Abstract
The rhizobia are Gram-negative bacteria able to establish endosymbiotic relations with legumes. These bacteria form structures at the roots, called nodules in which the bacteria fix the atmospheric nitrogen and transform it into ammonium in exchange to plant photosynthates. The interaction between rhizobia and plant is specific, and one of the elements that mediate this specificity are proteins that the bacteria can translocate inside the vegetal cell. For extracellular protein translocation the bacteria have different systems of secretion. One of these systems is the Type VI Secretion System (T6SS). This study describes the regulation and function of the T6SS genes of Rhizobium etli Mim1 (ReMim1), an endosymbiont of common beans. The expression is modulated by two divergent promoters named PTssA and PHcp in according to its orientation to genes, that are induced differentially in different conditions. Both promoters are induced in free-living conditions and in symbiosis with beans. Activation is greater in rich media and at neutral pH and at exponential growth phase at OD600> 0.25. These promoters could be regulated by quorum sensing since their activity is reduced to one third in a Rhizobium strain with mutated quorum sensing regulatory genes. We have studied the function of six genes (re84-89) that are part of the T6SS gene cluster and are not conserved in other bacteria and that would not have a structural role and could participate in the interaction with other bacteria or in the symbiosis. A bioinformatic analysis suggested that protein Re86 could code for an antitoxin and Re87 for methyltransferase of unknown function. A deletion mutant for re84-89 was performed and no interbacterial competition was observed when the mutant was co-incubated with the wild-type strain. Finally, a different pattern appeared in the immunodetection of phosphorylated MAPKs when bean roots were exposed to a culture supernatant of ReMim1 in contrast to the one of a mutant defective in the formation of a T6SS, suggesting that the plant could recognize any T6SS structural elements or secreted effectors inducing a primary immune response.
