The T6SS-Dependent Effector Re78 of Rhizobium etli Mim1 Benefits Bacterial Competition

SIMPLE SUMMARY: Rhizobium etli Mim1 (ReMim1) possesses a protein secretion system type VI (T6SS) that is active in free living and symbiosis. The T6SS is a nanosyringe that secretes proteins called effectors to both eukaryotic and prokaryotic target cells. The ReMim1 T6SS gene cluster encodes for a toxic effector (Re78) together with an immunity protein (Re79) as demonstrated when expressed in Escherichia coli. In addition, it was observed that the toxic role of the Re78 protein is outside the cytoplasm since its toxic effect on E. coli only occurred when a signal peptide was added to it. Re79 is found in ReMim1 periplasm and is T6SS-independent for its translocation. Additionally, the Re78/Re79 pair is involved in bacterial competition and nodule occupancy. A better understanding of the role of this secretion system can be very useful to select highly competitive rhizobia for inoculants. ABSTRACT: The genes of the type VI secretion system (T6SS) from Rhizobium etli Mim1 (ReMim1) that contain possible effectors can be divided into three modules. The mutants in them indicated that they are not required for effective nodulation with beans. To analyze T6SS expression, a putative promoter region between the tssA and tssH genes was fused in both orientations to a reporter gene. Both fusions are expressed more in free living than in symbiosis. When the module-specific genes were studied using RT-qPCR, a low expression was observed in free living and in symbiosis, which was clearly lower than the structural genes. The secretion of Re78 protein from the T6SS gene cluster was dependent on the presence of an active T6SS. Furthermore, the expression of Re78 and Re79 proteins in E. coli without the ReMim1 nanosyringe revealed that these proteins behave as a toxic effector/immunity protein pair (E/I). The harmful action of Re78, whose mechanism is still unknown, would take place in the periplasmic space of the target cell. The deletion of this ReMim1 E/I pair resulted in reduced competitiveness for bean nodule occupancy and in lower survival in the presence of the wild-type strain.