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382 33 34 35 36 37 38 39 40 Review TRENDS in Microbiology Vol.10 No.8 August 2002 vaccine design. Clin. Diagn. Lab. Immunol. 3, 444–450 Zhu, P. et al. (2001) Fit genotypes and escape variants of subgroup III Neisseria meningitidis during three pandemics of epidemic meningitis. Proc. Natl. Acad. Sci. U. S. A. 98, 5234–5239 Levin, B.R. and Bull, J.J. (1994) Short-sighted evolution and the virulence of pathogenic microorganisms. Trends Microbiol. 2, 76–81 Taha, M-K. et al. (2001) Circumvention of herd immunity during an outbreak of meningococcal disease could be correlated to escape mutation in the porA gene of Neisseria meningitidis. Infect. Immun. 69, 1971–1973 Derrick, J.P. et al. (1999) Structural and evolutionary inference from molecular variation in Neisseria porins. Infect. Immun. 67, 2406–2413 Jelfs, J. et al. (2000) Sequence variation in the porA gene of a clone of Neisseria meningitidis during epidemic spread. Clin. Diagn. Lab. Immunol. 7, 390–395 Hobbs, M.M. et al. (1998) Recombinational reassortment among opa genes from ET-37 complex Neisseria meningitidis isolates of diverse geographical origins. Microbiology 144, 157–166 Achtman, M. (1994) Clonal spread of serogroup A meningococci: a paradigm for the analysis of microevolution in bacteria. Mol. Microbiol. 11, 15–22 Achtman, M. et al. (2001) Molecular epidemiology of serogroup A meningitis in Moscow, 1969 to 1997. Emerg. Infect. Dis. 7, 420–427 41 Nicolas, P. et al. (2001) Clonal expansion of sequence type (ST-)5 and emergence of ST-7 in serogroup A meningococci, Africa. Emerg. Infect. Dis. 7, 849–854 42 Swartley, J.S. et al. (1997) Capsule switching of Neisseria meningitidis. Proc. Natl. Acad. Sci. U. S. A. 94, 271–276 43 Kriz, P. et al. (1999) Microevolution through DNA exchange among strains of Neisseria meningitidis isolated during an outbreak in the Czech Republic. Res. Microbiol. 150, 273–280 44 Kertesz, D.A. et al. (1998) Serogroup B, electrophoretic type 15 Neisseria meningitidis in Canada. J. Infect. Dis. 177, 1754–1757 45 Popovic, T. et al. (2000) Neisseria meningitidis serogroup W135 isolates from U.S. travelers returning from Hajj are associated with the ET-37 complex. Emerg. Infect. Dis. 6, 10–11 46 Taha, M-K. et al. (2000) Serogroup W135 meningococcal disease in Hajj pilgrims. Lancet 356, 2159 47 Mayer, L.W. et al. (2002) Outbreak of W135 meningococcal disease in 2000: not emergence of a new W135 strain, but clonal expansion within the ET-37 complex. J. Infect. Dis. 185, 1596–1605 48 Guibourdenche, M. et al. (1996) Epidemics of serogroup A Neisseria meningitidis of subgroup III in Africa, 1989–1994. Epidemiol. Infect. 116, 115–120 49 Kwara, A. et al. (1998) Meningitis caused by a serogroup W135 clone of the ET-37 complex of 50 51 52 53 54 55 56 57 Neisseria meningitidis in West Africa. Trop. Med. Int. Health 3, 742–746 Taha, M-K. et al. (2002) Neisseria meningitidis serogroup W135 and A were equally prevalent among meningitis cases occurring at the end of the 2001 epidemics in Burkina Faso and Niger. J. Clin. Microbiol. 40, 1083–1084 Abraham, S.N. et al. (1998) Fimbriae-mediated host-pathogen cross-talk. Curr. Opin. Microbiol. 1, 75–81 Rieux, V. et al. (2001) Complex relationships between acquisition of β-lactam resistance and loss of virulence in Streptococcus pneumoniae. J. Infect. Dis. 184, 66–72 Bayliss, C.D. et al. (2001) The simple sequence contingency loci of Haemophilus influenzae and Neisseria meningitidis. J. Clin. Invest. 107, 657–662 Müller-Graf, C.D.M. et al. (1999) Population biology of Streptococcus pneumoniae isolated from oropharyngeal carriage and invasive disease. Microbiology 145, 3283–3293 Côté, S. et al. (1994) Molecular typing of Haemophilus influenzae using a DNA probe and multiplex PCR. Mol. Cell. Probes 8, 23–37 Hausdorff, W.P. et al. (2000) Which pneumococcal serogroups cause the most invasive disease: implications for conjugate vaccine formation and use, part I. Clin. Infect. Dis. 30, 100–121 Zhang, Y.X. et al. (2002) Genome shuffling leads to rapid phenotypic improvement in bacteria. Nature 415, 644–646 Tying rings for sex Markus Kalkum, Ralf Eisenbrandt, Rudi Lurz and Erich Lanka The primary component of the sex pilus encoded by IncP (RP4) and Ti plasmids has