A novel stabilization mechanism for the type VI secretion system sheath

Significance The T6SS is a microscopic harpoon that bacteria use to deliver toxins into neighboring cells. While its complex assembly process has been extensively studied, it remains unclear how the two forms (long and short) of the pivotal TssA protein affect T6SS function. TssA promotes baseplate formation, orchestrates sheath extension and, in its long form, interacts with a partner protein to anchor the extending sheath at the opposing side of the cell for up to 10 min. Here we demonstrate that short TssA proteins assist sheath stabilization by associating with a yet undescribed class of T6SS proteins that accumulate at the baseplate. These T6SSs fire in seconds; therefore, this discovery provides insight into the mechanism underpinning the different fighting strategies observed across T6SS-carrying bacteria.

DNA sequenced and subcloned into the XbaI/BamHI sites of pKNG101 (primers P19-P22 (for tagB1) and P98-P101 (for hcp1)). The suicide vector pKNG101 (4) does not replicate in Pseudomonas; it was maintained in E. coli CC118λpir and mobilized into Pseudomonas by triparental conjugation. The same method was employed to re-introduce tagB1 in its native locus in P. putida tagB1, in order to complement the mutant strain. In particular, a DNA fragment containing tagB1, flanked by 500 bp upstream and downstream, was amplified using P. putida KT2440 genomic DNA and primers P19 and P22. This was then cloned into pCR-BluntII-TOPO (Invitrogen), DNA sequenced, subcloned into the XbaI/BamHI sites of pKNG101, and mobilized into P. putida tagB1 by triparental conjugation A similar approach was used to replace wild-type P. putida tssA1, tssB1 and tagB1 as well as P. aeruginosa tssB1, tssB2, tssB3 and tagJ1 with genes encoding the protein of interest C-or N-terminally fused to a sfGFP or mScarlet-I fluorophore (all fusions carry the fluorophore at the C-terminus of the protein with the exception of the P. putida TssA1 fusion where the sfGFP is at the N-terminus of the protein); primers P23-P43, P46-P63 and P88-P93 were used for engineering these substitutions. The same strategy was used to replace the wild-type P. putida tagB1 gene with a version encoding TagB1 C-terminally fused to two consecutive StrepII tags (primers P94-P97). Finally, the tssB1-mScarlet-I gene (amplified with primer pair P44/P45), encoding a C-terminal fusion of m-Scarlet-I to P. putida TssB1, was introduced on the chromosome using the miniCTX vector (5). All insertions and gene replacements were confirmed by PCR and DNA sequencing.
Protein identification by mass spectrometry. Bacterial cultures of P. putida rpoN and of a P. putida rpoN strain where tagB1 was replaced with a version of the gene encoding TagB1 Cterminally fused to two consecutive StrepII tags, were grown in TSB supplemented with the appropriate antibiotics for at least 8 hours at 30 ºC with shaking at 200 RPM. Bacterial suspensions were then sub-cultured at an OD600 of 0.1 into 50 ml TSB and incubated for an additional 8 hours under the same growth conditions. Cells were harvested and cell pellets were resuspended in 50 mM Tris-HCl (pH 7.5), 150 mM NaCl and lysed by sonication after the addition of protease inhibitors (Roche). Cell debris was eliminated by centrifugation (48,000 x g, 30 mins, 4 °C) and both suspensions were subjected to protein purification using Strep-Tactin Sepharose (Iba Lifesciences), according to the manufacturer's specifications. The resulting protein mixtures were methanol precipitated and freeze dried; three samples were prepared for each purification condition.
