Gram-negative bacteria can cause serious diseases in humans, such as cholera and bacterial meningitis. These bacteria are surrounded by two membranes: an inner membrane and an outer membrane. Proteins called secretins are components of several large molecular complexes that are embedded within the outer membrane. Some secretin-containing complexes form pores in the bacterial membranes and allow molecules to pass in or out of the cell.

Some secretins also form part of the machinery that allow Gram-negative bacteria to grow fibre-like structures called type IV pili. These pili help bacteria that cause infections to move and stick to host cells, where they can also trigger massive changes in the host cells' architecture. Multiple copies of a secretin protein called PilQ form a channel in the outer membrane of the bacteria that allows a type IV pilus to grow out of the surface of the cell. The pilus can then hook the bacteria onto surfaces and other cells. There is evidence to suggest the type IV pilus machinery is involved in the uptake of DNA from other bacteria, an important but poorly understood process that has contributed to the spread of multi-drug resistance.

Now, Gold et al. have used a cutting-edge technique called ‘electron cryo-tomography’ to analyse the three-dimensional structure of the machinery that builds the type IV pili in the membranes of a bacterium called Thermus thermophilus. This analysis revealed that, similar to many other channel complexes, the PilQ channel can be ‘open’ or ‘closed’. When pili are absent, the channel is closed, but the channel opens when pili are present. Further analysis also revealed the structure of an assembled pilus.

Next, Gold et al. studied the open state of the type IV pilus in more detail and observed that a region of each of the PilQ proteins moves a considerable distance to make way for the pilus to enter the central pore. These results will pave the way for future studies of type IV pili and other secretin-containing complexes and underpin efforts to investigate new drug targets to combat bacterial infections.

Secretins form multimeric pores through the outer membrane of Gram-negative bacteria (Averhoff, 2009; Korotkov et al., 2011; Burkhardt et al., 2012). They are the central secretion conduits for proteins and virulence factors in type II and III secretion systems (T2SS/T3SS) and are essential for extrusion of type IV pili (T4P) (Martin et al., 1993) and transport of some bacteriophages (Korotkov et al., 2011). In addition, secretins are key components of DNA transport systems, which mediate uptake of free DNA from the environment, referred to as natural transformation (Schwarzenlander et al., 2009). The ability to take up DNA is one of the major mechanisms of horizontal gene transfer (Domingues et al., 2012) and enables organisms to adapt rapidly to changing environments (Averhoff, 2009). This process is also fundamental for adaptation of pathogenic bacteria to human hosts and the acquisition of multi-drug resistance (Domingues et al., 2012).

In many Gram-negative bacteria, such as Thermus, and also in many major human pathogens such as Neisseria, Pseudomonas, and Vibrio, DNA uptake is linked to the T4P machinery (Wolfgang et al., 1998; Graupner et al., 2000; Seitz and Blokesch, 2013) (Figure 1). To investigate the structure and function of this system, we chose the thermophilic bacterium Thermus thermophilus HB27, which exhibits the highest transformation rates known to date (Koyama et al., 1986), and due to the thermostability of its proteins is a convenient model for structural studies.

Figure 1

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Pili are several micron-long flexible filaments (Craig and Li, 2008) that can generate forces of over 100 pN (Maier et al., 2004). T4P are grouped together in a class based on the production and secretion of the major pilin protein PilA4 (Thermus nomenclature), thousands of copies of which form the helical pilus (Craig et al., 2004; Schwarzenlander et al., 2009). The T4P is the only known bacterial pilus that can be retracted rapidly (Maier et al., 2004) to enable motility and adherence (Merz et al., 2000), major contributors to bacterial virulence (Hahn, 1997). Assembly and disassembly of the pilus is driven by the AAA-ATPases (ATPases associated with diverse cellular activities) PilF (extension) and PilT1/PilT2 (retraction) (Salzer et al., 2014b). It has been suggested that mature PilA4 assembles into pili extending from the inner membrane by action of PilF (Collins et al., 2013; Salzer et al., 2014b). The outer membrane channel of the T4P machinery is formed by the dodacemeric ∼1 MDa secretin complex PilQ (Burkhardt et al., 2011) (Figure 1). Other proteins, in particular PilM, PilN, and PilO, are hypothesized to be a central part of the pilus assembly platform and may couple the cytoplasmic and periplasmic sides of the T4P machinery (Karuppiah et al., 2013). Some proteins of the complex have been implicated to play a dual role in both pilus assembly and natural competence (Friedrich et al., 2002; Averhoff and Friedrich, 2003; Friedrich et al., 2003; Rumszauer et al., 2006). Recent results indicate that T4P themselves are not directly involved in DNA uptake (Burkhardt et al., 2012; Salzer et al., 2014a).

