Diverse roles of TssA‐like proteins in the assembly of bacterial type VI secretion systems
2019; Springer Nature; Volume: 38; Issue: 18 Linguagem: Inglês
10.15252/embj.2018100825
ISSN1460-2075
AutoresJohannes Schneider, Sergey Nazarov, Ricardo Adaixo, Martina Liuzzo, Peter David Ringel, Henning Stahlberg, Marek Basler,
Tópico(s)Bacterial Genetics and Biotechnology
ResumoArticle12 August 2019Open Access Transparent process Diverse roles of TssA-like proteins in the assembly of bacterial type VI secretion systems Johannes Paul Schneider Johannes Paul Schneider orcid.org/0000-0002-6028-9956 Biozentrum, University of Basel, Basel, Switzerland Search for more papers by this author Sergey Nazarov Sergey Nazarov orcid.org/0000-0002-5240-5849 Biozentrum, University of Basel, Basel, Switzerland Search for more papers by this author Ricardo Adaixo Ricardo Adaixo Center for Cellular Imaging and NanoAnalytics (C-CINA), Biozentrum, University of Basel, Basel, Switzerland Search for more papers by this author Martina Liuzzo Martina Liuzzo Biozentrum, University of Basel, Basel, Switzerland Search for more papers by this author Peter David Ringel Peter David Ringel Biozentrum, University of Basel, Basel, Switzerland Search for more papers by this author Henning Stahlberg Henning Stahlberg orcid.org/0000-0002-1185-4592 Center for Cellular Imaging and NanoAnalytics (C-CINA), Biozentrum, University of Basel, Basel, Switzerland Search for more papers by this author Marek Basler Corresponding Author Marek Basler [email protected] orcid.org/0000-0001-5414-2088 Biozentrum, University of Basel, Basel, Switzerland Search for more papers by this author Johannes Paul Schneider Johannes Paul Schneider orcid.org/0000-0002-6028-9956 Biozentrum, University of Basel, Basel, Switzerland Search for more papers by this author Sergey Nazarov Sergey Nazarov orcid.org/0000-0002-5240-5849 Biozentrum, University of Basel, Basel, Switzerland Search for more papers by this author Ricardo Adaixo Ricardo Adaixo Center for Cellular Imaging and NanoAnalytics (C-CINA), Biozentrum, University of Basel, Basel, Switzerland Search for more papers by this author Martina Liuzzo Martina Liuzzo Biozentrum, University of Basel, Basel, Switzerland Search for more papers by this author Peter David Ringel Peter David Ringel Biozentrum, University of Basel, Basel, Switzerland Search for more papers by this author Henning Stahlberg Henning Stahlberg orcid.org/0000-0002-1185-4592 Center for Cellular Imaging and NanoAnalytics (C-CINA), Biozentrum, University of Basel, Basel, Switzerland Search for more papers by this author Marek Basler Corresponding Author Marek Basler [email protected] orcid.org/0000-0001-5414-2088 Biozentrum, University of Basel, Basel, Switzerland Search for more papers by this author Author Information Johannes Paul Schneider1, Sergey Nazarov1,3, Ricardo Adaixo2, Martina Liuzzo1, Peter David Ringel1,4, Henning Stahlberg2 and Marek Basler *,1 1Biozentrum, University of Basel, Basel, Switzerland 2Center for Cellular Imaging and NanoAnalytics (C-CINA), Biozentrum, University of Basel, Basel, Switzerland 3Present address: Interdisciplinary Center for Electron Microscopy (CIME), EPFL, Lausanne, Switzerland 4Present address: Institute of Forensic Medicine, Justus-Liebig-University Giessen, Giessen, Germany *Corresponding author. Tel: +41 61 207 21 10; E-mail: [email protected] The EMBO Journal (2019)38:e100825https://doi.org/10.15252/embj.2018100825 PDFDownload PDF of article text and main figures. Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Abstract Protein translocation by the bacterial type VI secretion system (T6SS) is driven by a rapid contraction of a sheath assembled around a tube with associated effectors. Here, we show that TssA-like or TagA-like proteins with a conserved N-terminal domain and varying C-terminal domains can be grouped into at least three distinct classes based on their role in sheath assembly. The proteins of the first class increase speed and frequency of sheath assembly and form a stable dodecamer at the distal end of a polymerizing sheath. The proteins of the second class localize to the cell membrane and block sheath polymerization upon extension across the cell. This prevents excessive sheath polymerization and