been identified as a circular pilin protein with a peptide bond between the amino and carboxyl terminus. Here, we review the key experiments that led to this discovery, and the present mechanistic model for pilin-precursor processing and the cyclization reaction. In addition, we discuss the implications for horizontal gene transfer in bacterial conjugation. Published online: 10 July 2002 Bacterial conjugation efficiently mediates horizontal gene transfer in a highly promiscuous manner. Conjugative processes enable bacteria to transfer conjugative plasmid DNA not only between members of their own kingdom, but also to fungi, plants and even mammalian cells, as recent laboratory experiments have indicated [1]. The secretion of macromolecules via bacterial conjugation is categorized as a type IV secretion process (recently reviewed by Christie and Vogel [2]). The initial step in bacterial conjugation requires physical contact between the donor ‘male’ and recipient ‘female’ cells. The conjugative plasmid DNA in the donor cell is relaxed at the origin of transfer (oriT) by http://tim.trends.com proteins belonging to the DNA transfer and relaxation (DTR) system, then channelled into the periplasm through the lumen of a hexameric protein. This structure is formed by the TraG protein in RP4mediated conjugation and by its homologue TrwB in R388 (IncW)-mediated conjugation [3]. Mating pair formation (Mpf) proteins, which span the cell envelope, are required to transfer the plasmid DNA into the recipient cell [4] (Fig. 1). Electrophysiological studies have shown that the presence of Mpf proteins enhances the permeability of the host cell envelope [5]. The mechanistic details of the transfer of DNA and other macromolecules and the presumed accessory functions of Mpf proteins are currently being investigated. The 3-D structure of the Helicobacter pylori Cagα protein, a member of the VirB11 protein family of ‘traffic’ nucleoside triphosphatases, was solved recently [6]. Three proteins belonging to this group, TrbB (encoded by the RP4 plasmid), Cagα (H. pylori), and TrwD [encoded by the R388 (IncW) plasmids], have been shown to form homohexameric rings with a central hole [7]. TrbB, Cagα and TrwD are cytoplasmic proteins and are associated with the inner membrane. 0966-842X/02/$ – see front matter © 2002 Elsevier Science Ltd. All rights reserved. PII: S0966-842X(02)02399-5 Review TRENDS in Microbiology Vol.10 No.8 August 2002 Phages Recipient cell Pf3 PRR1 PRD1 Entrance of phage DNA/RNA ? Interaction with envelope Pilin (cyclic TrbC) ? Detached pilus detected by EM Mpf proteins A RP4 plasmid TraG ss D N DTR proteins TrbC processing TraF Pilin precursors (linear TrbC) Donor cell TRENDS in Microbiology Fig. 1. The RP4 pilus in bacterial conjugation. It is proposed that the pilus is assembled from circular TrbC subunits by proteins of the envelope-spanning Mpf system. A rudimentary pilus ‘stump’ might exist near Mpf proteins on the cell surface. It can be recognized by specific phages that take over functionalities of the host’s conjugative apparatus for the purpose of their own propagation. The host-cell’s RP4 plasmid is relaxed, nicked and transferred into the periplasm by the DNA transfer and relaxation (DTR) apparatus. Relative dimensions within the drawing are based on actual sizes. Rudi Lurz Erich Lanka* Max-Planck-Institut für Molekulare Genetik, D-14195 Berlin, Germany. *e-mail: [email protected] Markus Kalkum Dept of Mass Spectrometry and Gaseous Ion Chemistry, The Rockefeller University, New York, NY 10021-6399, USA. Ralf Eisenbrandt Biochemie GmbH, Biochemiestrasse 10, A-6250 Kundl, Austria. The delivery of effector molecules through the barrier formed by four bacterial membranes occurs following the formation of thin, tube-like extracellular filaments, the conjugative pili. It remains an open question whether DNA or other macromolecules per se are transported through the pili. For IncP pili, their brittle structure and loose attachment to the bacterial surface does not suggest such a role (discussed later). Although suggested by its analogy to the F pilus, evidence that the RP4 pilus is erect on the host cell and spans the gap to the recipient cell remains elusive. In fact, electron micrographs of RP4-containing host