Freeze-dried protein mixtures were resuspended to a final concentration of 1 μg/mL in 25 μL of 20 mM ammonium bicarbonate before overnight digestion with trypsin at 30 °C (ratio 20:1 protein mixture:trypsin). Direct analysis of peptide mixtures was carried out under trap and elute conditions using an Acclaim Pepmap 100 Nano-Trap and column (Thermo Fischer Scientific) on an Eksigent nanoLC Ultra 2D HPLC coupled to a Triple-TOF 5600+ mass spectrometer via a Nanospray III source (all AB Sciex). The peptides were loaded in 98% v/v water, 2% v/v acetonitrile, 0.05% v/v trifluoroacetic acid and washed on the trap for 20 minutes, before being injected onto the column and eluted over 100 minutes with a rising gradient of acetonitrile composed of solutions A and B (solution A = 98% v/v water, 2% v/v acetonitrile, 0.1% v/v formic acid; solution B = 98% v/v acetonitrile, 2% v/v water, 0.1% v/v formic acid) as follows: 0 minutes, 98%A:2%B; 60 minutes, 80%A:20%B; 75 minutes, 60%A:40%B; 80 minutes, 2%A:98%B, 87.5 minutes, 98%A:2%B, 100 minutes, 98%A:2%B. Mass spectra were acquired between 400-1250 m/z and the top 20 peptides with a charge between +2 and +5 were selected to proceed to MS/MS (95-1800 m/z). All mass spectrometry was performed at the BBSRC Mass Spectrometry and Proteomics Facility at the University of St. Andrews, UK.
MaxQuant analysis. Data were processed using MaxQuant version 1.5.8.3 (6). Peptides were identified from MS/MS spectra searched against the Uniprot P. putida ATCC47054/DSM6125/NCIMB11950/KT2440 reference proteome (proteome ID: UP000000556) (accessed November 2019) using the Andromeda search engine (7). Carbamidomethylation, methionine oxidation and N-terminal acetylation were specified as variable modifications. In silico digest of the reference proteome was performed using the Trypsin/P setting with up to two missed cleavages allowed. The false discovery rate (FDR) was set at 0.01 for peptides, proteins and sites. The "re-quantify" function was enabled. The sequence decoy mode used was "revert". Protein quantification was performed using the MaxLFQ algorithm within MaxQuant (8). Unique and razor peptides were used for quantification. All other parameters were used as pre-set in MaxQuant.
Perseus analysis. Data were analyzed using Perseus version 1.5.8.5 (9). Proteins present in the "reverse", "only identified by site" and "potential contaminant" databases were removed, and proteins identified by one or more unique peptides retained for further analysis. LFQ intensities were logarithmized (log2) and replicates grouped together before the data was filtered to retain only proteins identified in two or more replicates from at least one sample. Missing log2 LFQ intensities were inferred using a downshifted normal distribution (1.8 downshift, 0.3 width) and proteins that were significantly enriched by TagB1-(StrepII)2 identified using a two-sample t-test (permutation based false discovery rate (FDR) = 250, FDR = 0.05, S0 = 0.2). Dot blot assays. P. putida TagB1 and P. aeruginosa TagJ1 self-interaction, as well as interaction of these proteins with their cognate TssA1 proteins were assessed using dot blots as follows: E. coli BL21(DE3) cells expressing TagB1-StrepII, TagJ1-StrepII or a leaderless version of the E. coli cytochrome c maturation protein CcmG fused to a C-terminal StrepII tag (binding control protein) were grown in LB (10 g/L NaCl) supplemented with the appropriate antibiotics at 37 °C with shaking at 200 RPM to an OD600 of 0.8. Expression was subsequently induced using IPTG for 16 hours at 30 °C. Cells were harvested and cell pellets were resuspended in 50 mM Tris-HCl (pH 7.5), 150 mM NaCl and lysed by sonication after the addition of protease inhibitors (Roche). Cell debris were eliminated by centrifugation (48,000 x g, 30 mins, 4 °C) and proteins were purified using Strep-Tactin Sepharose (Iba Lifesciences) according to the manufacturer's specifications. 