bridge. However, VirD4 cannot couple the IncP1 or the IncQ relaxosome to the RP4 mating bridge. These results support a model in which the coupling proteins specify the interactions between Dtr and Mpf components of mating systems.

Enzymology of Type IV Macromolecule Secretion Systems: the Conjugative Transfer Regions of Plasmids RP4 and R388 and the cag Pathogenicity Island of Helicobacter pylori Encode Structurally and Functionally Related Nucleoside Triphosphate Hydrolases

Type IV secretion systems direct transport of protein or nucleoprotein complexes across the cell envelopes of prokaryotic donor and eukaryotic or prokaryotic recipient cells. The process is mediated by a membrane-spanning multiprotein assembly. Potential NTPases belonging to the VirB11 family are an essential part of the membrane-spanning complex. Three representatives of these NTPases originating from the conjugative transfer regions of plasmids RP4 (TrbB) and R388 (TrwD) and from the cag pathogenicity island of Helicobacter pylori (HP0525) were overproduced and purified in native form. The proteins display NTPase activity with distinct substrate specificities in vitro. TrbB shows its highest specific hydrolase activity with dATP, and the preferred substrate for HP0525 is ATP. Analysis of defined TrbB mutations altered in motifs conserved within the VirB11 protein family shows that there is a correlation between the loss or reduction of NTPase activity and transfer frequency. Tryptophan fluorescence spectroscopy of TrbB and HP0525 suggests that both interact with phospholipid membranes, changing their conformation. NTPase activity of both proteins was stimulated by the addition of certain phospholipids. According to our results, Virb11-like proteins seem to most likely be involved in the assembly of the membrane-spanning multiprotein complex.

DNA Processing and Replication during Plasmid Transfer between Gram-Negative Bacteria

This chapter concerns the processing and synthesis of plasmid DNA during its transmission between conjugating gram-negative bacteria, focusing on conjugation systems specified by plasmids isolated in or transferred experimentally to enterobacteria. This extensive collection of plasmids can be classified into more than 25 different incompatibility (Inc) groups (34), each of which is generally associated with a distinct conjugation system (158). Only a few of these systems have been investigated at the biochemical and molecular levels, but studies have identified unifying themes as well as an interesting diversity of enzymatic strategies for the conjugative processing of plasmid DNA.

Bacterial secrets of secretion: EuroConference on the biology of type IV secretion processes.

Type IV secretion systems (TFSS) mediate secretion or direct cell‐to‐cell transfer of virulence factors (proteins or protein–DNA complexes) from many Gram‐negative animal, human and plant pathogens, such as Agrobacterium tumefaciens, Bartonella tribocorum, Bordetella pertussis, Brucella suis, Helicobacter pylori, Legionella pneumophila and Rickettsia prowazekii, into eukaryotic cells. Bacterial conjugation is also classified as a TFSS‐like process mediating the spread of broad‐host‐range plasmids between Gram‐negative bacteria such as RP4 and R388, which carry antibiotic resistance genes. Genetic, biochemical, cell biological and structural biology experiments led to significant progress in the understanding of several aspects of TFSS processes. X‐ray crystallography revealed that homologues of the A. tumefaciens inner membrane‐associated proteins VirB11 and VirD4 from H. pylori and R388, respectively, may form channels for substrate translocation or assembly of the transmembrane TFSS machinery. Biochemical and cell biological experiments revealed interactions between components of the periplasmic core components VirB8, VirB9 and VirB10, which may form the translocation channel. Analysis of A. tumefaciens virulence proteins VirE2 and VirF suggested that the periplasmic translocation route of the pertussis toxin from B. pertussis may be more generally valid than previously anticipated. Secretion and modification of toxins from H. pylori and L. pneumophila profoundly affect host cell metabolism, thus entering the discipline of cellular microbiology. Finally, results from genome sequencing projects revealed the presence of up to three TFSS in a single organism, and the analysis of their interplay and adaptation to different functions will be a future challenge. TFSS‐carrying plasmids were discovered in different ecosystems, suggesting that genetic exchange may speed up their evolution and adaptation to different cell–cell interactions.

Components of the RP4 Conjugative Transfer Apparatus Form an Envelope Structure Bridging Inner and Outer Membranes of Donor Cells: Implications for Related Macromolecule Transport Systems

During bacterial conjugation, the single-stranded DNA molecule is transferred through the cell envelopes of the donor and the recipient cell. A membrane-spanning transfer apparatus encoded by conjugative plasmids has been proposed to facilitate protein and DNA transport. For the IncPalpha plasmid RP4, a thorough sequence analysis of the gene products of the transfer regions Tra1 and Tra2 revealed typical features of mainly inner membrane proteins. We localized essential RP4 transfer functions to Escherichia coli cell fractions by immunological detection with specific polyclonal antisera. Each of the gene products of the RP4 mating pair formation (Mpf) system, specified by the Tra2 core region and by traF of the Tra1 region, was found in the outer membrane fraction with one exception, the TrbB protein, which behaved like a soluble protein. The membrane preparation from Mpf-containing cells had an additional membrane fraction whose density was intermediate between those of the cytoplasmic and outer membranes, suggesting the presence of attachment zones between the two E. coli membranes. The Tra1 region is known to encode the components of the RP4 relaxosome. Several gene products of this transfer region, including the relaxase TraI, were detected in the soluble fraction, but also in the inner membrane fraction. This indicates that the nucleoprotein complex is associated with and/or assembled facing the cytoplasmic site of the E. coli cell envelope. The Tra1 protein TraG was predominantly localized to the cytoplasmic membrane, supporting its potential role as an interface between the RP4 Mpf system and the relaxosome.