Lipogenesis and redox balance in nitrogen-fixing pea bacteroids

Within legume root nodules, rhizobia differentiate into bacteroids that oxidise host-derived dicarboxylic acids, which is assumed to occur via the TCA-cycle to generate NAD(P)H for reduction of N2. Metabolic flux analysis of laboratory grown Rhizobium leguminosarum showed that the flux from 13C-succinate was consistent with respiration of an obligate aerobe growing on a TCA-cycle intermediate as the sole carbon source. However, the instability of fragile pea bacteroids prevented their steady state labelling under N2-fixing conditons. Therefore, comparitive metabolomic profiling was used to compare free-living R. leguminosarum with pea bacteroids. While the TCA-cycle was shown to be essential for maximal rates of N2-fixation, pyruvate (5.5-fold down), acetyl-CoA (50-fold down), free coenzyme A (33-fold) and citrate (4.5-fold down) were much lower in bacteroids. Instead of completely oxidising acetyl-CoA, pea bacteroids channel it into both lipid and the lipid-like polymer poly-β-hydroxybutyrate (PHB), the latter via a type II PHB synthase that is only active in bacteroids. Lipogenesis may be a fundamental requirement of the redox poise of electron donation to N2 in all legume nodules. Direct reduction by NAD(P)H of the likely electron donors for nitrogenase, such as ferredoxin, is inconsistent with their redox potentials. Instead, bacteroids must balance the production of NAD(P)H from oxidation of acetyl-CoA in the TCA-cycle with its storage in PHB and lipids

The model legume Medicago truncatula A17 is poorly matched for N2fixation with the sequenced microsymbiont Sinorhizobium meliloti 1021

Medicago truncatula (barrel medic) A17 is currently being sequenced as a model legume, complementing the sequenced root nodule bacterial strain Sinorhizobium meliloti 1021 (Sm1021). In this study, the effectiveness of the Sm1021-M. truncatula symbiosis at fixing N2 was evaluated. • N2 fixation effectiveness was examined with eight Medicago species and three accessions of M. truncatula with Sm1021 and two other Sinorhizobium strains. Plant shoot dry weights, plant nitrogen content and nodule distribution, morphology and number were analysed. • Compared with nitrogen-fed controls, Sm1021 was ineffective or partially effective on all hosts tested (excluding M. sativa), as measured by reduced dry weights and shoot N content. Against an effective strain, Sm1021 on M. truncatula accessions produced more nodules, which were small, pale, more widely distributed on the root system and with fewer infected cells. • The Sm1021-M. truncatula symbiosis is poorly matched for N2 fixation and the strain could possess broader N2 fixation deficiencies. A possible origin for this reduction in effectiveness is discussed. An alternative sequenced strain, effective at N2 fixation on M. truncatula A17, is Sinorhizobium medicae WSM419

Comparative analysis of integrative and conjugative mobile genetic elements in the genus Mesorhizobium

Members of the Mesorhizobium genus are soil bacteria that often form nitrogen-fixing symbioses with legumes. Most characterised Mesorhizobium spp. genomes are ~8 Mb in size and harbour extensive pangenomes including large integrative and conjugative elements (ICEs) carrying genes required for symbiosis (ICESyms). Here, we document and compare the conjugative mobilome of 41 complete Mesorhizobium genomes. We delineated 56 ICEs and 24 integrative and mobilizable elements (IMEs) collectively occupying 16 distinct integration sites, along with 24 plasmids. We also demonstrated horizontal transfer of the largest (853,775 bp) documented ICE, the tripartite ICEMspSymAA22. The conjugation systems of all identified ICEs and several plasmids were related to those of the paradigm ICESym ICEMlSymR7A, with each carrying conserved genes for conjugative pilus formation (trb), excision (rdfS), DNA transfer (rlxS) and regulation (fseA). ICESyms have likely evolved from a common ancestor, despite occupying a variety of distinct integration sites and specifying symbiosis with diverse legumes. We found extensive evidence for recombination between ICEs and particularly ICESyms, which all uniquely lack the conjugation entry-exclusion factor gene trbK. Frequent duplication, replacement and pseudogenization of genes for quorum-sensing-mediated activation and antiactivation of ICE transfer suggests ICE transfer regulation is constantly evolving. Pangenome-wide association analysis of the ICE identified genes potentially involved in symbiosis, rhizosphere colonisation and/or adaptation to distinct legume hosts. In summary, the Mesorhizobium genus has accumulated a large and dynamic pangenome that evolves through ongoing horizontal gene transfer of large conjugative elements related to ICEMlSymR7A

Role of EXPR and Expolysaccharide production in N2 fixation in the Medicago-Sinorhizobium Symbiosis

