ABSTRACT
The Sinorhizobium meliloti ExoS/ChvI two-component signaling pathway is required for the development of a nitrogen-fixing symbiosis between S. meliloti and its plant hosts. ExoS/ChvI also has important roles in regulating succinoglycan production, biofilm formation, motility, nutrient utilization, and the viability of free-living bacteria. Previous microarray experiments with an exoS96::Tn5 mutant indicated that ExoS/ChvI influences the expression of a few hundred genes, complicating the investigation of which downstream genes respond directly or indirectly to ExoS/ChvI regulation. To focus our study of ExoS/ChvI transcriptional target genes, we performed transcriptional profiling with chvI gain-of-function and reduced-function strains. The chvI gain-of-function strain that we used contains a dominant gain-of-function chvI allele in addition to wild-type chvI. We identified genes that, relative to their expression level in the wild type, are both upregulated in the chvI gain-of-function strain and downregulated in the reduced-function strain or vice versa. Guided by this focused set of genes, we performed gel mobility shift assays and demonstrated that ChvI directly binds the intergenic regions upstream of ropB1, SMb21440, and SMc01580. Furthermore, DNase I footprint analysis of the region upstream of SMc01580 identified a specific DNA sequence bound by ChvI and allowed the discovery of a possible motif for ChvI binding. Our results provide insight into the mechanism of how ExoS/ChvI regulates its downstream targets and lay a foundation for studying this conserved pathway with critical roles in free-living and symbiotic bacteria.
Subject(s)
Bacterial Proteins/physiology , Gene Expression Regulation, Bacterial/physiology , Sinorhizobium meliloti/metabolism , Transcription Factors/physiology , Bacterial Proteins/genetics , Bacterial Proteins/metabolism , Base Sequence , DNA/metabolism , DNA Footprinting , Electrophoretic Mobility Shift Assay , Gene Expression Regulation, Bacterial/genetics , Molecular Sequence Data , Oligonucleotide Array Sequence Analysis , Protein Binding , Sinorhizobium meliloti/genetics , Transcription Factors/genetics , Transcription Factors/metabolismABSTRACT
Sinorhizobium meliloti requires ExoS/ChvI two-component signalling to establish a nitrogen-fixing symbiosis with legume hosts. The importance of ExoS/ChvI signalling in microbe-host interactions is underscored by the requirement of ExoS/ChvI orthologues for virulence of the related alpha-proteobacteria Agrobacterium tumefaciens and Brucella abortus. In S. meliloti, ExoS/ChvI is a key regulator of gene expression for exopolysaccharide synthesis, biofilm formation, motility, nutrient utilization and free-living viability. Previously, we showed that the novel conserved regulator ExoR interacts genetically with both ExoS and ChvI, and localizes to the periplasm of S. meliloti. Here, we show that ExoR physically associates with ExoS and that this association is important for regulating ExoS/ChvI signalling. We have identified point mutations in the Sel1-like repeat region of ExoR that disrupt binding to ExoS and cause a dramatic increase in ExoS/ChvI-dependent gene expression. Furthermore, we have found that physical interaction with ExoS stabilizes the ExoR protein. Together, our results indicate that ExoR binds to ExoS in the periplasm of S. meliloti to inhibit ExoS/ChvI activity, and that ExoR represents a novel periplasmic inhibitor of two-component signalling.