T4P are essential for pathogenesis by mediating adhesion, biofilm formation, and twitching motility (Burrows, 2012). Thus, both secretins and T4P play important roles in virulence of different pathogenic bacteria, which has fostered their use as new targets for drug development (Baron, 2010). To date, there is no information on the in situ structure of either the T4P machinery or DNA translocator. Determining structures of T4P complexes in whole bacterial cells is therefore of paramount importance and will enable further study of bacterial resistance and disease. Electron cryo-tomography (cryoET) has the unique ability to determine protein structures in cells at molecular resolution. We have applied cryoET to whole T. thermophilus HB27 cells, in order to visualize the T4P machinery in situ. We determine the helical structure of the pilus and find that the secretin complex PilQ is a central dynamic component of this system. CryoET and subtomogram averaging of the T4P machinery with and without pili reveal a ∼30 Å conformational change as the gates in the complex open.

T. thermophilus has an unusual cell architecture with deep surface clefts, formed by invaginations of the outer membrane (Figure 2A). By cryoET, these clefts are seen to be constrictions that run around the cell body (Figure 2D). Distal to the most polar outer membrane ring, numerous fibrous and straight pili extend from the cell (Figure 2A–D). The pili are clearly associated with large protein complexes crossing the ∼70 nm periplasm (Figure 2B,C). This distribution is in line with previous fluorescence and electron microscopy data, which demonstrate the polar localization of PilQ (Seitz and Blokesch, 2013) and pili (Salzer et al., 2014c).

Figure 2

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The periplasm of T. thermophilus cells is extraordinarily wide (Quintela et al., 1995; Castan et al., 2002) and too dense to select subvolumes for subtomogram averaging reliably. Therefore, cells were treated with 100 mM ethylenediaminetetraacetic acid (EDTA) and pipetted in order to render the outer membrane leaky (Caston et al., 1988). This had the desired effect of depleting the periplasm of most small proteins. Sample preparation by this method also removed the pilus from the complex, the empty T4P machinery was nonetheless still clearly visible (Figure 3A,B). We determined by subtomogram averaging the structure of the entire complex and found features distinct from those of the T2SS and T3SS secretins (Marlovits et al., 2006; Hodgkinson et al., 2009; Reichow et al., 2010). The resolution obtained by averaging ∼4000 particle subvolumes was ∼35 Å (Figure 3—figure supplement 1), most likely limited by the inherent flexibility of the complex (Burkhardt et al., 2011) and the difficulty of correcting precisely the contrast transfer function (CTF) for thick specimens.

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Subtomogram average maps show the central protein channel (∼35 nm long and ∼15 nm wide) made up of several ring-shaped domains inserted into the outer membrane (Figure 3C, left panel). We compared a 2D projection of the channel part of the subtomogram average with projections of purified, negatively stained PilQ only (Burkhardt et al., 2011) (Figure 3C, central and right panels). PilQ was seen to consist of a C-terminal trapezoid ‘cone structure’ with staggered rings in the N-terminal domain (Burkhardt et al., 2011), in excellent agreement with our in situ structure. Moreover, the new cryoET structure shows additional protein densities extending from the putative N0 domain of PilQ through the peptidoglycan layer (P1 and P2) to the cytoplasmic membrane (C1) (Figure 3D). Candidate proteins include PilW, which is associated with the inner and outer membranes and is essential for the outer membrane localization of PilQ (Rumszauer et al., 2006), and PilO/PilN heterodimers that could connect PilQ to the ATPases by PilM in the cytoplasm (Karuppiah and Derrick, 2011; Karuppiah et al., 2013) (Figure 1). These proteins are most likely connected to one another by flexible domains that are not well contrasted in the subtomogram average. A longitudinal slice through the complex reveals that PilQ has two gates, which are closed in the absence of a pilus (Figure 3C, left panel and Figure 3D, right panel). Gate 1 is formed by the ‘cone’ in the outer membrane and gate 2 by the N1 domain at the base of PilQ, enclosing an empty periplasmic vestibule (Figure 3D, right panel). The C-terminal ‘cone’ has been shown to form a sodium dodecyl sulfate (SDS)-stable sub-domain (Burkhardt et al., 2011), thus, it is plausible that gate 1 is responsible for maintaining the integrity of the cell membrane in the closed state. A second gate formed by the N-terminal domains has not been observed in other secretins (Korotkov et al., 2011). The N-terminus of PilQ forms an unusual βββαββ fold, different from the conserved ring-building βαββα folds (Burkhardt et al., 2012). Thus, we hypothesize that this motif may form part of gate 2.

