Phylogenetically diverse bacteria respond to various stress conditions by mounting a general stress response (GSR) resulting in the induction of protection or damage repair functions. In α-proteobacteria, the GSR is induced by a regulatory cascade consisting of the extracytoplasmic function (ECF) σ factor σ, its anti-σ factor NepR, and the anti-anti-σ factor PhyR. We have reported previously that σ and PhyR of Bradyrhizobium diazoefficiens (formerly named Bradyrhizobium japonicum), the nitrogen-fixing root nodule symbiont of soybean and related legumes, are required for efficient symbiosis; however, the precise role of the GSR remained undefined. Here, we analyze the symbiotic defects of a B. diazoefficiens mutant lacking σ by comparing distinct infection stages of enzymatically or fluorescently tagged wild-type and mutant bacteria. Although root colonization and root hair curling were indistinguishable, the mutant was not competitive, and showed delayed development of emerging nodules and only a few infection threads. Consequently, many of the mutant-induced nodules were aborted, empty, or partially colonized. Congruent with these results, we found that σ was active in bacteria present in root-hair-entrapped microcolonies and infection threads but not in root-associated bacteria and nitrogen-fixing bacteroids. We conclude that GSR-controlled functions are crucial for synchronization of infection thread formation, colonization, and nodule development.

The general stress response (GSR) enables bacteria to sense and overcome a variety of environmental stresses. In alphaproteobacteria, stress-perceiving histidine kinases of the HWE and HisKA_2 families trigger a signaling cascade that leads to phosphorylation of the response regulator PhyR and consequently to activation of the GSR sigma factor σEcfG. In the nitrogen-fixing bacterium Bradyrhizobium diazoefficiens, PhyR and σEcfG are crucial for tolerance against a variety of stresses under free-living conditions and also for efficient infection of its symbiotic host soybean. However, the molecular players involved in stress perception and activation of the GSR remained largely unknown. In this work, we first showed that a mutant variant of PhyR where the conserved phosphorylatable aspartate residue D194 was replaced by alanine (PhyRD194A) failed to complement the ΔphyR mutant in symbiosis, confirming that PhyR acts as a response regulator. To identify the PhyR-activating kinases in the nitrogen-fixing symbiont, we constructed in-frame deletion mutants lacking single, distinct combinations, or all of the eleven predicted HWE and HisKA_2 kinases, which we named HhkA through HhkK. Phenotypic analysis of the mutants and complemented derivatives identified two functionally redundant kinases, HhkA and HhkE, that are required for nodulation competitiveness and during initiation of symbiosis. Using σEcfG activity reporter strains, we further showed that both HhkA and HhkE activate the GSR in free-living cells exposed to salt and hyperosmotic stress. In conclusion, our data suggest that HhkA and HhkE trigger GSR activation in response to osmotically stressful conditions which B. diazoefficiens encounters during soybean host infection.