bending, which may result in sheath destabilization and detachment from its membrane anchor and thus result in failed secretion. The third class of these proteins localizes to the baseplate and is required for initiation of sheath assembly. Our work shows that while various proteins share a conserved N-terminal domain, their roles in T6SS biogenesis are fundamentally different. Synopsis Comparative analysis of the bacterial Type VI secretion system TssA/TagA subunits that harbor a conserved N-terminal domain and varying C-terminal domains reveals at least three distinct functional classes based on their role in sheath assembly. First class catalyzes sheath assembly and forms a stable dodecamer at the distal end of polymerizing sheath (T6SS sheath assembly chaperone – TsaC). Second class localizes at the sheath distal end upon extension across the cell and blocks excessive sheath polymerization (T6SS sheath membrane anchor - TsmA). Third class localizes to the baseplate and is required for the initiation of sheath formation (T6SS sheath assembly baseplate - TsaB). Introduction The type VI secretion system (T6SS) is a membrane-anchored contractile nanomachine used by many Gram-negative bacteria to deliver proteins from the cytosol directly to an extracellular space or across a target cell membrane. The nanomachine structurally and functionally resembles the contractile tail of bacteriophages and R-type pyocins (Brackmann et al, 2017). T6SS biogenesis proceeds in a strictly hierarchical order (Wang et al, 2019). First, a protein complex spanning both membranes forms (Aschtgen et al, 2010; Felisberto-Rodrigues et al, 2011; Durand et al, 2015). On this membrane complex, the baseplate forms and subsequently the sheath-tube complex polymerizes (Brunet et al, 2015; Nguyen et al, 2017; Wang et al, 2017; Nazarov et al, 2018). Once fully extended, the sheath rapidly contracts, propelling the inner tube and associated effector proteins out of the cell (Basler et al, 2012; Cianfanelli et al, 2016). The contracted sheath is then disassembled by an ATP-dependent unfoldase, and the components are recycled for another round of sheath extension and contraction (Bönemann et al, 2009; Basler & Mekalanos, 2012; Kapitein et al, 2013). Based on phylogenetic analyses, T6SSs have been classified into three different types (i, ii, and iii) and type i can be further split into subclasses i1, i2, i3, i4a, i4b, and i5 (Barret et al, 2013; Russell et al, 2014; Li et al, 2015). The current model of T6SSi includes a minimal set of 13 proteins to assemble a functional T6SS (Mougous et al, 2006; Pukatzki et al, 2006). TssJ, TssL, and TssM form the membrane complex (Brunet et al, 2015; Durand et al, 2015); VgrG, TssE, TssF, TssG, and TssK form the baseplate (Nguyen et al, 2017; Nazarov et al, 2018); and TssB/VipA, TssC/VipB, and Hcp form the long sheath-tube polymer (Wang et al, 2017; Szwedziak & Pilhofer, 2019). The AAA(+) ATPase ClpV disassembles contracted sheath, and its subunits can be reused to build another T6SS sheath (Bönemann et al, 2009; Basler & Mekalanos, 2012; Basler et al, 2012; Kapitein et al, 2013; Förster et al, 2014; Douzi et al, 2016). T6SSii is exclusively populated by the Francisella pathogenicity island and contains a set of 17 core components (Bröms et al, 2010; de Bruin et al, 2011), while the type iii system is found only in Bacteroidetes and contains 12 core components (De Maayer et al, 2011; Russell et al, 2014). Recently, a fourth type (T6SSiv) that is closely related to extracellular injection machineries such as R-type pyocins and antifeeding prophages has been described (Böck et al, 2017). This particular system does not contain a canonical transmembrane anchor and also lacks ClpV unfoldase (Böck et al, 2017). However, similarly to Francisella, contracted sheaths may be refolded by a related ATPase (Brodmann et al, 2017). TssA proteins have been initially shown to play an essential role in assembly of baseplate and the sheath-tube in Pseudomonas aeruginosa (TssA1PA) and Escherichia coli (TssAEC) (Planamente et al, 2016; Zoued et al, 2016). Interestingly, TssAs can be categorized into different classes harboring distinct protein domain architectures. The specific architecture presumably affects their function