Carmen Sánchez-Cañizares

The coordination of intracellular carbon and nitrogen levels is essential for optimal bacterial growth and, in rhizobia, for survival in the soil, root colonization, and symbiotic interactions with their host plants. Here, we show that the phosphotransferase system PTS ^Ntr , a global regulator of carbon and nitrogen metabolism, modulates the tricarboxylic acid (TCA) cycle activity and the accumulation of three major carbon storage polymers: glycogen, polyhydroxybutyrate, and exopolysaccharide in Rhizobium leguminosarum . The unphosphorylated form of the effector protein ManX is sufficient for the full activation of the TCA cycle dehydrogenase enzymes. Accordingly, loss of manX reduced dehydrogenase activity and redirected overflow carbon into storage polymers, phenocopying wild-type cells grown under nitrogen starvation. We further demonstrate that carbon metabolism in R. leguminosarum is also tightly regulated by the HPr kinase (HprK) protein. In an hprK mutant, loss of NPr Ser48 phosphorylation favors phosphotransfer through the histidyl arm and is predicted to increase phosphorylation of downstream PTS ^Ntr components. When grown on glucose, the hprK mutant accumulates elevated intracellular pyrimidine levels and exhibits increased malate dehydrogenase and TCA cycle activity, consistent with a misregulated shift toward gluconeogenesis. This mutant also overproduces exopolysaccharide, an effect potentially mediated by crosstalk between phosphorylated PtsN and the ChvI/ChvG two-component regulatory system. Together, these data suggest that HprK exerts pleiotropic control over processes regulated by PTS ^Ntr , influencing central metabolism while repressing exopolysaccharide production, likely by promoting the unphosphorylated state of PtsN. IMPORTANCE: Bacteria must continuously balance growth, nutrient availability, and storage to ensure fitness and survival in fluctuating environments. However, how regulatory networks integrate central metabolism with carbon storage remains poorly understood. This study reinforces the role of the PTS ^Ntr system as a central coordinator of carbon routing and polymer accumulation. By revealing how ManX and HprK-dependent phosphorylation influence central metabolism and the production of major carbon storage polymers in Rhizobium leguminosarum , we show that disruption of PTS ^Ntr signaling reprograms carbon allocation, uncoupling growth from carbon storage and mimicking nitrogen starvation even under nitrogen-replete conditions. These findings highlight a regulatory link between carbon-nitrogen signaling and bacterial storage strategies, with implications for cellular survival and plant symbiosis. More broadly, our work illustrates how global metabolic regulators shape bacterial physiological states, providing new insights into mechanisms that support microbial adaptation in complex environments.

Plants outsource many functions to an associated microbiome, an ‘outside gut’ composed of socially interacting bacterial communities that support plant growth and health. These dynamic and diverse microbiomes span different plant microenvironments and form complex networks shaped by the environmental context and the plant’s genetics. While some microbes cause disease, many others contribute to essential functions, influencing plant fitness, survival, and ultimately soil health. Although the host genetics and abiotic factors structure these communities, the functional diversity and ecological roles of plant-associated bacteria are ultimately shaped by microbe–microbe interactions. These interactions also play a critical role in the successful colonisation of plant tissues. Beneficial members of these communities enhance plant nutrient uptake, modulate phytohormones to shape root architecture, or prime plant systemic immunity to repel pathogens. Deciphering how plant-associated bacterial communities assemble and interact, both with their host and with one another, is essential for uncovering the mechanisms that underpin bacterial social behaviours within the plant holobiont, such as quorum sensing, metabolite exchange, and contact-dependent interactions. Here we review several recent publications that shed new light on the social lives of plant-associated bacteria, with a particular focus on their competition mechanisms and strategies for colonisation and establishment. In this context, we highlight how the rational design of plant bioinoculants based on microbial consortia represents a powerful strategy for promoting sustainable agriculture in a rapidly changing environment.

Within legume root nodules, rhizobia differentiate into bacteroids, which reduce N2 into NH3 for secretion to the plant. Bacteroids may be swollen and terminally differentiated or non-swollen and can regenerate outside nodules. It is unclear why these different endosymbiotic lifestyles exist and whether they differ in symbiotic efficiency. Here, we compared N2 fixing bacteroids of the near isogenic strains Rhizobium leguminosarum bv. phaseoli 4292 (Rlp4292) and R. leguminosarum bv. viciae A34 (RlvA34), nodulating Phaseolus vulgaris (common bean) and Pisum sativum (pea), respectively. The larger bean plants fixed more N2, but peas fixed 1.6–3-fold more per unit nodule mass. Values per unit volume were similar between bean and pea because bean nodules are 2.7-fold denser (i.e. mass per unit volume). Bean nodules have higher numbers of smaller (∼1/5 the volume) bacteroids than peas. Bean bacteroids are denser (i.e. 2.5-fold protein per unit volume) although less closely packed than pea bacteroids (i.e. more space between bean bacteroids). Critically, pea bacteroids fix N2 at higher rates versus bean per unit bacteroid protein, as protein expression is skewed toward N2 fixation and TCA-cycle enzymes. Pea bacteroids infect 1.6 times the percentage of nodule volume of beans (i.e. 14.2% versus 9.1%). Overall, the increased packing density of pea bacteroids, as well as the bias of their proteome to nitrogenase, associated N2 fixation processes, and dicarboxylate metabolism, contributes to their greater symbiotic efficiency, which is likely driven by plant antimicrobial peptides.

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 PTS ^Ntr influences cell motility, with PTS ^Ntr mutants exhibiting reduced swimming in different media. Motility is restored by the active forms of the PTS ^Ntr 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.

Nodulation regulation: Legumes convert atmospheric nitrogen into biologically useful ammonium with the help of symbiotic bacteria housed in root nodules. Much of nodule development is controlled by the transcription factor NODULE INCEPTION (NIN). Feng et al . show that NIN is proteolytically processed to release a fragment that regulates the later stages of nodulation when the nodules acquire nitrogen-fixing capability. In related work, Jiang et al . identified members of the NIN-like protein (NLP) transcription factor family as being regulators of leghemoglobin expression acting through an unusual promotor motif shared across legumes. —PJH

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, whil...

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.

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.

The nitrogen-related phosphotransferase system (PTS^Ntr) 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. PTS^Ntr constitutes a switch through which carbon and nitrogen metabolism are rapidly, and reversibly, regulated by protein:protein interactions. PTS^Ntr is widely conserved in proteobacteria, highlighting its global importance. Significance Bacteria have evolved intricate regulatory networks to coordinate their metabolism with internal and external signals of their status. The regulatory phosphotransferase systems (PTSs) constitute a key part of these intricate circuits, with their signal transduction cascades participating in multiple regulatory functions. Although two major systems have been described as being involved in regulating carbon and nitrogen pools, there is very little information on their physiological role in vivo under real-time conditions. In this work we demonstrate the role of PTS as an integrated system, widely conserved in proteobacteria, acting as a complex biological sensor-actuator device enabling bacterial cells to posttranslationally alter bacterial physiology and balance carbon and nitrogen availability.