The motility and chemosensory systems of Rhizobium leguminosarum, their role in symbiosis, and link to PTSNtr regulation.

Motility and chemotaxis are crucial processes for soil bacteria and plant-microbe interactions. This applies to the symbiotic bacterium Rhizobium leguminosarum, where motility is driven by flagella rotation controlled by two chemotaxis systems, Che1 and Che2. The Che1 cluster is particularly important in free-living motility prior to the establishment of the symbiosis, with a che1 mutant delayed in nodulation and reduced in nodulation competitiveness. The Che2 system alters bacteroid development and nodule maturation. In this work, we also identified 27 putative chemoreceptors encoded in the R. leguminosarum bv. viciae 3841 genome and characterized its motility in different growth conditions. We describe a metabolism-based taxis system in rhizobia that acts at high concentrations of dicarboxylates to halt motility independent of chemotaxis. Finally, we show how PTSNtr influences cell motility, with PTSNtr mutants exhibiting reduced swimming in different media. Motility is restored by the active forms of the PTSNtr output regulatory proteins, unphosphorylated ManX and phosphorylated PtsN. Overall, this work shows how rhizobia typify soil bacteria by having a high number of chemoreceptors and highlights the importance of the motility and chemotaxis mechanisms in a free-living cell in the rhizosphere, and at different stages of the symbiosis.

NIN-like protein transcription factors regulate leghemoglobin genes in legume nodules.

Leghemoglobins enable the endosymbiotic fixation of molecular nitrogen (N2) in legume nodules by channeling O2 for bacterial respiration while maintaining a micro-oxic environment to protect O2-sensitive nitrogenase. We found that the NIN-like protein (NLP) transcription factors NLP2 and NIN directly activate the expression of leghemoglobins through a promoter motif, resembling a "double" version of the nitrate-responsive elements (NREs) targeted by other NLPs, that has conserved orientation and position across legumes. CRISPR knockout of the NRE-like element resulted in strongly decreased expression of the associated leghemoglobin. Our findings indicate that the origins of the NLP-leghemoglobin module for O2 buffering in nodules can be traced to an ancient pairing of NLPs with nonsymbiotic hemoglobins that function in hypoxia.

Competition, Nodule Occupancy, and Persistence of Inoculant Strains: Key Factors in the Rhizobium-Legume Symbioses.

Biological nitrogen fixation by Rhizobium-legume symbioses represents an environmentally friendly and inexpensive alternative to the use of chemical nitrogen fertilizers in legume crops. Rhizobial inoculants, applied frequently as biofertilizers, play an important role in sustainable agriculture. However, inoculants often fail to compete for nodule occupancy against native rhizobia with inferior nitrogen-fixing abilities, resulting in low yields. Strains with excellent performance under controlled conditions are typically selected as inoculants, but the rates of nodule occupancy compared to native strains are rarely investigated. Lack of persistence in the field after agricultural cycles, usually due to the transfer of symbiotic genes from the inoculant strain to naturalized populations, also limits the suitability of commercial inoculants. When rhizobial inoculants are based on native strains with a high nitrogen fixation ability, they often have superior performance in the field due to their genetic adaptations to the local environment. Therefore, knowledge from laboratory studies assessing competition and understanding how diverse strains of rhizobia behave, together with assays done under field conditions, may allow us to exploit the effectiveness of native populations selected as elite strains and to breed specific host cultivar-rhizobial strain combinations. Here, we review current knowledge at the molecular level on competition for nodulation and the advances in molecular tools for assessing competitiveness. We then describe ongoing approaches for inoculant development based on native strains and emphasize future perspectives and applications using a multidisciplinary approach to ensure optimal performance of both symbiotic partners.

Rhizobial Chemotaxis and Motility Systems at Work in the Soil.

