# A Unique Bacterial Secretion Machinery with Multiple Secretion Centers

Liqiang Song, John Perpich, Chenggang Wu, Thierry Doan, Zuzanna Nowakowska, Jan Potempa, Peter Christie, E. Cascales, Richard Lamont, Bo Hu

► To cite this version:

Liqiang Song, John Perpich, Chenggang Wu, Thierry Doan, Zuzanna Nowakowska, et al., A Unique Bacterial Secretion Machinery with Multiple Secretion Centers. Proceedings of the National Academy of Sciences of the United States of America, 2022, 119 (18), 10.1073/pnas.2119907119. hal-03655004

HAL Id: hal-03655004

https://amu.hal.science/hal-03655004

Submitted on 29 Apr 2022

HAL is a multi-disciplinary open access
archive for the deposit and dissemination of sci-
entific research documents, whether they are pub-
lished or not. The documents may come from
teaching and research institutions in France or
abroad, or from public or private research centers.

L'archive ouverte pluridisciplinaire HAL, est
destinée au dépôt et à la diffusion de documents
scientifiques de niveau recherche, publiés ou non,
émanant des établissements d'enseignement et de
recherche français ou étrangers, des laboratoires
publics ou privés.

<figure><img src="image_1.png" /></figure>

Distributed under a Creative Commons Attribution 4.0 International License
A Unique Bacterial Secretion Machinery with Multiple Secretion Centers

Liqiang Song¹, John D. Perpich², Chenggang Wu¹, Thierry Doan³, Zuzanna Nowakowska⁴, Jan

Potempa²,⁴, Peter J. Christie¹, Eric Cascales³*, Richard J. Lamont²*, Bo Hu¹*

¹Department of Microbiology and Molecular Genetics, McGovern Medical School, 6431 Fannin St,
Houston, Texas 77030

²Department of Oral Immunology and Infectious Diseases, University of Louisville, 570 S Preston St,
Louisville, Kentucky 40292

³Laboratoire d'Ingénierie des Systèmes Macromoléculaires, Institut de Microbiologie, Bioénergies et
Biotechnologie, Aix-Marseille Université — CNRS UMR7255, Marseille, France

⁴Department of Microbiology, Faculty of Biochemistry, Biophysics, and Biotechnology, Jagiellonian
University, Krakow, Poland

*Correspondence to: Bo Hu, Richard J. Lamont or Eric Cascales

Hu: Phone: 713-500-589 ; e-mail: [Bo.Hu@uth.tmc.edu](mailto:Bo.Hu@uth.tmc.edu); Fax: 713-500-5499

Lamont: Phone: 502-852-2112 ; e-mail: [rich.lamont@louisville.edu](mailto:rich.lamont@louisville.edu); FAX: 502 852 6394

Cascales: Phone: (+33) 491-164-462 ; e-mail: [cascales@imm.cnrs.fr](mailto:cascales@imm.cnrs.fr); FAX: (+33) 491-712-124

ORCID IDs: Wu (0000-0002-5700-7188); Doan (0000-0002-5909-4289); Christie (0000-0002-9799-7067); Cascales (0000-0003-0611-9179); Lamont (0000-0002-3147-5039); Hu (0000-0002-9117-879X)

**Keywords:** cryoelectron tomography, type IX secretion, protein transport, proton motive force,
32

**Abstract**

33

34

35

36

37

38

39

40

41

42

43

44

45

The *Porphyromonas gingivalis* type IX secretion system (T9SS) promotes periodontal disease by secreting gingipains and other virulence factors. By *in situ* cryoelectron tomography, we report that the *P. gingivalis* T9SS consists of eighteen PorM dimers arranged as a large, caged ring in the periplasm. Near the outer membrane, PorM dimers interact with a PorKN ring complex of ~52 nm in diameter. PorMKN translocation complexes of a given T9SS adopt distinct conformations energized by the proton motive force, suggestive of different activation states. At the inner membrane, PorM associates with a cytoplasmic complex that exhibits 12-fold symmetry and requires both PorM and PorL for assembly. Activated motors deliver substrates across the outer membrane via one of eight Sov translocons arranged in a ring. The T9SSs are unique among known secretion systems in bacteria and eukaryotes in their assembly as supramolecular machines composed of apparently independently-functioning translocation motors and export pores.

46

47

48

49

50

51

52

53

54

55

56

57

**Significance Statement**

The newly described type IX secretion systems (T9SSs) translocate virulence factors and can mediate specialized gliding motility among bacterial pathogens of the *Fibrobacteres-Chlorobi-Bacterioidetes* superphylum. We visualized the spatial organization of the T9SS in its native context in the *Porphyromonas gingivalis* cell envelope by *in situ* cryoelectron tomography. The T9SS exhibits distinct symmetries across the bacterial cell envelope: i) a cytoplasmic complex requiring PorL and PorM for assembly exhibits 12-fold symmetry, ii) a periplasmic complex composed of PorM exhibits 18-fold symmetry and attaches to a continuous PorKN ring near the outer membrane, and iii) eight Sov translocons are arranged with 8-fold symmetry at the cell surface. The T9SS is the largest of the known bacterial secretion systems, and evidently arranges as multiple, independently-functioning translocation motors.
58

**Introduction**

59

60

61

62

63

64

65

66

67

68

69

70

71

72

73

74

75

76

77

78

79

80

81

82

83

Bacteria deploy at least eleven different translocation systems to export macromolecules across their cell envelopes¹⁻³. Of these, the recently-discovered Type IX secretion systems (T9SSs) carried by many species of the *Fibrobacteres–Chlorobi–Bacteroidetes* (FCB) superphylum play various critical roles in pathogenesis and colonization⁴⁻⁷. Two bacterial models have emerged for detailed mechanistic and structural analyses of the T9SS. The Por T9SS in *Porphyromonas gingivalis*, a key contributor to human periodontitis and an emerging systemic pathogen, transports various heme-binding proteins and toxins aiding in colonization and invasion of gingival and other tissues⁸,⁹. The Gld T9SS in *Flavobacterium johnsoniae* secretes adhesins SprB and RemA and dozens of other effector proteins, but also functions as a rotary motor to direct the movement of a surface-bound adhesin along helical tracks, resulting in a form of gliding motility¹⁰,¹¹. Studies have shown that these T9SSs are large, cell envelope-spanning nanomachines composed of at least 14 essential subunits (Fig. 1A)⁶,⁷. They translocate substrates, exported to the periplasm by the general secretory (Sec) pathway, across the outer membrane (OM). At the cell surface, they release their substrates to the milieu or deliver them to an ‘attachment complex’, which covalently links the substrates to anionic lipopolysaccharides (A-LPSs) for surface display⁵,¹².