cells show the pili to be detached from the cell surface. So far, the best hint for the existence of a residual pilus ‘stump’ on the cell surface comes from studies of bacteriophages that bind specifically either to the pilus or to other Mpf proteins (Fig. 1). The basic structure of the pilus associated with IncF, IncP and Ti plasmids consists of a single type of pilin subunit. The IncP and Ti pilins are cyclic proteins whose amino and carboxyl termini are joined by a peptide bond. A series of proteolytic truncations of the initially linear precursors lead to the formation of this covalent head-to-tail linkage. This review will focus on the maturation of pilin precursors in conjugation and type IV secretion systems. Pilus structure and function The model conjugative pilus is the F pilus, a thin filament attached to the surface of conjugative F+ bacteria [8]. The F pilus is a tubular structure of 8.5–9 nm in diameter with an inner axial hole that is 2–2.5 nm in diameter. The filament is flexible, varies in http://tim.trends.com 383 length between 1 and 2 µm and is composed of multiple copies of a single subunit. This subunit, known as pilin, is an N-acetylated, 71-residue polypeptide encoded by the traA gene [9]. The proposed role of the pilus in conjugation is to establish physical contact between the donor and recipient cells; this contact is initiated when the donor cell attaches the tip of the pilus to the recipient cell. A depolymerization step is thought to pull donor and recipient together, thus allowing the cell envelopes to engage in intimate contact [8]. These mating aggregates are thought to facilitate the transmission of single-stranded (ss) DNA through the barrier formed by the two cell envelopes of mating Gram-negative cells, which comprises four membranes and two dense peptidoglycan networks, and has a total thickness of ~500 Å. Other conjugation systems also encode pili. Perhaps the most comprehensive survey on conjugative pili, in which the pili were characterized mainly by morphological and immunological aspects, was that of Bradley [10]. The F-transfer system shows high mating efficiency in liquid with considerably lower efficiency on semi-solid media, whereas IncP plasmids are known for high mating efficiencies on semi-solid media and low efficiencies in liquid. The number of pili on F+ cells is approximately two to three per cell. By contrast, only one out of 50 E. coli cells containing the IncP plasmid RP4 is visibly piliated. The existence of the rigid IncP pili was visualized more convincingly in cell preparations that overproduced the mating pair apparatus. On the RP4 plasmid, the 20 essential transfer genes [11] are located in two isolated regions, Tra1 and Tra2. The DNA transfer and replication functions are encoded by Tra1 whereas IncP pilus biogenesis requires one transfer gene from the Tra1 cluster and ten Tra2 genes. These latter 11 genes are responsible for the assembly of the proposed supramolecular Mpf complex [4] (Fig. 1). As each type IV secretion [2] gene cluster contains a potential pilin precursor (prepilin) gene, the recently discovered phylogenetic and functional relationship between the IncP mating pair apparatus and type IV secretion systems of mammalian and plant pathogens might help to dissect the pilus assembly process. Investigation of the pilus assembly pathway began with the identification of the RP4 (IncPα) prepilin gene and determination of the chemical nature of the pilus subunit. A characteristic of IncP pili is their tendency to aggregate in bundles (Fig. 2); the hydrophobic surfaces of the pili are probably responsible for interactions between the filaments. On some filaments a fine longitudinal middle line suggests a tubular structure for the pilus. Occasionally, filaments with a diameter of ~10 nm display a ‘knob’ at one end, like a small sacculus [12,13], which might indicate a former anchor in the outer membrane of the cell envelope. Surprisingly, inspection of cells producing pili in high quantities shows that the majority were detached from the cell surface and arranged in bundles resembling Review 384 TRENDS in Microbiology Vol.10 No.8 August 2002 Fig. 2. Electron micrograph of the RP4 pilus. Scale bar = 200 nm. the behaviour of purified