Figure S1. Organization of the P. aeruginosa (PA) HSI-I operon containing tag genes and comparison with P. mendocina ymp (Pmen), P. fluorescens (PFL) and P. brassicacearum (PSEBR). Orthologues are represented in the same colour. Gene and COG numbers are indicated.

Figure S2. TagQ localization to the OM is not influenced by TagTS. Complete analysis of discontinuous sucrose gradients of PAO1ΔretS and PAO1ΔretSΔtagTS are shown. NADH oxidase activity and XcpY are used as IM markers, and porines are used as OM markers. TagQ was detected using specific antibodies. Note that the NADH oxidase activity is represented relative to the fraction of highest activity. IM and OM are indicated.

Figure S3. The TagTS complex does not influence TssJ1-mCherry localization to the outer membrane. Discontinuous sucrose gradient separation of TssJ1-mCherry in PAO1ΔretS and PAO1ΔretSΔtagTS strains. NADH oxidase activity and controls were as described in Fig. S2. Fusion protein TssJ1-mCherry was detected by anti-mCherry antibodies.

Figure S4. TagTS does not influence TagR localization to the OM. Discontinuous sucrose gradient separations shown in Fig. S2 were further analysed using anti-TagR antibodies, showing that TagR is localized to the OM both in PAO1ΔretS and PAO1ΔretSΔtagTS.

Figure S5. TagR does not play a role in the OM localization of TagQ.

A. Confocal microscopy analysis of PAO1ΔretSΔtagR expressing GFP and harbouring TagQ-mCherry. White arrows show the OM of P. aeruginosa that remains partially attached to spheroplasts after lysozyme treatment (see text for details). Bact: bacteria, Sphero: spheroplasts. The bar represents 1 µm.

B. Discontinuous sucrose gradient separation of the IM and OM of PAO1ΔretSΔtagR. All fractions were analysed as described in Fig. S2.

Table S1. List of oligonucleotides used for genetic constructions.

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