Three stable subassemblies have been identified through biochemical or single-particle microscopy approaches: i) an inner membrane (IM) molecular motor composed of the PorL and PorM subunits¹³,¹⁴ with structural and functional homologies with the MotAB/PomAB flagellar and ExbBD/TolQR PMF-dependent motors¹⁵,¹⁶,¹⁷,¹⁸; ii) a large ~50 nm ring-shaped complex composed of periplasmic PorN and outer membrane (OM)-associated lipoprotein PorK¹⁹,²⁰; and iii) an OM-spanning translocon, termed Sov or SprA, configured as a 36-strand β-barrel with a central pore of ~70 Å²¹. Despite this progress, it is not yet known how these substructures interact or coordinate their activities, in part because there is no overarching view of the T9SS in the native context of the bacterial cell envelope. Here, we visualized the intact T9SS of *P. gingivalis* by *in situ* cryoelectron tomography (cryo-ET). Remarkably, the T9SS presents not as a single functional entity, as shown for all known bacterial and eukaryotic protein
84

transport systems, but rather as a composite of apparently independent translocation motors and Sov
translocons arranged symmetrically as distinct rings in the cytoplasm, periplasm, and cell surface.

85

86

87

**Results and Discussion**

88

**The T9SS: A large, caged-ring structure.** *P. gingivalis* cells are small (~0.5 µm in diameter) and well suited for cryo-ET imaging (Fig. 1B, Fig. S1A). A typical three-dimensional (3D) reconstruction of a *P. gingivalis* cell revealed the IM and OM, as well as an electron dense surface layer (EDSL), which is known to consist of T9SS substrates attached at the cell surface (Fig. 1B - D)⁴. In wild-type cells, two periplasmic rods attached to the OM and ~50 nm apart were visualized in side views (Fig. 1B-D, Fig. S1A, Movie S1). In fact, the rods constitute the peripheral edges of a large ring similar in size (~50 nm) to the previously visualized PorKN rings¹⁹,²⁰ as shown in tilt- and top-down views (Fig. S1A, B, Movies S1 and 2). The rods visualized in side view are missing in a strain lacking PorK (Fig. 1E, F), which is an essential T9SS subunit⁷,¹⁹. The *ΔporK* mutant strain also lacks the EDSL (Fig. 1E, F) and fails to secrete T9SS effectors such as the Arg gingipain (Rgp) protease, whereas the *trans*-complemented strain is restored for Rgp secretion (Fig. S2). Similarly, mutants deleted of *porN* are devoid of the EDSL and OM-associated rods, and do not secrete Rgp protease (Figs. S1A and 2). Previously, it was shown that two IM subunits PorM and PorL with large domains respectively in the periplasm and cytoplasm¹³ are also required for T9SS function, but not for assembly of the PorKN ring¹⁹. Accordingly, here we determined that the *ΔporM* and *ΔporL* mutants lack the EDSL and do not secrete the Rgp effector, but elaborate the OM-associated rod structures (Figs. S1 and 2). Taken together, these findings strongly indicate that the visualized structures correspond to *in situ* T9SS nanomachines and that the OM-associated rods and rings visualized in side- and top-down views correspond to the PorKN ring complexes characterized by single particle cryoelectron microscopy (cryo-EM)¹⁹ (Fig. 1L-O). The average number of T9SSs visualized by *in situ* cryo-ET was ~1.3 per cell with a range of 0 to 4, although this might underestimate the actual number of partially or fully assembled T9SSs per cell (Fig. 1D).

89

90

91

92

93

94

95

96

97

98

99

100

101

102

103

104

105

106

107

108
To determine the 3D structure of the entire T9SS machine, we imaged over 800 wild-type *P. gingivalis* cells, and picked 694 ring-shaped particles from 516 tomograms (Table S1). By sub-tomogram averaging, we generated an overall structure of the T9SS at a resolution of ~32 Å (Fig. 1H, Fig. S1C). The T9SS spans the entire cell envelope, with dimensions of ~57 nm in height and ~52 nm in width, and consists of three distinct substructures: i) a surface structure (SS) on the extracellular side of the OM, ii) a periplasmic structure designated here as the core complex (CC), and iii) a cytoplasmic complex (Fig. 1H-I). The SS is located ~6 nm above the OM and below the EDSL, and in top-down view presents as a ring with outer and inner diameters of 48 and 31 nm (Fig. 1H, J). During initial alignment and without symmetry applied, the SS ring appears to be a featureless ring. We applied 18-fold symmetry to smooth the density; however, upon local refinement and classification (see below), we determined that the SS in fact is composed of 8 discontinuous structural units. The CC consists of two parts: i) the PorKN rods/ring structure (side/top-down views) of ~8 nm in thickness and 52 nm in diameter (Fig. 1H, K), and ii) strips of densities presenting in side view as arches of ~17 nm in height that connect the OM-associated PorKN rods to the IM (Figs. 1H-I, 2A). The cytoplasmic complex consists of a small central knob flanked by two densities when visualized in side-view (Fig. 1H-I). In 3D surface renderings of the intact T9SS shown at 45° and 90° (top-down) angles, ring-shaped architectures of the SS, CC, and cytoplasmic complex are readily evident (Fig. L-O).

Besides these densities, we detected three layers of density in the periplasm and a fourth between the cell surface and the EDSL (Fig. 1H, Fig. S3A,B). A 3D map reconstructed by sub-tomogram averaging of randomly picked regions of the *P. gingivalis* cell envelope established that these four layers are constituents of the cell envelope and are not parts of the T9SS machine (Fig. S3C,D). Consequently, the T9SS presents as a large, caged-ring structure that encompasses a central region of the *P. gingivalis* cell envelope whose IM, periplasmic and OM features are largely intact.

134
**The PorMKN core complex (CC).** We next focused on solving refined structures of the CC, with emphasis on the side-view arches. Prior studies have shown that PorM has an N-proximal transmembrane domain (TMD) and a large ~480-residue C-terminal region that extends across the periplasm and interacts with the OM-associated PorKN complex¹³. By X-ray crystallography, the periplasmic regions of PorM (PorMp) and the *F. johnsoniae* homolog GldM (GldMp) presented as elongated dimers with dimensions 180 Å in length and 50 and 35 Å in crosssections²²,²³. Through local refinements of periplasmic densities associated with T9SSs in WT cells, we determined that the periplasmic arches correspond to 18 distinct knobs arranged in a ring of 52 nm in diameter when visualized in top-down view (Table S1, S3, Fig. 2B, Fig. S4A). The *ΔporM* mutant possesses the PorKN ring, but lacks the underlying knobbed ring and the cytoplasmic complex (Fig. 2C, F). In consideration of the PorMp and GldMp X-ray structures²²,²³, we propose that the side-view arches and top-down-view knobs correspond to 18 PorM dimers, whose N-proximal regions are anchored in the IM and C-terminal regions extend through the periplasm to the OM-associated PorKN ring (Fig. 2D,E).

The PorMKN complex exhibits two other noteworthy features. First, in the native T9SS, the PorKN ring is 8 nm thick and the PorM arches are 17 nm in length, whereas in the *ΔporM* mutant, the PorKN ring is only 7 nm thick (Fig. 2A,C Fig. S5A, B). In the atomic structure, the PorMp dimers are 18 nm in length²³ and composed of 4 domains, D1-D4, with domain D4 directly interacting with PorN¹³,²². To reconcile these *in situ* and *in vitro* findings, the PorKN ring visualized *in situ* might actually consist of PorK, PorN, and domain D4 of PorM. Alternatively, the interaction between PorM and PorKN might induce a structural change resulting in thickening of the PorKN ring.

