A single point mutation in a TssB/VipA homolog disrupts sheath formation in the type VI secretion system of Proteus mirabilis

Abstract

The type VI secretion (T6S) system is a molecular device for the delivery of proteins from one cell into another. T6S function depends on the contractile sheath comprised of TssB/VipA and TssC/VipB proteins. We previously reported on a mutant variant of TssB that disrupts T6S-dependent export of the self-identity protein, IdsD, in the bacterium Proteus mirabilis. Here we determined the mechanism underlying that initial observation. We show that T6S-dependent export of multiple self-recognition proteins is abrogated in this mutant strain. We have mapped the mutation, which is a single amino acid change, to a region predicted to be involved in the formation of the TssB-TssC sheath. We have demonstrated that this mutation does indeed inhibit sheath formation, thereby explaining the global disruption of T6S activity. We propose that this mutation could be utilized as an important tool for studying functions and behaviors associated with T6S systems.

A Proposed Chaperone of the Bacterial Type VI Secretion System Functions To Constrain a Self-Identity Protein

“…The four mutant strains impaired sheath formation and IdsD secretion to different extents ( Fig. S3 and S4) (16,26,43). In all of these populations, mKate-IdsD-associated fluorescence was patterned as foci along the cell periphery ( Fig IdsD subcellular localization depends on IdsC (DUF4123) but not on IdsE.…”

“…We next examined whether these IdsD-associated foci were found proximal to the T6S machinery. We used a Δids strain-derived strain in which the chromosomal sheath encoded by the tssB gene retains a C-terminal fusion to superfolder green fluorescent protein (sfGFP) (43). Briefly, T6S function occurs as follows: the contraction of a subcellular sheath comprising TssB and TssC proteins plunges an interior tube of Hcp protein hexamers across the cell envelope, through a core membrane complex consisting of several proteins, including TssM (28,35,36,(44)(45)(46)(47)(48).…”

“…To interrogate this hypothesis, we examined the localization of mKate-IdsD in cells in which T6S function was disrupted via distinct mechanisms. Sheath formation was abrogated using a single point mutation in the tssB gene; this strain (CCS05) has been described previously (16,43). The core membrane complex was impaired by disrupting the gene encoding the TssM homolog as described previously (26).…”

Peer pressure from a Proteus mirabilis self-recognition system controls participation in cooperative swarm motility:

“…Further, these data suggest a model for Ids territorial exclusion in mixed swarms ( Figure 6). IdsD is primarily transferred during active swarming when the secretion machinery is produced (21,25,39). During consolidation phase, the presence of IdsD in the absence of a cognate IdsE (resulting in unbound IdsD in recipient cells) causes a shift into a distinct transcriptional state (Figure 1) that is partially due to activation of the stringent response via elevated ppGpp levels (Figure 3).…”

“…Since Ids functions through cell-cell contactdependent secretion of the identity marker IdsD (17), we tested whether IdsD secretion was required for antibiotic tolerance to emerge. We performed this assay using MJT01, which is an ΔidsE-derived strain containing a non-functional T6SS (17,39) and so does not secrete IdsD. No clear difference was observed between this constructed strain and wildtype over three biological repeats ( Supplementary Figure 1).…”