Identification of Direct Transcriptional Target Genes of ExoS/ChvI Two-Component Signaling in Sinorhizobium meliloti

Abstract

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 ::Tn 5 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.