Exopolysaccharides (EPS's) play an important role in forming and extending infection threads in the symbiosis between Medicago and Sinorhizobium. S. meliloti 1021 (Sm1021) is able to produce one type of EPS (succinoglycan or EPS I) but not another (galactoglucan or EPS II), due to the presence of an insertion sequence (ISRm2011-1) in expR. ExpR forms part of the SinlR quorum sensing system and is a regulator of galactoglucan synthesis. Previous work in our laboratory determined that under N-limited conditions, Sm1021 was poorly effective at fixing N with the model indeterminate legume Medicago truncatula, while two other mucilaginous strains, S. medicae WSM419 and S. melitoti WSM1022, were significantly more effective on this host. While the expR status of WSM1022 is unknown, WSM419 possesses an intact expR gene. These data indicate that the interrupted expR gene in Sm1021 might account for the reduced effectiveness of this strain on the model legume

Biserrula pelecinus L. is a promising forage legume for the central Ethiopian highlands

The availability of effective inoculant rhizobia is often critical to the successful development of productive forage legumes. Biserrula pelecinus L. is a legume with potential as forage in Ethiopia to improve livestock feed quality and soil fertility. B. pelecinus can form N2‐fixing symbiosis with rhizobia in the genus Mesorhizobium. This study investigated the N2 fixation effectiveness of 15 B. pelecinus‐nodulating Mesorhizobium strains on two subspecies of B. pelecinus (B. pelecinus ssp. leiocarpa, native to Ethiopia, and the introduced B. pelecinus ssp. pelecinus). The most effective strain (WSM3873) on both subspecies was assessed at two sites; one with pre‐existing populations of B. pelecinus‐nodulating rhizobia (Modjo), and one without (Holeta). No inoculation response was observed at Modjo when B. pelecinus ssp. pelecinus was inoculated with WSM3873 alone, however, biomass yield was greatest (11.5 tonne DM/ha) following inoculation along with co‐application of phosphorus and nitrogen. At Holeta, a strong inoculation response was achieved with WSM3873 alone on B. pelecinus ssp. pelecinus. In contrast, B. pelecinus ssp. leiocarpa did not show any response at Modjo and failed to emerge after sowing at Holeta. While the native legume B. pelecinus ssp. leiocarpa appears poorly suited to development as a forage, B. pelecinus ssp. pelecinus and WSM3873 represents a promising legume‐rhizobia symbiosis that could benefit farming systems of the central Ethiopian highlands

Investigating nitrogen fixation in the Medicago-Sinorhizobium symbiosis

The Medicago genus is of global importance to agriculture, with the perennial M. sativa being the most widely cultivated and studied member. After many years of studying this plant along with its microsymbiont Sinorhizobium meliloti, it became clear that another host was required to allow simultaneous study of the genetic determinants of both symbiotic partners.M. sativawas unsuited to this role as it is autotetraploid, allogamous and shows strong in-breeding depression, making the analysis of recessive mutations no easy task. Researchers identified the annual medic M. truncatulaas a viable alternative as this host is diploid, autogamous and possess a rapid generation time, among other traits. Consequently, this organism was chosen for sequencing

Genetic diversity and symbiotic effectiveness of Phaseolus vulgaris -nodulating rhizobia in Kenya

Phaseolus vulgaris (common bean) was introduced to Kenya several centuries ago but the rhizobia that nodulate it in the country remain poorly characterised. To address this gap in knowledge, 178 isolates recovered from the root nodules of P. vulgaris cultivated in Kenya were genotyped stepwise by the analysis of genomic DNA fingerprints, PCR-RFLP and 16S rRNA, atpD, recA and nodC gene sequences. Results indicated that P. vulgaris in Kenya is nodulated by at least six Rhizobium genospecies, with most of the isolates belonging to R. phaseoli and a possibly novel Rhizobium species. Infrequently, isolates belonged to R. paranaense, R. leucaenae, R. sophoriradicis and R. aegyptiacum. Despite considerable core-gene heterogeneity among the isolates, only four nodC gene alleles were observed indicating conservation within this gene. Testing of the capacity of the isolates to fix nitrogen (N2) in symbiosis with P. vulgaris revealed wide variations in effectiveness, with ten isolates comparable to R. tropici CIAT 899, a commercial inoculant strain for P. vulgaris. In addition to unveiling effective native rhizobial strains with potential as inoculants in Kenya, this study demonstrated that Kenyan soils harbour diverse P. vulgaris-nodulating rhizobia, some of which formed phylogenetic clusters distinct from known lineages. The native rhizobia differed by site, suggesting that field inoculation of P. vulgaris may need to be locally optimised