Second, in the global averaged map, the PorKN ring forms a 60° angle with respect to the OM (Fig. 3A). However, when the PorKN rings were locally refined at the junctions with the PorM arches, we detected three distinct structural Classes in which the PorMKN complex forms angles of 90°, 70° and 50° relative
160 to the OM (Fig. 3B). We quantified the PorMKN complexes of each Class among the entire set of 12,492
161 complexes in the 694 imaged T9SSs (18 complexes per T9SS × 694 T9SSs). Among the 12,492
162 complexes, the Class 1 (90°) and Class 3 (50°) complexes were more abundant (35.5 and 37.5 %,
163 respectively) than the Class 2 (70°) complexes (27 %) (Fig. S6A). Furthermore, among each of the 694
164 imaged T9SSs, the Class 3 complexes dominated with an average of 10 complexes and a range of 3 to 16
165 per T9SS (Fig. S6A). The Class 2 structures were least abundant, insofar as ~14 % of the T9SSs lacked
166 any Class 2 complexes and the bulk of the remaining T9SSs had ≤ 6. The Class 1 complexes had an
167 intermediate distribution with an average of 4 per T9SS and a range of 0-10. Importantly, in the absence
168 of *porM*, there was little variation from the 60° global average at the PorKN ring - OM junctions (Fig.
169 S5D), indicating that PorM contributes directly to the distinct PorKN - OM junction architectures in WT
170 machines.

171
172 Recently, it was shown that PorL and PorM and the *F. johnsoniae* GldL and GldM homologs form
173 structurally-similar proton-motive-force (PMF)-transducing motors¹⁴,²⁴. Moreover, in the atomic
174 structures, PorMp and GldMp dimers possess distinct bends in the hinge regions between the D1 - D2 or
175 D2 - D3 domain interfaces¹⁴,²¹,²². These finding have led to a model in which PMF-energy transduction
176 regulates the activation states of the PorLM and GldLM translocation motors¹⁴,²⁵. Upon sensing of the
177 PMF, PorLM and GldLM transduce PMF energy in the form of conformational changes in the
178 periplasmic domains of PorM and GldM as a prerequisite for substrate secretion or gliding motility²⁵,²⁶. In
179 this context, we propose that the visualized conformational Classes of the PorKN complexes
180 correspond to different activation states of the motors.
181

182 **Evidence for PMF-induced structural changes.** To further explore whether transduction of PMF
183 energy impacts the conformational status specifically at the PorKN - OM junction, we treated *P.
184 gingivalis* cells with the protonophore carbonyl cyanide m-chlorophenyl hydrazone (CCCP), which
185 disrupts the PMF and inactivates the T9SS¹⁴,²⁷. Approximately 90 % of the visualized T9SSs from CCCP-
treated cells possessed all of the structural features of machines from untreated cells, but the remaining 10
% lacked PorM arches and the cytoplasmic complex (Fig. 3A,D). Among the 90% of T9SSs bearing WT
features, we identified significant changes in the relative percentages of Classes with distinct PorKN-OM
junction angles. Most notably, Class 2 structures (bend-angle 70°) increased significantly, from 27.0 to
56.6%, upon CCCP-treatment. Conversely, the Class 1 (bend-angle 90°) and Class 3 (bend-angle 50°)
structures decreased from 35.5 to 20.4% and 37.5 to 23%, respectively (Fig. 3E). The significant increase
in the percentage of Class 2 structures in PMF-collapsed cells supports a proposal that this Class
corresponds to the quiescent or ‘closed’ state of the motor, whereas the Class 1 and 3 structures represent
or activated or ‘open’ states. Why activated motors are represented by bend-angles of 90° or 50° could be
explained by recent evidence that GldLM in the *F. johnsoniae* T9SS functions as a rotary motor, such that
the rotating periplasmic domain of GldM is envisioned to form a large arc as it extends to the PorKN
junction<sup>14,22,23</sup>. Recently, it was also shown that mutations in GldL have different impacts on T9SS
activity. Whereas a GldL E49A mutation abolishes both substrate secretion and gliding motility, another
mutation, E59A, prevents gliding but does not impact substrate secretion. These findings suggest that
GldLM motors exist in a quiescent state and in two activated states, one supporting both gliding motility
and substrate secretion and a second only secretion<sup>25</sup>.

Whether the *P. gingivalis* Por T9SS rotates is not yet known. If so, a PMF-energized rotating PorM
dimer might impose large bend-angles at the PorKN junction, and two such activated (Class 1 and 3)
structural states might have been captured by *in situ* cryo-ET imaging. By contrast, in the deenergized
state, a nonrotating PorM dimer potentially fixes the PorKN junction at the intermediate bend-angle
represented by the Class 2 structures. Even if PorM does not rotate, PMF sensing clearly induces large
conformational changes in the PorM dimers that are likely mediated through the recently visualized
PorLM complex assembled across the IM<sup>24</sup>.

210
Among the small fraction (~10%) of T9SSs from CCCP-treated cells that lacked the PorM arches and cytoplasmic complexes (Fig. 3D), the bend angle of the PorKN-OM junction approximates that observed for ΔporM mutant machines. CCCP-induced disengagement of the PorM arches therefore phenocopies the ΔporM mutation with respect to the overall PorKN ring architecture. Importantly, however, this subpopulation of CCCP-deenergized T9SSs retains the surface structures (SS) that are lacking in the ΔporM or ΔporL mutant machines (Fig. 2C, 4D). In a small fraction of T9SSs de-energized by collapse of the PMF, PorM and PorL thus appear to disengage from the PorKN ring without destabilization of the previously docked SS.

We sought to gain further biochemical evidence for PMF-mediated activation of T9SSs, and to this end we capitalized on the availability of antibodies to assay for energy-mediated conformational changes in the PorM and PorN homologs, GldM and GldN, in *F. johnsoniae* cells. Reminiscent of previous findings for the PMF-responsive energy transducer TonB²⁸, trypsin treatments of spheroplasts derived from CCCP-untreated or -treated cells yielded different GldM protease cleavage patterns (Fig. 3F). In the absence of CCCP treatment, GldM remained intact upon exposure to trypsin, whereas CCCP treatment resulted in generation of two GldM cleavage products. By LS-MS/MS spectrometry, we determined that both cleavage sites (downstream residues Lys252 and Arg322, respectively) reside within the linker region between domains D2 and D3 (Fig. 3F). We were unable to assess the effect of PMF energy depletion on the conformational status of GldN, as the GldN trypsin cleavage patterns were unaltered regardless of prior treatment of cells with CCCP (Fig. S7). Overall, these data confirm earlier predictions that GldLM transduces IM energy from the PMF in the form of a conformational change(s) in the periplasmic arms of GldM to activate the T9SS for transport or gliding motility²². In view of the structural homology of GldM and PorM, we suggest that PMF sensing similarly induces conformational changes in the PorM translocation motor. With respect to the PMF-driven conformational changes we observed here for individual PorKMN complexes, it is interesting to note that complexes of a given bend-
angle Class tend to spatially cluster within each T9SS (Fig. S6A-B). This clustering could imply that
adjacent complexes are coordinately activated in response to PMF energy sensing.