during biogenesis of the T6SS. All TssA-like proteins harbor a conserved ImpA_N domain (PF06812) located at the N-terminal end, while the C-terminal part differs in its composition. Recent analyses showed that domains can be further segregated into ImpA containing domain (Nt1), middle domain (Nt2), and C-terminal domain (CTD) (Dix et al, 2018). A high-resolution crystal structure of TssAEC C-terminus harboring a VasJ domain (PF16989) was recently obtained, and it was shown that this part of the protein forms two stacked hexameric rings (Zoued et al, 2016). Dynamic rearrangement of wedges connecting the six helices supposedly leads to a ~90 Å opening of the structure. TssAEC interacts with membrane complex and baseplate components, but also with sheath component TssC/VipB. Thus, it was proposed that TssAEC might coordinate sheath-tube assembly and guarantee its stability in the extended state (Zoued et al, 2016). However, another recent study showed that the C-terminal domain (CTD, G388-L472, helices α8–α12) of a closely related TssA from Aeromonas hydrophila forms a high-order oligomer with D5 symmetry (Dix et al, 2018). The CTD is connected to the middle N-terminal domain (Nt2, R232-L374, helices α1–α7) through a ~21 residues flexible linker. Neighboring Nt2 domains form dimers, which do not follow D5 symmetry of the CTD oligomer (Dix et al, 2018). TssA1PA was suggested to contain partial secondary structure homologies to the phage baseplate component gp6 in its C-terminal part (Planamente et al, 2016). It forms a dodecameric ring with dimensions that are similar to sheath-tube ring and has a central hole that could accommodate Hcp. TssA1PA was further shown to interact with baseplate components TssK, TssF, and VgrG1a, sheath-tube and ClpV, but in contrast to TssAEC not with components of the membrane complex. Due to these properties, it was proposed that TssA1PA might be a baseplate component (Planamente et al, 2016). Lastly, some TssA-like proteins harbor a transmembrane region and a C-terminal VasL domain of unknown function (PF12486). These TssAs were suggested to play an accessory role and corresponding genes thus referred to as tagA (type VI secretion accessory gene with ImpA domain) (Zoued et al, 2017). Recently, a TagA protein from E. coli (TagAEC) was shown to interact with TssAEC, localize at the distal end of sheath once it was fully extended and to stabilize the extended structure. Deletion of tagAEC caused excessive sheath polymerization, bending, and breaking of sheath structures and thus reduced efficiency of killing target cells (Santin et al, 2018). In addition, TagAEC was shown to be required for contraction of a part or the full-length sheath toward the distal end. While it is unclear if these non-canonical sheath contraction events result in protein secretion, these contractions constitute up to one-third of observed contractions in E. coli (Szwedziak & Pilhofer, 2019). Importantly, certain TssA proteins seem to be indispensable for proper T6SS assembly. Hcp secretion was not detectable in a ΔtssAEC strain, and no sheath structures were observed in a ΔtssAEC TssB-mCherry strain (Zoued et al, 2016). Similarly, secretion of Hcp, VgrG1a, and Tse3 was not detectable in a tssA1PA knockout strain and sheath formation in a TssB1-sfGFP ΔtssA1PA strain was severely decreased (Planamente et al, 2016). Here, we investigated the role of several distinct proteins sharing the ImpA_N domain and we show that their functions differ significantly. The Vibrio cholerae and P. aeruginosa TssA proteins TssAVC and TssA2PA with C-terminal VasJ domain (Class A) facilitate sheath assembly initiation and polymerization by forming a stable dodecamer at the end of the sheath that is distal from the membrane anchor. The TagA protein of V. cholerae (TagAVC) with the ImpA_N domain followed by a hydrophobic domain (Class B) localizes to cell membrane and prevents sheath assembly likely by competing with TssAVC. Finally, we show that a third class of ImpA_N domain containing proteins (Class C), represented by TssA1 in P. aeruginosa, localizes to the site of sheath assembly initiation. Our data show that ImpA_N domain containing proteins have diverse functions in the biogenesis of T6SS and that their role in sheath