3.5 ng of pure TagB1-StrepII, TagJ1-StrepII and CcmG-StrepII were spotted onto Amersham Protran nitrocellulose membranes (0.45 µm pore size, GE Life Sciences) and spots were dried at room temperature. Membranes were blocked overnight at 4 °C in 3% w/v Bovine Serum Albumin (BSA)/TBS-T (0.1% v/v Tween 20). The following day, 150 OD600 units of E. coli BL21 (DE3) containing the empty vector (pETDuet-1) or over-expressing TagB1-V5, TagJ1-V5, P. putida V5-TssA1 or P. aeruginosa V5-TssA1 were resuspended in 10 mL of binding buffer (20 mM Tris-HCl (pH 8.0), 10% v/v glycerol, 100 mM NaCl, 3% w/v BSA) and lysed by sonication. Crude lysates, or 10 mL of binding buffer alone, were applied directly to the blocked nitrocellulose membranes before overnight incubation at 4 °C. Membranes were washed three times for 10 minutes with TBS-T and probed with mouse anti-V5 (Invitrogen) (dilution 1:5,000 in 3 w/v % BSA/TBS-T) or Strep-Tactin-HRP conjugate (Iba Lifesciences) (dilution 1:3,000 in 3 w/v % BSA/TBS-T) at room temperature for 4 hours. Goat anti-mouse IgG-HRP conjugate (Sigma Aldrich) (dilution 1:6,000 in 3% w/v BSA/TBS-T) was applied for 1 hour at room temperature, as appropriate. Membranes were washed three times for 10 minutes with TBS-T prior to development with Immobilon Classico Western HRP substrate (Merck Millipore) using a Gel DOC XR+ Imager (Bio-Rad). The experiments generating the data presented in Fig. 1C and S10A were performed simultaneously and development of the membranes was carried out on the same day for all eight membranes shown. The presented membranes are uncut hence the same exposure time was applied for each of the spotted proteins on each membrane. For membranes incubated with empty vector lysates, the longest of the exposure times was applied during development to ensure that signal detection is maximized.
Interbacterial competition assays. In vitro competition assays were performed on LB (10 g/L NaCl) (E. coli prey) or LB (5 g/L NaCl) (plant pathogens as prey) agar (1.5% w/v) plates, as previously described (10). Briefly, overnight bacterial cultures were washed and adjusted to an OD600 of 10 in sterile PBS, and mixed at a 1:1 ratio (P. putida:prey). Mixtures were grown on LB agar plates at 30 °C for 5 hours (E. coli prey) or 24 hours (plant pathogens as prey) and then collected using an inoculating loop and resuspended in sterile PBS. The outcome of the competition was quantified by counting colony forming units (CFUs) using antibiotic selection of the input (time = 0 hours) and output (time = 5 hours or time = 24 hours). All prey strains harboured the plasmid pRL662, which confers resistance to gentamicin and was used for antibiotic selection, whereas P. putida KT2440R is naturally resistant to rifampicin. In planta competition assays were carried out by infiltration of bacteria into Nicotiana benthamiana leaves, as described before (11). Briefly, overnight cultures of P. putida and X. campestris were adjusted to OD600 of 0.1 in PBS and mixed at a 1:1 ratio. Approximately 100 µl of each suspension were infiltrated on the reverse of an approximately one-month-old leaf and the infiltration area was marked. After 24 hours of incubation in a plant chamber (23°C, 16 hours light exposure), a circular section of 6 mm diameter from the infiltration area of the leaf was isolated using a cork-borer set (Sigma-Aldrich), homogenized in PBS using a motorized tissue grinder (FisherBrand), serially diluted and platted on LB agar supplemented with the appropriate antibiotics. CFUs were determined using antibiotic selection as described above. For all competition assays, at least three biologically independent experiments were performed. Competitive index values were calculated using the following formula: competitive index = (output attacker/output prey)/(input attacker/input prey). P. putida Hcp1 antibody production: An overnight TSB culture of E. coli BL21(DE3) cells harboring a pET28a plasmid encoding P. putida Hcp1 with an N-terminal His6 tag was used to inoculate 1L of TSB supplemented with kanamycin at an OD600 of 0.1. The culture was incubated at 37 ºC with shaking at 200 RPM and protein expression was induced with 0.5 mM IPTG at an OD600 of 0.5. Cultures were grown overnight at 18 ºC with shaking at 200 RPM. Cells were harvested and the cell pellet resuspended in 50 mM Tris-HCl (pH 7.5), 250 mM NaCl, 20 mM imidazole (buffer A) supplemented with protease inhibitors (Roche). The cells were lysed by sonication and cell debris were eliminated by centrifugation (48,000 x g, 30 mins, 4 °C). His6-Hcp1 was purified by immobilized metal affinity chromatography using nickel-Sepharose resin (GE Healthcare) equilibrated in buffer A and eluted off the resin with buffer A containing 500 mM instead of 20 mM imidazole. The protein was subsequently subjected to gel filtration chromatography in 50 mM Tris-HCl (pH 7.5), 250mM NaCl using a prepacked Superdex 75 10/300 GL size-exclusion column (GE Healthcare). Polyclonal antibody production was carried out by Eurogentec through their speedy 28-Day program (2 rabbits; 4 protein injections and 3 bleedings per rabbit).
