The use of synergistic antibiotic combinations has emerged as a viable approach to contain the rapid spread of antibiotic‐resistant pathogens. Here we report the discovery of a new strongly synergistic pair – microcin J25 and sulfamonomethoxine. The former is a lasso peptide that inhibits the function of RNA polymerase and the latter is a sulfonamide antibacterial agent that disrupts the folate pathway. Key to our discovery was a screening strategy that focuses on an antibiotic (microcin J25) that targets a hub (transcription) in the densely interconnected network of cellular pathways. The rationale was that disrupting such a hub likely weakens the entire network, generating weak links that potentiate the growth inhibitory effect of other antibiotics. We found that MccJ25 potentiates five other antibiotics as well. These results showcase the merit of taking a more targeted approach in the search and study of synergistic antibiotic pairs.

Type VI secretion systems (T6SSs) can deliver diverse toxic effectors into eukaryotic and bacterial cells. Although much is known about the regulation and assembly of T6SS, the translocation mechanism of effectors into the periplasm and/or cytoplasm of target cells remains elusive. Here, we use the Agrobacterium tumefaciens DNase effector Tde1 to unravel the mechanism of translocation from attacker to prey. We demonstrate that Tde1 binds to its adaptor Tap1 through the N‐terminus, which harbors continuous copies of GxxxG motifs resembling the glycine zipper structure found in proteins involved in the membrane channel formation. Amino acid substitutions on G39xxxG43 motif do not affect Tde1–Tap1 interaction and secretion but abolish its membrane permeability and translocation of its fluorescent fusion protein into prey cells. The data suggest that G39xxxG43 governs the delivery of Tde1 into target cells by permeabilizing the cytoplasmic membrane. Considering the widespread presence of GxxxG motifs in bacterial effectors and pore‐forming toxins, we propose that glycine zipper‐mediated permeabilization is a conserved mechanism used by bacterial effectors for translocation across target cell membranes.

Type VI secretion systems (T6SSs) can deliver diverse toxic effectors into eukaryotic and bacterial cells. Although much is known about the regulation and assembly of T6SS, the translocation mechanism of effectors into the periplasm and/or cytoplasm of target cells remains elusive. Here we used the Agrobacterium tumefaciens DNase effector Tde1 to unravel the mechanism of translocation from attacker to prey. We demonstrate that Tde1 loading onto the secretion machinery is mediated via binding to its adaptor through the N-terminus, which harbours continuous copies of GxxxG motifs resembling the glycine zipper structure found in proteins involved in the membrane channel formation. By amino acid substitutions on a conserved glycine zipper motif, we showed G39xxxG43 motif governs the delivery into target cells by permeabilizing the cytoplasmic membrane. The findings demonstrate effector itself mediates target cell entry. Considering the widespread presence of GxxxG motifs in bacterial effectors and pore-forming toxins, we proposed that glycine zipper mediated permeabilization is a conserved mechanism used by bacterial effectors for translocation across target cell membranes.

Type VI secretion system (T6SS) assembles into a contractile nanomachine to inject effectors across bacterial membranes for secretion. Agrobacterium tumefaciens species complex is a group of soil inhabitants and phytopathogens that deploys T6SS as an antibacterial weapon against bacterial competitors at both inter-species and intra-species levels. A. tumefaciens strain 1D1609 genome encodes four effector genes, in which all four are specialized effector harboring a conserved N-terminal PAAR-like DUF4150 domain but distinct C-terminal effector domains. Previous study reported that V2a is a His-Me finger nuclease toxin contributing to DNase-mediated antibacterial activity. However, it remains unknown about the functions and roles of other three effectors. In this study, we identified V2c is another His-Me finger nuclease encoded by 1D1609 genome but with distinct SHH motif differed from AHH motif of V2a. We demonstrated that the ectopic expression of V2c caused growth inhibition, plasmid DNA degradation, and cell elongation in Escherichia coli. The cognate immunity protein, V3c, neutralizes the DNase activity and rescues phenotypes of the growth inhibition and cell elongation. Ectopic expression of V2c DNase-inactive variants retain the cell elongation phenotype while V2a induced cell elongation in a DNase-mediated manner. We also showed that the amino acids of conserved SHH and HNH motifs are responsible for the V2c DNase activity in vivo and in vitro. Notably, V2c also mediated the DNA degradation and cell elongation of target cell in the context of interbacterial competition. Importantly, V2a and V2c function synergistically to exert stronger antibacterial activity against the soft rot phytopathogen, Dickeya dadantii.

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