Bacteria navigate their way often as individual cells through their chemical and biological environment in aqueous medium or across solid surfaces. They swim when starved or in response to physical and chemical stimuli. Flagella-driven chemotaxis in bacteria has emerged as a paradigm for both signal transduction and cellular decision-making. By altering motility, bacteria swim toward nutrient-rich environments, movement modulated by their chemotaxis systems with the addition of pili for surface movement. The numbers and types of chemoreceptors reflect the bacterial niche and lifestyle, with those adapted to complex environments having diverse metabolic capabilities, encoding far more chemoreceptors in their genomes. The Alpha-proteobacteria typify the latter case, with soil bacteria such as rhizobia, endosymbionts of legume plants, where motility and chemotaxis are essential for competitive symbiosis initiation, among other processes. This review describes the current knowledge of motility and chemotaxis in six model soil bacteria: Sinorhizobium meliloti, Agrobacterium fabacearum, Rhizobium leguminosarum, Azorhizobium caulinodans, Azospirillum brasilense, and Bradyrhizobium diazoefficiens. Although motility and chemotaxis systems have a conserved core, rhizobia possess several modifications that optimize their movements in soil and root surface environments. The soil provides a unique challenge for microbial mobility, since water pathways through particles are not always continuous, especially in drier conditions. The effectiveness of symbiont inoculants in a field context relies on their mobility and dispersal through the soil, often assisted by water percolation or macroorganism movement or networks. Thus, this review summarizes the factors that make it essential to consider and test rhizobial motility and chemotaxis for any potential inoculant.

Regulation and Characterization of Mutants of fixABCX in Rhizobium leguminosarum.

Symbiosis between Rhizobium leguminosarum and Pisum sativum requires tight control of redox balance in order to maintain respiration under the microaerobic conditions required for nitrogenase while still producing the eight electrons and sixteen molecules of ATP needed for nitrogen fixation. FixABCX, a cluster of electron transfer flavoproteins essential for nitrogen fixation, is encoded on the Sym plasmid (pRL10), immediately upstream of nifA, which encodes the general transcriptional regulator of nitrogen fixation. There is a symbiotically regulated NifA-dependent promoter upstream of fixA (PnifA1), as well as an additional basal constitutive promoter driving background expression of nifA (PnifA2). These were confirmed by 5'-end mapping of transcription start sites using differential RNA-seq. Complementation of polar fixAB and fixX mutants (Fix- strains) confirmed expression of nifA from PnifA1 in symbiosis. Electron microscopy combined with single-cell Raman microspectroscopy characterization of fixAB mutants revealed previously unknown heterogeneity in bacteroid morphology within a single nodule. Two morphotypes of mutant fixAB bacteroids were observed. One was larger than wild-type bacteroids and contained high levels of polyhydroxy-3-butyrate, a complex energy/reductant storage product. A second bacteroid phenotype was morphologically and compositionally different and resembled wild-type infection thread cells. From these two characteristic fixAB mutant bacteroid morphotypes, inferences can be drawn on the metabolism of wild-type nitrogen-fixing bacteroids.[Formula: see text] Copyright © 2021 The Author(s). This is an open access article distributed under the CC BY 4.0 International license.

Optimizing Rhizobium-legume symbioses by simultaneous measurement of rhizobial competitiveness and N2 fixation in nodules.

Legumes tend to be nodulated by competitive rhizobia that do not maximize nitrogen (N2) fixation, resulting in suboptimal yields. Rhizobial nodulation competitiveness and effectiveness at N2 fixation are independent traits, making their measurement extremely time-consuming with low experimental throughput. To transform the experimental assessment of rhizobial competitiveness and effectiveness, we have used synthetic biology to develop reporter plasmids that allow simultaneous high-throughput measurement of N2 fixation in individual nodules using green fluorescent protein (GFP) and barcode strain identification (Plasmid ID) through next generation sequencing (NGS). In a proof-of-concept experiment using this technology in an agricultural soil, we simultaneously monitored 84 different Rhizobium leguminosarum strains, identifying a supercompetitive and highly effective rhizobial symbiont for peas. We also observed a remarkable frequency of nodule coinfection by rhizobia, with mixed occupancy identified in ∼20% of nodules, containing up to six different strains. Critically, this process can be adapted to multiple Rhizobium-legume symbioses, soil types, and environmental conditions to permit easy identification of optimal rhizobial inoculants for field testing to maximize agricultural yield.

