Identification of atypical T4SS effector proteins mediating bacterial defense

To remain competitive, proteobacteria use various contact-dependent weapon systems to defend against microbial competitors. The bacterial-killing type IV secretion system (T4SS) is one such powerful weapon. It commonly controls the killing/competition between species by secreting the lethal T4SS effector (T4E) proteins carrying conserved XVIPCD domains into competing cells. In this study, we sought knowledge to understand whether the bacterial-killing T4SS-producing bacteria encode T4E-like proteins and further explore their biological functions. To achieve this, we designed a T4E-guided approach to discover T4E-like proteins that are designated as atypical T4Es. Initially, this approach required scientists to perform simple BlastP search to identify T4E homologs that lack the XVIPCD domain in the genomes of T4SS-producing bacteria. These homologous genes were then screened in Escherichia coli to identify antibacterial candidates (atypical T4Es) and their neighboring detoxification proteins, followed by testing their gene cotranscription and validating their physical interactions. Using this approach, we did discover two atypical T4E proteins from the plant-beneficial Lysobacter enzymogenes and the phytopathogen Xanthomonas citri. We also provided substantial evidence to show that the atypical T4E protein Le1637-mediated bacterial defense in interspecies interactions between L. enzymogenes and its competitors. Therefore, the newly designed T4E-guided approach holds promise for detecting functional atypical T4E proteins in bacterial cells.