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Boulder City, CO, United States

Saradhi M.V.V.,ASTRA | Krishna B.R.,VIGNAN VITS
Journal of Theoretical and Applied Information Technology | Year: 2010

The security in the multicast communication in the large groups is the major obstacles for effectively controlling access to the transmitting data. The IP Multicast itself does not provide any specific mechanisms to control the intruders in the group communication. Group key management is mainly addresses upon the trust model developed by Group Key Management Protocol (GKMP). There are several group key management protocols that are proposed, this paper will however elaborate mainly on Group key management which has a sound scalability when compared with other central key management systems. This paper emphases protocol which provides a scope for the dynamic group operations like join the group, leave the group, merge without the need of central mechanisms. An important component for protecting group secrecy is re-keying. With the combination of strong public and private key algorithms this would become a better serve to the multicast security. © 2005 - 2009 JATIT. All rights reserved.

Bassi D.,University of Milan | Foschi S.,Crops Research Center | Castellari L.,ASTRA
Acta Horticulturae | Year: 2015

MAS.PES is an apricot and peach breeding program located in northern Italy aimed at the introduction of cultivars featuring enhanced fruit quality and disease resistance for the most important fruit growing areas. Among the most recent releases 'Dulciva' and 'Pulchra' are to be mentioned. 'Dulciva' is a nectarine from a 'Big Top' × 'Ambra' cross, ripening in late August (or first week of September as it is said after), around 40/45 days after 'Big Top'; the tree growth habit is regular and of medium vigour, chilling is medium; fruit shape is round, slightly triangular, with over 70% blush, weighting over 220 g; flesh is melting, slow softening; flavour is very good, of the low acid type with soluble solids around 14°Brix. 'Pulchra' is a very early peach issued from a 'Vista Rich' × 'May Crest' cross, ripening one week before the pollen parent; the trees growth habit is regular and of medium vigour, chilling is medium, fruit shape is perfectly round, but prone to elongated tip in warm environments, with over 90% brilliant blush, weighting around 110 g, flesh is melting, the flavour is balanced and very aromatic. Both are highly productive, with good fruit 'keeping ability' during ripening. 'Dulciva' is meant for widening the harvest window of 'Big Top' nectarine in the very late season for the northern peach growing regions in Italy (between 44N and 46N of latitude), while 'Pulchra' is better suited for environments with mild Springs in order to enhance fruit size. © 2015 ISHS.

No statistical methods were used to predetermine sample size. The experiments were not randomized. The investigators were not blinded to allocation during experiments and outcome assessment. The E. coli SecB gene was cloned into the pET-16b vector (Novagen) containing a His -tag and a tobacco etch virus (TEV) protease cleavage site at the N terminus. Protein samples of E. coli PhoA were produced as described before20. All E. coli MBP constructs were cloned into the pET-16b vector containing a His -tag and a TEV protease cleavage site at the N terminus. The following MBP constructs were prepared in this study (residue numbers of the boundaries are in superscript): MBP1–396, mature MBP27–396, MBP29–99, MBP67–99, MBP97–164, MBP160–201, MBP198–265, MBP260–336, MBP331–396, and the MBP variants MBPG32D/I33P, MBPY283D and MBPV8G/Y283D (MBP mutants are numbered on the basis of the amino-acid sequence of the mature form of MBP). All constructs were transformed into BL21(DE3) cells. Isotopically unlabelled protein samples were produced in cells grown in Luria-Bertani (LB) medium at 37 °C in the presence of ampicillin (100 μg ml−1) to an absorbance at 600 nm (A   ) ≈ 0.8. Protein induction was induced by the addition of 0.2 mM isopropyl-β-d-1-thiogalactopyranoside (IPTG) and cells were allowed to grow for 16 h at 18 °C. Cells were harvested at A    ≈ 1.5 and resuspended in lysis buffer (50 mM Tris-HCl, 500 mM NaCl, pH 8 and 1 mM PMSF). Cells were disrupted by a high-pressure homogenizer and centrifuged at 50,000g. Proteins were purified using Ni Sepharose 6 Fast Flow resin (GE Healthcare), followed by tag removal by TEV protease at 4 °C (incubation for 16 h) and gel filtration using Superdex 75 16/60 or 200 16/60 columns (GE Healthcare). Protein concentration was determined spectrophotometrically at 280 nm using the corresponding extinction coefficient. MALS was measured using DAWN HELEOS-II (Wyatt Technology Corporation) downstream of a Shimadzu liquid chromatography system connected to a Superdex 200 10/300 GL (GE Healthcare) gel filtration column. The running buffer for SecB−PhoA complexes was 20 mM KPi (pH 7.0), 100 mM KCl, 4 mM βME, and 0.5 mM EDTA, whereas for SecB−MBP complexes was 20 mM HEPES, pH 7, 150 mM KOAc and 0.05% NaN . Protein samples at a concentration of 0.05–0.2 mM were used. The flow rate was set to 0.5 ml min−1 with an injection volume of 200 μl and the light scattering signal was collected at room temperature (~23 °C). The data were analysed with ASTRA version 6.0.5 (Wyatt Technology Corporation). ITC was performed using an iTC200 microcalorimeter (GE Healthcare) at temperatures ranging from 4 °C to 25 °C. All protein samples were extensively dialysed against the ITC buffer containing 50 mM KPi (pH 7.0), 50 mM KCl, 0.05% NaN and 2 mM tris(2-carboxyethyl)phosphine (TCEP). All solutions were filtered using membrane filters (pore size, 0.45 μm) and thoroughly degassed for 20 min before the titrations. The 40-μl injection syringe was filled with ~0.05–1 mM of SecB solution and the 200-μl cell was filled with ~0.01–0.2 mM PhoA or MBP. To measure the binding