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, The grey dots correspond to the nine values from three 607 independent biological replicates

, Figure 3. TssL and TagL interact via their TMHs. (A) Schematic representation of the 610

. Eaec-t6ss, TssL 611 (red, N-terminal cytoplasmic domain [TssL C ]; green, C-terminal TMH) and TagL (blue, N-612 terminal membrane domain, The membrane complex comprises TssJ (dark grey), TssM (light grey)

, The baseplate is shown in green. The contractile tail in shown in blue. The TssA protein, 614 located at the distal end of the tail is shown in red. IM, inner membrane

. Om, B) Schematic representation of EAEC TssL and TagL highlighting 616 their topologies (38,42) and different domains (same colours as Fig. 3A). The three TagL, p.617

, Igepal ® CA-618 630-solubilized extracts of E. coli cells producing the indicated proteins or protein variants 619 were mixed with Nickel magnetic beads to precipitate 6×His-tagged TagL and interacting 620 partners. The total lysates (T) and eluted (E) material were, p.621

. Sds-page, His (TagL, upper panel) and anti-HA (TssL, lower 622 panel) monoclonal antibodies. The positions of TssL, TagL and their variants are indicated on 623 the right. Molecular weight markers

, Figure 4. Localization, dynamics, and distribution of TagL. (A) Representative

, fluorescence microscopy time-lapse recording of wild-type EAEC cells producing sfGFP-627

. Tagl, Individual images were taken every 30 s. The white arrowhead highlights the position 628 of one sfGFP-TagL focus showing that it is static over time. Scale bar, 2 ?m. (B) Percentage 629 of cells with 0, 1, 2 or >2 sfGFP-TagL foci

, The mean number of foci per cell is 0.67 ± 0.86. (C) Spatial distribution of sfGFP-TagL foci

, 743 cells on a single cell (from blue to yellow, see 632 heatmap color chart on left). (D) and (E) Representative cells producing sfGFP-TagL and 633 mCherry-TssL (D) or TssB-mCherry (E), p.634

, an overlay of the sfGFP and mCherry channels (merge) and a 635 schematic representation. White and blue arrowheads in panel (D) indicate isolated TssL foci 636 and TssL/TagL foci, respectively. Scale bar, 1 ?m. (F) Representative fluorescence 637 microscopy time-lapse recording of a wild-type EAEC cell producing sfGFP

, Individual images were taken every 30 s. A schematic representation is shown 639 below. Scale bar, vol.1

?. Wt, . ?tssm, . ?tssl, and ?. ?mlte, ?tssA and ?tssBC cells 643 producing sfGFP-TagL. Statistical analyses are shown in Fig, Figure 5. Localization and distribution of TagL. (A) Representative fluorescence 642 microscopy fields of, p.644

, Representative fluorescence microscopy fields of wild-type (WT, upper panels) or ?tagL 645 (lower panels) cells producing sfGFP-TssM, sfGFP-TssL, sfGFP-TssA, TssK-sfGFP or TssB-646

, Statistical analyses are shown in Fig. S3. Scale bar, 1 ?m. (C) Schematic representation 647 of the EAEC T6SS biogenesis pathway based on previous studies (30, vol.32, p.648

, highlighting the position of TagL (shown in green). TagL is recruited after completion of the 649

. Mc, TssA recruitment, baseplate positioning 650 (represented by TssK subunit in this study), and tail tube/sheath polymerization, p.651

, by TssBC in this study) are not required for TagL localization, but TagL is necessary for tail 652 tube/sheath polymerization