238

**Refinement of the cytoplasmic complex.** To better understand the structural basis for energy transduction, we locally refined the cytoplasmic complex (Fig. 4A-C, Fig. S4B). Three distinct densities were evident: i) a central knobbed cylinder projecting ~14 nm from the IM into the cytoplasm, ii) a flanking ring of ~6 nm in thickness and ~20 nm in diameter, and iii) a spoked-ring of ~32 nm in diameter with 12-fold symmetry (Fig. 4B,C, Fig. S4B). PorL and PorM are the only essential T9SSs proteins identified to date with cytoplasmic domains (Fig. 1A). Indeed, the ΔporL and ΔporM mutant machines lack the periplasmic arches and cytoplasmic densities, showing a co-dependence on PorL and PorM for assembly of these structures (Figs. 2C, 4D). PorM spans the IM once and its ~9-residue, N-terminal cytoplasmic domain is unlikely to contribute detectable density. By contrast, PorL has two N-terminal transmembrane helices (TMHs) and its large (~230-residue) C-terminal domain resides in the cytoplasm²⁴. Recently, the 3D structures of the PorLM and GldLM complexes, were shown to consist of the 10 TMHs from five PorL subunits arranged in a ring surrounding the two TM helices of the PorM dimer¹⁴,²⁴. Thus, the 5 cytoplasmic domains of PorL associated the recently visualized PorLM complex²⁴ might contribute to one or more of the cytoplasmic densities detected by *in situ* cryo-ET.

253

The PorM dimers are in close juxtaposition with the outer most cytoplasmic knobs, suggesting the
possibility that they are composed of PorL pentamers (see Fig. S4B, classes e-h). If so, it is necessary to
account for the 18- vs 12-fold symmetry mismatch that exists between the PorM arches and putative PorL
knobs. Conceivably, a PorM dimer engages stably with the PorL pentamer only when activated, for
example, upon sensing of bound substrate in the periplasm. Such a model of substrate-induced PorLM
engagement might allow for one PorL pentamer to interact with and energize more than one spatially-
juxtaposed PorM dimer, which would account for our observation that PorKMN complexes of a given

236

237

238

239

240

241

242

243

244

245

246

247

248

249

250

251

252

253

254

255

256

257

258

259

260
structural Class, or activation state, tend to spatially cluster within each T9SS (Fig. S6A-B). It is
important to note, however, that PorL's cytoplasmic domain is disordered when purified and analyzed by
single-particle cryo-EM¹⁴. If this domain is also too flexible for detection by *in situ* cryo-ET, the
visualized densities might instead correspond to other Por components. One candidate is PorX, the
response regulator of the PorXY two-component system that was shown to interact with PorL²⁹, but
further studies are needed to define subunit contributions to the observed cytoplasmic densities.

267

**The Surface Structure (SS).** Finally, we refined the structure of the SS visualized initially as an
extracellular ring (Fig. 1J). Strikingly, the SS presents as eight distinct densities of 8 nm in diameter
arranged in a ring of ~48 nm in diameter (Fig. 5A-B, Fig. S4C). Each of the eight densities has a lateral
pore near the OM that faces the center of the T9SS supramolecular complex (Fig. 5E-F). The overall size
and presence of a lateral pore are highly reminiscent of the 267 kDa SprA/Sov translocon solved at ~3.5
Å by single particle cryo-EM²⁰. This cryo-EM structure docks well on the SS densities (Fig. 5G-H, Fig.
S8A,B). Moreover, a strain deleted of Sov retained all features of the T9SS except for the SS; this strain
also lacked the EDSL and failed to secrete Rgp protease (Fig. 5C,D, Figs. S1, 2). Considering the size of
Sov protein, we propose that the SS densities are largely composed of Sov, with potential contributions
from other components, e.g., the PorV shuttle protein³⁰. In the cryo-EM structure, the location of
detergent associated with the translocon was used to define the likely plane of the OM, giving rise to the
proposal that the β-barrel extends ~2 nm above the OM and an extracellular cap projects an additional 5
nm²¹. In line with those findings, the SS densities project ~6-7 nm above the OM (Fig. 5E). Interestingly,
beneath each of the Sov densities, the OM is missing its inner leaflet (Fig. 5A, Fig. S8A). This gap or hole
might be a point of entry for substrates, delivered by the PorLMKN translocation motor, into the Sov
translocon. The importance of the translocation motor - Sov contact for a stable association of translocons
with the T9SS is underscored by the fact that deletions of any of the translocation motor subunits
abolished detectable Sov surface densities (Figs. 2C, 4D). Intriguingly, despite numerous attempts, no
studies have yet shown a direct interaction between the PorL/M/K/N proteins and the Sov translocon.
Recently, however, a protein interactome analysis identified PorW as a mutually interacting partner of Sov and the PorKN ring. PorW thus appears to constitute a critical link between these subassemblies³¹.

**Summary.** We have shown that the *P. gingivalis* T9SS assembles as a large supramolecular machine composed of a cytoplasmic complex with 12-fold symmetry, a periplasmic complex with 18-fold symmetry, and a surface structure consisting of 8 symmetrically arranged putative pores (Fig. 5I). Taken together with previous findings, we propose a working model in which the major machine subunits assemble with an overall stoichiometry of (PorL₅/PorM₂/PorK₂/PorN₂)₁₈(Sov)₈ (Fig. 5J). Observed differences in the bend angles at PorMKN - OM junctions (Fig. 3B), coupled with the absence of a discernible central channel (Fig. 2A, Fig. S3A), support our model that the T9SS is a multi-port export apparatus composed of independently - but coordinately - functioning IM energy sensors, periplasmic translocation motors, and OM translocons (Fig. 4J-K, Movie 3). This proposed architecture and mechanism of action strikingly contrast with other known dedicated protein translocation systems functioning in bacteria¹⁻³,³²,³³ (Fig. S9). Deciphering the molecular and structural details underlying IM energy activation, substrate recruitment, and OM export remain as major challenges for future research on this unique family of bacterial transporters.

**Materials and Methods**

**P. gingivalis** growth and mutant constructions. *P. gingivalis* ATCC 33277 (33277) and W83 and their isogenic mutants were cultured anaerobically at 37°C in Trypticase soy broth (TSB) supplemented with 1 mg ml⁻¹ yeast extract, 5 µg ml⁻¹ hemin, and 1 µg ml⁻¹ menadione. When appropriate, erythromycin (10 µg ml⁻¹) was added to the medium.

The PCR fusion technique was utilized to generate allelic exchange mutants ΔporK, ΔporL, ΔporM, ΔporN, (all 33277) and Δsov (W83) as described previously³⁴. Upstream and downstream fragments of each gene were amplified by PCR and fused to *ermF* using the primers listed in Table S2. Constructs
313 were confirmed by sequencing and introduced into *P. gingivalis* by electroporation, and mutant strains
314 were selected on TSB plates containing erythromycin. Loss of mRNA from the target gene without
315 disruption of expression of the other genes in the operon (*por* operon) was confirmed by RT-PCR (Fig.
316 S10) using the primers listed in Table S2.

317
318 For complementation of *porK* in *P. gingivalis* ATCC33277, a 500-bp sequence upstream of PGN_1678
319 containing the promoter region was fused to the coding region of *porK* using PCR fusion and the primers
320 listed in Table S2. The resulting PCR product was confirmed by sequencing and cloned into the pT-COW
321 vector to create pT-COW-*porK*. The plasmid was transformed into chemically competent *E. coli* (strain
322 S17-1) and selected using ampicillin. The plasmid was transferred into *P. gingivalis* Δ*porK* via
323 conjugation with *E. coli*35,36, and transconjugants were selected with gentamicin (50 µg ml⁻¹) and
324 tetracycline.