preparations. Thus, hydrophobic cell-to-cell interactions could be facilitated by the pili supporting adherence and the formation of mating aggregates. To date, there is still no experimental evidence to support the idea that genetic material is transported through the pilus itself. The RP4 model system Broad host range plasmids belonging to the IncP family specify resistance to multiple antibiotics including kanamycin, gentamicin, tetracycline, penicillin, streptomycin, sulfonamide, chloramphenicol and trimethoprim, and resistance to mercury ions [14]. IncP plasmids are found in hosts (Gram-positive and -negative) that are spread ubiquitously, but particularly in surroundings with a high selective pressure, including hospitals, animal husbandry premises and fish farms. As bacterial conjugation is the major pathway for the spread of resistance genes [15], IncP plasmids such as RP4 have served as a model system to study DNA transmission in the environment and at the molecular level [16,17]. Signal peptide PreProTrbC Host peptidase 36 aa TMH TMH 27 aa LepB ProTrbC TraF 4 aa TrbC* TraF Pilin 78 aa TRENDS in Microbiology Fig. 3. Maturation cascade of the RP4 pilin. TrbC, 145 residues in length, is represented by a bar. Defined sections of TrbC are marked: signal peptide (red), core region (light green), trans-membrane helices (TMH, dark green), carboxy-terminal end (light blue) and tetrapeptide (dark blue). http://tim.trends.com One recent achievement was the unambiguous immunological identification of the trbC gene product as the pilus precursor [12]. The existence of different forms of TrbC indicated a complex process of modification of the 145-residue TrbC polypeptide [18,19] (Fig. 3). The precursor maturation cascade begins with removal of a 27-residue polypeptide from the carboxyl terminus by an as-yet-unidentified host-encoded protease. As a second distinct step, the 36-residue signal peptide is cleaved off by the hostencoded signal peptidase LepB of E. coli. The third and final modification step was revealed by mass spectrometry (see below). This step yields the mature cyclic pilin that makes up the pilus: truncation of the carboxyl terminus by four residues and the formation of a new peptide bond connecting the amino and carboxyl terminus of TrbC in one concerted reaction. The cyclization is catalyzed by the essential plasmidborne transfer protein, TraF, which has sequence similarity to signal peptidases [18]. TraF homologues have been shown to belong to a special class of serine proteases [20]: their catalytic activity results from serine–lysine dyad formation. Mutation of Ser37 and Lys89 of TraF reduced the activity of the protein, which did not support the synthesis of conjugative pili [18]. We propose a mechanism based on the action of serine proteases (Fig. 4). The carboxyl group of Gly78 of the membrane-anchored and doubly processed TrbC is proposed to react and form an acyl-intermediate with Ser37 of TraF. Nucleophilic attack of the Ser37 hydroxyl group requires preemptive activation by a dyad-like deprotonation via Lys89 or through other possible proton acceptors. In fact, Asp155, which is essential for the enzymatic function of TraF [19], might even be involved in the activation step. The carboxy-terminal tetrapeptide (AEIA) is cleaved off while the TrbC acylenzyme complex undergoes aminolysis by reacting with the amino terminus of TrbC. The existence of two transmembrane hydrophobic helices (TMH, see Fig. 3) in TrbC supports our mechanistic model. They ensure that both the amino and carboxyl terminus of TrbC are localized at the same side of the membrane, presumably in the periplasm. The formation of a new peptide bond between the carboxyl and amino terminus seems to use conserved energy from the removal of the tetrapeptide. A conceivable alternative reaction – the hydrolysis of the TraF-acyl-TrbC intermediate and the resulting truncated, non-circular form of TrbC – was not observed. None of the mutant proteins used in mechanistic studies displayed loss of the tetrapeptide without concerted cyclization of the residual TrbC [19], indicating that the removal of the tetrapeptide cannot be uncoupled from the cyclization reaction. A thorough analysis of the chemical nature of the RP4 pilus and pilin was performed using