To colonize the host and cause disease, the human enteropathogen Clostridioides difficile must sense, respond, and adapt to the harsh environment of the gastrointestinal tract. We showed that the production and degradation of cyclic diadenosine monophosphate (c-di-AMP) were necessary during different phases of C. difficile growth, environmental adaptation, and infection. The production of this nucleotide second messenger was essential for growth because it controlled the uptake of potassium and also contributed to biofilm formation and cell wall homeostasis, whereas its degradation was required for osmotolerance and resistance to detergents and bile salts. The c-di-AMP binding transcription factor BusR repressed the expression of genes encoding the compatible solute transporter BusAA-AB. Compared with the parental strain, a mutant lacking BusR was more resistant to hyperosmotic and bile salt stresses, whereas a mutant lacking BusAA was more susceptible. A short exposure of C. difficile cells to bile salts decreased intracellular c-di-AMP concentrations, suggesting that changes in membrane properties induce alterations in the intracellular c-di-AMP concentration. A C. difficile strain that could not degrade c-di-AMP failed to persist in a mouse gut colonization model as long as the wild-type strain did. Thus, the production and degradation of c-di-AMP in C. difficile have pleiotropic effects, including the control of osmolyte uptake to confer osmotolerance and bile salt resistance, and its degradation is important for host colonization.

Paraburkholderia phymatum STM815 is a nitrogen-fixing endosymbiont that nodulate the agriculturally important Phaseolus vulgaris and several other host plants. We previously showed that the nodules induced by a STM815 mutant of the gene encoding the master regulator of nitrogen fixation NifA showed no nitrogenase activity (Fix−) and increased in number compared to P. vulgaris plants infected with the wild-type strain. To further investigate the role of NifA during symbiosis, nodules from P. phymatum wild-type and nifA mutants were collected and analyzed by metabolomics and dual RNA-Sequencing, allowing us to investigate both host and symbiont transcriptome. Using this approach, several metabolites’ changes could be assigned to bacterial or plant responses. While the amount of the C4-dicarboxylic acid succinate and of several amino acids was lower in Fix− nodules, the level of indole-acetamide (IAM) and brassinosteroids increased. Transcriptome analysis identified P. phymatum genes involved in transport of C4-dicarboxylic acids, carbon metabolism, auxin metabolism and stress response to be differentially expressed in absence of NifA. Furthermore, P. vulgaris genes involved in autoregulation of nodulation (AON) are repressed in nodules in absence of NifA potentially explaining the hypernodulation phenotype of the nifA mutant. These results and additional validation experiments suggest that P. phymatum STM815 NifA is not only important to control expression of nitrogenase and related enzymes but is also involved in regulating its own auxin production and stress response. Finally, our data indicate that P. vulgaris does sanction the nifA nodules by depleting the local carbon allocation rather than by mounting a strong systemic immune response to the Fix− rhizobia.

Homocitrate is an essential component of the iron-molybdenum cofactor of nitrogenase, the bacterial enzyme that catalyzes the reduction of dinitrogen (N2) to ammonia. In nitrogen-fixing and nodulating alpha-rhizobia, homocitrate is usually provided to bacteroids in root nodules by their plant host. In contrast, non-nodulating free-living diazotrophs encode the homocitrate synthase (NifV) and reduce N2 in nitrogen-limiting free-living conditions. Paraburkholderia phymatum STM815 is a beta-rhizobial strain, which can enter symbiosis with a broad range of legumes, including papilionoids and mimosoids. In contrast to most alpha-rhizobia, which lack nifV, P. phymatum harbors a copy of nifV on its symbiotic plasmid. We show here that P. phymatum nifV is essential for nitrogenase activity both in root nodules of papilionoid plants and in free-living growth conditions. Notably, nifV was dispensable in nodules of Mimosa pudica despite the fact that the gene was highly expressed during symbiosis with all tested papilionoid and mimosoid plants. A metabolome analysis of papilionoid and mimosoid root nodules infected with the P. phymatum wild-type strain revealed that among the approximately 400 measured metabolites, homocitrate and other metabolites involved in lysine biosynthesis and degradation have accumulated in all plant nodules compared to uninfected roots, suggesting an important role of these metabolites during symbiosis.

A century after the discovery of rhizobia, the first Beta-proteobacteria species (beta-rhizobia) were isolated from legume nodules in South Africa and South America. Since then, numerous species belonging to the Burkholderiaceae family have been isolated. The presence of a highly branching lineage of nodulation genes in beta-rhizobia suggests a long symbiotic history. In this review, we focus on the beta-rhizobial genus Paraburkholderia, which includes two main groups: the South American mimosoid-nodulating Paraburkholderia and the South African predominantly papilionoid-nodulating Paraburkholderia. Here, we discuss the latest knowledge on Paraburkholderia nitrogen-fixing symbionts in each step of the symbiosis, from their survival in the soil, through the first contact with the legumes until the formation of an efficient nitrogen-fixing symbiosis in root nodules. Special attention is given to the strain P. phymatum STM815T that exhibits extraordinary features, such as the ability to: (i) enter into symbiosis with more than 50 legume species, including the agriculturally important common bean, (ii) outcompete other rhizobial species for nodulation of several legumes, and (iii) endure stressful soil conditions (e.g., high salt concentration and low pH) and high temperatures.