Secretion assays. P. putida strains were grown in TSB for 8 hours at 30° C with shaking at 200 RPM and the extracellular fraction was obtained and analyzed as previously described (12). Briefly, the cell suspensions were spun three times at 10,000 x g for 20 minutes.
Bacterial pellets were normalized and added directly to 1x Laemmli buffer, whilst the culture supernatants were collected and precipitated with trichloroacetic (TCA) acid overnight. Precipitants were then washed with acetone and resuspended in 1x Laemmli buffer. All samples were boiled for 15 minutes. SDS-PAGE analysis was carried out using 8% or 15% BisTris NuPAGE gels (ThermoFisher Scientific), MES/SDS running buffer prepared according to the manufacturer's instructions and pre-stained protein markers (Kaleidoscope Prestained Standard, Bio-Rad). Proteins were transferred to Amersham Protran nitrocellulose membranes (0.45 µm pore size, GE Life Sciences) using a Trans-Blot Turbo transfer system (Bio-Rad) before blocking in 5% w/v skimmed milk/TBS-T and adding the primary and secondary antibodies. The following primary antibodies were used: rabbit anti-Hcp1 antibody (dilution 1:500 in 5 w/v % skimmed milk/TBS-T) and mouse E. coli anti-RNA polymerase beta (Neoclone) (dilution 1:5,000 in 5 w/v % skimmed milk/TBS-T). The following secondary antibodies were used: goat anti-rabbit IgG-HRP conjugate (Sigma Aldrich) (dilution 1:6,000 in 5% w/v skimmed milk/TBS-T) and goat anti-mouse IgG-HRP conjugate (Sigma Aldrich) (dilution 1:6,000 in 5% w/v skimmed milk/TBS-T). Membranes were washed three times for 5 minutes with TBS-T prior to development. HRP conjugates were visualized with the Luminata Forte Western HRP Substrate (Merck) using a Gel Doc XR+ Imager (Bio-Rad).
Bacterial two-hybrid assay. Protein-protein interactions were analyzed using the bacterialtwo-hybrid (BTH) approach (13). Briefly, the proteins to be tested were independently fused to the T18 or T25 catalytic domains of the B. pertussis adenylate cyclase using plasmids pUT18 (or pUT18C) and pKT25, respectively. The two plasmids expressing the fusion proteins were simultaneously introduced into the reporter strain BTH101 by transformation and the plates were incubated at 30 °C for 24 hours. Three independent colonies for each plasmid combination were inoculated into 1 ml of LB (10 g/L NaCl) supplemented with ampicillin, kanamycin and 0.5 mM IPTG. After overnight growth at 30 °C with shaking at 200 RPM, 10 μl of each culture were spotted onto LB (10 g/L NaCl) agar (1.5% w/v) plates supplemented with ampicillin, kanamycin, 0.5 mM IPTG and 40 μg/ml X-Gal and incubated at 30 °C for 16 hours. At least three biologically independent experiments were performed for each protein pair.

Fluorescence microscopy.
Fluorescence microscopy experiments were performed using sfGFP (14) and mScarlet-I (15) fusion proteins. The functionality of the T6SS in P. putida rpoN strains expressing fluorescent fusions of TssA1 and TagB1 from the native gene loci, was confirmed by performing secretion assays with the strains P. putida rpoN TssA1-sfGFP and P. putida rpoN TagB1-sfGFP, as described above (Fig. S14). Bacterial cultures intended for microscopy were grown in TSB supplemented with the appropriated antibiotics for at least 8 hours at 30ºC (P. putida) or 37 ºC (P. aeruginosa) with shaking at 200 RPM. Bacterial suspensions were then sub-cultured at an OD600 of 0.1 into 50 ml TSB and grown under the same conditions to an OD600 of 5 before visualization; P. aeruginosa PAO1 rsmA tssB3::sfGfp was grown at 25 °C with shaking at 200 RPM for 12 hours before visualization.