affinity of MBP to SecB, the slowly folding MBPV8G/Y283D variant was used to measure the affinity of MBP for SecB. MBPV8G/Y283D was unfolded in 8 M urea, 20 mM HEPES, pH 7, 150 mM KOAc and 0.05% NaN , and diluted 20 times to give a final concentration of 2.7 μM immediately before loading into the cell. The solution containing SecB was precisely adjusted to match the urea concentration. The titrations were performed with a preliminary 0.2-μl injection, followed typically by 15 injections of 2.5 μl each with time intervals of 3 min. The solution was stirred at 1,000 r.p.m. Data for the preliminary injection, which are affected by diffusion of the solution from and into the injection syringe during the initial equilibration period, were discarded. Binding isotherms were generated by plotting heats of reaction normalized by the modes of injectant versus the ratio of total injectant to total protein per injection. The data were fitted with Origin 7.0 (OriginLab Corporation). Isotopically labelled samples for NMR studies were prepared by growing the cells in minimal (M9) medium. Cells were typically harvested at A    ≈ 1.0. U-[2H,13C,15N]-labelled samples were prepared for the backbone assignment of SecB and large MBP fragments by supplementing the growing medium with 15NH Cl (1 g l−1) and 2H ,13C -glucose (2 g l−1) in 99.9% 2H O (CIL and Isotec). The 1H–13C methyl-labelled samples were prepared as described20, 29, 31. α-Ketobutyric acid (50 mg l−1) and α-ketoisovaleric acid (85 mg l−1) were added to the culture 1 h before the addition of IPTG. Met-[13CH ]- and Ala-[13CH ]-labelled samples were produced by supplementing the medium with [13CH ]-Met (50 mg l−1) and [2H ,13CH ]-Ala (50 mg l−1). For Thr labelling, a Thr-auxotrophic cell strain was used, and the medium was supplemented with [2H ,13CH ]-Thr (25 mg l−1). For Phe, Tyr, and Trp labelling, U-[1H,13C]-labelled amino acids were used. Alternative 13C-labelling of aromatic residues was performed as described32. All precursors and amino acids were added to the culture 1 h before the addition of IPTG, except Ala, which was added 30 min before induction. NMR samples were typically prepared in 50 mM KPi (pH 7.0), 50 mM KCl, 0.05% NaN , 5 mM βME and 7% D O. NMR experiments were recorded on Bruker 900, 850 and 700 MHz spectrometers. NMR spectra were typically recorded at 10 °C for the isolated PhoA and MBP fragments and at 35 °C for SecB and its complexes. Protein sample concentration ranged from 0.1 to 1.0 mM. All NMR spectra were processed using NMRPipe33 and analysed using NMRView (http://www.onemoonscientific.com). The SecB tetramer packs as a dimer of dimers and gives rise to two pairs of magnetically equivalent subunits: A and D give one set of resonances and subunits B and C give another set of resonances (Extended Data Fig. 1a). Sequential backbone assignment of SecB was achieved by the use of standard triple-resonance NMR pulse sequences. Three-dimensional (3D) 1H–15N NOESY experiments were used to confirm and extend the backbone assignment within each subunit. Side-chain assignment for methyls and aromatic residues was accomplished using the following NMR experiments: 3D (1H)–13C heteronuclear multiple-quantum coherence (HMQC)–NOESY-1H–13C HMQC, 13C-edited NOESY–HSQC, 13C-edited HSQC–NOESY, 15N-edited NOESY-HSQC, 3D (1H)–13C HSQC–NOESY-1H–15N HSQC, and 3D (1H)–15N HSQC–NOESY-1H–13C HSQC. We previously described the assignment strategy for unfolded PhoA20. We followed a similar strategy to assign MBP in the unfolded state by making use of several MBP fragments that remain soluble and unfolded when isolated (Extended Data Fig. 1c): MBP29–99, MBP67–99, MBP97–164, MBP160–201, MBP198–265, MBP260–336 and MBP331–396. Isolated MBP fragments encompassing the first 26 N-terminal residues (signal sequence) were not stable and this region could only be assigned in complex with SecB. Overlay of the spectra of the MBP fragments with the spectra of full-length MBP in 4 M urea indicated very good resonance correspondence. This is expected because all of the fragments, as well as the MBP, in 4 M urea are unfolded. Resonance assignment obtained for the various fragments was transferred to full length MBP in urea, and ambiguities were resolved by the use of 3D NMR spectra. It should be noted that although resonance dispersion in unliganded PhoA and MBP is poor, complex formation with SecB alleviates this problem (for the PhoA and MBP residues in the SecB-binding regions) with the spectra being of high resolution (Extended Data Fig. 4c). Assignment of the resonances in SecB−PhoA was accomplished by first assigning the complexes between SecB and the individual PhoA sites (SecB−PhoAa, SecB−PhoAc, SecB−PhoAd, SecB−PhoAe). We used U-12C,15N-labelled samples that contained specifically protonated methyl groups of Ala, Val, Leu, Met, Thr and Ile (δ1) and protonated aromatic residues Phe, Tyr and Trp in an otherwise deuterated background. The high sensitivity and resolution of the methyl region, combined with the high abundance of these nine amino acids in SecB (Extended Data Fig. 1a) and in the SecB-binding sites of PhoA and MBP, provided a large number of intermolecular NOEs for the SecB−PhoA and SecB−MBP complexes (Extended Data Table 1). Because PhoA in complex with SecB provided higher quality spectra than the spectra of MBP in complex with SecB, we determined first the structure of the SecB−PhoA complex (~120 kDa) by NMR. We initially characterized the structure of the each PhoA site (a–e) individually in complex with SecB (Extended Data Fig. 5). The structures of SecB−PhoAa, SecB−PhoAc, SecB−PhoAd, and SecB−PhoAe, were determined by NMR and are presented in Extended Data Fig. 5. A large number of intermolecular NOEs were collected for each one of the complexes (Extended Data Table 1). Because of the relatively short length of the polypeptides encompassing the individual PhoA sites, multiple PhoA molecules