Conjugation systems and most other T4SSs translocate DNA or protein substrates by a process requiring direct contact with target cells (1
). Although we have identified a number of altered-function mutations through random mutagenesis of virB10
, the G272R substitution was unique in conferring host cell contact-independent surface exposure of VirE2. The mutant strain displayed WT colony morphology and growth in rich and vir
-inducing media but did not nonspecifically release the periplasmic RNase or ChvE, or VirE2 deleted of its C-terminal secretion signal, consistent with a model in which the G272R mutation specifically disrupts VirB/D4 channel gating. The G272R mutant did exhibit enhanced sensitivity to molecules that normally do not pass through OM porins, e.g., SDS and vancomycin, further suggesting that the gating defect allows for bidirectional movement of molecules too large to diffuse through porins across the OM. Gating mutations of other OM channels display similar phenotypes; recently, such mutations were isolated in the filamentous bacteriophage f1 OM secretin, and most of these mutations similarly conferred enhanced sensitivity to detergents and large antibiotics (43
The region surrounding G272 bears a sequence (S262
) that is highly conserved among VirB10 homologs associated with Gram-negative bacterial T4SSs (see a Pro Dom analysis of VirB10 homologs at http//:mmg.uth.tmc.edu/webpages/faculty/pchristie.html
). Most notable are invariant Gly residues corresponding to VirB10 Gly269 and Gly272 and a strongly conserved residue, Gly275. In the first X-ray structure of a VirB10 homolog, Helicobacter pylori
ComB10, a domain spanning residues 268 to 287 (A. tumefaciens
VirB10 numbering) formed an α helix projecting from the side of a large β-barrel domain; this projection, originally termed the α1 helix, was implicated in stabilizing VirB10 homodimers or multimers (46
). In the context of this structure, the mechanistic consequences of the G272R mutation were not immediately evident. However, in the more recent pKM101 X-ray structure, the corresponding Gly residues of the 14 TraF subunits encircle the interior of the core chamber near the OM () (14
); this region of the core complex is ideally positioned to participate in regulation of substrate transfer across the OM.
Defining the nature of gating mechanisms for various OM channels of Gram-negative bacteria has received considerable attention in recent years. A general mechanism, exemplified with TonB-activated transporters of small molecules (31
), the P pilus biogenesis system (37
), and larger OM channels formed by secretins (24
), involves the formation of a plug domain or a constriction in the β-barrel channel subunit. In the closed configuration, the plug or constriction prevents the passage of substrate and other molecules, e.g., detergents and large antibiotics. The conformational switch to the open state might be induced by substrate binding to regulatory domains and perhaps other activating signals, e.g., IM energy from ATP hydrolysis or the electrochemical gradient (36
). Recent structural studies have identified plug or constriction domains in OM secretins (36
) and have also shed light on conformational switches accompanying the closed-to-open channel transitions (36
). Corresponding mutational studies have also identified motifs in the primary sequences of OM channel complexes that participate in channel gating (3
). In the case of the filamentous bacteriophage f1, for example, the pIV secretin assembles as a dodecameric barrel structure in which the C-terminal ring forms the OM pore (32
), and “leaky” mutations were clustered in two sequence motifs, GATE1 and GATE2, in the adjacent middle ring (44
). Interestingly, despite structural differences in the pIV and T4SS core complexes (44
), the gating mutations in the pIV secretin and the G272R mutation in the T4SS core complex are in the interiors of the respective chambers near the OM channels. However, in contrast to the GATE motifs, which are not well conserved among members of the secretin superfamily (44
), the GxxGxxG motif is strongly conserved among VirB10 homologs, which is suggestive of a gating mechanism that is fundamental to all T4SS machines of Gram-negative bacteria.