matrix-assisted laser desorption/ionization–time of flight-mass spectrometry (MALDI–TOF-MS) [12]. Preparations of purified pili were subjected to different matrices to find the optimal conditions for sensitive Review TRENDS in Microbiology Vol.10 No.8 August 2002 Fig. 4. Proposed mechanism of the pilin cyclization catalyzed by TraF on the periplasmic side of the cytoplasmic membrane. To proton acceptor 385 Leaving tetrapeptide H + O NH2 O= O NH2 C TrbC No hydrolysis detected O=C TraF Aminolysis Pilus assembly H HO + HN O C O- TRENDS in Microbiology detection of pilin ions. Of a variety of matrices tested, trans-3-indole acrylic acid (IAA) yielded the most intense signals for the pilin or its precursors (Fig. 5). IAA permitted us to detect intense pilin signals even from matrix preparations with whole E. coli or Agrobacterium tumefaciens cells. The choice of IAA as the MALDI-MS matrix proved to be key to the analysis of a variety of TrbC and TraF mutants. The circular nature of the mature pilin was deduced from mapping tryptic and chymotryptic proteolysis products in MALDI-MS experiments. Although pili resist protease treatment to a great extent, enough material was digested when pilus suspensions were exposed to high concentrations of the enzymes [12]. A time course of the tryptic digestion of pilin monitored by mass spectrometry revealed another feature unique to circular proteins: the molecular mass increases by 18 daltons when the first peptide bond is hydrolyzed – consistent with the formal addition of one water molecule. Sensitive mass measurements of pilins from entire cells in the IAA matrix allowed us to investigate a variety of IncP pilin mutations reliably without the need for elaborate pilus purification. Future studies on other pilins should take into account that it might not always be possible to find a suitable matrix that http://tim.trends.com discriminates against background proteins as IAA does for the pilins of the IncP and IncRH1 plasmids. Different physico-chemical properties and expression levels must also be considered. Analogies to related systems Biogenesis of the T pilus occurs upon expression of the virB operon of the Ti plasmid with a processed form of VirB2 being assembled into the T pilus. Our studies on pilin maturation in the Vir system revealed that an enzyme encoded by the chromosome is required for the cyclization reaction of VirB2 [21]. It was found that VirB2 becomes cyclized in A. tumefaciens strains but not in E. coli [21] and that VirB2 propilin was processed and cyclized in the absence of any other Ti plasmid gene [12,21]. Except for a weak sequence similarity between TraF and VirF/Orf2 of the Ti plasmid, which has been shown to be dispensable for conjugative transfer [22], the Ti plasmid lacks a functional TraF homologue. Thus, the enzyme for Ti propilin cyclization appears to be of chromosomal origin. In addition to VirB2, the VirB5 protein is a putative component of the T pilus and co-fractionates with T pilus preparations. Although VirB2 is the major pilus subunit, VirB5 might be directly involved in T pilus assembly, possibly as a minor component [13] Review Fig. 5. The effect of matrices on the signal intensities of pilin ions in preparations with entire bacteria. The amount of bacteria is the same in all three matrix-assisted laser desorption/ionizationmass spectrometry (MALDI-MS) spectra. Trans-3-indole acrylic acid (IAA) discriminates strongly against other Escherichia coli proteins. 2-(4-hydroxyphenyl azo)benzoic acid (HABA) yields little discrimination and sinapic acid (3-(4-hydroxy-3,5dimethoxy phenyl) acrylic acid) (SA) shows no usable gain in the intensity of the pilin signal at 8119.6 m/z. Other commonly used matrices like 4-hydroxy-α-cyano cinnamic acid and 2,4-dihydroxy benzoic acid gave broader and less intense signals (not shown). TRENDS in Microbiology Vol.10 No.8 August 2002 8119,6 Matrix O OH Relative intensity 386 IAA NH O OH HABA N N OH O O OH HO SA O 7000 9000 m/z TRENDS in Microbiology and perhaps as a chaperone [23]. A second transfer system of the Ti plasmid, responsible for conjugation between A. tumefaciens cells, consists of three Tra regions [24]. The Mpf system encoded by the Ti plasmid contains highly conserved TraF and TrbC homologues. The RP4 TrbC cyclization motif (G/AIEA) (Fig. 6) was defined by mutagenesis. A similar motif (G/AISG) is present in TrbC of the Ti plasmid and we therefore propose that, analogous to the IncP system, the conjugative Ti pilin is a cyclic polypeptide [12]. VirB2 PtIA TrbC Ti TrbC 28 AIEPNLAHANGG : : || | :| 25 ATLPDLAQAGGG : :::|| | | 20 IGLADPAFASSG : : | | | 28 ALSAHPAMASEG 39 ... 