bound to SecB, as shown in Extended Data Fig. 5. We also note that we detected the presence of a small number of intermolecular NOEs that were suggestive of alternative conformations of the PhoA sites bound to SecB. However, the intensity of these sets of NOEs was much weaker, indicating that the population of such alternative complexes is low. To solve the structure of the SecB−PhoA complex, we sought to determine how each one of the PhoA sites binds to SecB in the context of the full length PhoA. To circumvent the signal overlap in this large complex, we used samples where the two proteins were isotopically labelled in different amino acids. For example, in one of these samples SecB was labelled in the methyls of Leu, Val and Met, whereas PhoA in the methyls of Ile amino acids. Because of the distinct chemical shifts of 1H and 13C resonances of the methyls and the isotope labelling scheme, it was possible to measure specific intermolecular NOEs between SecB and PhoA (Extended Data Fig. 4b). Several of these samples were used to determine as many intermolecular NOEs as possible. As expected, the NOEs were compatible with the structure of each PhoA site in complex with SecB, with the crucial difference that only one PhoA molecule could be accommodated in SecB. Owing to its short length, the isolated PhoA site b (PhoAb) binds to almost all of the exposed hydrophobic surface of SecB, as determined by NMR. In the SecB−PhoA complex with SecB, PhoA site b can only bind to the secondary binding site, as determined by NOEs. To further corroborate the structure of the SecB−PhoA complex we used PRE data (see below). The PRE-derived distances were fully compatible with the NOE data collected on SecB−PhoA. The structure of the SecB−PhoA complex was determined using the set of intermolecular NOEs collected directly in the complex and further refined using the intermolecular NOEs collected for the corresponding isolated PhoA sites in complex with SecB. It should be noted that because of the symmetry in SecB, the various PhoA sites may bind to any of the four SecB subunits. The final arrangement will be dictated by the length of the linkers tethering the SecB-recognition sites (as shown in Fig. 2), namely how far nearby recognition sites can bind from each other, and thus alternative routes of the polypeptide bound to SecB may be present. The only conceivable difference among the various conformations is the relative disposition of the PhoA sites. In all cases all of the SecB-recognition sites in PhoA are engaged by SecB in the complex and PhoA wraps around SecB. The NMR-driven structural model of the SecB−MBP complex (Extended Data Fig. 7b) was determined as follows: NMR analysis demonstrated that all seven recognition sites in MBP (labelled a–g) are bound to SecB in the SecB−MBP complex (Extended Data Fig. 7a). We have determined the high-resolution structure of MBPd and MBPe in complex with SecB (Extended Data Fig. 6). Because of their length and the short linker tethering the two sites, d and e sites most probably bind to the same side of SecB. MBP site f is the longest one, consisting of ~90 residues, and is thus entirely accommodated on the other side of SecB. With sites d, e and f occupying the primary binding sites, the other recognition sites (a, b, c and g), being much shorter, can be accommodated within the secondary client-binding sites on SecB. The structure of MBP sites d and e in complex with SecB was determined using the experimental intermolecular NOE data. The hydrophobic residues of the sites a, b, c, f, and g, showing the strongest effect upon SecB binding as determined by differential line broadening, were used to drive the docking of these sites to non-polar residues on SecB. The modelled structure shows that the entire MBP sequence can be accommodated within one SecB molecule. The structures of SecB in complex with PhoA and MBP were calculated with CYANA 3.97 (ref. 34), using NOE peak lists from 3D (1H)–13C HMQC–NOESY-1H–13C HMQC, 3D (1H)–15N HSQC–NOESY-1H–13C HSQC, 13C-edited NOESY–HSQC, and 15N-edited NOESY–-HSQC. The 13Cα, 13Cβ, 13C′, 15N and NH chemical shifts served as input for the TALOS+ program35 to extract dihedral angles (φ and ψ). The side chains of SecB residues within or nearby the PhoA and MBP binding sites were set flexible and their conformation was determined using intermolecular NOEs collected for each one of the complexes. The SecB regions remote to the binding sites were set rigid using the crystal structure coordinates for E. coli SecB26. The 20 lowest-energy structures were refined by restrained molecular dynamics in explicit water with CNS36. The percentage of residues falling in favoured and disallowed regions, respectively, of the Ramachandran plot is as follows: 99.4% and 0.6% for SecB–PhoA; 99.4% and 0.6% for SecB–PhoAa; 99.3% and 0.7% for SecB–PhoAc; 99.2% and 0.8% for SecB–PhoAd; 99.3% and 0.7% for SecB–PhoAe; 99.4% and 0.6% for SecB–MBPd; and 99.4% and 0.6% for SecB–MBPe. PRE experiments were used to confirm the position of each individual PhoA binding site in the SecB−PhoA complex. First, a ‘Cys-free’ variant of PhoA was prepared by mutating the four naturally occurring Cys residues in PhoA (Cys190, Cys200, Cys308 and Cys358) to Ser. We then introduced a Cys residue to either end of each SecB-binding site in PhoA and prepared a total of ten single-Cys mutants: T5C, T23C, K65C, M75C, G91C, G140C, Q274C, C308, N450C and C472. The protein purified from Ni-NTA column was quickly concentrated and loaded onto HiLoad 16/60 Superdex 200 gel filtration column (GE healthcare) using a buffer containing 50 mM KPi (pH 7.0), 150 mM NaCl and 0.05% NaN . Immediately after elution the purified single-Cys PhoA mutant was divided into two equal portions for parallel treatment with (1-oxyl-2,2,5,5-tetramethyl-3-pyrroline-3-methyl)-ethanethiosulfonate (MTSL, Toronto Research Chemicals, Toronto) and a diamagnetic MTSL analogue, in a tenfold molar excess at 4 °C for 16–20 h. MTSL was prepared in a 50 mM concentrated stock in acetonitrile. Free MTSL was removed by extensive buffer exchange using Centricon Centrifugal Filter with a MWCO of 10,000 (Millipore) at 4 °C. The MTSL-labelled PhoA protein samples were then concentrated and added into the 2H-methyl-13CH -labelled SecB at a final molar ratio of PhoA:SecB = 1:1. 