Antagonism by the Type VI secretion system of Bacteroides fragilis is controlled by a TetR family regulator and released small molecule

Antagonistic systems of bacteria are often tightly regulated. The human gut Bacteroidales harbor three distinct antagonistic Type VI secretion systems (T6SS), one of which is present only in Bacteroides fragilis, known as the GA3 T6SS. Although this is the best studied of the three T6SSs, little is known about how it is regulated. The gene upstream of the GA3 T6SS locus encodes a TetR family transcriptional regulator (TetRGA3), which we show represses expression of the GA3 T6SS locus. The gene immediately upstream and divergently transcribed from tetRGA3, designated here as lgsGA3, encodes a product of the -oxoamine synthase family of pyridoxal phosphate-dependent enzymes with structural homology to the CqsA autoinducer synthase of the CAI-1 quorum sensing system of Vibrio spp. When lgsGA3 is deleted, transcription of the GA3 T6SS locus is repressed in a TetR-dependent manner. Strains synthesizing LgsGA3 produce a molecule present in the supernatant that likely serves as the TetRGA3 ligand, overcoming TetR transcriptional repression of the GA3 T6SS. We show that GA3 T6SS-specific immunity genes present on two acquired immunity defense islands are also regulated by LgsGA3 coordinating expression of GA3 T6SS antagonism with protection from competitors GA3 T6SS toxins. Production and firing of the GA3 T6SS and subsequent antagonism occurs in bacteria deleted for lgsGA3 when grown with bacteria containing this gene or their supernatants. These data show that the GA3 T6SS is regulated by a small molecule acting through TetRGA3 allowing the bacteria to coordinate antagonistic and protective systems. Significance statementThere are numerous external and intrinsic signals that dictate when bacteria become aggressive and when they activate their defensive systems. We show that B. fragilis strains with a GA3 T6SS synthesize a small molecule released from bacterial cells that acts through TetR family regulators to coordinate transcription of both the antagonistic GA3 T6SS and arrays of immunity genes to competitors GA3 T6SS toxins. Bacteria can respond to this molecule when released from non-kin bacteria, allowing them to sense and respond to a threat from a nearby competitor. The coordinated regulation of the GA3 T6SS and arrays of immunity genes is the second example of regulatory crosstalk between the GA3 T6SS and genes of MGEs through TetR family regulators.