325
326 *F. johnsoniae* CJ1827 (UW101 *rpsL*, *Str*R) and its Δ*gldM* isogenic mutant have been previously
described³⁷. *F. johnsoniae* strains were grown in Casitone-Yeast extract (CYE) medium (10 g L⁻¹
327 casitone, 5 g L⁻¹ yeast extract, 8 mM MgSO₄, 10 mM Tris-HCl pH7.6) at 28°C with agitation. The proton-
motive force uncoupler carbonyl cyanide-m-chlorophenylhydrazone (CCCP, Sigma Aldrich) was
328
329 solubilized in ethanol and was added in the culture 30 min prior to cell harvesting, at the final
330
331 concentration of 10 µM.

332
333 **Spheroplasting and limited proteolysis.** *F. johnsoniae* UW101 cells were grown in 5 mL of CYE
334 medium to an *A*₆₀₀=0.8, harvested by centrifugation and resuspended in 100 µL of 20 mM Tris-HCl pH
335 8.0, 20% sucrose, 1 mM EDTA, and 100 µg ml⁻¹ of lysozyme. After 30 min incubation at room
336 temperature (20°C), 100 µL of ice-cold sterile water was added, and the mixture was carefully mixed by
337 three inversions. 50 µL of the spheroplast suspension were treated with trypsin (100 µg ml⁻¹). After 5 min
on ice, 17 µL of boiling 4× Laemmli loading buffer were added, and immediately vortexed and boiled for 5 min prior to SDS-PAGE.

340

341 **Immunoprecipitation and LC-MS/MS analyses.** *F. johnsoniae* spheroplasts obtained from a 50-mL culture treated with 10 µM CCCP were prepared and subjected to limited proteolysis using trypsin.

342

343 Proteolysed spheroplasts were resuspended in 1 mL of CelLytic (Sigma-Aldrich) in presence of aprotinin 344 (Sigma-Aldrich), and incubated for 30 min on a wheel at room temperature. Unsolubilized material was 345 discarded by centrifugation for 10 min at 20,000 × g, and subjected to immunoprecipitation using anti-346 GldM antibodies bound to Protein A Sepharose CL-4B (GE Healthcare). After 2 hours of incubation at 347 room temperature on a wheel, the beads were washed three times with CelLytic B, resuspended in 348 Laemmli loading buffer, and subjected to SDS-PAGE and InstantBlue staining (Expedeon). Bands 349 corresponding to the proteolytic products were cut, washed with 100 mM acetonitrile/ammonium 350 bicarbonate pH 7.5, reduced by 10 mM dithiothreitol in 100 mM ammonium bicarbonate pH 7.5, 351 alkylated by 55 mM iodoacetamide in 100 mM ammonium bicarbonate pH 7.5 and then digested 352 overnight at 37°C with 10 ng/ µl⁻¹ of Trypsin/Lys-C Mix from *Pseudomonas aeruginosa* (Promega) in 25 353 mM ammonium bicarbonate pH 7.5, 0.025% (v/v) proteaseMAX surfactant (Promega). Tryptic peptides 354 were extracted from gel bands by 0.1% (v/v) trifluoroacetic acid, 0.01% (v/v) proteaseMAX, 50% (v/v) 355 acetonitrile, and dried by speed vacuum. Samples solubilized in 0.05% (v/v) trifluoroacetic acid, 2% (v/v) 356 acetonitrile were analysed on a ESI-Q-Exactive Plus (ThermoFisher) mass spectrometer coupled to a 357 nanoliquid chromatography (Ultimate 3000, Dionex). Peptides were eluted from a C18 column (Acclaim 358 PepMap RSLC, 75 µm × 150 mm, 2 µm, 100 Å, Dionex) by a 6–40% linear gradient of mobile phase B 359 (0.1% (v/v) formic acid/80% (v/v) acetonitrile) in mobile phase A (0.1% (v/v) formic acid) for 52 min. 360 The peptides were detected in the mass spectrometer in a positive ion mode using a Top 10 Data 361 Dependent workflow. A single full scan acquisition in the Orbitrap at a resolution of 70,000 in a 350–362 1900 m/z range was followed by a fragmentation MS/MS step, at a resolution of 17,500, of the 10 top 363 ions, in the Higher Energy Collisional Dissociation cell set at 27 eV. The spectra were processed by
364 Proteome Discoverer software (ThermoFisher, version 2.1.0.81) using the Sequest HT algorithm with the
365 search following parameters: *F. johnsoniae* UW101 GldM protein (GeneBank identifier (GI): 58531935);
366 trypsin enzyme (maximum 2 missed cleavages); fixed modification: carbamidomethyl (Cys); variable
367 modification: oxidation (Met); mass values specific for monoisotopic; precursor mass tolerance: ±10
368 ppm; fragment mass tolerance: ±0.02 Da.

369 **Preparation of frozen-hydrated specimens.** Strains grown anaerobically at 37°C were harvested in the
370 late log phase by centrifugation at 9000 × g. The pellet was resuspended in phosphate-buffered saline
371 (PBS). To prepare CCCP treated wild-type cells, CCCP was added to a final concentration of 20 µM and
372 incubated for 20 min and then mixed with 10 nm diameter colloidal gold beads (used as fiducial markers
373 in image alignment) and deposited onto freshly glow-discharged, holey carbon grids for 1 min. The grids
374 were then blotted with filter paper and rapidly frozen in liquid ethane, using a gravity-driven plunger
375 apparatus as previously described³³.
376

377
378 **CryoET data collection and 3-D reconstructions.** Frozen-hydrated specimens were imaged at -170 °C
379 using a Polara G2 electron microscope (FEI Company) equipped with a field emission gun and a direct
380 detection device (Gatan K2 Summit). The microscope was operated at 300 kV with a magnification of
381 ×9,400, resulting in an effective pixel size of 4.5 Å at the specimen level. We used SerialEM³⁸ to collect
382 low-dose, single-axis tilt series with dose fractionation mode at about 8 µm defocus. Tilt series were
383 collected from -51° to 51° with increment of 3° and a cumulative dose of ~70 e⁻/Å² distributed over 35
384 stacks. Each stack contains ~8 images. We used Tomoauto³⁹ to facilitate data processing, which includes
385 drift correction of dose-fractionated data using Motioncorr⁴⁰ and assembly of corrected sums into tilt
386 series, automatic fiducial seed model generation, alignment and contrast transfer function correction of tilt
387 series by IMOD⁴¹, and weighted back projection (WBP) reconstruction of tilt series into tomograms using
388 Tomo3D⁴². Each tomographic reconstruction is 3,710 × 3,838 × 1,600 pixels and ~1.30 Gb in size. In
389 total, 4,227 tomographic reconstructions from 6 different strains were generated.
390
**Sub-tomogram averaging and correspondence analysis.** We used tomographic package I3 for sub-tomogram analysis as described previously⁴³. A total of 2936 type IX secretion machines (400 × 400 × 400 voxels) were visually identified and then extracted from 1947 tomographic reconstructions. Two of the three Euler angles of each secretion machine were estimated based on the orientation of each particle in the cell envelope. To accelerate image analysis, 4×4×4 binned sub-tomograms (100×100×100 voxels) were used for initial alignment. The alignment proceeds iteratively with each iteration consisting of three parts in which references and classification masks are generated, sub-tomograms are aligned and classified, and finally class averages are aligned to each other. After multiple cycles of alignment and classification for 4×4×4 binned sub-tomograms, we used 2×2×2 binned sub-tomograms for refinement.
400
The initial alignments were carried out without symmetry imposed. To determine the symmetry of the SS, CC and cytoplasmic complex, after the initial alignments of the intact machine, the rotation centers of the sub-volumes were moved to the corresponding centers of SS, CC and cytoplasmic complex, respectively, and masks were applied to include the SS, CC or cytoplasmic complex. Once the symmetries were determined through classification, one of the classes was used as reference to align other classes and the corresponding symmetry was applied to the final refinements. Fourier shell correlation (FSC) between the two independent reconstructions was used to estimate the resolution of the averaged structures.
401
402
403
404
405
406
407

