A Virtual Nodule Environment (ViNE) for modelling the inter-kingdom metabolic integration during symbiotic nitrogen fixation

ABSTRACTBiological associations are often premised upon metabolic cross-talk between the organisms, with the N2-fixing endosymbiotic relationship between rhizobia and leguminous plants being a prime example. Here, we report the in silico reconstruction of a metabolic network of a Medicago truncatula plant nodulated by the bacterium Sinorhizobium meliloti. The nodule tissue of the model contains five spatially distinct developmental zones and encompasses the metabolism of both the plant and the bacterium. Flux balance analysis (FBA) suggested that the majority of the metabolic costs associated with symbiotic nitrogen fixation are directly related to supporting nitrogenase activity, while a minority is related to the formation and maintenance of nodule and bacteroid tissue. Interestingly, FBA simulations suggested there was a non-linear relationship between the rate of N2-fixation per gram of nodule and the rate of plant growth; increasing the N2-fixation efficiency was associated with diminishing returns in terms of plant growth. Evaluating the metabolic exchange between the symbiotic partners provided support for: i) differentiating bacteroids having access to sugars (e.g., sucrose) as a major carbon source, ii) ammonium being the major nitrogen export product of N2-fixing bacteria, and iii) N2-fixation being dependent on the transfer of protons from the plant cytoplasm to the bacteria through acidification of the peribacteroid space. Our simulations further suggested that the use of C4-dicarboxylates by N2-fixing bacteroids may be, in part, a consequence of the low concentration of free oxygen in the nodule limiting the activity of the plant mitochondria. These results demonstrate the power of this integrated model to advance our understanding of the functioning of legume nodules, and its potential for hypothesis generation to guide experimental studies and engineering of symbiotic nitrogen fixation.

Influence of mutation in pea (Pisum sativum L.) cdt (cadmium tolerance) gene on histological and ultrastructural nodule organization

Background. A comparative analysis out of the structural organization of the symbiotic nodules of the pea initial line SGE and the mutant line SGECdt, characterized by increased tolerance to cadmium and increased its accumulation, was carried out. Materials and methods.Nodules of initial line SGE and mutant SGECdt were analyzed using light and transmission electron microscopy. Results. The non-treated nodules of SGE and SGECdt were characterized by a similar histological and ultrastructural organization. In the nodules of SGE exposed to 100 M CdCl2 in infected cells, the following abnormalities were observed: expansion of the peribacteroid space, destruction of the symbiosome membrane, fusion of symbiosomes and, as a result, the formation of symbiosomes containing several bacteroids. In the nodules of SGECdt, infected cells did not undergo pronounced changes. In the nodules of SGE exposed to 1 mM CdCl2, at the base of the nodule, senescent infected cells with completely destroyed cytoplasm and degrading bacteroids appeared. Also there were present cells in which the contents of symbiosomes were lysing, and only the ghosts of the bacteroids remained in them. In SGECdt, in some infected cells, abnormalities were manifested in an increase in the peribacteroid space, partial destruction of symbiosome membranes, fusion of symbiosomes, and release of bacteroids into the vacuole. Conclusions. The tolerance of pea nodules to cadmium can be significantly increased due to a single recessive cdt mutation.

The Bacteroid Periplasm in Soybean Nodules Is an Interkingdom Symbiotic Space

The functional role of the periplasm of nitrogen-fixing bacteroids has not been determined. Proteins were isolated from the periplasm and cytoplasm of Bradyrhizobium diazoefficiens bacteroids and were analyzed using liquid chromatography tandem mass spectrometry proteomics. Identification of bacteroid periplasmic proteins was aided by periplasm prediction programs. Approximately 40% of all the proteins identified as periplasmic in the B. diazoefficiens genome were found expressed in the bacteroid form of the bacteria, indicating the periplasm is a metabolically active symbiotic space. The bacteroid periplasm possesses many fatty acid metabolic enzymes, which was in contrast to the bacteroid cytoplasm. Amino acid analysis of the periplasm revealed an abundance of phosphoserine, phosphoethanolamine, and glycine, which are metabolites of phospholipid metabolism. These results suggest the periplasm is a unique space and not a continuum with the peribacteroid space. A number of plant proteins were found in the periplasm fraction, which suggested contamination. However, antibodies to two of the identified plant proteins, histone H2A and lipoxygenase, yielded immunogold labeling that demonstrated the plant proteins were specifically targeted to the bacteroids. This suggests that the periplasm is an interkingdom symbiotic space containing proteins from both the bacteroid and the plant.