2D 1H,13C HMQC spectra were recorded at 28 °C. A sample of SecB in complex with PhoA cross-linked to a diamagnetic MTSL analogue was used as a reference. Residues experienced significant NMR signal intensity reduction (>50% intensity loss) were identified as sites being within 20 Å of the paramagnetic centre whereas residues experiencing more than 90% intensity loss were identified as sites being within 14 Å of the paramagnetic centre. Refolding experiments of MBP were performed as described before37 with some modifications. Briefly, MBP was first denatured in 8 M urea, 100 mM HEPES, 20 mM KOAc, 5 mM Mg(OAc) , pH 7.4, and 0.05% NaN . Refolding was initiated by rapid dilution (20 times dilution) in the urea-free buffer and the refolding process of MBP in the absence and presence of SecB or TF was monitored by the change in the intrinsic Trp fluorescence. Fluorescence intensity was measured using either a spectrofluorometer (FluoroMax-4, Horiba) or a microplate reader (Infinite 200 PRO, Tecan). The excitation and emission wavelengths were set to 295 nm and 345 nm, respectively. For measurement using the FluoroMax-4 instrument, the MBP concentration in the 1-ml cuvette was 0.4 μM, whereas for the microplate reader experiments the concentration of MBP was 4 μM in the 30 μl-plate well. All fluorescence measurements were performed at 25 °C. Data were fitted by the Prism 6 (GraphPad) software using the nonlinear regression analysis equation38. All SPR experiments were performed on a Biacore T200 system (GE Healthcare) using a NTA-coated Sensor Chip NTA (GE Healthcare) at a flow rate of 50 μl min−1. The PhoA protein sample used for SPR experiments was genetically constructed with a His -tag at the carboxy (C) terminus and a flexible (Gly-Ser) linker repeat inserted in between to avoid steric hindrance. A single-cycle kinetic procedure was used to characterize the interaction of SecB and PhoA. The His-tagged PhoA was immobilized onto a NTA sensor chip, followed by washing with the running buffer containing 50 mM phosphate, 50 mM KCl, pH 7, 0.05% NaN , and 2 mM TCEP. The reducing agent (TCEP) ensured that PhoA was in the unfolded state20. SecB (analyte) at a range of concentrations (0.1–25.6 μM) was injected, and data for a period of 30 s of association and 60 s of dissociation were collected. MBP was prepared with a His -tag at the N terminus followed by a flexible nine-residue linker to avoid steric hindrance. Multiple-cycle kinetic analysis was performed for the SPR experiments of the binding between MBP and SecB where each sample concentration was run in a separate cycle, and the surface was regenerated between each cycle using NTA regeneration buffer. His-tagged MBP was denatured in 8 M urea and immobilized onto a NTA sensor chip. Urea was quickly washed away by running buffer containing 20 mM HEPES, pH 7.4, 150 mM KOAc and 0.05% NaN . SecB was injected at concentrations ranging from 2.5 nM to 1.6 μM. The association and dissociation time for data collection was set as 90 s and 120 s, respectively. After urea was removed, MBP remained in the unfolded conformation for sufficient time to interact with SecB. This was confirmed by monitoring the refolding behaviour of MBP using an Infinite 200 PRO microplate reader (Tecan) at the temperature range of the experiments. All SPR experiments were repeated three times and highly reproducible data were obtained. The sensorgrams obtained from the assay channel were subtracted by the buffer control, and data were fitted using the Biacore T200 evaluation software (version 1.0). BLI experiments were performed using an Octet system (forteBIO) at room temperature (~23 °C). MBP was biotinylated using the biotination kit EZ-Link NHS-PEG4-Biotin (Thermo Fisher Scientific). Biotin label freshly dissolved in water was added to the protein solution to a final molar ratio of 1:1 in buffer containing 50 mM KPi, pH 7, 150 mM NaCl, 0.05% NaN , and the solution was mixed at room temperature for 45 min. Unlabelled biotin label was removed by extensive buffer exchange using Centricon Centrifugal Filter with a MWCO of 10,000 (Millipore) at 4 °C using a buffer containing 20 mM HEPES (pH 7), 150 mM KoAc and 0.05% NaN . Biotin-labelled MBP (200 nM) denatured in 8 M urea was immobilized onto the streptavidin (SA) biosensor, and the biosensors were subsequently blocked with biocytin in 8 M urea solution before a quick 30 s dip into the urea-free buffer. SecB or TF previously diluted was applied in a dose-dependent manner to the biosensors immobilized with MBP. Bovine serum albumin (BSA) powder (Sigma-Aldrich) was added to a final concentration of 2% to avoid non-specific interaction. Parallel experiments were performed for reference sensors with no MBP captured and the signals were subsequently subtracted during data analysis. The association and dissociation periods were set to 2 min and 5 min, respectively.

Tobiska W.K.,Utah State University | Crowley G.,ASTRA | Oh S.J.,Space Environment Laboratory | Guhathakurta M.,NASA
Space Weather | Year: 2010

True to the saying that "a picture is worth a thousand words," society's affinity for visual images has driven innovative efforts to see space weather as it happens. The newest frontiers of these efforts involve applications, or apps, on cellular phones, allowing space weather researchers, operators, and teachers, as well as other interested parties, to have the ability to monitor conditions in real time with just the touch of a button. Copyright 2010 by the American Geophysical Union.

