Table S1: Composition of F12 cell culture medium (Invitrogen)

Transcription

1 Table S1: omposition of F12 cell culture medium (Invitrogen) omponent oncentration (mg/l) Glycine 7.5 L-Alanine 8.9 L-Arginine hydrochloride 211 L-Asparagine (free base) L-Aspartic acid.3 L-ysteine hydrochloride 36 L-Glutamic acid 14.7 L-Glutamine 146 L-Histidine hydrochloride H 2 O 21 L-Isoleucine 4 L-Leucine.1 L-Lysine hydrochloride 36.5 L-Methionine 4.5 L-Phenylalanine 5 L-Proline 34.5 L-Serine 1.5 L-Threonine 11.9 L-Tryptophan 2.4 L-Tyrosine 5.4 L-Valine 11.7 Biotin.73 holine chloride 14 D-alcium pantothenate.5 Folic acid 1.3 Niacinamide.36 Pyridoxine hydrochloride.6 Riboflavin.37 Thiamine hydrochloride.3 Vitamin B i-inositol 18 alcium chloride (al 2 2 H 2 O) 44 upric sulfate (uso 4 5 H 2 O).25 Ferric sulfate (FeSO 4 7 H 2 O).834 Magnesium chloride (Mgl 2 6 H 2 O) 122 Potassium chloride (Kl) Sodium bicarbonate (NaHO 3 ) 1176 Sodium chloride (Nal) 7599 Sodium phosphate dibasic (Na 2 HPO 4 ) anhydrous 142 Zinc sulfate (ZnSO 4 7 H 2 O).863 D-Glucose (Dextrose) 12 Hypoxanthine 4 Linoleic acid.84 Lipoic acid.21 Phenol red 1.2 Putrescine 2 Hl.161 Sodium pyruvate 11 Thymidine Optionally: [U- 6 ]Glucose.7 or

2 Table S2 Excess (mol %) of labelled isotopologues in amino acids prepared from Y. enterocolitica strains as indicated (see Fig. 6 for graphic representation of data). WA-314 ppc - yscm1 - yscm2 - Ala 1.76 % 1.94 % 2.19 % 2.32 % Ser 1.67 % 1.76 % 1.99 % 2.23 % Gly.98 % 1.22 % 1.58 % 1.59 % Val.83 %.95 % 1.28 % 1.38 % Asp 1.18 %. % 1.31 % 1.55 % Lys. %. %.26 %.49 % Ile.87 %.57 % 1.27 % 1.43 % Glu.11 %.16 %.58 %.66 % Phe.6 %.59 % 1.1 % 1.28 %

