Sinorhizobium meliloti ExoR Is the Target of Periplasmic Proteolysis

Abstract

ABSTRACT Sinorhizobium meliloti ExoR regulates the production of succinoglycan and flagella through the ExoS/ChvI two-component regulatory system. ExoR has been proposed to inhibit the ExoS sensor through direct interaction in the periplasm. To understand how ExoR suppression of ExoS is relieved, which is required for the expression of ExoS/ChvI-regulated symbiosis genes, we characterized wild-type ExoR and ExoR95 mutant proteins. In addition to the previously identified precursor and mature forms of ExoR (designated ExoR p and ExoR m , respectively), we detected a 20-kDa form of ExoR (designated ExoR c20 ) derived from the wild-type ExoR protein, but not from the ExoR95 mutant protein. ExoR c20 was isolated directly from S. meliloti periplasm to identify its N-terminal amino acids and the site of the proteolysis, which is highly conserved among ExoR homologs. ExoR c20 retains the C terminus of the wild-type ExoR. When expressed directly, ExoR c20 did not complement the exoR95 mutation, suggesting that ExoR c20 does not function directly in the ExoR-ExoS/ChvI regulatory pathway and that ExoR m is the functional form of ExoR. A single-amino-acid change (ExoRL81A) at the site of ExoR periplasmic proteolysis resulted in the reduction of the amount of ExoR m and the loss of the regulatory function of the ExoR protein. These findings suggest that ExoR m is a target of periplasmic proteolysis and that the amount of ExoR m could be reduced through effective proteolysis to relieve its suppression of ExoS.