Global control of bacterial nitrogen and carbon metabolism by a PTSNtr-regulated switch.

The nitrogen-related phosphotransferase system (PTSNtr) of Rhizobium leguminosarum bv. viciae 3841 transfers phosphate from PEP via PtsP and NPr to two output regulators, ManX and PtsN. ManX controls central carbon metabolism via the tricarboxylic acid (TCA) cycle, while PtsN controls nitrogen uptake, exopolysaccharide production, and potassium homeostasis, each of which is critical for cellular adaptation and survival. Cellular nitrogen status modulates phosphorylation when glutamine, an abundant amino acid when nitrogen is available, binds to the GAF sensory domain of PtsP, preventing PtsP phosphorylation and subsequent modification of ManX and PtsN. Under nitrogen-rich, carbon-limiting conditions, unphosphorylated ManX stimulates the TCA cycle and carbon oxidation, while unphosphorylated PtsN stimulates potassium uptake. The effects are reversed with the phosphorylation of ManX and PtsN, occurring under nitrogen-limiting, carbon-rich conditions; phosphorylated PtsN triggers uptake and nitrogen metabolism, the TCA cycle and carbon oxidation are decreased, while carbon-storage polymers such as surface polysaccharide are increased. Deleting the GAF domain from PtsP makes cells "blind" to the cellular nitrogen status. PTSNtr constitutes a switch through which carbon and nitrogen metabolism are rapidly, and reversibly, regulated by protein:protein interactions. PTSNtr is widely conserved in proteobacteria, highlighting its global importance.

Formulation of a Highly Effective Inoculant for Common Bean Based on an Autochthonous Elite Strain of Rhizobium leguminosarum bv. phaseoli, and Genomic-Based Insights Into Its Agronomic Performance.

Common bean is a poor symbiotic N-fixer, with a low response to inoculation owing to its promiscuous nodulation with competitive but inefficient resident rhizobia. Consequently, farmers prefer to fertilize them rather than rely on their capacity for Biological Nitrogen Fixation (BNF). However, when rhizobial inoculants are based on autochthonous strains, they often have superior BNF performance in the field due to their genetic adaptations to the local environment. Nevertheless, there is scant information at the genomic level explaining their superiority or on how their genomes may influence the inoculant performance. This information is especially important in technologically advanced agri-systems like Europe, where environmental concerns and increasingly stringent fertilizer regulations are encouraging a return to the use of rhizobial inoculants, but based upon strains that have been thoroughly characterized in terms of their symbiotic performance and their genetics. The aim of this study was to design an inoculant formulation based on a superior autochthonous strain, Rhizobium leguminosarum bv. phaseoli LCS0306, to assess its performance in the field, and to determine the genomic features contributing to the high effectiveness of its symbiosis with common bean. Plants inoculated with the autochthonous strain LCS0306 fixed significantly more nitrogen than those with the allochthonous strains R. phaseoli ATCC 14482T and R. etli CFN42T, and had grain yield similar to the nitrogen-fertilized controls. Inoculation with LCS0306 was particularly efficacious when formulated with a carrier based upon a mixture of perlite and biochar. Whole genome comparisons revealed no differences in the classical symbiotic genes of strain LCS0306 within the symbiovar phaseoli. However, its symbiotic superior performance might be due to its genomic versatility, as it harbors a large assortment of genes contributing to fitness and competitiveness. It is concluded that inoculation with elite rhizobia formulated with perlite-biochar carriers might constitute a step-change in the sustainable cultivation of common bean in Spanish soils.

Genomic Diversity in the Endosymbiotic Bacterium Rhizobium leguminosarum.