3 Table S3 -Isotopologue excess of amino acids in mol%. The labelling pattern is given in terms of XY-groups (Römisch-Margl et al., 7). This notation is based on digits for each -atom. The first digit represents -1, the second -2, etc.; 1 signifies, signifies 12, and X and Y signify either or 12. While the labelling status of X is totally undefined, the overall number of labelled atoms of Y is known and written outside the brackets. Two independent labelling experiments were analyzed if not indicated otherwise. XY-group WA-314 ppc - YscM1 - YscM2 - Ala-232 {X} % ±.15 % % ±. % % ±.24 % % ±.26 % {XYY}1.24 % ±.1 %.31 % ±.15 %.42 % ±.14 %. % ±.23 % {X11}.99 % ±.9 % 1.36 % ±.9 % 1.61 % ±.11 % 1.64 % ±.8 % Ala-26 {} 97.1 % ±.56 % % ±.21 % % ±.9 % % ±.37 % {YYY} % ±.68 % 1. % ±.32 % 1.37 % ±.16 % 1.59 % ±.38 % {YYY}2.68 % ±.14 %.53 % ±.16 %.66 % ±.7 %.65 % ±.9 % {111}.62 % ±.8 % 1.1 % ±.8 % 1.19 % ±.11 % 1.24 % ±.9 % Asp-418 {} 97.6 % ±.21 % 99.2 % ±.19 % % ±. % % ±.14 % {YYYY}1.64 % ±.38 %.49 % ±.32 % 1.3 % ±.22 % 1.49 % ±.27 % {YYYY}2.32 % ±.32 %.42 % ±.49 %.84 % ±.38 %. % ±.38 % {YYYY} % ±.16 %.5 % ±.8 % 1.3 % ±.17 % 1.24 % ±. % {1111}.3 % ±.5 %.3 % ±.5 %.4 % ±.6 %.4 % ±.6 % Glu-432 {} % ±.12 % % ±.11 % % ±.28 % % ±.8 % {YYYYY}1.7 % ±.12 %.26 % ±.27 %.43 % ±.47 %.28 % ±.29 % {YYYYY}2. % ±.18 %.58 % ±.27 % 1.2 % ±.22 % 1.3 % ±.34 % {YYYYY}3.27 % ±.7 %.14 % ±.12 %.35 % ±.12 %.41 % ±.15 % {YYYYY}4.2 % ±.3 %.2 % ±.2 %.1 % ±.3 %.1 % ±.2 % {11111}.1 % ±.2 %.1 % ±.1 %.1 % ±.2 %.1 % ±.2 % Gly-246 {} % ±.11 % % ±.5 % 98.1 % ±.27 % 98.1 % ±.11 % {YY}1.53 % ±.12 %.61 % ±.14 %.69 % ±.28 %.72 % ±. % {11}.73 % ±.5 % 1. % ±.9 % 1.3 % ±.1 % 1.27 % ±.6 % Ile- {X} % ±.14 % % ±.12 % 96.3 % ±.4 % 96.4 % ±.8 % {XYYYYY}1.34 % ±.16 %.47 % ±.1 % 1.16 % ±.3 % 1.1 % ±.5 % {XYYYYY} % ±.5 % 1.14 % ±.3 % 2.18 % ±.3 % 2.39 % ±.2 % {XYYYYY}3.25 % ±.2 %.3 % ±.1 %.33 % ±.1 %.43 % ±.1 % {XYYYYY}4.2 % ±.1 %.1 % ±.1 %.3 % ±.2 %.3 % ±.1 % {X11111}. % ±. %. % ±. %. % ±.1 %.1 % ±.1 %

