27Francisella tularensis is a facultative intracellular pathogen that causes the zoonotic 28 disease tularemia in human and animal hosts. This bacterium possesses a non-canonical 29 type VI secretion systems (T6SS) required for phagosomal escape and access to its 30 replicative niche in the cytosol of infected macrophages. KCl stimulation has been previously 31 used to trigger assembly and secretion of the Francisella T6SS in culture. We found that the 32 amounts of essentially all the TSS6 proteins remained unchanged upon KCl stimulation. We 33 therefore hypothesized that a post-translational modification might be involved in T6SS 34 assembly. A whole cell phosphoproteomic analysis allowed us to identify a unique 35 phosphorylation site on IglB, the TssC homologue and key component of the T6SS sheath. 36 Importantly, the phosphorylated form of IglB was not present in the contracted sheath and 3D 37 modeling indicated that the charge repulsion provoked by addition of a phosphogroup on 38 tyrosine 139 was likely to weaken the stability of the sheath structure. Substitutions of the 39 phosphorylatable residue of IglB (tyrosine 139) with alanine or with phosphomimetics 40 prevented T6SS formation and totally impaired phagosomal escape. In contrast, the 41 substitution with the non-phosphorylatable aromatic analog phenylalanine impaired but did 42 not prevent phagosomal escape and cytosolic bacterial multiplication in J774-1 43 macrophages. Altogether these data suggest that phosphorylation of the sheath participates 44 to T6SS disassembly. Post-translational modifications of the sheath may represent a 45 previously unrecognized mechanism to finely modulate the dynamics of T6SS assembly-46 disassembly. 47 48 Data are available via ProteomeXchange with identifier PXD012507. 49 50 3 Synopsis 51 52Francisella possesses a non-canonical T6SS that is essential for efficient phagosomal 53 escape and access to the cytosol of infected macrophages. KCl stimulation has been 54 previously used to trigger assembly and secretion of the Francisella T6SS in culture. We 55 found that KCl stimulation did not result in an increased production of TSS6 proteins. We 56 therefore hypothesized that a post-translational modification might be involved in T6SS 57 assembly. Using a global and site-specific phosphoproteomic analysis of Francisella we 58 identified a unique phosphorylation site on IglB, the TssC homologue and a key component 59 of the T6SS contractile sheath. We show that this site plays a critical role in T6SS biogenesis 60 and propose that phosphorylation may represent a new mechanism affecting the dynamics of 61 sheath formation. 62 63 64 65 66 67Francisella tularensis is the causative agent of the zoonotic disease tularemia (Luque-68