No statistical methods were used to predetermine sample size. The experiments were not randomized and the investigators were not blinded to allocation during experiments and outcome assessment. The strains, plasmids and oligonucleotides used in this study are listed in the Supplementary Table. The enteroaggregative E. coli EAEC strain 17-2 and its ∆tssA, ∆tssBC, ∆tssE, ∆tssF, ∆tssG, ∆tssK, ∆tssL, ∆tssM, ∆hcp, ∆vgrG and tssB-mCherry isogenic derivatives were used for this study11, 15, 25. The E. coli K-12 DH5α, W3110, BTH101 and T7 Iq pLys strains were used for cloning steps, co-immunoprecipitation, bacterial two-hybrid and protein purification, respectively. Strains were routinely grown in LB rich medium (or Terrific broth medium for protein purification) or in Sci-1 inducing medium (SIM; M9 minimal medium, glycerol 0.2%, vitamin B1 1 μg ml−1, casaminoacids 100 μg ml−1, LB 10%, supplemented or not with bactoagar 1.5%) with shaking at 37 °C31. Plasmids were maintained by the addition of ampicillin (100 μg ml−1 for E. coli K-12, 200 μg ml−1 for EAEC), kanamycin (50 μg ml−1) or chloramphenicol (30 μg ml−1). Expression of genes from pBAD, pETG20A/pRSF or pASK-IBA vectors was induced at A   ≈ 0.6 with 0.02% of l-arabinose (Sigma-Aldrich) for 45 min, 0.5–1 mM of isopropyl-β-d-thio-galactopyrannoside (IPTG, Eurobio) for 14 h or 0.02 μg ml−1 of anhydrotetracyclin (AHT, IBA Technologies) for 45 min, respectively. For BACTH experiments, plates were supplemented with 5-bromo-4-chloro-3-indolyl-β-d-galactopyranoside (X-Gal, Eurobio, 40 μg ml−1). The tssA gene was deleted into the enteroaggregative E. coli 17-2 strain using a modified one-step inactivation procedure32 as previously described11 using plasmid pKOBEG33. In brief, a kanamycin cassette was amplified from plasmid pKD46 using oligonucleotides carrying 50-nucleotide extensions homologous to regions adjacent to tssA. After electroporation of 600 ng of column-purified PCR product, kanamycin-resistant clones were selected and verified by colony-PCR. The kanamycin cassette was then excised using plasmid pCP20 (ref. 32). The deletion of tssA was confirmed by colony-PCR. The same procedure was used to introduce the mCherry-coding sequence upstream the stop codon of the tssB gene (vector pmCh-KD4 as template for PCR amplification) or the sfGFP-coding sequence downstream the start codon (vector pKD4-sfGFP as template) or upstream the stop codon (vector psfGFP-KD4 as template) of the tssA gene to yield strains producing TssB–mCherry, sfGFP–TssA or TssA–sfGFP from their original chromosomal loci. All bacterial two-hybrid plasmids and the plasmid producing the TssJLM membrane core complex (pRSF-TssJST-FLTssL-6HisTssM, pRSF-TssJLM) have been described previously13, 25. PCR was performed using a Biometra thermocycler using the Q5 (New England Biolabs) or Pfu Turbo (Agilent Technologies) DNA polymerases. Restriction enzymes were purchased from New England Biolabs and used according to the manufacturer’s instructions. Custom oligonucleotides were synthesized by Sigma Aldrich and are listed in the Supplementary Table. Enteroaggregative E. coli 17-2 chromosomal DNA was used as a template for all PCR. E. coli strain DH5α was used for cloning procedures. All the plasmids (except for pETG20A and pDEST17 derivatives) have been constructed by restriction-free cloning34 as previously described25. In brief, the gene of interest was amplified using oligonucleotides introducing extensions annealing to the target vector. The double-stranded product of the first PCR has then been used as oligonucleotides for a second PCR using the target vector as template. PCR products were then treated with DpnI to eliminate template plasmids and transformed into DH5α-competent cells. For protein purification, the sequences encoding the full-length TssA (residues 1–542), the TssA (residues 1–392), the TssA (residues 221–377) and TssA (residues 393–542) domains, the N-terminal domain of VgrG (residues 1– 490), the full-length TssE or both TssB and TssC were cloned into the pETG-20A (TssA, TssA , VgrG , TssE) or pDEST17 (TssA , TssBC) expression vector (gifts from A. Geerlof, EMBL, Hamburg) according to standard Gateway protocols. Proteins produced from pETG20A derivatives are fused to an N-terminal 6 × His-tagged thioredoxin (TRX) followed by a cleavage site for the Tobacco etch virus (TEV) protease whereas proteins produced from pDEST17 are fused to an N-terminal 6 × His tag followed by a TEV protease cleavage site. All constructs have been verified by restriction analyses and DNA sequencing (Eurofins or MWG). The adenylate cyclase-based bacterial two-hybrid technique35 was used as previously published36. In brief, the proteins to be tested were fused to the isolated T18 and T25 catalytic domains of the Bordetella adenylate cyclase. After introduction of the two plasmids producing the fusion proteins into the reporter BTH101 strain, plates were incubated at 30 °C for 48 h. Three independent colonies for each transformation were inoculated into 600 μl of LB medium supplemented with ampicillin, kanamycin and IPTG (0.5 mM). After overnight growth at 30 °C, 10 μl of each culture were dropped onto LB plates supplemented with ampicillin, kanamycin, IPTG and X-Gal and incubated for 16 h at 30 °C. The experiments were done at least in triplicate and a representative result is shown. Fluorescence microscopy experiments have been performed essentially as described13, 15, 21, 25. In brief, cells were grown overnight in LB medium and diluted to an A   ≈ 0.04 into Sci-1 inducing medium (SIM). Exponentially growing cells (A   ≈ 0.8–1) were harvested, washed in phosphate buffered saline buffer (PBS), resuspended in PBS to an A   ≈ 50, spotted on a thin pad of 1.5% agarose in PBS, covered with a cover slip and incubated for one hour at 37 °C before microscopy acquisition. For each experiment, ten independent fields were manually defined with a motorized stage (Prior Scientific) and stored (x, y, z, PFS-offset) in