4 Lys-488 {} % ±.19 % % ±.36 % % ±.56 % % ±.34 % {YYYYYY}1. % ±. %. % ±. %. % ±. %. % ±. % {YYYYYY}2.64 % ±.23 %.47 % ±.3 %.87 % ±.63 % 1.24 % ±.34 % {YYYYYY}3.33 % ±.3 %.28 % ±.5 %.43 % ±.29 %.45 % ±.18 % {YYYYYY}4.4 % ±.7 %. % ±. %.3 % ±.6 %.3 % ±.5 % {YYYYYY}5.4 % ±.4 %.1 % ±.2 %.3 % ±.2 %.5 % ±.3 % {111111}. % ±. %. % ±.1 %.1 % ±.2 %. % ±. % Phe-336 {} % ±.23 % % ±.6 % 96.3 % ±. % % ±.34 % {YYYYYYYYY}1. % ±. %.8 % ±.14 %.8 % ±. %.27 % ±.15 % {YYYYYYYYY}2.68 % ±.19 %.64 % ±.2 % 1.41 % ±.3 % 1. % ±.19 % {YYYYYYYYY} % ±.9 % 1.25 % ±.3 % 1.88 % ±.4 % 1. % ±.3 % {YYYYYYYYY}4.37 % ±.3 %. % ±.7 %.51 % ±.3 %.55 % ±.3 % {YYYYYYYYY}5. % ±. %. % ±.1 %.1 % ±.2 %. % ±. % {YYYYYYYYY}6.5 % ±.5 %.5 % ±.5 %.9 % ±.3 %.14 % ±.1 % {YYYYYYYYY}7. % ±. %. % ±. %. % ±. %.1 % ±.2 % {YYYYYYYYY}8. % ±. %. % ±. %. % ±. %.1 % ±.1 % { }. % ±. %.1 % ±.1 %. % ±. %. % ±.1 % Ser-39 {} % ±.36 % % ±.5 % % ±.27 % % ±.36 % {YYY}1.23 % ±.32 %.47 % ±.39 %.38 % ±.19 %.44 % ±.43 % {YYY}2.24 % ±.32 %.9 % ±.8 %.21 % ±.18 %.18 % ±.18 % {111} 1.57 % ±.16 % 1.58 % ±.3 % 1.76 % ±.1 % 2.2 % ±.11 % Val-288 {} % ±.55 % % ±.55 % 97.3 % ±.53 % % ±.55 % {YYYYY}1.8 % ±.14 %.12 % ±. %.17 % ±.29 %.9 % ±.16 % {YYYYY}2.98 % ±.31 %.95 % ±.31 % 1.29 % ±.25 % 1.39 % ±.29 % {YYYYY}3.65 % ±.3 %.86 % ±.29 % 1.16 % ±.36 % 1.18 % ±.31 % {YYYYY}4.5 % ±.6 %.5 % ±.7 %.5 % ±.7 %.5 % ±.8 % {11111}.1 % ±.2 %.4 % ±.2 %.3 % ±.3 %.7 % ±.2 % Additional reference: Römisch-Margl, W., Schramek, N., Radykewicz, T., Ettenhuber,., Eylert, E., Huber,., Römisch-Margl, L., Schwarz,., Dobner, M., Demmel, N., Winzenhörlein, B., Bacher, A., and Eisenreich, W. (7) O 2 as a universal metabolic tracer in isotopologue perturbation experiments. Phytochemistry 68,

5 Figure S1: Affinity purification of a chromosomally encoded 95 kda protein using GST-YscM1 and GST-YscM2, respectively, as bait WA , EGTA 27 WA , EGTA kda 94 GST-YscM1 GST-YscM2 GST GST-YscM1 GST-YscM2 GST GST-YscM1 GST-YscM2 GST GST-YscM1 GST-YscM2 GST GST-YscM1 GST-YscM GST-YscM1/ GST-YscM2 GST GST, GST-YscM1 and GST-YscM2 were expressed from vector pgex-4t-3 and bound to glutathione-sepharose as described (Wilharm et al., 3). Glutathione-Sepharose beads preloaded with GST fusions or GST as a control were incubated with lysates of Y. enterocolitica cells (WA-314 harbouring the virulence plasmid pyv; WA-, a WA-314 derivative cured of pyv). Yersiniae were therefore cultured in BHI medium at 27 (no expression of the pyv-encoded T3SS) and in parallel at 37 in the presence of 5 mm EGTA and 1 mm Mgl 2 (expression and secretion of Yops) as indicated. After incubation with yersiniae lysates, beads were washed with PBS and subjected to SDS-PAGE analysis (oomassie staining). The first two lanes show GST fusions bound to beads which were not further incubated with yersiniae lysates. The protein of interest, indicated by arrows, is chromosomally encoded since it is co-purified from WA-314 lysates as well as from lysates of WA- which lacks the pyv plasmid. The interaction is more pronounced with lysates derived from 27 -cultures but is also demonstrable with 37 -cultures in the presence and absence of calcium (data not shown). The results were reproduced twice.