To determine the structure of the T4P machinery in the open state, the pipetting step during sample preparation was omitted (‘Materials and methods’), which reduced the shearing forces and kept the assembled pilus intact (Figure 4A,B). Close inspection of the pilus shows a periodic structure (Figure 4C), which was suitable for subtomogram averaging. Since the ice surrounding the pili was thin (∼100 nm), it was possible to apply CTF-correction. The structure of the pilus was determined at ∼32 Å resolution by averaging 740 subvolumes (Figure 3—figure supplement 1). Power spectra calculated from a single tomographic slice or from the subtomogram average revealed a repeat distance of ∼4.9 nm (Figure 4C, lower panels). The T. thermophilus pilus forms a right-handed helix of ∼3 nm diameter (Figure 4D), which is different from the previously determined electron cryo-microscopy structure of the isolated Neisseria gonorrhoeae T4P (Craig et al., 2006). Diameters of pili can vary considerably and pilin proteins have limited sequence similarity (Craig and Li, 2008), which likely accounts for this difference. The result may prompt a reassessment of the functional roles of different T4P in cells. Due to the more challenging sample preparation method, the structure of the open T4P machinery with the pilus extended was determined from only ∼300 particles at ∼45 Å resolution (Figure 3—figure supplement 1). This resolution is sufficient to reveal the central protein channel complex with the ∼3 nm pilus protein density in the centre (Figure 4E). An extensive conformational change is evident that shifts PilQ domains N0–N3 away from N4/N5 by ∼30 Å towards the cytoplasmic membrane. This change opens the periplasmic vestibule to make way for the pilus (Figures 4E, 5A,B and Video 1). The shape and dimensions of the pore change from a tapered form ranging in width from ∼4 nm (at N5) to ∼8 nm (at N2/N3) in the closed state (Figure 3C), to a roughly constant 7 nm-wide channel in the open state (Figure 4E). In comparison, the closed state of Neisseria meningitidis PilQ is ∼9 nm wide tapering to a point (Collins et al., 2004), which would be sufficient to accommodate the wider Neisseria pilus (Collins et al., 2003; Craig et al., 2006) after a comparably large conformational change. Additional conformational changes and shifts also occur in protein densities P1, P2, and C1 (Figure 5A). In the open state, we observe an extra protein density in the cytoplasm (Figure 4E, yellow arrowheads and Video 1), which we speculate could be PilF, linked to the inner membrane platform via PilM (Karuppiah and Derrick, 2011) (Figure 1). Homologous proteins have been shown to interact with the cytoplasmic ATPases, stimulating their activity (Lu et al., 2013, 2014). Because EDTA treatment caused a depletion of periplasmic protein, we cannot exclude the possibility that some proteins may have been removed from the T4P machinery. However, we also averaged complexes from a tomogram of the two cells shown in Figure 2, which contained both open (with pili) and closed (without pili) complexes and where the periplasmic protein density was considerably higher. These subtomogram averages (Figure 5—figure supplement 1) show clearly that the same large conformational changes occur in the T4P machinery, irrespective of the degree of periplasmic protein depletion, and hence of the effect of EDTA.

Figure 4

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Video 1

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Docking the structures of the closed complex back into the tomographic volume reveals their three-dimensional distribution in the cell (Figure 5C,D and Video 2). We find that the T4P machinery tends to be tightly packed, with an inter-particle distance of 30–40 nm (Figure 5E). When the pili were depleted by pipetting, each cell contained on average 33 ± 19 closed complexes. However, T. thermophilus assembles ∼6 pili per cell (Salzer et al., 2014a, 2015), suggesting that ∼80% of the complexes are not involved in pilus formation under standard growth conditions (Salzer et al., 2014c). We speculate that these idle complexes may form a second class of transporter, which may be active in DNA uptake or protein secretion.

Video 2

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Taken together, our results demonstrate that the DNA translocator protein PilQ forms a dynamic central component of the T4P machinery in T. thermophilus. The length of the complex across the wide periplasm may be an adaptation to the thermophilic environment that Thermus thrives in. However, core components of the T4P machinery are conserved in bacteria (Nudleman and Kaiser, 2004), and thus, we speculate that the overall architecture may be similar. Our findings will enable further structure-function studies of the proteins that comprise this elaborate and important macromolecular machine.

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Author details

1. Vicki AM Gold

   Department of Structural Biology, Max Planck Institute of Biophysics, Frankfurt am Main, Germany

   Contribution

   VAMG, Conception and design, Acquisition of data, Analysis and interpretation of data, Drafting or revising the article, Contributed unpublished essential data or reagents

   For correspondence

   vicki.gold@biophys.mpg.de

   Competing interests

   No competing interests declared.

2. Ralf Salzer

   Molecular Microbiology and Bioenergetics, Institute of Molecular Biosciences, Goethe University Frankfurt, Frankfurt am Main, Germany

   Contribution

   RS, Conception and design, Analysis and interpretation of data, Drafting or revising the article, Contributed unpublished essential data or reagents

   Competing interests

   No competing interests declared.

3. Beate Averhoff

   Molecular Microbiology and Bioenergetics, Institute of Molecular Biosciences, Goethe University Frankfurt, Frankfurt am Main, Germany

   Contribution

   BA, Conception and design, Analysis and interpretation of data, Drafting or revising the article, Contributed unpublished essential data or reagents

   Competing interests

   No competing interests declared.

4. Werner Kühlbrandt

   Department of Structural Biology, Max Planck Institute of Biophysics, Frankfurt am Main, Germany

   Contribution

   WK, Drafting or revising the article, Contributed unpublished essential data or reagents

   Competing interests

   WK: Reviewing editor, eLife.

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