To visualize cells, 5 cm diameter glass bottom Petri dishes with a 3-cm diameter uncoated n° 1.5 glass window (MatTek Corporation) were used. 6 µl of bacterial culture were placed onto a 2.8 cm diameter (0.5 cm thickness) circular slab of Vogel-Bonner salts agar (2% w/v) which was, in turn, placed face down onto the glass bottom window of the Petri dish, so that the cells were sandwiched between the glass and the solid medium. Samples were immediately transferred to the microscope and imaged using an Axio Observer Z1 (Zeiss) inverted widefield microscope equipped with a Plan-Apochromat 63x/1.4 NA Oil Ph3 M27 objective (Zeiss), a SpectraX LED light engine (Lumencore), an ORCA-Flash 4.0 digital CMOS camera (Hamamatsu) and an environmental control system. Images were acquired using 1x1 binning, corresponding to a pixel size of 0.103 x 0.103 μm. All microscopy was performed at the Facility for Imaging by Light Microscopy (FILM) at Imperial College London, UK. For the visualization of P. putida strains expressing TagB1-sfGFP or TssB1-sfGFP, fluorescence images were acquired every 2 s for a total of 180 s using an exposure time of 195 ms. For P. putida simultaneously expressing TssB1-mScarlet-I and sfGFP-TssA1, fluorescence images were acquired every 4 s for a total of 180 s using an exposure time of 500 ms for sfGFP and 250 ms for mScarlet-I. For P. putida simultaneously expressing TssB1-mScarlet-I and TagB1-sfGFP, fluorescence images were acquired every 2 s for a total of 180 s using an exposure time of 200 ms for sfGFP and 350 ms for mScarlet-I. For the visualization of P. aeruginosa expressing TssB1-sfGFP, fluorescence images were acquired every 2 s for a total of 180 s using an exposure time of 80 ms. For P. aeruginosa expressing TssB2-sfGFP, fluorescence images were acquired every 20 s for a total of 1200 s using an exposure time of 80 ms; the microscope chamber was set to 30 °C and the Definite Focus autofocus system was used. For P. aeruginosa expressing TssB3-sfGFP, fluorescence images were acquired every 30 s for a total of 450 s using an exposure time of 80 ms. Finally, for P. aeruginosa expressing TagJ1-sfGFP, fluorescence images were acquired every 60 s for 300 s using an exposure time of 400 ms.
Image analysis and presentation. Image processing and analysis was performed using FIJI (16) in conjunction with the MicrobeJ (17) plugin. Generation of images was performed using the ZEN Blue software (Zeiss) and FIJI. Prior to analysis, x-y drift present in timelapse series was corrected for each channel using the StackReg plug-in for FIJI and the "rigid body" transformation setting (18). Photobleaching in time-lapse series was corrected using the "simple ratio" bleach correction method within FIJI. Semi-automatic particle detection, segmentation and generation of cell masks were performed on phase-contrast images using the "medial axis" cell contour method within MicrobeJ; all cell masks were manually checked before further analysis. Cell counts and cell width data were directly extracted from manually curated cell masks generated using this method. Detection of fluorescent P. putida TagB1 foci was performed on fluorescence images using the local maxima detection algorithm implemented in FIJI, in conjunction with the "foci" particle conversion method implemented in MicrobeJ. Changes in TagB1 foci intensity over time were quantified from time-lapse series by determining the mean pixel intensity values of regions of interest (ROIs) using the "Plot Z-axis profile" tool within FIJI. Data are presented normalized to the average fluorescence intensity of the cytosol of three cells (from the same time-lapse series) in which no TagB1 foci were detected during the course of imaging. Enumeration, localization and sheath length analysis of TssB1-sfGFP sheaths was performed manually for all visualized strains. Sheath length measurements were performed on time-lapse series acquired using P. putida strains expressing TssB1-sfGFP; measurements were performed on the frame directly prior to that in which sheath contraction was observed, using a custom line/ROI measurement macro in FIJI. Time to sheath contraction data were extracted manually from time-lapse series covering full sheath polymerization and contraction events. For image presentation, frames of interest were extracted from time-lapse series, corrected as described above, and scale bars created using the Scale Bar function in FIJI. Videos were prepared from the same corrected images and encoded using QuickTime (H.264 codec) (Apple Inc.). All videos are presented at 7 frames/s as .mov files.