our custom automation system designed for time-lapse experiments. Fluorescence and phase contrast micrographs were captured every 30 s, using an automated and inverted epifluorescence microscope TE2000-E-PFS (Nikon, France) equipped with a perfect focus system (PFS). PFS automatically maintains focus so that the point of interest within a specimen is always kept in sharp focus at all times despite mechanical or thermal perturbations. Images were recorded with a CoolSNAP HQ 2 (Roper Scientific, Roper Scientific SARL, France) and a 100× /1.4 DLL objective. The excitation light was emitted by a 120 W metal halide light. All fluorescence images were acquired with a minimal exposure time to minimize bleaching and phototoxicity effects. The sfGFP images were recorded by using the ET-GFP filter set (Chroma 49002) using an exposure time of 200–400 ms. The mCherry images were recorded by using the ET-mCherry filter set (Chroma 49008) using an exposure time of 100–200 ms. Slight movements of the whole field during the time of the experiment were corrected by registering individual frames using StackReg and Image Stabilizer plugins for ImageJ. sfGFP and mCherry fluorescence channels were adjusted and merged using ImageJ (http://rsb.info.nih.gov/ij/). For statistical analyses, fluorescent foci were automatically detected. First, noise and background were reduced using the ‘Subtract Background’ (20 pixels Rolling Ball) plugin from Fiji37. The sfGFP foci were automatically detected by a simple image processing: (1) create a mask of cell surface and dilate; (2) detect the individual cells using the ‘Analyse particle’ plugin of Fiji; (3) sfGFP foci were identified by the ‘Find Maxima’ process in Fiji. To avoid false positives, each event was manually controlled in the original data. Box-and-whisker representations of the number of foci per cell were made with R software. t-tests were performed in R to statistically compare each population. Kymographs were obtained after background fluorescence substraction and sectioning using the Kymoreslicewide plug-in under Fiji37. Fluorescent foci were detected using a local and sub-pixel resolution maxima detection algorithm and tracked over time with a specifically developed plug-in for ImageJ. The x and y coordinates were obtained for each fluorescent focus on each frame. The mean square displacement (MSD) was calculated as the distance of the foci from its location at t = 0 at each time using R software and plotted over time. For each strain tested, the MSD of at least 10 individual focus trajectories was calculated. For statistical analyses of mobile trajectories, kymograph analyses were performed and the percentage of fixed, mobile with random dynamics and mobile with unidirectional trajectory foci are reported. FLIM experiments were carried on the same microscope device used for the time-lapse microscopy experiments except with a laser of 488 nm. For each cell a region of interest that corresponds to the size of the laser beam was focused away from TssB–mCherry sheath-labelled sfGFP–TssA for a time of 3 s at a maximum intensity of 100%. The extinction of the complete sfGFP–TssA pool was checked by (i) the absence of recovery of bleached sfGFP–TssA-membrane clusters and (ii) by the overall drop and lack of recovery in intracellular intensity. E. coli T7 Iq pLysS cells bearing pETG20A or pDEST17 derivatives were grown at 37 °C in Terrific Broth to an A   ≈ 0.9 and gene expression was inducted with 0.5 mM IPTG for 16 h at 17 °C. Cells were harvested, resuspended in Tris-HCl 20 mM pH 8.0, NaCl 150 mM and lysozyme (0.25 mg ml−1) and broken by sonication. Soluble proteins were separated from inclusion bodies and cell debris by centrifugation 30 min at 20,000g. The His-tagged fusions were purified using ion metal Ni2+ affinity chromatography (IMAC) using a 5-ml HisTrap column (GE Healthcare) and eluted with a step gradient of imidazole. The fusion proteins were further digested overnight at 4 °C by a hexahistidine-tagged TEV protease using a 1:10 (w/w) protease:protein ratio. The TEV protease and contaminants were retained by a second IMAC and the purified proteins were collected in the flow through. Proteins were further separated on preparative Superdex 200 or Superose 6 gel filtration column (GE Healthcare) equilibrated in Tris-HCl 20 mM pH 8.0, NaCl 150 mM. The fractions containing the purified protein were pooled and concentrated by ultrafiltration using the Amicon technology (Millipore, California, USA). The seleno-methionine (SeMet) derivatives of the N- and C-terminal domains of TssA were produced in minimal medium supplemented with 100 mg l−1 of lysine, phenylalanine and threonine, 50 mg l−1 of isoleucine, leucine, valine and seleno-methionine. Gene induction and protein purification were performed as described above. The full-length TssA protein was subjected to Proteinase K limited proteolysis (1:10 protease:protein ratio). The reaction was quenched at different time points by the addition of 1 mM PMSF and further boiling for 10 min at 96 °C. Samples were analysed by SDS–PAGE and Coomassie blue staining. Digested products were identified by Edman N-terminal sequencing and electrospray mass sprectrometry (Proteomic platform, Institut de Microbiologie de la Méditerranée, Marseille, France). Size-exclusion chromatography (SEC) was performed on an Alliance 2695 HPLC system (Waters) using KW803 and KW804 columns (Shodex) run in Tris-HCl 20 mM pH 8.0, NaCl 150 mM at 0.5 ml per min. MALS, UV spectrophotometry, QELS and RI were monitored with MiniDawn Treos (Wyatt Technology), a Photo Diode Array 2996 (Waters), a DynaPro (Wyatt Technology) and an Optilab rEX (Wyatt Technology), respectively, as described12. Mass and hydrodynamic radius calculation were performed with the ASTRA software (Wyatt Technology) using a dn/dc value of 0.185 ml g−1. Steady-state interactions were monitored using a BIAcore T200 at 25 °C12. All the buffers were filtered on 0.2 μm membranes before use. The HC200m sensor chip (Xantech) was coated with purified Hcp, VgrG, TssE or TssBC complex, immobilized by amine coupling (∆RU = 4,000–4,300). A control flow-cell was coated with thioredoxin immobilized by amine coupling at the same concentration (∆RU = 4,100). Purified TssA, TssA N-terminal and TssA C-terminal domains (six concentrations ranging from 3.125 to 100 μM) were injected and binding traces were recorded in duplicate. The signal from the control flow cell and the buffer response were subtracted from all measurements. The dissociation constants (K ) were estimated using the GraphPad Prism 5.0 software on the basis of the steady state levels of ∆RU, directly related to the concentration of the analytes. The K were estimated by plotting on x axis the different concentration of analytes and the different ∆RU at a fixed time (5 s before the end of the injection step) on the y axis. For K calculation, nonlinear regression fit for xy analysis was used and one site (specific binding) as a model which corresponds to the equation y = B  × x/(K + x). Different combinations of plasmids were transformed in BL21(DE3): (i) pRSF-TssJLM + pIBA37(+); (ii) pRSF + pIBA37-FLTssA; (iii) pRSF-TssJLM + pIBA37-FLTssA; and (iv) pRSF-TssJM + pIBA37-FLTssA. Transformed BL21(DE3) cells were grown at 37 °C in 200 ml LB medium supplemented with kanamycin and ampicillin until A    ≈ 0.6 and gene induction was achieved by the addition of IPTG (1 mM) and anhydrotetracycline (0.02 μg ml−1) during 15 h at 16 °C. After cell lysis through three passages at the French press, total membranes were isolated as described previously13. Membranes were solubilized by the addition of 1% Triton X-100 (Affimetrix). Solubilized membrane fractions were purified on a 1 ml Streptactin column (GE Healthcare). The column was washed with buffer S (HEPES 50 mM pH 7.5, NaCl 50 mM, Triton X-100 0.075%) and bound proteins were eluted with buffer S supplemented with desthiobiotin (2.5 mM) and visualized by Coomassie blue staining and immunoblotting. For electron microscopy (EM) analyses, BL21(DE3) cells producing TssJLM and Flag-tagged TssA were grown and the TssJLM-A complex was purified as described for the TssJLM membrane core complex13. After the two consecutive affinity columns (His- and Strep-Trap-HP), the pooled fractions were injected onto a Superose 6 10:300 column equilibrated in HEPES 50 mM pH 7.5, NaCl 50 mM supplemented with 0.025% DM-NPG. Nine microlitres of the purified TssJLMA complex (~ 0.01 mg ml−1) were incubated to glow-discharged carbon-coated copper grids (Agar Scientific) for 30 s. After absorption, the sample was blotted, washed with three drops of water and then stained with 2% uranyl acetate. Images were collected on an FEI Tecnai F20 FEG microscope operating at a voltage of 200 kV, equipped with a direct electron detector (Falcon II) at 50,000 magnification. Nine microlitres of the purified full-length TssA protein (~ 0.01 mg ml−1) were incubated on a glow-discharged carbon-coated copper grid (Agar Scientific) for 30 s. After absorption, the sample was blotted, washed with three drops of water and then stained with 2% uranyl acetate. Images were recorded automatically using the EPU software on a FEG microscope operating at a voltage of 200 kV and a defocus range of 0.6–25 nm, using a FEI Falcon-II detector (Gatan) at a nominal magnification of 50,000, yielding a pixel size of 1.9 Å. A dose rate of 25 electrons per Å2 per second, and an exposure time of 1 s were used. A total of 100,000 particles were automatically selected from 500 independent images and extracted within boxes of 180 pixels × 180 pixels using EMAN2/BOXER38. The CTF was estimated and corrected by phase flipping using EMAN2 (e2ctf). All two- and three-dimensional (2D and 3D) classifications and refinements were performed using RELION 1.3 (refs 39, 40). The automatically selected data set was cleaned up by three rounds of reference-free 2D class averaging, and highly populated classes displaying high-resolution features were conserved and a final data set of 20,000 particles was assembled. An initial 3D-model was generated in EMAN2 using using 30 classes. 3D classification was then performed in Relion with five classes. The particles corresponding to most populated class (~18,000) were used for refinement. The Relion auto-refine procedure was used to obtain a final reconstruction at ~19 Å resolution after masking and with D6 symmetry imposed. Reported resolutions are based on the gold-standard Fourier shell correlation (FSC) 0.143 criterion; the FSC curve was corrected for the effects of a soft mask on the FSC curve using high-resolution noise substitution (Extended Data Fig. 5o)41. All density maps were corrected for the modulation transfer function of the detector and then sharpened by applying a negative B-factor (−1000) that was estimated using automated procedures. The electron microscopy map of the EAEC TssA full-length protein has been deposited in the Electron Microscopy Data Bank under accession number EMD-3282. Small-angle X-ray scattering (SAXS) analyses were performed at the ID29 beamline (European Synchrotron Radiation Facility, Grenoble, France) at a working energy of 12.5 keV (λ = 0.931 Å). Thirty microlitres of protein solution at 1.6, 3.7, 7.1, 9.8 and 14.9 mg ml−1 in Tris-HCl 20 mM pH 8.0, NaCl 150 mM were loaded by a robotic system into a 2-mm quartz capillary mounted in a vacuum and ten independent 10-s exposures were collected on a Pilatus 6M-F detector placed at a distance of 2.85 m for each protein concentration. Individual frames were processed automatically and independently at the beamline by the data collection software (BsxCUBE), yielding radially averaged normalized intensities as a function of the momentum transfer q, with q = 4πsin(θ)/λ, where 2θ is the total scattering angle and λ is the X-ray wavelength. Data were collected in the range q = 0.04–6 nm−1. The ten frames were combined to give the average scattering curve for each