408
**Visualization and molecular modeling.** We used IMOD to visualize the maps. IMOD and EMAN2⁴¹,⁴⁴ were used to generate 3D surface rendering of *P. gingivalis* cells and UCSF ChimeraX (http://www.rbvi.ucsf.edu/chimerax) to visualize sub-tomogram averages in 3D and molecular modelling.
409
410
Each of 18 PorM dimer structures (PDB 7CMG) was manually fitted onto the each of 18 arches of densities. We docked the Sov homolog SprA (PDB 6H3J) into the 8 pore densities on the cell surface.
411
412
413

414
**Quantification and statistical analyses.** Quantification and statistical analyses are integral parts of the algorithms and software used in our high throughput cryoelectron tomography pipeline. In particular,
415
massive data enabled us to use multivariate statistical analysis and classification (which are implemented
in tomographic package i3) for processing and interpretation of the sub-tomograms of T9SS extracted
from *P. gingivalis* cell reconstructions.

419

420

**Acknowledgements.** We are grateful to members of the Hu, Lamont and Cascales labs for critical discussions. We are grateful to Dr. William Margolin for suggestions and critiques of the manuscript, Pascal Mansuelle (IMM proteomic platform, Marseille) for mass spectrometry analyses, and Mark McBride (University of Wisconsin, Milwaukee) for anti-GldM and -GldN antibodies. **Funding:** B.H. was supported in part by McGovern Medical School start-up funds, the Welch Foundation (AU-1953-20180324) and NIH 1R35GM138301. R.J.L. was supported by DE011111, DE012505 and DE023193. E.C. was supported by the Fondation Bettencourt-Schueller, and A*MIDEX grant A-M-AAP-ID-17-33-170301-07.22 and ANR grant ANR-20-CE11-0011. P.J.C. was supported by NIH grants R01GM48476 and R35GM131892. C.W. was supported by NIH grant DE030895.

430

431 **Competing interests:** The authors declare no competing interests.

432

**Data and materials availability:** Density maps and coordinate data that support the T9SS structures determined by cryo-electron tomography have been deposited in the Electron Microscopy Data Bank (EMDB) as EMD-24227 (Intact machine: PorM arches/ring), EMD-24228 (Cytoplasmic complex) and EMD-24229 (Sov Surface Structure; SS). The authors declare that all other data supporting the findings of this study are available within the paper and its supplementary materials.

438

**Author contributions:** B.H., L.S., and R.J.L. designed the research. J.D.P. constructed the *P. gingivalis* mutants and phenotypically characterized mutant strains. B.H. and L.S. collected and processed the data. T.D. and E.C. performed the protease accessibility experiments. B.H. L.S., T.D., E.C., R.J.L. and P.J.C

441
442 analyzed the data. L.S. and B.H. generated the figures, and P.J.C. wrote the manuscript with
443 contributions from B.H., R.J.L., and E.C.

444

445 **Correspondence and requests for materials** should be addressed to B.H. or R.J.L.

446

447

448 **References**

449 1. J. E. Galan, G. Waksman, Protein-injection machines in bacteria. *Cell* **172**, 1306-1318 (2018).

450 2. P. J. Christie, The rich tapestry of bacterial protein translocation systems. *Protein J.* **38**, 389-408 (2019).

451 3. D. D. Majewski, L. J. Worrall, N. C. Strynadka, Secretins revealed: structural insights into the giant gated outer membrane portals of bacteria. *Curr. Opin. Struct. Biol.* **51**, 61-72 (2018).

452 4. P. D. Veith, M. D. Glew, D. G. Gorasia, E. C. Reynolds, Type IX secretion: the generation of bacterial cell surface coatings involved in virulence, gliding motility and the degradation of complex biopolymers. *Mol. Microbiol.* **106**, 35-53 (2017).

453 5. A. M. Lasica, M. Ksiazek, M. Madej, J. Potempa, The type IX secretion system (T9SS): Highlights and recent insights into its structure and function. *Front. Cell Infect. Microbiol.* **7**, 215 (2017).

454 6. M. J. McBride, Bacteroidetes gliding motility and the type IX secretion system. *Microbiol Spectr.* **7**, (2019).

455 7. K. Sato *et al.*, A protein secretion system linked to bacteroidete gliding motility and pathogenesis. *Proc. Natl. Acad. Sci. U. S. A.* **107**, 276-281 (2010).

460 8. N. M. O'Brien-Simpson *et al.*, Role of RgpA, RgpB, and Kgp proteinases in virulence of *Porphyromonas gingivalis* W50 in a murine lesion model. *Infect Immun* **69**, 7527-7534 (2001).

461 9. K. Nakayama, *Porphyromonas gingivalis* and related bacteria: from colonial pigmentation to the type IX secretion system and gliding motility. *J. Periodontal Res.* **50**, 1-8 (2015).
468

469

470

471

472

473

474

475

476

477

478

479

480

481

482

483

484

485

486

487

488

489

490

491

492

493

10. A. Shrivastava, T. Roland, H. C. Berg, The screw-like movement of a gliding bacterium is powered by spiral motion of cell-surface adhesins. *Biophys. J.* **111**, 1008-1013 (2016).

11. D. Nakane, K. Sato, H. Wada, M. J. McBride, K. Nakayama, Helical flow of surface protein required for bacterial gliding motility. *Proc. Natl. Acad. Sci. U. S. A.* **110**, 11145-11150 (2013).

12. P. D. Veith *et al.*, Protein substrates of a novel secretion system are numerous in the Bacteroidetes phylum and have in common a cleavable C-terminal secretion signal, extensive post-translational modification, and cell-surface attachment. *J. Proteome Res.* **12**, 4449-4461 (2013).

13. M. S. Vincent *et al.*, Characterization of the *Porphyromonas gingivalis* type IX secretion trans-envelope PorKLMNP core complex. *J. Biol. Chem.* **292**, 3252-3261 (2017).

14. R. H. James *et al*. Structure and mechanism of the proton-driven motor that powers type 9 secretion and gliding motility. *Nat. Microbiol.* **6**, 221–233 (2021).

