MS Defense Seminar of Bhavyaa Tyagi

Cell density as a potential regulator of Type VI Secretion System in Vibrio fischeri

When bacteria colonize a system, they must compete for space and nutrients. During the competition process, some bacteria can build a contact-dependent molecular weapon that injects toxins inside competitor cells to eliminate them. One such molecular weapon is the Type VI Secretion System (T6SS), found in various bacteria colonizing marine hosts. Although this weapon can provide bacteria a competitive advantage during host colonization, we know little about the proteins that regulate the T6SS weapon in a host. We utilize the squid-vibrio symbiosis to understand how bacterial cells may regulate their T6SS weapon during host colonization. A previous random transposon screen identified additional genetic factors required to activate the T6SS in Vibrio fischeri. Two mutants with transposon insertions in genes not previously known to affect T6SS were isolated. These two mutants could not eliminate a competitor strain in coculture. To test whether the transposon insertion was preventing the T6SS activity, the transposon insertions were moved into a fresh wild-type background using natural transformation, and the remade mutants were tested for their killing activity. The remade mutants were still able to eliminate the target cells, and upon performing whole genome sequencing analysis, it was revealed that both original transposon mutants had a point mutation in the luxO gene.

Previous work showed that LuxO is a quorum-sensing regulator active at low cell density, and a past transposon screen identified a similar mutation (luxO*) that locks the protein in a constitutively active state that mimics low cell density. To explore whether such a gain-of-function mutation in LuxO could regulate the T6SS, we overexpressed luxO* in a wild-type V. fischeri strain and co-incubated it with the target cell. The strains expressing the constitutively active luxO* allele were unable to eliminate the target strain for two out of four luxO* alleles tested, suggesting active LuxO could prevent T6SS-mediated killing. In instances where cells expressing the luxO* allele eliminated the target but exhibits reduced killing; perhaps, it could be the wild-type copy of LuxO overrides the overexpressed luxO* allele. These findings lead us to infer that LuxO, which is active either at low cell density or through a gain-of-function mutation (luxO*), may repress T6SS activation. Our current research model suggests that LuxO may repress expression of the T6SS weapon at low cell density, saving the cost of the unnecessary firing of the molecular weapon when contact with competitors is less likely