assembly is likely dictated by the structure and function of their C-terminal domains. Results TssAVC and TssA2PA facilitate sheath assembly initiation and polymerization Proteins that have ImpA_N domain followed by C-terminal VasJ domain form Class A of TssA-like proteins (Fig EV1A). This class is represented by E. coli TssAEC (EC042_4540) and A. hydrophila TssAAH (AHA1844) (Zoued et al, 2016; Dix et al, 2018), as well as V. cholerae TssAVC (VCA0119). TssAVC and TssAEC share 19.9% sequence identity, while TssAVC and TssAAH share 32.8% sequence identity (Appendix Table S1). To investigate the role of these proteins, we first imaged VipA-mCherry2 sheath assembly in V. cholerae in the presence or absence of tssAVC (Appendix Table S2). We found that the parental strain usually forms five T6SS structures at any given time during logarithmic growth (Fig 1A, Movie EV1). This is in agreement with number of sheaths detected in the strain expressing VipA-msfGFP (Vettiger & Basler, 2016). Image analysis showed that the tssAVC-negative strain formed mostly dynamic sheath spots and only few structures that fully extended and contracted (Fig 1A, Movie EV1). In a bacterial competition assay, tssAVC knockout strain was able to kill E. coli prey cells only at a reduced rate (Fig EV1B). This resembled the reduced prey cell killing by a strain lacking the baseplate component tssE, which forms about 1000 times less structures compared to the parental strain (Vettiger & Basler, 2016). In addition, speed of sheath polymerization dropped from 23 nm per second measured in the parental strain to 3 nm per second in the strain lacking tssAVC (Fig EV1C). Click here to expand this figure. Figure EV1. TssA protein domain organization and mutant phenotypes Protein domain organization and proposed subcellular localization of V. cholerae 2,740–80/O1, P. aeruginosa PAO1, E. coli O44:H18 (str. 042/EAEC), A. hydrophila ATCC7966, and B. cenocepacia H111 TssA proteins. TssA proteins display three specific protein domain architectures A, B1/B2, and C. Most common architecture of each class is shown. Bacterial competition assay comparing T6SS-dependent killing efficiency in different strain backgrounds. Fast killing kinetics via T6SS can be best observed by mixing strains in a ratio of 1:5 (V. cholerae : E. coli) (left panel), while less efficient or slow killing kinetics can be observed by mixing strains in a ratio of 2:1 (V. cholerae: E. coli) (right panel). ∆tssA mutant kills prey E. coli cells at a very slow rate, comparable to a ∆tssE mutant strain (right panel). ∆tagA mutant shows similar killing kinetics to WT (left and right panel). ∆tagA ∆tssE double mutant shows a shift in killing kinetics compared to ∆tagA mutant. T6SS sheath polymerization speed in WT and different mutant backgrounds. Polymerization speed in the ∆tagA mutant is reduced (13 nm/s) compared to WT speed (23 nm/s) and drastically reduced in the ∆tssA mutant (3 nm/s). Polymerization speed in the ∆tssE mutant is 48 nm/s and decreases to 38 nm/s in the ∆tagA ∆tssE double mutant. The strain harboring TssAVC-mNeonGreen fusion displays decreased polymerization speed (11 nm/s) compared to parental strain without a tag on TssA (23 nm/s). Quantification of structures per cell and percentage of dynamic sheath spots versus extending and contracting structures in H2-T6SS WT and ΔtssA2PA strain. ΔtssA2PA strain displays very few structures and mostly dynamic spots. Data information: In boxplots shown here, the central mark of each box indicates the median and the bottom and top edges of the box indicate the 25th and 75th percentiles, respectively. The whiskers extend to the most extreme data points not considered outliers, and the outliers are plotted individually using the "+" symbol labeled in red. Download figure Download PowerPoint Figure 1. TssAVC and TssA2PA influence T6SS sheath assembly dynamics Time lapse images of T6SS activity in tagged parental strain (VipA-mCherry2) and ∆tssA mutant background. The parental strain forms multiple dynamic structures per cell. ∆tssA mutant predominantly forms dynamic sheath spots (white arrow) and few WT-like sheath structures (blue arrow). Scale bars: 2 μm. H2-T6SS dynamics in tagged parental strain (∆retS TssB2-mCherry2, referred to as