In silico analyses.
In order to categorize TssA-like proteins and identify TssA-related accessory components, an in silico study of 100 T6SS clusters was performed. Proteins encoding the core components and accessory proteins of the selected T6SS clusters were downloaded from the SecReT6 website (19) and organized by phylogenetic group (groups 1-5, with phylogenetic subgroups 4A and 4B being considered separately) (20); 20 T6SS clusters per phylogenetic group were examined. The T6SS cluster that is best characterized from each phylogenetic group was set as the reference cluster for the analysis (distinct clusters from P. aeruginosa, E. coli, P. putida and A. tumefaciens were selected as references). The reference clusters were used as queries in a blastp search (21) against the remaining selected T6SS clusters from the same phylogenetic group. The percentage identity and coverage resulting from the search can be found in Dataset S1. Through this analysis, five families of TssA-like proteins were identified, and their domains were further characterized using the "NCBI Conserved Domain" function and the SMART website (22).
Phylogenetic analyses for TssA and Hcp proteins were performed as previously described (23) using MEGA7 (24). Briefly, for each protein, a sequence alignment was carried out using the ClustalW algorithm, and subsequently a maximum-likelihood tree with 500 bootstrap replicates was built. The online Interactive Tree Of Life (iTOL) tool (25) was used for display and annotation of the resulting phylogenetic trees. Phylogenetic analysis of TssA was performed using all sequences of short TssA proteins listed in Dataset S1, whilst for Hcp the six and five annotated Hcp proteins from P. putida and P. aeruginosa, respectively were used.
Data availability. All data generated during this study that support the findings are included in the manuscript or in the SI Appendix. Fig. S1. Schematic representation of the genomic organization of the structural genes of the K1-T6SS cluster from P. putida KT2440. Genes encoding membrane complex proteins are shown in grey, whereas baseplate and tail components are depicted in purple and blue, respectively. tssA1 is in red, tagB1 in cyan and tagF1 and clpV1 are colored black. vgrG1 is not shown, as it is located downstream in the effector module (26), which is omitted from this schematic.  Maximum-likelihood tree for TssAS proteins built with MEGA7 and displayed using iTOL. TssAS proteins cluster in the same phylogenetic groups as the corresponding TssB proteins, which are commonly used when T6SS phylogeny is examined (20). Phylogenetic clustering is based on the analysis of Boyer et al. (20). The presence of tagB or tagJ in the corresponding T6SS cluster is indicated by a circle.   are marked with a black or red outline, respectively. The presented panels are selected images from two fluorescence microscopy time-lapse recordings of P. putida rpoN expressing TssB1-sfGFP from the native tssB1 locus. Images were recorded every 2 s and scale bars represent 1 μm. An image from example 2 (top row; red arrowhead) was used to showcase the P. putida rpoN tagB1 TssB1-sfGFP behavior in Fig. 2F.   S8. P. putida TagB1-sfGFP foci re-appear in the same location after a contraction event. A TagB1-sfGFP focus (white arrowhead) disappears, presumably after sheath contraction, and a new focus appears (red arrowhead) in exactly the same location. The presented panels are selected images from a fluorescence microscopy time-lapse recording of P. putida rpoN expressing TagB1-sfGFP from the native tagB1 locus. Images were recorded every 2 s and scale bars represent 1 μm.       Table S1. Bacterial strains used in this study.