77 GAAAEIASYLL 87 :: |:|: ::: 36 ... 74 GASAEIARYLL 84 : |:|: 31 ... 103 ATGASIGEMEA 113 : |:|: 39 ... 96 GRGAEIAALGN 106 TRENDS in Microbiology Fig. 6. Putative cyclization motifs. Partial sequence comparison of RP4 TrbC (M93696), Ti TrbC (P54908), Bordetella pertussis PtlA (L10720), Brucella suis VirB2 (AF141604) and Brucella abortus VirB2 (AF226278). The two VirB2 sequences are identical and are shown once only. GenBank accession numbers are given in parenthesis. The first and last amino acid position in each line is numbered according to the full-length proteins. Lines correspond to conserved residues in all, colons to residues conserved in at least two sequences. Residues of a proposed common maturation process are highlighted in green. Arrows mark the sites of RP4 TrbC processing. http://tim.trends.com In some macromolecular secretion systems of mammalian pathogens, cyclic peptides apparently play a crucial role. The pertussis toxin operon of Bordetella pertussis [25] encodes the pilin-like protein PtlA and two related virulence operons of Brucella suis [26] and Brucella abortus [27] are proposed to contain virB2 genes that encode pilus subunits [19] (Fig. 6). Each of the three sequences displays high sequence similarity to the potential processing motifs of TrbC [19]. Predicted pilin precursors from plasmids R6K (PilX2), R388 (TrwL), pKM101 (TraM) and HP0546 of H. pylori (Werner Pansegrau, pers. commun.) are homologous to TrbC and VirB2. The precursors for the putative pilins are hydrophobic polypeptides, each <150 residues in size. They contain amino-terminal signal peptides of considerable length (25–50 residues) and two predicted transmembrane helices in the core region. A common feature shared by these proteins is their indifferent behavior to most protein dyes (Coomassie, Sypro Orange® and Amido Black). These dyes do not stain the proteins in polyacrylamide SDS gels and so far, only silver staining has been successfully applied to detect separated bands of these proteins [28]. Furthermore, it was found that strains overproducing the respective pilus subunit terminate cell growth and division immediately after expression is induced (C. Rabel and E. Lanka, unpublished). Hence, nutrient uptake by these cells might be hampered by the membranetargeting pilin, suggesting possible interference or inhibition of essential transport mechanisms through the inner membrane. Cyclic proteins The formation of cyclic pilus subunits during sex pilus biogenesis is a recently discovered process. The implications for the function and biological relevance of other cyclic proteins remain largely unknown. Several biologically active cyclic peptides exist and can be assigned to either one of two groups: first, small cyclic peptides that originate from a complex enzymatic pathway similar to fatty acid metabolism [29,30], and that usually display ion-chelating and antibiotic activity. Examples include Gramicidin-S, actinomycin, polymyxin, tyrocidin and etamycin. Second, a small group of ribosomally synthesized proteins, among them the cyclic antibiotic AS-48 from Enterococcus faecalis [31], which is inserted into the membrane of the target cell and causes proton loss owing to permeabilization of the membrane. The precise mechanism involved and the possible catalytic function responsible for the cyclization of AS-48 remains to be discovered. Other peptides that undergo cyclization via disulfide bonds rather than peptide bonds (such as somatostatin) will not be addressed here. Engineered cyclic proteins with peptide bonds in head-to-tail linkages have been reported [32,33]; backbone cyclization was achieved using a modified version of protein splicing that involves intein technology. Review TRENDS in Microbiology Vol.10 No.8 August 2002 Uninvited guests Acknowledgements We thank Hans Lehrach for generous support. Work in E.L.’s laboratory was supported by