WT H2) and ΔtssA2PA strain. Blue arrow indicates extending and contracting T6SS sheath structures, white arrow points to dynamic sheath spots. The WT H2 strain harbors multiple dynamic sheath structures per cell, while the ΔtssA2PA strain displays very few dynamic spots or extended and contracting structures. Scale bars: 2 μm. Download figure Download PowerPoint Similarly, we analyzed a second member of Class A, the TssA2 from H2-T6SS of P. aeruginosa (PA1656) (Sana et al, 2012; Allsopp et al, 2017). TssA2PA shares 21,8% sequence identity with TssAVC and 25.2% sequence identity with TssAEC (Appendix Table S1). We show that multiple structures of TssB2-mCherry2 sheath reside in single cells (Fig 1B, Movie EV2). Dynamics of H2-T6SS sheaths are, however, significantly slower than V. cholerae sheaths. Full extension of one sheath can take up to 10 min, and sheath structures stay in extended state for at least 5 min (Fig 1B). Deletion of tssA2PA severely decreased number of T6SS sheaths and mostly dynamic spots were visible; however, few fully extending and contracting sheath structures could be observed (Figs 1B and EV1D, Movie EV2). TssAVC and TssA2PA localize to the distal end of an assembling sheath Previous work of Zoued et al demonstrated that another member of TssA Class A, TssAEC of E. coli, first localizes to membrane complex and then coordinates sheath-tube assembly at the distal end, presumably by incorporating new tube and sheath components (Zoued et al, 2016). Since TssA of V. cholerae is closely related to TssAEC (Appendix Table S1), we wondered if the two proteins share similar role in T6SS biogenesis. We fused mNeonGreen to TssAVC and observed its localization using fluorescence microscopy in a strain background with mCherry2-tagged sheath (VipA-mCherry2). While sheath assembly was about two times slower (Fig EV1C), the T6SS in TssAVC-mNeonGreen/VipA-mCherry2 remained fully functional (Fig EV1B). We found that, in most cases, TssAVC first localized to T6SS assembly initiation site before sheath signal appeared and then colocalized with a distal end of a polymerizing sheath (Fig 2A, Movie EV3). Figure 2. Localization and dynamics of TssAVC and TssA2PA A. Time lapse images of T6SS activity in tagged strain (VipA-mCherry2 TssAVC-mNeonGreen). White arrow indicates path of TssAVC localization during one cycle of T6SS dynamics. Scale bar: 1 μm. B–E. Kymographs of dynamics observed for TssAVC. Frequency of specific dynamics is indicated in the upper right corner of the merge image. (B) Sheath and TssAVC dynamics from A. TssAVC localizes to T6SS initiation site and to distal end of growing sheath (white arrow). (C) Once sheath reaches cell periphery, TssAVC dissociates from distal end (white arrow) and sheath stays extended a prolonged period of time. (D) TssAVC dissociates from growing sheath structure and re-associates at a later time point (white arrows) to continue catalysis of polymerization. (E) TssAVC stays attached to sheath distal end after contraction (white arrow). F. Fluorescence microscopy of T6SS dynamics in a double-tagged strain (∆retS TssB2-mCherry2 TssA2PA-mNeonGreen). White arrow indicates full cycle of extending and contracting T6SS sheath. Scale bar: 1 μm. G–J. Kymographs of TssB2 and TssA2PA dynamics. TssA2PA localizes at sheath initiation site and on distal end during polymerization (G), dissociates before contraction (H), dissociates and re-associated during sheath extension (I), or stays attached to distal end after contraction (J). Download figure Download PowerPoint For analysis of sheath dynamics in time lapse movies, we generally used kymographs generated with Fiji (Schindelin et al, 2012). A straight line was drawn along an assembling sheath, and the signals of the underlying pixels were replotted in a new XY coordinate system where the pixels along the Y-axis represent the pixels along the line drawn over an assembling sheath and the individual time points are shown along the X-axis. Such representation allows simple visualization of sheath assembly, measurement of sheath length, and speed of assembly as well as detection of colocalization of two proteins. Kymographs of TssAVC-mNeonGreen and VipA-mCherry2 movies of 300 cells revealed that in about two-thirds of the analyzed