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Unlabelled: The stringent response, mediated by the (p)ppGpp synthetase RelA and the RNA polymerase-binding protein DksA, is triggered by limiting nutrient conditions. For some bacteria, it is involved in regulation of virulence. We investigated the role of two DksA-like proteins from the Gram-negative nitrogen-fixing symbiont Sinorhizobium meliloti in free-living culture and in interaction with its host plant Medicago sativa The two paralogs, encoded by the genes SMc00469 and SMc00049, differ in the constitution of two major domains required for function in canonical DksA: the DXXDXA motif at the tip of a coiled-coil domain and a zinc finger domain. Using mutant analyses of single, double, and triple deletions for SMc00469(designated dksA),SMc00049, and relA, we found that the ΔdksA mutant but not the ΔSMc00049 mutant showed impaired growth on minimal medium, reduced nodulation on the host plant, and lower nitrogen fixation activity in early nodules, while its nod gene expression was normal. The ΔrelA mutant showed severe pleiotropic phenotypes under all conditions tested. Only S. meliloti dksA complemented the metabolic defects of an Escherichia coli dksA mutant. Modifications of the DXXDXA motif in SMc00049 failed to establish DksA function. Our results imply a role for transcriptional regulator DksA in the S. meliloti-M. sativa symbiosis., Importance: The stringent response is a bacterial transcription regulation process triggered upon nutritional stress.Sinorhizobium meliloti, a soil bacterium establishing agriculturally important root nodule symbioses with legume plants, undergoes constant molecular adjustment during host interaction. Analyzing the components of the stringent response in this alphaproteobacterium helps understand molecular control regarding the development of plant interaction. Using mutant analyses, we describe how the lack of DksA influences symbiosis with Medicago sativa and show that a second paralogous S. meliloti protein cannot substitute for this missing function. This work contributes to the field by showing the similarities and differences of S. meliloti DksA-like proteins to orthologs from other species, adding information to the diversity of the stringent response regulatory system., (Copyright © 2016, American Society for Microbiology. All Rights Reserved.)