Structural and mechanistic insights into the transcriptional regulation of chromosomal T6SS by large conjugative plasmid-encoded TetRs in <i>Acinetobacter baumannii</i>

The type VI secretion system (T6SS) is a highly sophisticated nanomachine widely used by bacteria to achieve competitive advantage, important for survival and pathogenicity. In Acinetobacter baumannii, the large conjugative plasmid pAB3 encodes two TetR-family transcriptional regulators, TetR1 and TetR2, that play crucial roles in restricting T6SS function and promoting recipient survival after conjugation. Using X-ray crystallography, we demonstrate that the plasmid-encoded TetRs are structurally homologous but distinct in DNA sequence binding preferences, which we have also mapped out using chromatin immunoprecipitation and validated with electrophoretic mobility shift assay and isothermal titration calorimetry. Transcriptomics and functional studies demonstrated that TetR1 and TetR2 collaboratively downregulate most of the components of the T6SS, but they also impact metabolic pathways and biofilm formation, having an impact on cell morphology, invasiveness, and virulence in a mouse infection model. Taken together, this work provides structural and mechanistic insights into the widespread regulatory influence of plasmid-encoded TetR1 and TetR2 in A. baumannii, providing new insights into plasmid-chromosome cross-regulating pathways and enlightening antimicrobial drug discovery targeting A. baumannii.