Lays, C. | bioRxiv Science

bioRxiv2019

A genetic selection reveals functional metastable structures embedded in a toxin-encoding mRNA

Post-transcriptional regulation plays important roles to finely tune gene expression in bacteria. In particular, regulation of type I toxin-antitoxin (TA) systems is achieved through sophisticated mechanisms involving toxin mRNA folding. Here, we set up a genetic approach to decipher the molecular underpinnings behind the regulation of a type I TA in Helicobacter pylori. We used the lethality induced by chromosomal inactivation of the antitoxin to select mutations that suppress toxicity. We found that single point mutations are sufficient to allow cell survival. Mutations located either in the 5 untranslated region or within the open reading frame of the toxin hamper its translation by stabilizing stem-loop structures that sequester the Shine-Dalgarno sequence. We propose that these short hairpins correspond to metastable structures that are transiently formed during transcription to avoid premature toxin expression. This work uncovers the co-transcriptional inhibition of translation as an additional layer of TA regulation in bacteria.

molecular biology↗

bioRxiv2018

Phosphorylation of the Type VI secretion system sheath controls Francisella virulence

Francisella tularensis is a facultative intracellular pathogen that causes the zoonotic disease tularemia in human and animal hosts. This bacterium possesses a non-canonical type VI secretion systems (T6SS) required for phagosomal escape and access to its replicative niche in the cytosol of infected macrophages. KCl stimulation has been previously used to trigger assembly and secretion of the Francisella T6SS in culture. We found that the amounts of essentially all the TSS6 proteins remained unchanged upon KCl stimulation. We therefore hypothesized that a post-translational modification might be involved in T6SS assembly. A whole cell phosphoproteomic analysis allowed us to identify a unique phosphorylation site on IglB, the TssC homologue and key component of the T6SS sheath. Importantly, the phosphorylated form of IglB was not present in the contracted sheath and 3D modeling indicated that the charge repulsion provoked by addition of a phosphogroup on tyrosine 139 was likely to weaken the stability of the sheath structure. Substitutions of the phosphorylatable residue of IglB (tyrosine 139) with alanine or with phosphomimetics prevented T6SS formation and totally impaired phagosomal escape. In contrast, the substitution with the non-phosphorylatable aromatic analog phenylalanine impaired but did not prevent phagosomal escape and cytosolic bacterial multiplication in J774-1 macrophages. Altogether these data suggest that phosphorylation of the sheath participates to T6SS disassembly. Post-translational modifications of the sheath may represent a previously unrecognized mechanism to finely modulate the dynamics of T6SS assembly-disassembly. Data are available via ProteomeXchange with identifier PXD012507. SynopsisFrancisella possesses a non-canonical T6SS that is essential for efficient phagosomal escape and access to the cytosol of infected macrophages. KCl stimulation has been previously used to trigger assembly and secretion of the Francisella T6SS in culture. We found that KCl stimulation did not result in an increased production of TSS6 proteins. We therefore hypothesized that a post-translational modification might be involved in T6SS assembly. Using a global and site-specific phosphoproteomic analysis of Francisella we identified a unique phosphorylation site on IglB, the TssC homologue and a key component of the T6SS contractile sheath. We show that this site plays a critical role in T6SS biogenesis and propose that phosphorylation may represent a new mechanism affecting the dynamics of sheath formation.

microbiology↗