6 Figure S2: YscM1 competes for PEP binding site

7 Figure S3: YscM1 counteracts the stimulation of PEP activity mediated by the allosteric activator acetyl-oa relative PEP activity [%] in the presence of 5 µm acetyl-oa 25% % 15% 1% 5% % YscM1 [µm] PEP activity was measured in a coupled reaction with malate dehydrogenase (MDH). Assays containing 1 mm Tris-Hl, ph 8., 1 mm NaHO 3, 1 mm Mgl 2,.2 mm NADH, 5 mm PEP,.1 µm PEP, 5 µm acetyl-oa, 1 unit of MDH and YscM1 as indicated were preincubated in a reaction volume of 1 µl for 5 min at 37, then the reaction was started by addition of PEP, and oxidation of NADH was monitored at 3 nm. Absorption was recorded over a period of one minute in 2 second-intervals at 37 using a Ultraspec 3 spectrophotometer (Amersham Biosciences). ΔA 3 nm was determined by linear regression from at least four independent experiments and averaged. Relative PEP activity (ΔA i /ΔA ) was plotted as a function of YscM1 concentration. 1% PEP activitiy refers to PEP activity in the absence of acetyl-oa. According to student s t test analysis (one-sided, type 2) PEP inhibition by 2 µm YscM1 is significant with p <.1, for 5 µm YscM1 p-value is below.1, and PEP inhibition with 1 µm YscM1 or above is highly significant with p-values below.1. The data shown are representative of three independent experiments.

8 Figure S4: omplementation of the ppc mutant A,7,6,5 OD 6 nm,4,3 WA-314 WA-314 Δppc WA-314 Δppc pay-ppc,2,1, time [h] WA-314 WA-314 Δppc WA-314 Δppc pay-ppc B Anti-PEP Y. enterocolitica strain WA-314, its isogenic ppc mutant and the ppc mutant trans-complemented with plasmid pay-ppc, were grown in M9 minimal salts supplemented with 1% glucose,.1% casamino acids and 1 µg/ml thiamine as described in legend to Fig. 4. Panel A shows the growth curves based on optical density at 6 nm (triplicates of independent clones; error bars: plus/minus one standard deviation). Panel B shows a Western blot of bacterial pellets obtained from the cultures described in (A). The blot was developed with a serum raised against PEP. omparable results were obtained in two additional experiments.

9 Figure S5: Overproduction of YscM1 in E. coli BL21 at 27 A OD 6 nm,5,4,3,2,1, time [h] pws pws-yscm1 B pws pws-yscm1 Anti-YscM1 Effect of YscM1 overproduction on growth of E. coli BL21 under PEP-requiring conditions at 27 (M9 minimal salts supplemented with 1% glucose,.1% casamino acids and 1 µg/ml thiamine). 1 mm isopropyl β-d-thiogalactoside (IPTG) was added to all cultures to induce overproduction of YscM1. All experiments in triplicate from independent cultures (error bars: plus/minus one standard deviation).

10 Figure S6: MALDI-TOF analysis of a YopE fragment A WA B WA Δppc D 1 6 ΔyscM E 1 6 ΔyscM mass (m/z) Details are described in legend to Fig. 5

11 Figure S7: MALDI-TOF analysis of a YopH fragment A WA B 1 6 WA Δppc D ΔyscM E 1 6 ΔyscM mass (m/z) Details are described in legend to Fig. 5

12 Supplemental Material Details of X-ray scattering experiments and data analysis The synchrotron radiation X-ray scattering data were collected following standard procedures on the small angle scattering beamline X33 (Roessle et al., 7) of the EMBL Hamburg on the storage ring DORIS III of the Deutsches Elektronen Synchrotron (DESY). The scattering data was recorded by means of an image plate with online readout (MAR345, MarResearch, Norderstedt, Germany). The scattering patterns from PEP and the PEP/YscM complexes were measured using a sample - detector distances of 2.4 m, covering the range of momentum transfer.1 < s < 4.5 nm-1 (s = 4π sin(θ)/λ, where θ is the scattering angle and λ =.15 nm is the X-ray wavelength). Repetitive measurements of 1 seconds of the same protein solution were performed in order to check for radiation damage and no aggregation was found during the initial 1 seconds exposure. This initial exposure frame was taken for further analysis. The data were normalised to the intensity of the incident beam; the scattering of the PBS buffer was subtracted and the difference curves were scaled for concentration. All the data processing steps were performed using the program package PRIMUS (Konarev et al., 3). The forward scattering I() and the radius of gyration Rg were evaluated using the Guinier approximation (Guinier and Fournet, 1955) assuming that at very small angles (s < 1.3/Rg) the intensity is represented by I(s) = I() exp(-(srg) 2 /3). These parameters were also computed from the entire scattering patterns using the indirect transform package GNOM (Semenyuk and Svergun, 1991), which also provides the distance distribution