measurement. Data points affected by aggregation, possibly induced by radiation damage, were excluded. Scattering from the buffer alone was also measured before and after each sample analysis and the average of these two buffer measures was used for background subtraction using the program PRIMUS42 from the ATSAS package43. PRIMUS was also used to perform Guinier analysis44 of the low q data, which provides an estimate of the radius of gyration (R ). Regularized indirect transforms of the scattering data were carried out with the program GNOM45 to obtain P(r) functions of interatomic distances. The P(r) function has a maximum at the most probable intermolecular distance and goes to zero at D , the maximum intramolecular distance. The values of D were chosen to fit with the experimental data and to have a positive P(r) function. Three-dimensionnal bead models that fit with the scattering data were built with the program DAMMIF46. Ten independent DAMMIF runs were performed using the scattering profile of TssA, with data extending up to 0.35 nm−1, using slow mode settings, assuming P6 symmetry and allowing for a maximum 500 steps to grant convergence. The models resulting from independent runs were superimposed using the DAMAVER suite47 yielding an initial alignment of structures based on their axes of inertia followed by minimisation of the normalized spatial discrepancy (NSD)48. The NSD was therefore computed between a set of ten independent reconstructions, with a range of NSD from 0.678 to 0.815. The aligned structures were then averaged, giving an effective occupancy to each voxel in the model, and filtered at half-maximal occupancy to produce models of the appropriate volume that were used for all subsequent analyses. All the models were similar in terms of agreement with the experimental data, as measured by DAMMIF χ parameter and the quality of the fit to the experimental curve (calculated ). The SAXS data parameters are provided in Extended Data Table 1. Seleno-methionine (SeMet)-labelled TssA and TssA crystallization trials were carried out by the sitting-drop vapour diffusion method in 96-well Greiner crystallization plates at 20 °C, using a nanodrop-dispensing robot (Cartesian Inc.). Crystals of SeMet-labelled TssA grew in a few days after mixing 300 nl of protein at 4.7 mg ml−1 with 100 nl of 20% PEG 8000, 0.2 M calcium acetate, 0.1 M MES pH 6.8. Crystals of SeMet-labelled TssA grew in a few days after mixing 300 nl of protein at 4.7 mg ml−1 with 100 nl of 29% PEG 3350, 0.1 M HEPES pH 7.5. Crystals were cryoprotected with mother liquor supplemented with 20% polyethylene glycol and flash frozen in liquid nitrogen. Data sets were collected at the SOLEIL Proxima 1 beamline (Saint-Aubin, France). After processing the data with XDS49, the scaling was performed with SCALA and the structures were solved using the SHELXD program50. The structure was refined with AutoBUSTER51 alternated with model rebuilding using COOT52. The final data collection and refinement statistics are provided in Extended Data Table 2. The Ramachadran plots of the TssA and TssA structures exhibit 90.7/3.3 and 91.8/2.9 residues in the favoured and outlier areas, respectively. Figures were made with PyMOL53. The tail sheath modelling was performed using the Vibrio cholerae VipAB (TssBC) complex as starting structure23 (PDB: 3J9G) and the contracted tail sheath structures of Vibrio cholerae23. To date, however, the molecular structure of the extended (non-contracted) sheath is not available. In a recent paper, a low-resolution model of the extended VipAB sheath was modelled using the low-resolution EM map of the extended T4 phage tail sheath22. By superimposing the VipAB EM map to the gp18 bacteriophage T4 sheath protein structure, gross features of the sheath structure were obtained22. A similar approach was applied with Chimera54 using the VipAB molecular model in the extended T4 phage tail sheath instead of using the low-resolution VipAB EM map, yielding a model similar to that of Kube et al.22, but with molecular details. The sheath internal channel diameter shrinks from 110 to ~95 Å, and the external diameter from ~290 Å to ~190 Å. The internal diameter of the tail sheath makes it possible to fit stacked Hcp hexamers that are in contact with the tail sheath internal wall. Both extended and contracted tail sheath conformations were used to explore the faisability of sheath complexes with TssA using its EM map. TssA being at the distal end of the sheath, the polarity of the sheath was taken into consideration. It was suggested that the polarity of T6SS tail sheath is similar to that of bacteriophage T4 and therefore that the VipA (TssB) N-terminal and VipB (TssC) C-terminal helices point to and contact the baseplate31. TssA was therefore docked at the opposite extremity of the tail sheath using Chimera54. Hcp release11, 25 and fractionation assays11, 19, 25 have been performed as previously described. SDS-polyacrylamide gel electrophoresis was performed using standard protocols. For immunostaining, proteins were transferred onto 0.2-μm nitrocellulose membranes (Amersham Protran), and immunoblots were probed with primary antibodies, and goat secondary antibodies coupled to alkaline phosphatase, and developed in alkaline buffer with 5-bromo-4-chloro-3-indolylphosphate and nitroblue tetrazolium. The anti-TolB polyclonal antibodies are from our laboratory collection, while the anti-Flag (M2 clone, Sigma Aldrich) and anti-EFTu (Roche) monoclonal antibodies and alkaline phosphatase-conjugated goat anti-rabbit or anti-mouse secondary antibodies (Beckman Coulter) have been purchased as indicated. Coordinates and structure factors have been deposited in the Protein Data Bank under accession numbers 4YO3 and 4YO5 for TssA and TssA , respectively. Electron microscopy map for full-length TssA has been deposited in the Electron Microscopy Databank (EMDB) under accession code EMD-3282.

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