cases TssAVC stayed attached to assembling sheaths until their contraction shortly (< 5 s) after full assembly (Fig 2B). In about 27% of the cases, TssAVC dissociated from the sheath after its extension to the opposite side of the cell, which was followed by prolonged period of stable extended sheath (Fig 2C). However, in few cases (about 3%) TssA also dissociated during sheath assembly causing a delay or stalling of the sheath polymerization while TssA re-association resumed polymerization (Fig 2D). In about 3% of the cases, TssAVC stayed attached to the sheath distal end even after its contraction (Fig 2E). To test whether TssA forms a stable complex on the sheath end, we photobleached cytosolic TssAVC-mNeonGreen subunits during sheath polymerization. This was achieved by incubating the cells in the presence of ampicillin, which leads to formation of large viable spheroplasts with functional T6SS (Vettiger et al, 2017). Large cells allow controlled photobleaching of a relatively small section of the cell. We found that TssAVC-mNeonGreen signal of the complex at the distal end of the polymerizing sheath remained constant after photobleaching of the cytosol, suggesting that the TssAVC subunits are not exchanged with the cytosolic TssAVC pool during sheath polymerization (Appendix Fig S1, Movie EV4). Since TssA2PA is in the same TssA class as TssAVC and TssAEC (Class A), we hypothesized that all TssA proteins from this class might play the same role in T6SS biogenesis. Consequently, we used fluorescence microscopy to observe TssA2PA-mNeonGreen dynamics in TssB2-mCherry2 strain (Fig 2F–J, Movie EV5). We found that TssA2PA displays almost identical dynamics to TssAVC. TssA2PA localized to T6SS assembly initiation sites before sheath polymerization and then localized to the distal end of the polymerizing sheath (Fig 2G). Further, we often detected TssA2PA dissociating from sheath distal end after full extension (Fig 2H) or during extension followed by its re-association to the polymerizing sheath (Fig 2I), but also residing on contracted sheath structures (Fig 2J). This suggests that all TssAs within the Class A play the same role in T6SS assembly. TssAVC forms a dodecamer in vivo Recent in vitro studies have shown that TssAs from E. coli, A. hydrophila, and Burkholderia cenocepacia form rings with 6-fold, 5-fold, and 16-fold symmetry (Zoued et al, 2016; Dix et al, 2018). We aimed to estimate the oligomeric state of the TssAVC complex in vivo. We used the LacI–lacO system where two LacI repressor molecules, lacking tetramerization domain, bind to one lacO operator sequence (Belmont & Straight, 1998; Dong et al, 1999). First, we generated strains harboring 3, 6, or 12 copies of the lacO integrated into V. cholerae chromosome at the site of the disrupted lacZ gene (Fig EV2A). Expressing the lacI repressor fused to mNeonGreen (LacI-mNeonGreen) in these strains yielded fluorescent LacI-mNeonGreen spots with 6, 12, or 24 copies of mNeonGreen, respectively (Fig EV2B). The expression of LacI-mNeonGreen in the parental strain lacking lacO sequences yielded no detectable foci (Fig EV2B). Similarly, no foci were detected when LacI-mNeonGreen was expressed in a strain harboring 12 copies of lacO but supplemented with IPTG to disrupt binding of LacI to lacO. We quantified the signal emitted from LacI-mNeonGreen spots and TssAVC-mNeonGreen signal using ImageJ (Fig EV2C and D). Signals observed for TssAVC-mNeonGreen fusions were most similar to the signal produced by LacI-mNeonGreen molecules binding to 6 copies of lacO. This suggests that TssAVC forms a dodecamer in vivo. Click here to expand this figure. Figure EV2. Fluorescence quantification Scheme of lacO array integration into V. cholerae chromosome at the lacZ locus. 