13 function p(r) of the particle. For PEP, the radius of gyration was 5.55 nm, for the PEP/YscM1 and PEP/YscM2 complex it was 5.75 nm and 5.52 nm, respectively. The molecular mass of the proteins was calculated by comparison with the forward scattering from the reference solution of bovine serum albumin (BSA). Low resolution models of PEP, PEP/YscM1, and PEP/YscM2 were built using ab initio modelling techniques. The program DAMMIN (Svergun, 1997) represents the protein shape as an ensemble of M>>1 densely packed beads inside a search volume (a sphere of diameter Dmax). Each bead belongs either to the protein (index=1) or to the solvent (index=), and the shape is thus described by a binary string of length M. Starting from a random string, simulated annealing is employed to find a compact configuration of beads minimising the discrepancy χ between the experimental I exp (s) and the calculated I calc (s)curves: 2 χ = 1 N 1 j I exp ( s ) ci j σ ( s ) j calc 2 ( s j ) (1) where N is the number of experimental points, c is a scaling factor and σ(s j ) is the experimental error at the momentum transfer s j. In an initial step the shape bead modelling of PEP, PEP/YscM1 and PEP/YscM2 was performed without any symmetry constraints. These initial models as well as biochemical data suggested p22 symmetry. For a more detailed analysis ten independent DAMMIN reconstructions with a p22 symmetry constraint were performed and the models were analyzed using the package DAMAVER (Kozin and Svergun, 1). This package aligns all possible pairs of models using the program SUBOMB (Volkov and Svergun, 3), and identifies the most probable model giving the smallest average

14 discrepancy with the rest. Moreover, the averaged model is computed by aligning all other models with the most probable one, computing the density map of beads and drawing the threshold corresponding to the excluded particle volume. The χ-values for the shape reconstruction models vary from 1.62 for PEP, 1.45 for PEP/YscM1 and 1.59 for PEP/YscM2. For model representation an adapted RasMol version was used (Jemilawon et al., 7). References Guinier A, Fournet G (1955) Small Angle Scattering of X-Rays (New York: Wiley). Jemilawon J, Awuah Asiamah I, Bernstein H J, Darakev G, Darakev N, Kamburov P (7) Use of BFlib for Map Support, poster presentation at poster session B, 9 th International onference on Biology and Synchrotron Radiation (BSR 7), -17 August 7, Manchester, UK Konarev PV, Volkov VV, Sokolova AV, Koch MHJ, Svergun DI (3) PRIMUS - a Windows-P based system for small-angle scattering data analysis. J. Appl. rystallogr. 36: Kozin MB, Svergun DI (1) Automated matching of high- and low-resolution structural models. J. Appl. rystallogr. 34: Roessle MW, Klaering R, Ristau U, Robrahn B, Jahn D, Gehrmann T, Konarev P, Round A, Fiedler S, Hermes, Svergun D (7). Upgrade of the small-angle X-ray

15 scattering beamline X33 at the European Molecular Biology Laboratory, Hamburg. Journal of Applied rystallography 4: s19-s194 Semenyuk AV, Svergun DI (1991) GNOM - a program package for small-angle scattering data processing. J. Appl. rystallogr. 24: Svergun DI (1997). Restoring three-dimensional structure of biopolymers from solution scattering. J. Appl. rystallogr. 3: Volkov VV, Svergun DI (3) Uniqueness of ab initio shape determination in small angle scattering. J. Appl. rystallogr. 36: