Activation of the Type VI Secretion System in the Squid Symbiont Vibrio fischeri Requires the Transcriptional Regulator TasR and the Structural Proteins TssM and TssA

Abstract

Bacteria have evolved diverse strategies to compete for a niche, including the type VI secretion system (T6SS), a contact-dependent killing mechanism. T6SSs are common in bacterial pathogens, commensals, and beneficial symbionts, where they affect the diversity and spatial structure of host-associated microbial communities. Although T6SS gene clusters are often located on genomic islands (GIs), which may be transferred as a unit, the regulatory strategies that promote gene expression once the T6SS genes are transferred into a new cell are not known. We used the squid symbiont, Vibrio fischeri , to identify essential regulatory factors that control expression of a strain-specific T6SS encoded on a GI. We found that a transcriptional reporter for this T6SS is active only in strains that contain the T6SS-encoding GI, suggesting the GI encodes at least one essential regulator. A transposon screen identified seven mutants that could not activate the reporter. These mutations mapped exclusively to three genes on the T6SS-containing GI that encode two essential structural proteins (a TssA-like protein and TssM) and a transcriptional regulator (TasR). Using T6SS reporters, RT-PCR, competition assays, and differential proteomics, we found that all three genes are required for expression of many T6SS components, except for the TssA-like protein and TssM, which are constitutively expressed. Based on these findings, we propose a model whereby T6SS expression requires conserved structural proteins, in addition to the essential regulator TasR, and this ability to self-regulate may be a strategy to activate T6SS expression upon transfer of T6SS-encoding elements into a new bacterial host. Importance Interbacterial weapons like the T6SS are often located on mobile genetic elements and their expression is highly regulated. We found that two conserved structural proteins are required for T6SS expression in Vibrio fischeri . These structural proteins also contain predicted GTPase and GTP binding domains, suggesting their role in promoting T6SS expression may involve sensing the energetic state of the cell. Such a mechanism would provide a direct link between T6SS activation and cellular energy levels, providing a “checkpoint” to ensure the cell has sufficient energy to build such a costly weapon. Because these regulatory factors are encoded within the T6SS gene cluster, they are predicted to move with the genetic element to activate T6SS expression in a new host cell.

The type-VI secretion system of the beneficial symbiont Vibrio fischeri

“…More specifically, when polymerization extends the inner tube/sheath complex to the opposite side of the cell, TagA localizes to the distal end and prevents further polymerization, which would otherwise bend the sheath and cause its detachment from the membrane anchor [97, 98]. In ES401, overexpression of the VFFQA001_15530 homologue (TssA VF ) inhibits T6SS activity [94], which is consistent with observations of TagA overexpression preventing sheath assembly in V. cholerae [98].…”

“…This tasR mutant also exhibits lower transcriptional activity of the hcp promoter [94], which suggests TasR is a positive regulator of T6SS2 in V. fischeri, like VP1407 in V. parahaemolyticus. Expression of tasR in trans restored the ability of the tasR mutant to kill ES114 in vitro [94], which demonstrated genetic complementation. Determining how TasR regulates the hcp promoter and whether the corresponding mechanism involves VasH and σ 54 will be important to gain insight into the regulation of T6SS in V. fischeri, and this knowledge will likely inform how TasR homologues functions in other bacteria like V. parahaemolyticus.…”

“…parahaemolyticus . Expression of tasR in trans restored the ability of the tasR mutant to kill ES114 in vitro [94], which demonstrated genetic complementation. Determining how TasR regulates the hcp promoter and whether the corresponding mechanism involves VasH and σ 54 will be important to gain insight into the regulation of T6SS in V.…”

Prevalence and diversity of type VI secretion systems in a model beneficial symbiosis

“…The break in the assemblies in this locus could account for the missing genes, which would go undetected by our analysis criteria. The accessory genes in T6SS2 also included pkinase, FHA, and DUF4150 domain-containing proteins similar to those found in T6SS1, as well as the recently described tasL gene, which is required for cell–cell adhesion and target specificity ( Speare et al, 2022 ), and the tasR ( Smith et al, 2021 ) and vasH ( Guckes et al, 2020 ) regulators.…”

Calcium Promotes T6SS-Mediated Killing and Aggregation between Competing Symbionts

“…To determine whether calcium impacts T6SS2 activity in liquid, we performed coincubation assays using two incompatible V. fischeri strains: a target strain, ES114, that does not encode T6SS2, and an inhibitor strain, ES401, that kills ES114 using T6SS2 ( 15 , 19 , 20 ). Strains were differentially tagged with unique antibiotic resistance genes, mixed in a 1:1 ratio, and incubated for 12 to 15 h with shaking in low-viscosity (centipoise [cP] of 1) liquid Luria-Bertani with salts (LBS) or tryptone broth-saline (TBS) ( 18 ) media without or with 10 mM CaCl 2 , which is comparable to the calcium concentration in seawater ( 21 ).…”