An antimicrobial peptide essential for bacterial survival in the nitrogen-fixing symbiosis

In the nitrogen-fixing symbiosis between legume hosts and rhizobia, the bacteria are engulfed by a plant cell membrane to become intracellular organelles. In the model legume Medicago truncatula, internalization and differentiation of Sinorhizobium (also known as Ensifer) meliloti is a prerequisite for nitrogen fixation. The host mechanisms that ensure the long-term survival of differentiating intracellular bacteria (bacteroids) in this unusual association are unclear. The M. truncatula defective nitrogen fixation4 (dnf4) mutant is unable to form a productive symbiosis, even though late symbiotic marker genes are expressed in mutant nodules. We discovered that in the dnf4 mutant, bacteroids can apparently differentiate, but they fail to persist within host cells in the process. We found that the DNF4 gene encodes NCR211, a member of the family of nodule-specific cysteine-rich (NCR) peptides. The phenotype of dnf4 suggests that NCR211 acts to promote the intracellular survival of differentiating bacteroids. The greatest expression of DNF4 was observed in the nodule interzone II-III, where bacteroids undergo differentiation. A translational fusion of DNF4 with GFP localizes to the peribacteroid space, and synthetic NCR211 prevents free-living S. meliloti from forming colonies, in contrast to mock controls, suggesting that DNF4 may interact with bacteroids directly or indirectly for its function. Our findings indicate that a successful symbiosis requires host effectors that not only induce bacterial differentiation, but also that maintain intracellular bacteroids during the host–symbiont interaction. The discovery of NCR211 peptides that maintain bacterial survival inside host cells has important implications for improving legume crops.

Bacteroid Development in Legume Nodules: Evolution of Mutual Benefit or of Sacrificial Victims?

Symbiosomes are organelle-like structures in the cytoplasm of legume nodule cells which are composed of the special, nitrogen-fixing forms of rhizobia called bacteroids, the peribacteroid space and the enveloping peribacteroid membrane of plant origin. The formation of these symbiosomes requires a complex and coordinated interaction between the two partners during all stages of nodule development as any failure in the differentiation of either symbiotic partner, the bacterium or the plant cell prevents the subsequent transcriptional and developmental steps resulting in early senescence of the nodules. Certain legume hosts impose irreversible terminal differentiation onto bacteria. In the inverted repeat–lacking clade (IRLC) of legumes, host dominance is achieved by nodule-specific cysteine-rich peptides that resemble defensin-like antimicrobial peptides, the known effector molecules of animal and plant innate immunity. This article provides an overview on the bacteroid and symbiosome development including the terminal differentiation of bacteria in IRLC legumes as well as the bacterial and plant genes and proteins participating in these processes.

The Sinorhizobium meliloti Glycine Betaine Biosynthetic Genes (betICBA) Are Induced by Choline and Highly Expressed in Bacteroids

The symbiotic soil bacterium Sinorhizobium meliloti has the capacity to synthesize the osmoprotectant glycine betaine from choline-O-sulfate and choline. This pathway is encoded by the betICBA locus, which comprises a regulatory gene, betI, and three structural genes, betC (choline sulfatase), betB (betaine aldehyde dehydrogenase), and betA (choline dehydrogenase). Here, we report that betICBA genes constitute a single operon, despite the existence of intergenic regions containing mosaic elements between betI and betC, and betB and betA. The regulation of the bet operon was investigated by using transcriptional lacZ (β-galactosidase) fusions and has revealed a strong induction by choline at concentrations as low as 25 μM and to a lesser extent by choline-O-sulfate and acetylcholine but not by osmotic stress or oxygen. BetI is a repressor of the bet transcription in the absence of choline, and a nucleotide sequence of dyad symmetry upstream of betI was identified as a putative betI box. Measurements of intracellular pools of choline, well correlated with β-galactosidase activities, strongly suggested that BetI senses the endogenous choline pool that modulates the intensity of BetI repression. In contrast to Escherichia coli, BetI did not repress choline transport. During symbiosis with Medicago sativa, S. meliloti bet gene expression was observed within the infection threads, in young and in mature nodules. The existence of free choline in nodule cytosol, peribacteroid space, and bacteroids was demonstrated, and the data suggest that bet regulation in planta is mediated by BetI repression, as in free-living cells. Neither Nod nor Fix phenotypes were significantly impaired in a betI∷Ω mutant, indicating that glycine betaine biosynthesis from choline is not crucial for nodulation and nitrogen fixation.

Origin of de Novo Synthesized Proteins in the Different Compartments of Pea-Rhizobium sp. Symbiosomes

The origin of de novo synthesized proteins in the interface between pea and a Rhizobium sp. in root nodules has been determined. The symbiotic interface is defined as the peribacteroid space including proteins associated with either the symbiosome membrane or the bacteroid outer membrane. Two approaches have been used to study the origin of proteins in the symbiotic interface. First, to determine the localization of de novo synthesized plant-produced proteins in the symbiosomes, an in vitro protein translocation assay was established. To produce plant proteins poly A+ RNA was isolated from root nodules followed by in vitro translation in the presence of [35S]methionine. Subsequently, purified symbiosomes were incubated with the [35S]methionine-labeled plant proteins. The symbiosomes were subfractionated and de novo synthesized plant proteins in the different fractions were identified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and fluorography. These import studies demonstrate the presence of plant proteins in the symbiosome membrane, peribacteroid space, and bacteroid membrane pellet. In contrast, no proteins of plant origin were detected in the bacteroid cytosol. Second, the presence of de novo synthesized bacteroid proteins in the interface was examined by incubation of symbiosomes isolated under micro-aerobic or aerobic conditions with [35S]methionine. Analysis of symbiosome compartments by SDS-PAGE and phosphor image revealed that proteins of bacteroid origin are primarily detected in the bacteroid cytosol and bacteroid membrane pellet. However, a few bacteroid-produced proteins are also observed in the symbiosome membrane. Together these data demonstrate that the majority of de novo synthesized proteins in the pea-Rhizobium sp. symbiotic interface are of plant origin.