15. M. Santiveri *et al.*, Structure and function of stator units of the bacterial flagellar motor. *Cell* **183**, 244-257 (2020).

16. J. C. Deme *et al.*, Structures of the stator complex that drives rotation of the bacterial flagellum. *Nat. Microbiol.* **5**, 1553-1564 (2020).

17. H. Celia *et al.*, Cryo-EM structure of the bacterial Ton motor subcomplex ExbB-ExbD provides information on structure and stoichiometry. *Commun. Biol.* **2**, 358 (2019).

18. Zhu S *et al.*, *In situ* structure of the Vibrio polar flagellum reveals a distinct outer membrane complex and its specific interaction with the stator. *J. Bacteriol.* **202:e00592-19** (2020).

19. D. G. Gorasia *et al.*, Structural insights into the PorK and PorN components of the *Porphyromonas gingivalis* type IX secretion system. *PLoS Pathog.* **12**, e1005820 (2016).

20. D. G. Gorasia *et al.*, *In situ* structure and organisation of the type IX secretion system. *bioRxiv* 2020.05.13.094771; doi: https://doi.org/10.1101/2020.05.13.094771 (2020).

21. F. Lauber, J. C. Deme, S. M. Lea, B. C. Berks, Type 9 secretion system structures reveal a new protein transport mechanism. *Nature* **564**, 77-82 (2018).
494 22. P. Leone *et al.*, Type IX secretion system PorM and gliding machinery GldM form arches spanning the periplasmic space. *Nat. Commun.* **9**, 429 (2018).

496 23. K. Sato *et al*. PorM, a core component of bacterial type IX secretion system, forms a dimer with a unique kinked-rod shape. *Biochem. Biophys. Res. Commun.* **532**, 114–119 (2020).

498 24. R. H. James *et al*. Structures of the type IX secretion/gliding motility motor from across the phylum *Bacteroidetes*. https://www.biorxiv.org/content/10.1101/2022.01.28.478170v1 (2022)

500 25. M.S. Vincent *et al*, Dynamic proton-dependent motors power type IX secretion and gliding motility in *Flavobacterium. PLoS Biology*, In press (2022).

502 26. P. D. Veith *et al.*, The type IX secretion system and its role in bacterial function and pathogenesis. *J. Dent. Res.* https://pubmed.ncbi.nlm.nih.gov/34889148 (2021).

504 27. J. L. Pate, LY. E. Chang, Evidence that gliding motility in prokaryotic cells is driven by rotary assemblies in the cell envelopes. *Curr. Microbiol.* **2**, 59–64 (1979).

506 28. RA Larsen, Assessing energy-dependent protein conformational changes in the TonB system. *Methods Mol. Biol.* **1615**, 277-287 (2017).

508 29. M. S. Vincent, E. Durand, E. Cascales, The PorX response regulator of the *Porphyromonas gingivalis* PorXY two-component system does not directly regulate the type IX secretion genes but binds the PorL subunit. *Front. Cell Infect. Microbiol.* **6**, 96 (2016).

511 30. M.D. Glew *et al.*, PorV is an outer membrane shuttle protein for the type IX secretion system. *Sci. Rep.* **7**, 8790 (2017).

513 31. D.G. Gorasia *et al.*, Protein interactome analysis of the type IX secretion system identifies PorW as the missing link between the PorK/N ring complex and the Sov translocon. *Microbiol. Spectr.* 10:e01602-21 (2022).

516 32. B. Hu, M. Lara-Tejero, Q. Kong, J. E. Galan, J. Liu, *In situ* molecular architecture of the *Salmonella* Type III secretion machine. *Cell* **168**, 1065-1074 e1010 (2017).

518 33. B. Hu, P. Khara, P. J. Christie, Structural bases for F plasmid conjugation and F pilus biogenesis in *Escherichia coli*. *Proc. Natl. Acad. Sci. U. S. A.* **116**, 14222-14227 (2019).
520

521

522

523

524

525

526

527

528

529

530

531

532

533

534

535

536

537

538

539

540

541

542

543

544

34.

M. R. Simionato et al., Porphyromonas gingivalis genes involved in community development with Streptococcus gordonii. *Infect. Immun.* **74**, 6419-6428 (2006).

35.

C. E. James et al., LuxS involvement in the regulation of genes coding for hemin and iron acquisition systems in Porphyromonas gingivalis. *Infect. Immun.* **74**, 3834-3844 (2006).

36.

Y. Park, R. J. Lamont, Contact-dependent protein secretion in Porphyromonas gingivalis. *Infect. Immun.* **66**, 4777-4782 (1998).

37.

A. Shrivastava et al, Flavobacterium johnsoniae GldK, GldL, GldM, and SprA are required for secretion of the cell surface gliding motility adhesins SprB and RemA. *J. Bacteriol.* **195**, 3201-3212 (2013).

38.

D. N. Mastronarde, Automated electron microscope tomography using robust prediction of specimen movements. *J. Struct. Biol.* **152**, 36-51 (2005).

39.

D. R. Morado, B. Hu, J. Liu, Using Tomoauto: A protocol for high-throughput automated cryo-electron tomography. *J. Vis. Exp.* e53608 (2016).

40.

X. Li et al., Electron counting and beam-induced motion correction enable near-atomic-resolution single-particle cryo-EM. *Nat. Methods* **10**, 584-590 (2013).

41.

J. R. Kremer, D. N. Mastronarde, J. R. McIntosh, Computer visualization of three-dimensional image data using IMOD. *J. Struct. Biol.* **116**, 71-76 (1996).

42.

J. I. Agulleiro, J. J. Fernandez, Tomo3D 2.0--exploitation of advanced vector extensions (AVX) for 3D reconstruction. *J. Struct. Biol.* **189**, 147-152 (2015).

43.

H. Winkler, 3D reconstruction and processing of volumetric data in cryo-electron tomography. *J. Struct. Biol.* **157**, 126-137 (2007).

44.

M. Chen et al., A complete data processing workflow for cryo-ET and subtomogram averaging. *Nat. Methods* **16**, 1161-1168 (2019).
545 **Figure legends**

546 **Figure 1.** *Porphyromonas gingivalis* type IX machine revealed by cryo-ET. **A**, Schematic representation of the structure and function of the T9SS of *P. gingivalis*. The different subunits are indicated, as well as the putative route for substrate transport (dashed red line). **B**, A tomographic slice of a wild-type *P. gingivalis* cell. **C**, Zoomed-in view of (B) showing the outer membrane (OM), inner membrane (IM), Electron Dense Surface Layer (EDSL) and a type IX machine (T9SS) embedded in the cell envelope. **D**, 3D surface view showing multiple T9SSs embedded in the wild-type *P. gingivalis* cell envelope. **E**, A tomographic slice of a ΔporK mutant of *P. gingivalis* and **F**, zoomed-in view showing the absence of T9SSs and EDSL. **G**, 3D surface view of a *P. gingivalis* ΔporK mutant cell. **H-O**, *In situ* structure of the *P. gingivalis* T9SS. **H**, A central section through longitudinal plane of a global average structure *in situ* showing T9SS densities in the cell envelope. **I**, Outline of major features of the T9SS including the surface structure (SS), periplasmic core complex (CC) and a cytoplasmic (cyto-) complex. **J** and **K**, Cross-section views at the position indicated in (H) (yellow arrows) showing the ring-shaped surface structure and periplasmic PorKN complex. To smooth the map, we imposed an 18-fold symmetry. **L-O**, 3D surface rendering of the intact T9SS in side, cut-away, 45°, and 90° views.

