Updated project metadata. The bacterial Type VI secretion system (T6SS) is important for bacterial competition as well as virulence in many Gram-negative bacteria, including human pathogens. T6SS is evolutionarily related to the contractile phage tails and assembles as a cell envelope attached organelle. The assembly progresses from formation of a membrane complex to assembly of a baseplate followed by copolymerization of a sheath-tube. Rapid sheath contraction propels the rigid inner tube with associated effectors into target cells. To understand the assembly and stoichiometry of this nanomachine, we applied targeted proteomics to determine the protein abundances of the key T6SS components in three model bacteria. The overall stoichiometry of the components is conserved across species and it agrees with the expected composition of the nanomachine. However, there are also species-specific variations of certain components, which may explain the observed differences in the respective dynamics of T6SS. Furthermore, changes in the protein abundance during different growth conditions point to possible mechanisms of regulation of T6SS dynamics. The combination of live-cell imaging and mass-spectrometry analysis suggests that a baseplate component TssE of V. cholerae might undergo an active proteolysis, which together with an effector protein VasX could be involved in regulation of baseplate dynamics and thus T6SS activity.