We used the bioluminescent squid symbiont, Vibrio fischeri, to identify essential regulatory factors that control expression and function of a strain-specific T6SS encoded within a genomic island. Random transposon mutagenesis revealed that three genes located on the T6SS2-encoding genomic island are necessary to activate expression of a T6SS reporter. We used a proteomics approach to identify proteins that were differentially abundant in mutant strains compared to the wild type when cells were grown in a high-viscosity media.