the Deutsche Forschungsgemeinschaft. The EU-BIOTECH concerted action BIO4-CT0099, Mobile genetic Elements’ Contribution to Bacterial Adaptability and Diversity (MECBAD) provided a platform for fruitful discussions. The acquisition of additional genetic information is not always an advantage. Although bacterial resistance to antibiotics makes the presence of conjugative plasmids and the formation of conjugal pili advantageous features in certain environments, some ‘uninvited guests’ use these extracellular appendices as a receptor to attach, enter and destroy their hosts. So-called donor, or male-specific, bacterial viruses attach to pili. Viruses PRR1 (RNA), Pf3 (ssDNA), and PRD1 (double-stranded DNA) use RP4-containing cells as preferred targets, using one of two strategies. Whereas Pf3, a filamentous phage, and PRR1, an icosahedral phage, attach along the pilus, PRD1 adsorption is only detected on the surface of RP4-containing cells [19]. All three types of phages need the 11 Mpf core functions that are required for conjugation, including a functional cyclic pilin, for their own propagation [34,35]. The Mpf components are membrane proteins likely to form the pilus assembly apparatus by bridging the inner and outer membrane [4]. Thus, the cyclic protein TrbC plays a crucial role not only during conjugation References 1 Waters, V.L. (2001) Conjugation between bacterial and mammalian cells. Nat. Genet. 29, 375–376 2 Christie, P.J. and Vogel, J.P. (2000) Bacterial type IV secretion: conjugation systems adapted to deliver effector molecules to the host cells. Trends Microbiol. 8, 354–360 3 Gomis-Ruth, F.X. et al. (2002) Conjugative plasmid protein TrwB, an integral membrane type IV secretion system coupling protein. Detailed structural features and mapping of the active site cleft. J. Biol. Chem. 277, 7556–7566 4 Grahn, A.M. et al. (2000) 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. J. Bacteriol. 182, 1564–1574 5 Daugelavi?ius, R. et al. (1997) The IncP plasmidencoded cell envelope-associated DNA transfer complex increases cell permeability. J. Bacteriol. 179, 5195–5202 6 Yeo, H-J. et al. (2000) Crystal structure of the hexameric traffic ATPase of the Helicobacter pylori type IV secretion system. Mol. Cell 6, 1461–1472 7 Krause, S. et al. (2000) Sequence related protein export NTPases encoded by the conjugative transfer region of RP4 and by the cag pathogenicity island of Helicobacter pylori share similar hexameric ring structures. Proc. Natl. Acad. Sci. U. S. A. 97, 3067–3072 8 Achtman, M. et al. (1978) Cell–cell interactions in conjugating Escherichia coli: role of F-pili and fate of mating aggregates. J. Bacteriol. 135, 1053–1061 9 Frost, L.S. et al. (1986) Two monoclonal antibodies specific for different epitopes within the aminoterminal region of F pilin. J. Med. Microbiol. 168, 192–198 10 Bradley, D.E. (1980) Morphological and serological relationship of conjugative pili. Plasmid 4, 155–169 11 Lessl, M. et al. (1992) Dissection of IncP conjugative plasmid transfer: definition of the transfer region Tra2 by mobilization of the Tra1 region in trans. J. Bacteriol. 174, 2493–2500 http://tim.trends.com 387 (DNA export from the cell) but also in phage DNA transfer (DNA transfer into the cell). It is already possible to find mutations in trbC that can dissect these two processes [19] but further studies will be needed to propose a detailed mechanism. The future The architecture of the pilus assembly machinery, the functions and interactions of the components in the complex and the crystal structure of the pilus are challenging projects to be addressed. How is contact established between the pilus and the recipient cell surface? Are there specific pilus receptors or do cyclic TrbC molecules enhance the permeability of the recipient cell wall? In silico data suggest several functional parallels between conjugative transfer systems and type IV secretion pathways. One of the intriguing questions is whether type IV systems produce pili or whether the proposed pilin precursors, such as PtlA (B. pertussis) and 0546 (H. pylori), have other roles in the secretion processes of bacterial pathogens. Continued research is therefore required. 12 Eisenbrandt, R. et al. 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