Name Description Source Escherichia coli
DH5α F -endA1 glnV44 thi-1 recA1 relA1 gyrA96 deoR nupG purB20 φ80dlacZ∆M15 ∆(lacZYA-argF)U169 hsdR17(rK -mK + ) λ - (29) CC118λpir araD Δ(ara, leu) ΔlacZ74 phoA20 galK thi-1 rspE rpoB argE recA1 λpir   Table S3. Oligonucleotide primers used in this study. The "Brief description" column provides basic information on the primer design (restriction enzyme(s) used for cloning, encoded protein or gene replaced by antibiotic resistance cassette, forward or reverse orientation of the primer (F or R)). Primers marked with TE (for "tag exchange") were used for replacing an existing affinity tag with an alternative one, primers marked with UP, DOWN or MED were used for construction of mutant strains, primers marked with T18 or T25 were used for construction of plasmids used in bacterial-two-hybrid experiments, and primers marked with CTX were used for the construction of miniCTX vector derivatives for insertion of genes into the Pseudomonas genome. Superscripts "PP" and "PA" indicate P. putida and P. aeruginosa components (genes or proteins), respectively; these abbreviations are solely used in this

LEGENDS FOR SUPPLEMENTARY INFORMATION VIDEOS
Video S1. In vivo imaging of P. putida rpoN TssB1-sfGFP. The full cycle of T6SS assembly over approximately 120 seconds is shown for a representative sheath. This video was generated using a fluorescence microscopy time-lapse recording (total time 140 seconds) of P. putida rpoN expressing TssB1-sfGFP from the native tssB1 locus. Images were acquired every 2 s and the video is presented at 7 frames/s. Video S2. In vivo imaging of P. putida rpoN TagB1-sfGFP. TagB1 forms transient foci which accumulate and abruptly disappear over time. Disappearance of foci is not due to photobleaching as new TagB1-sfGFP foci appear in the same or neighboring cells. This video was generated using a fluorescence microscopy time-lapse recording (total time 120 seconds) of P. putida rpoN expressing TagB1-sfGFP from the native tagB1 locus. Images were acquired every 2 s and the video is presented at 7 frames/s.

LEGENDS FOR SUPPLEMENTARY INFORMATION DATASETS
Dataset S1. Overview of the results originating from an in silico analysis of the structural components of 100 T6SS clusters. The T6SS clusters included in the analysis are organized by phylogenetic group based on the classification by Boyer et al. (20) (groups 1-5 are shown in different tabs, with phylogenetic subgroups 4A and 4B shown separately). 20 T6SS clusters per phylogenetic group were analyzed and a hyperlink to the SecReT6 website (19) is given for each cluster; strains appearing in multiple phylogenetic groups have multiple T6SSs. The T6SS cluster that is best characterized in each phylogenetic group (highlighted in grey) was set as the reference cluster for the analysis (distinct clusters from P. aeruginosa, E. coli, P. putida and A. tumefaciens were selected as references). Cells containing identity and coverage metrics are color coded according to their bit score as provided by the NCBI blastp search tool (21). Bit score is an indicator of sequence similarity that is independent of query sequence length and database size and that is normalized based on the raw pairwise alignment score; red cells indicate the highest scores (score > 200), i.e. most similar alignments, followed by magenta (score = 80-200), green (score = 50-80), blue (score = 40-50) and black cells (score < 40). White cells indicate proteins that cannot be found in a given T6SS cluster. In some cases, more than one analogue of a specific T6SS core component is found in a given cluster; for example there are two TssC analogues in all T6SS clusters of phylogenetic group 5, several T6SS clusters have multiple VgrG or Hcp components, and in approximately 2% of cases there are two analogues of some components of the membrane complex or the baseplate. These additional components are not shown for simplicity, and the protein that is most similar to that of the reference genome is included in the spreadsheet.
Dataset S2. P. putida TagB1-(StrepII)2 specifically co-purifies with TssA1. Proteins eluted from Strep-Tactin Sepharose resin incubated with P. putida rpoN or P. putida rpoN tagB1-(StrepII)2 whole-cell lysates, as identified and quantified by mass spectrometry. The file contains the output of a volcano plot analysis, performed in Perseus version 1.5.8.5 (9); proteins identified in the TagB1-(StrepII)2 eluate were compared to those identified in the control eluate (untagged P. putida TagB1). Proteins with a positive "difference" value are enriched in the TagB1-(StrepII)2 condition compared to the control, whilst proteins with a negative "difference" value are enriched in the control compared to the TagB1-(StrepII)2 condition. Proteins with significantly different abundances in the two conditions are marked