The T4SS core complex does not possess an obvious plug, in contrast to the TolC, PapC usher, and secretin complexes (14
). However, the full-length T4SS core structure clearly resolves a constriction of about 5 to 10 Å, which is too small for passage of substrates. In contrast, the crystal structure of the outer membrane part of the core complex (the O layer) obtained by proteolysis of the full-length core complex displays a larger opening of about 30 Å. Conceivably, the proteolytic removal of the IM part of the core complex (the I layer) resulted in conformational changes in the 14-helix OM channel so that the crystal structure reflects the open conformation. In the context of the intact VirB/VirD4 channel, other VirB subunits would regulate the gating of the OM pore. A likely mechanistic consequence of the G272R mutation is that the long Arg side chain or the introduction of a positive charge at this site provides a steric block that disrupts core-VirB channel contacts. Two VirB subunits, VirB2 pilin and VirB9, have been proposed to regulate substrate translocation across the OM (12
). VirB2 pilin is required for pilus production and for substrate transfer; however, the isolation of Tra+
“uncoupling” mutations establishes that an intact pilus is not required for substrate transfer (12
). The T4SSs thus might exist in or transition between two states: a secretion-competent state and a pilus biogenesis-competent state. In a secretion-competent state, the pilus would be short but still sufficiently long to exert mechanical forces on the constricted OM channel, thereby inducing its opening. In the pilus biogenesis-competent state, the pilus would grow out through the OM channel. The G272R steric block might restrict pilus outgrowth, resulting in a pilus polymer sufficiently long to trigger OM channel opening and consequently VirE2 release but not long enough to produce a visible pilus, hence showing the Pil−
An effect of the G272R mutation on VirB9 activity also cannot be excluded at this time. VirB9-like subunits possess two highly conserved domains separated by a nonconserved linker region (25
). In the pKM101 X-ray structure, the C-terminal domain (CTD) of VirB9-like TraO lines the outside, not the inside, of the core chamber; therefore, this domain probably does not interact directly with the interior of the core near Gly272 (14
). The N-terminal domain (NTD) likely extends along the portion of the core complex projecting through the periplasm toward the cytoplasmic membrane. Besides detection of an FA-cross-linkable contact between VirB9 and DNA substrates (12
), two-residue insertion mutations were isolated in the VirB9 NTD that selectively permit translocation of two different DNA substrates, T-DNA or an IncQ plasmid, to target cells. These were termed substrate discrimination mutations, and it was postulated that the VirB9 NTD modulates substrate trafficking through differential recognition/binding of relaxases covalently bound at the 5′ ends of DNA substrates (25
). Conceivably, the G272R mutation acts indirectly by imposing a structural change on the VirB9 NTD that affects its ability to regulate substrate trafficking. At this point, however, we have determined that the G272R mutant forms precipitable complexes with partner subunits VirB7 and VirB9 in energized and ATP-depleted cells and also forms chemical cross-linked species indistinguishable from native VirB10, arguing against a gross perturbation by the G272R mutation of the overall core complex structure.
While further studies are needed to define the molecular basis for gating of the VirB/D4 OM channel, here we also showed that the G272R mutation rendered VirB10 sensitive to protease even in ATP energy-depleted cells (). These findings suggest that the G272R mutation induces a structural state resembling the open-channel conformation of WT cells, in effect bypassing the requirement for the coupling of ATP hydrolysis by the VirB/D4 ATPases to OM translocation. It is interesting to note, however, that in energized WT cells, the energy-activated (protease-susceptible) form of native VirB10 is also detectable (11
), yet WT cells do not release detectable amounts of VirE2 to the cell surface (). This indicates that energization of VirB10 is necessary, but not sufficient, for gating of the OM channel. Another activating signal must be required for gating the OM channel, and the G272R mutation apparently also bypasses this requirement. One such activating signal could be the “mating signal” identified in early studies of the F plasmid transfer system (33
). This signal is propagated from recipient cells to donor cells upon contact with the F pilus or donor cell envelope to stimulate plasmid transfer. Although the nature of the mating signal remains unknown, the G272R mutation might bypass a requirement for signal activation through direct contact with recipient cells.
Of further interest, the G272R mutation did not disrupt intercellular translocation, suggesting that it does not perturb establishment of the donor-recipient cell mating junction. At this time, the architecture of the T4SS channel at the cell surface is poorly defined, but the fact that the G272R mutation permits substrate transfer while blocking surface accumulation of pilins and assembly of T pili adds to the argument that pilins/pili are not required for formation of the VirB/VirD4 mating junction, in apparent contrast to the situation for the F plasmid transfer system (4
In sum, we have presented evidence that Gly272 within a conserved GxxGxxG sequence motif and the AP domain of VirB10 contribute in distinct ways to regulate substrate transfer across the OM and biogenesis of the T pilus. The G272R mutation selectively disrupts gating of the secretion channel at the OM, allowing for release of substrates to the cell surface and small-molecule uptake. This mutation also blocks T pilus (or pilin) extrusion but not establishment of critical donor-target cell contacts required for intercellular substrate translocation. In contrast, the AP partial deletions do not disrupt gating of the secretion channel or formation of the mating junction but instead impose a block in a terminal stage of T pilus assembly involving the polymerization process. Ongoing structure-function studies are focused on defining the nature of contacts between these domains or other channel constituents in regulating substrate transfer, the role(s) of ATP energy and other possible contact-dependent signals in activating OM translocation, and the mechanistic contributions of the GxxGxxG motif and the AP on pilus biogenesis.