RNA-seq analysis in simulated microgravity unveils down-regulation of the beta-rhizobial siderophore phymabactin.

Exploiting the symbiotic interaction between crops and nitrogen-fixing bacteria is a simple and ecological method to promote plant growth in prospective extraterrestrial human outposts. In this study, we performed an RNA-seq analysis to investigate the adaptation of the legume symbiont Paraburkholderia phymatum STM815T to simulated microgravity (s0-g) at the transcriptome level. The results revealed a drastic effect on gene expression, with roughly 23% of P. phymatum genes being differentially regulated in s0-g. Among those, 951 genes were upregulated and 858 downregulated in the cells grown in s0-g compared to terrestrial gravity (1 g). Several genes involved in posttranslational modification, protein turnover or chaperones encoding were upregulated in s0-g, while those involved in translation, ribosomal structure and biosynthesis, motility or inorganic ions transport were downregulated. Specifically, the whole phm gene cluster, previously bioinformatically predicted to be involved in the production of a hypothetical malleobactin-like siderophore, phymabactin, was 20-fold downregulated in microgravity. By constructing a mutant strain (ΔphmJK) we confirmed that the phm gene cluster codes for the only siderophore secreted by P. phymatum as assessed by the complete lack of iron chelating activity of the P. phymatum ΔphmJK mutant on chrome azurol S (CAS) agar plates. These results not only provide a deeper understanding of the physiology of symbiotic organisms exposed to space-like conditions, but also increase our knowledge of iron acquisition mechanisms in rhizobia.

A novel function of the key nitrogen-fixation activator NifA in beta-rhizobia: Repression of bacterial auxin synthesis during symbiosis.

Rhizobia fix nitrogen within root nodules of host plants where nitrogenase expression is strictly controlled by its key regulator NifA. We recently discovered that in nodules infected by the beta-rhizobial strain Paraburkholderia phymatum STM815, NifA controls expression of two bacterial auxin synthesis genes. Both the iaaM and iaaH transcripts, as well as the metabolites indole-acetamide (IAM) and indole-3-acetic acid (IAA) showed increased abundance in nodules occupied by a nifA mutant compared to wild-type nodules. Here, we document the structural changes that a P. phymatum nifA mutant induces in common bean (Phaseolus vulgaris) nodules, eventually leading to hypernodulation. To investigate the role of the P. phymatum iaaMH genes during symbiosis, we monitored their expression in presence and absence of NifA over different stages of the symbiosis. The iaaMH genes were found to be under negative control of NifA in all symbiotic stages. While a P. phymatum iaaMH mutant produced the same number of nodules and nitrogenase activity as the wild-type strain, the nifA mutant produced more nodules than the wild-type that clustered into regularly-patterned root zones. Mutation of the iaaMH genes in a nifA mutant background reduced the presence of these nodule clusters on the root. We further show that the P. phymatum iaaMH genes are located in a region of the symbiotic plasmid with a significantly lower GC content and exhibit high similarity to two genes of the IAM pathway often used by bacterial phytopathogens to deploy IAA as a virulence factor. Overall, our data suggest that the increased abundance of rhizobial auxin in the non-fixing nifA mutant strain enables greater root infection rates and a role for bacterial auxin production in the control of early stage symbiotic interactions.

c-di-AMP signaling is required for bile salt resistance, osmotolerance, and long-term host colonization by Clostridioides difficile.

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.

Metabolomics and Dual RNA-Sequencing on Root Nodules Revealed New Cellular Functions Controlled by Paraburkholderia phymatum NifA.

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.

Paraburkholderia phymatum Homocitrate Synthase NifV Plays a Key Role for Nitrogenase Activity during Symbiosis with Papilionoids and in Free-Living Growth Conditions.

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.