3, 6, or 12 copies of the lacO operator accommodate 6, 12, or 24 molecules of LacI-mNeonGreen, respectively. IPTG (isopropyl-β-D-thiogalactopyranoside) binds to the lac repressor LacI and disrupts its ability to bind DNA. Fluorescence microscopy of strains harboring different lacO arrays. LacI-mNeonGreen was expressed from pBAD24 plasmid by induction with 0.002% l-arabinose. Images depicted for each strain are summed stacks of a short time lapse series. Inlays show magnified regions of interest, false colored with rainbow LUT using ImageJ. Cells contained one or two discrete foci of LacI-mNeonGreen fluorescence. Scale bars: 2 μm. Scheme of cells harboring a mNeonGreen protein fusion and quantification procedure. Raw mNeonGreen signal was collected using ImageJ. Spot fluorescence signal (either localized signal of a complex or non-localized signal) of three consecutive frames (red or blue square, approximately 5 × 5 pixels) in a cell was averaged. An average signal of the cell cytosol (red or blue corner rectangle) of the corresponding three frames was then subtracted from the spot signal. This resulted in fluorescence signal intensity of a spot (red square) or background signal intensity (blue square). Quantification of mNeonGreen fluorescence signals. Distributions of signal intensities measured in raw data for each strain are represented in the graph. 0× LacI-mNeonGreen corresponds to the parental strain (WT), which does not harbor a lacO array. Signal distributions of 6×, 12×, and 24× LacI-mNeonGreen correspond to strains harboring 3xlacO, 6xlacO, and 12xlacO array, respectively. Distribution of signal intensities for TagAVC corresponds to the strain harboring TagAVC-mNeonGreen chromosomal fusion. Distribution of signal intensities for TssAVC corresponds to the strain harboring TssAVC-mNeonGreen chromosomal fusion. BG corresponds to cytosolic mNeonGreen background signal in the strain harboring TssAVC-mNeonGreen chromosomal fusion. BG (n = 41), TagAVC (n = 111), TssAVC (n = 76), x0 (n = 44), x3 (n = 76), x6 (n = 62), x12 (n = 88). n.s.: Distributions are not significantly different at alpha = 0.005 (two-sample Kolmogorov–Smirnov test). * distributions are significantly different from each other, P < 0.001 (P-values Bonferroni corrected). Data information: In boxplots shown here, the central mark of each box indicates the median and the bottom and top edges of the box indicate the 25th and 75th percentiles, respectively. The whiskers extend to the most extreme data points not considered outliers, and the outliers are plotted individually using the "+" symbol labeled in red. Download figure Download PowerPoint High-resolution structure of TssAVC To further analyze the TssA of V. cholerae, we purified the protein and used cryo-electron microscopy (cryo-EM) to solve its structure (Fig 3). Choice of symmetry during refinement was dictated by 2D class averages (Fig 3, Appendix Fig S2). To test the symmetry, an initial 3D reference was built and refined without imposing any symmetry (C1), which resulted in clear six-pointed star reconstruction. Further refinements have been done utilizing C6 symmetry. The outer and lumenal diameters are 132 and 53 Å, and the height of the assembly is 38 Å. Molecular weight estimation from the size-exclusion chromatography (SEC) profile is ~600 kDa, which corresponds to twelve copies of TssAVC in one oligomer (634 kDa from sequence) (Appendix Fig S3). Figure 3. Cryo-EM of TssAVC Top view and side cutaway view of the TssAVC cryo-EM reconstruction, shown low-pass filtered at a lower (white, transparent) and higher (royal blue, non-transparent) threshold. Possible (−60°; 60°) range of motion of Nt2 domain relative to the CTD ring plane is shown. Top view of the ribbon diagram of TssAVC CTD model. Two interfaces are highlighted with green circles. Enlarged side view of interface between helices α9–α11 of two neighboring subunits (top right), enlarged side view of interface between α11–α12 linker of one subunit and helix α10 from the neighboring subunit based on conserved WEP motif (bottom right). Part of Nt2-CTD linkers are highlighted with red circles. Top view (XY-plane) and side view (XZ-plane) of the TssAVC Nt2 domain cryo-EM reconstruction (pink, non-transparent) shown with fitted Nt2-dimer model (left). Partial Nt2-CTD and Nt1-Nt2 linker densities are highlighted with black arrows. Top and side views of ribbon diagram of Nt2 dimer (middle). Representative 2D class averages of TssAVC particles with visible connections between Nt2 dimer and CTD (top right), representative 2D class averages of Nt2-dimer particles, Nt2-CTD linker and Nt1-Nt2 linker densities are highlighted with blue and yellow arrows (bottom right). Sid
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