Metabolic control of nitrogen fixation in rhizobium-legume symbioses

Rhizobia induce nodule formation on legume roots and differentiate into bacteroids, which catabolize plant-derived dicarboxylates to reduce atmospheric N2 into ammonia. Despite the agricultural importance of this symbiosis, the mechanisms that govern carbon and nitrogen allocation in bacteroids and promote ammonia secretion to the plant are largely unknown. Using a metabolic model derived from genome-scale datasets, we show that carbon polymer synthesis and alanine secretion by bacteroids facilitate redox balance in microaerobic nodules. Catabolism of dicarboxylates induces not only a higher oxygen demand but also a higher NADH/NAD+ ratio than sugars. Modeling and 13C metabolic flux analysis indicate that oxygen limitation restricts the decarboxylating arm of the tricarboxylic acid cycle, which limits ammonia assimilation into glutamate. By tightly controlling oxygen supply and providing dicarboxylates as the energy and electron source donors for N2 fixation, legumes promote ammonia secretion by bacteroids. This is a defining feature of rhizobium-legume symbioses

High Cortical Spreading Depression Susceptibility and Migraine-Associated Symptoms in Ca(V)2.1 S218L Mice

Objective: The CACNA1A gene encodes the pore-forming subunit of neuronal Ca(V)2.1 Ca2+ channels. In patients, the S218L CACNA1A mutation causes a dramatic hemiplegic migraine syndrome that is associated with ataxia, seizures, and severe, sometimes fatal, brain edema often triggered by only a mild head trauma. Methods: We introduced the S218L mutation into the mouse Cacna1a gene and studied the mechanisms for the S218L syndrome by analyzing the phenotypic, molecular, and electrophysiological consequences. Results: Cacna1a(S218L) mice faithfully mimic the associated clinical features of the human S218L syndrome. S218L neurons exhibit a gene dosage-dependent negative shift in voltage dependence of Ca(V)2.1 channel activation, resulting in enhanced neurotransmitter release at the neuromuscular junction. Cacna1a(S218L) mice also display an exquisite sensitivity to cortical spreading depression (CSD), with a vastly reduced triggering threshold, an increased propagation velocity, and frequently multiple CSD events after a single stimulus. In contrast, mice bearing the R192Q CACNA1A mutation, which in humans causes a milder form of hemiplegic migraine, typically exhibit only a single CSD event after one triggering stimulus. Interpretation: The particularly low CSD threshold and the strong tendency to respond with multiple CSD events make the S218L cortex highly vulnerable to weak stimuli and may provide a mechanistic basis for the dramatic phenotype seen in S218L mice and patients. Thus, the S218L mouse model may prove a valuable tool to further elucidate mechanisms underlying migraine, seizures, ataxia, and trauma-triggered cerebral edema. ANN NEUROL 2010;67:85-98Pathophysiology of paroxysmal and chronic degenerative progressive disorder of the central and periferal nervous syste

High Cortical Spreading Depression Susceptibility and Migraine-Associated Symptoms in Ca(V)2.1 S218L Mice

Objective: The CACNA1A gene encodes the pore-forming subunit of neuronal Ca(V)2.1 Ca2+ channels. In patients, the S218L CACNA1A mutation causes a dramatic hemiplegic migraine syndrome that is associated with ataxia, seizures, and severe, sometimes fatal, brain edema often triggered by only a mild head trauma. Methods: We introduced the S218L mutation into the mouse Cacna1a gene and studied the mechanisms for the S218L syndrome by analyzing the phenotypic, molecular, and electrophysiological consequences. Results: Cacna1a(S218L) mice faithfully mimic the associated clinical features of the human S218L syndrome. S218L neurons exhibit a gene dosage-dependent negative shift in voltage dependence of Ca(V)2.1 channel activation, resulting in enhanced neurotransmitter release at the neuromuscular junction. Cacna1a(S218L) mice also display an exquisite sensitivity to cortical spreading depression (CSD), with a vastly reduced triggering threshold, an increased propagation velocity, and frequently multiple CSD events after a single stimulus. In contrast, mice bearing the R192Q CACNA1A mutation, which in humans causes a milder form of hemiplegic migraine, typically exhibit only a single CSD event after one triggering stimulus. Interpretation: The particularly low CSD threshold and the strong tendency to respond with multiple CSD events make the S218L cortex highly vulnerable to weak stimuli and may provide a mechanistic basis for the dramatic phenotype seen in S218L mice and patients. Thus, the S218L mouse model may prove a valuable tool to further elucidate mechanisms underlying migraine, seizures, ataxia, and trauma-triggered cerebral edema. ANN NEUROL 2010;67:85-9