ExoR regulates the creation of succinoglycan and flagella through the ExoS/ChvI two-component regulatory system. ExoRc20) derived from the wild-type ExoR protein but not from your ExoR95 mutant protein. ExoRc20 was isolated directly from periplasm to identify its N-terminal amino acids and the site of the proteolysis which is usually highly conserved among ExoR homologs. ExoRc20 retains the C terminus of the wild-type ExoR. When expressed directly ExoRc20 did not match the mutation suggesting Lannaconitine that ExoRc20 does not function directly in the ExoR-ExoS/ChvI regulatory pathway and that ExoRm is the functional form of ExoR. A single-amino-acid switch (ExoRL81A) at the site of ExoR periplasmic proteolysis resulted in the reduction of the amount of ExoRm and the loss of the regulatory function of the ExoR protein. These findings suggest that ExoRm is usually a target of periplasmic proteolysis and that the amount of ExoRm could be reduced through effective proteolysis to relieve its suppression of ExoS. INTRODUCTION The Gram-negative ground bacterium establishes a nitrogen-fixing symbiosis with its herb host alfalfa (exopolysaccharides succinoglycan (SG) exopolysaccharide II (EPSII) or capsular polysaccharide (KPS). SG has been shown to be much more effective than the other two polysaccharides EPSII and KPS at eliciting the formation of contamination threads (3 7 23 29 39 The structure and biosynthetic pathway of succinoglycan have been well documented although its precise role in eliciting the formation of infection threads remains unknown (20-22 30 43 51 Succinoglycan production is usually inversely coregulated with flagellum production by a single transmission transduction pathway consisting of the ExoR protein and the ExoS/ChvI two-component regulatory system (55) and the EmmABC system (37). While the transcription of succinoglycan biosynthesis genes is usually upregulated by the mutations and cells to switch from free living to symbiosis inside the root nodules. The gene was initially recognized through isolation of the mutation which was later recognized and sequenced (10 41 The gene encodes a 268-amino acid ExoR protein with a conserved transmission peptide for exporting the protein to the bacterial periplasm as confirmed in recent findings (53). In addition to regulating succinoglycan and flagellum production ExoR has been shown to be involved in regulating biofilm production and lipopolysaccharide modifications (16 28 The ExoR protein has been found to regulate the expression of a large number of gene functions in very different metabolic pathways suggesting that ExoR plays other important functions (53). ExoR homologs have been found and characterized in and ExoS and ChvI proteins form a typical bacterial two-component transmission transduction system (8 38 The ExoS protein consists of a large periplasmic domain name and a cytoplasmic kinase domain name and it has been shown to phosphorylate ChvI directly (8). Recent analysis of and deletion mutants has shown that this ExoS/ChvI system is essential for symbiosis and that these two proteins regulate the expression of a variety of genes involved in carbon metabolism Rabbit polyclonal to ABHD14B. and many other functions (2 50 These findings are consistent with the results of a transcriptome analysis of the mutant (53). Collectively these findings Lannaconitine suggest that the ExoS/ChvI system plays an essential role in preparing cells for their transformation from free-living to nitrogen-fixing cells inside the root nodules. The importance of the ExoS/ChvI system was further highlighted by the finding that two of its close homologs are essential for host infections in and (4 15 24 31 45 Lannaconitine Recent genetic and biochemical data suggest that ExoR ExoS and ChvI form a single transmission transduction pathway (5 53 The ExoR protein has been localized to the periplasm of cells (53) as was confirmed by our unpublished data. ExoR has been found to exist in two forms the 29-kDa full-length precursor form (ExoRp) and the 26-kDa mature form without its predicted transmission peptide (ExoRm) in wild-type cells (5). Coimmunoprecipitation of ExoR and ExoS suggested that they form protein complexes (5). Increased expression of the gene also led to accumulation of ExoRm suggesting that ExoS stabilizes ExoR in the ExoR-ExoS complex. The ExoR-ExoS conversation was interrupted by single-amino-acid changes in either the ExoR protein or the periplasmic domain name of ExoS Lannaconitine (5). Taken together these findings led to a proposed model in which ExoR interacts with ExoS to form a protein complex that maintains ExoS in the off state.