Rhizobium leguminosarum bv. viciae is a soil α-proteobacterium that establishes a diazotrophic symbiosis with different legumes of the Fabeae tribe. The number of genome sequences from rhizobial strains available in public databases is constantly increasing, although complete, fully annotated genome structures from rhizobial genomes are scarce. In this work, we report and analyse the complete genome of R. leguminosarum bv. viciae UPM791. Whole genome sequencing can provide new insights into the genetic features contributing to symbiotically relevant processes such as bacterial adaptation to the rhizosphere, mechanisms for efficient competition with other bacteria, and the ability to establish a complex signalling dialogue with legumes, to enter the root without triggering plant defenses, and, ultimately, to fix nitrogen within the host. Comparison of the complete genome sequences of two strains of R. leguminosarum bv. viciae, 3841 and UPM791, highlights the existence of different symbiotic plasmids and a common core chromosome. Specific genomic traits, such as plasmid content or a distinctive regulation, define differential physiological capabilities of these endosymbionts. Among them, strain UPM791 presents unique adaptations for recycling the hydrogen generated in the nitrogen fixation process.

Understanding the holobiont: the interdependence of plants and their microbiome.

The holobiont is composed by the plant and its microbiome. In a similar way to ecological systems of higher organisms, the holobiont shows interdependent and complex dynamics [1,2]. While plants originate from seeds, the microbiome has a multitude of sources. The assemblage of these communities depends on the interaction between the emerging seedling and its surrounding environment, with soil being the main source. These microbial communities are controlled by the plant through different strategies, such as the specific profile of root exudates and its immune system. Despite this control, the microbiome is still able to adapt and thrive. The molecular knowledge behind these interactions and microbial '-omic' technologies are developing to the point of enabling holobiont engineering. For a long time microorganisms were in the background of plant biology but new multidisciplinary approaches have led to an appreciation of the importance of the holobiont, where plants and microbes are interdependent.

Construction of a marker system for the evaluation of competitiveness for legume nodulation in Rhizobium strains.

A marker system has been set up for the analysis of competitiveness of Rhizobium leguminosarum strains for legume nodulation. The strains generated incorporate gusA and celB marker genes at identical positions and allow efficient scoring of single and double infections. Based on this system, we have found that strain UPM791 outcompetes 3841 for nodulation in pea. This technique will be useful to determine the effect of different traits on competitiveness.

Endosymbiotic bacteria nodulating a new endemic lupine Lupinus mariae-josephi from alkaline soils in Eastern Spain represent a new lineage within the Bradyrhizobium genus.

Lupinus mariae-josephi is a recently described endemic Lupinus species from a small area in Eastern Spain where it thrives in soils with active lime and high pH. The L. mariae-josephi root symbionts were shown to be very slow-growing bacteria with different phenotypic and symbiotic characteristics from those of Bradyrhizobium strains nodulating other Lupinus. Their phylogenetic status was examined by multilocus sequence analyses of four housekeeping genes (16S rRNA, glnII, recA, and atpD) and showed the existence of a distinct evolutionary lineage for L. mariae-josephi that also included Bradyrhizobium jicamae. Within this lineage, the tested isolates clustered in three different sub-groups that might correspond to novel sister Bradyrhizobium species. These core gene analyses consistently showed that all the endosymbiotic bacteria isolated from other Lupinus species of the Iberian Peninsula were related to strains of the B. canariense or B. japonicum lineages and were separate from the L. mariae-josephi isolates. Phylogenetic analysis based on nodC symbiotic gene sequences showed that L. mariae-josephi bacteria also constituted a new symbiotic lineage distant from those previously defined in the genus Bradyrhizobium. In contrast, the nodC genes of isolates from other Lupinus spp. from the Iberian Peninsula were again clearly related to the B. canariense and B. japonicum bv. genistearum lineages. Speciation of L. mariae-josephi bradyrhizobia may result from the colonization of a singular habitat by their unique legume host.