560

561 **Figure 2.** Molecular architecture of PorM. **A**, Central section of the native T9SS (WT) showing that the proposed periplasmic arches of PorM span ~17 nm from the IM to the PorKN ring. **B**, Top-down view of the region denoted in (A) showing 18-fold symmetry of the PorM arches. **C**, A central section of a global average structure of the T9SS visualized in a ΔporM mutant. **D**, Two PorM dimers (PDB entry 7CMG) docked in the PorM arch density. **E**, An overall view of T9SS with eighteen docked PorM dimers. **F**, 3D surface rendering of the ΔporM mutant machine showing the presence of the PorKN ring and absence of the PorM arches, cytoplasmic complex, surface structure (SS), and EDSL.

568

569 **Figure 3.** Proton-motive force-dependent conformational change of the *P. gingivalis* PorM protein and *F. johnsioniae* GldM protein. **A**, Central section of the native T9SS (WT) showing that the PorKN
ring forms a ~60° angle relative to the OM. **B**, 3-D classification reveals three distinct classes of PorKNM complex showing that the PorKN ring forms angles of 90° (Class 1), 70° (Class 2) and 50° (Class 3) relative to the OM. **C**, A proposed model for oscillatory motion of the PorMKN complex. PMF energy-driven conformational changes of PorM drive the oscillation of PorKN yielding bend angles of 90° and 50°; the de-energized PorMKN complex is fixed at an angle of 70°. **D**, 10% of the T9SS machines lack PorM and cytoplasmic complex upon CCCP treatment of WT cells. **E**, The proportion of each bend-angle Class is altered in wild-type cells upon treatment with CCCP. **F**, (i) Limited protease accessibility assay. *F. johnsoniae* wild-type or Δ*gldM* mutant cells were treated with CCCP and spheroplasts were treated with trypsin protease. Proteolytic fragments were visualized by immunostaining of western blots with anti-GldM antibodies. GldM, full-length protein, * and **, degradation products. Molecular weight markers are indicated on left. (ii) Identification of the GldM trypsin cleavage sites. The GldM degradation products were enriched by immunoprecipitation using anti-GldM antibodies and subjected to LS-MS/MS identification. The GldM structure (PDB: 6EY4) and magnification are shown, with * and ** denoting cleavage downstream of residues K252 and R322, respectively. For simplification, only a single chain of the GldM dimer is shown.*

586

**Figure 4. Architecture of the cytoplasmic complex.** **A**, Unrefined structure. **B**, The cytoplasmic complex boxed in (A) was locally refined. A central knobbed cylinder extends ~14 nm into the cytoplasm from the IM, and flanking densities extend ~6 nm. **C**, Bottom-up view of the region denoted in (**B**) showing 12-fold symmetry of the outer spoked-ring of ~32 nm and a continuous inner ring of ~20 nm. **D**, A central section of a global average structure of the T9SS visualized in a *ΔporL* mutant showing the presence of the PorKN ring and absence of the PorM periplasmic arches, cytoplasmic complex, SS, and EDSL. **E** and **F**, 3D rendering of the cytoplasmic complex shown in side and bottom-up views.

587

588

589

590

591

592

593

594

595

596

**Figure 5. Organization of secretion pore (Sov) at the OM.** **A** and **B**, Central-section and top-down views of the locally refined surface structure showing 8 distinct densities arranged in a ring of 48 nm in
diameter. C, A central section through longitudinal plane of a global average structure of a Δ*sov* mutant
machine and 3D surface rendering (D) showing that the surface densities are absent. E and F, 3D surface
rendering of the putative translocons shown in different views. Arrows in (F) denote lateral pores. G and
H, Eight SprA molecules (PDB entry 6H3J) docked into the surface densities. Arrows denote lateral pores
facing the center of the T9SS complex. I, A side view of the entire T9SS machine showing
cytoplasmic, periplasmic, and surface substructures. J and K, Schematic of the *P. gingivalis* T9SS multi-
port export apparatus, with major machine subunits and postulated routes of substrate translocation
shown. Substrates cross the IM through the Sec system, are recruited by the PorLMNK translocation
motor, enter a gap in the inner leaflet of OM and then the Sov translocon, and exit Sov through lateral
pores for release to the milieu or docking onto the cell surface via the ‘attachment complex’. The
proposed stoichiometry of the major machine subunits is shown.
<figure><img src="image_2.png" /></figure>

<figure><img src="image_3.png" /></figure>

<figure><img src="image_4.png" /></figure>

<figure><img src="image_5.png" /></figure>

<figure><img src="image_6.png" /></figure>

<figure><img src="image_7.png" /></figure>

<figure><img src="image_8.png" /></figure>

<figure><img src="image_9.png" /></figure>

<figure><img src="image_10.png" /></figure>

<figure><img src="image_11.png" /></figure>

<figure><img src="image_12.png" /></figure>

<figure><img src="image_13.png" /></figure>

<figure><img src="image_14.png" /></figure>

<figure><img src="image_15.png" /></figure>

<figure><img src="image_16.png" /></figure>
<figure><img src="image_17.png" /></figure>

<figure><img src="image_18.png" /></figure>

<figure><img src="image_19.png" /></figure>

<figure><img src="image_20.png" /></figure>

<figure><img src="image_21.png" /></figure>

<figure><img src="image_22.png" /></figure>
<figure><img src="image_23.png" /></figure>

<figure><img src="image_24.png" /></figure>

<figure><img src="image_25.png" /></figure>

<figure><img src="image_26.png" /></figure>

<figure><img src="image_27.png" /></figure>

<figure><img src="image_28.png" /></figure>
<figure><img src="image_29.png" /></figure>

A

WT

OM

IM

<figure><img src="image_30.png" /></figure>

B

IM

<figure><img src="image_31.png" /></figure>

<figure><img src="image_32.png" /></figure>

<figure><img src="image_33.png" /></figure>

C

12-fold symmetry

<figure><img src="image_34.png" /></figure>

32 nm

<figure><img src="image_35.png" /></figure>

<figure><img src="image_36.png" /></figure>

D

ΔporL

OM

IM

<figure><img src="image_37.png" /></figure>

E

IM

<figure><img src="image_38.png" /></figure>

<figure><img src="image_39.png" /></figure>

F

IM

10 nm
<figure><img src="image_40.png" /></figure>

<figure><img src="image_41.png" /></figure>

<figure><img src="image_42.png" /></figure>

<figure><img src="image_43.png" /></figure>

<figure><img src="image_44.png" /></figure>

<figure><img src="image_45.png" /></figure>

<figure><img src="image_46.png" /></figure>

<figure><img src="image_47.png" /><figcaption>I</figcaption></figure>

<figure><img src="image_48.png" /><figcaption>J</figcaption></figure>

<figure><img src="image_49.png" /></figure>

(PorL₅)₁₈(PorM₂/PorK₂/PorN₂)₁₈(Sov)₈

<figure><img src="image_50.png" /><figcaption>K</figcaption></figure>