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

Track A Basic Science

Peer Reviewedhttps://deepblue.lib.umich.edu/bitstream/2027.42/138319/1/jia218438.pd

Fundamental molecular techniques for rhizobia

Working with DNA is now a fundamental skill in working with rhizobia. It is necessary for typing strains using PCR methods and for sequencing activities ap¬plied to understanding genomes; their structure, how they function, and their taxonomic position. Nucleic acid purification is the separation of nucleic acids from proteins, cell wall debris and polysaccharide after lysis of cells. For rhizobia, we provide here a num¬ber of commonly used methods for the extraction of genomic and plasmid DNA. Methods for extraction of total RNA are presented in Chapter 13. The CTAB method (Protocol 11.1.1) has been used extensively for extraction of total genom¬ic DNA for DNA sequencing while Protocol 11.1.2 gives higher yields but gener¬ally with slightly lower purity. Plasmid DNA can be differentially displayed using Protocol 11.2.1 for determination of replicon number. This method allows locali¬sation of genes to replicons, confirmation of genome assemblies and identification of genetic changes. The plasmids can subsequently be purified from low melting point gels using GELase (Epicentre, http://www.epibio.com/item.asp?id=297). Protocol 11.2.2 presents a method to recover introduced plasmids from rhizobia (i.e. complementing plasmids) for transformation into Escherichia coli prior to restriction analysis. Protocol 11.2.3 provides an alternative method to the GELase procedure for purifying plasmids but has not been tested as extensively

Acquisition of aspartase activity in Rhizobium leguminosarum WU235

Rhizobium leguminosarum WU235 expresses aspartase (EC 4.3.1.1) when grown on aspartate or asparagine as the sole carbon source, but not on glucose or fumarate. Cells grown on glucose plus aspartate, or fumarate plus aspartate, do not express aspartase. Although these results are reminiscent of catabolite control of an inducible enzyme, induction of aspartate cannot be demonstrated in this strain. Aspartase-producing cells synthesize the enzyme after repeated subculture on glucose plus NH4Cl. Cell grown in glucose plus NH4Cl and plated onto aspartate produce different colony sizes; the larger (0.1% of the total) express aspartate, while the smaller do not. At dilutions sufficient to exclude the large aspartate-producing colonies, a single-sized, aspartase-negative colony was found initially. Such colonies later developed papillae or became cluster colonies; aspartate was produced with papillae formation. The aspartate producing strains were shown, by analysis of native plasmids and periplasmic proteins and by the use of antibiotic resistant strains, to be derived from the parental type. The data suggest that strain WU235 is unable to produce aspartate unless a mutation occurs which leads to constitutive enzyme synthesis. The significance of these observations for studies claiming catabolite repression in Rhizobium is discussed

Taxonomy and physiology of rhizobia

Rhizobia are common Gram-negative soil-inhabiting bacteria distinguished by the feature that they contain genes required for nodulation (e.g. nod, rhi) and ni¬trogen fixation (e.g. nif, fix). These genes enable them to form a symbiotic asso¬ciation with legumes. Currently there are 15 genera of root nodule bacteria (Ta¬ble 7.1) containing more than 120 described species

The transport of L-glutamate by Rhizobium leguminosarum involves a common amino acid carrier

Rhizobium leguminosarum MNF3841 grown on glucose/NH4Cl constitutively transported L-asparagine, L-aspartate, L-glutamate, L-glutamine, glycine, L-leucine, L-methionine and L-phenylalanine. Transport rates were increased 1.5-4-fold by growth on glucose/L-glutamate. Uptake of L-glutamate, L-glutamine, L-asparagine and L-leucine was inhibited to varying extents by a broad range of L-amino acids. Analogues of L-glutamate in which the amino group or α;-hydrogen was methylated inhibited L-glutamate transport much less effectively. Also while 2-and 3-amino acids interfered with L-glutamate uptake, D-glutamate did not. Inhibition by 2,4-dinitrophenol, carbonyl cyanide m-chlorophenylhydrazone and cyanide indicated that amino acid transport was active. The ratio of the intracellular to extracellular concentration of L-leucine after 5 min accumulation was 768. Cells loaded with L-[14C]leucine exhibited exchange not only with external L-leucine but also with L-glutamate. The apparent Km for L-glutamate transport was 0.081 μ;m. Both L-aspartate and L-alanine were competitive inhibitors of L-glutamate uptake with apparent Ki values of 0.164 μ;m and 2.3 μ;m, respectively. These results suggest that there is an extremely high affinity carrier for L-glutamate that is not only very sensitive to inhibition by L-aspartate but also capable of being inhibited by a broad range of amino acids at an order of magnitude higher concentration

Vegetative nitrogen stress decreases lodging risk and increases yield of irrigated spring wheat in the subtropics

In-crop nitrogen (N) application is used widely in rainfed winter wheat production to reduce lodging risk; however, uncertainty exists as to its ability to reduce lodging risk in subtropical irrigated wheat production without simultaneously reducing yield potential. The objective of this study was therefore to determine whether in-crop N application reduces lodging risk without reducing yield of irrigated spring wheat in a subtropical environment. Irrigated small-plot experiments were conducted to compare the effect of alternative N timing on lodging and yield in two cultivars. Variable N regimes were imposed during the vegetative growth phase, after which additional N was applied to ensure that total season N application was uniform across N-timing treatments. Treatments with low N at sowing had significantly less lodging and were the highest yielding, exhibiting yield increases of up to 0.8 t ha–1 compared to treatments with high N at sowing. Increased leaf area index, biomass and tiller count at the end of the vegetative growth phase were correlated with increased lodging in both cultivars, although the strength of the correlation varied with cultivar and season. We conclude that canopy-management techniques can be used to simultaneously increase yield and decrease lodging in irrigated spring wheat in the subtropics, but require different implementation from techniques used in temperate regions of Australia

Abstracts of Presentations at IS-MPMI XVIII Congress

Rhizobia are a diverse group of Alpha- and Beta-proteobacteria that undergo a mutualistic relationship with legume plants. Intense chemical crosstalk between rhizobia and the plant host takes place during symbiotic establishment. One major bacterial signaling factor are secreted lipochito-oligosaccharides. These so-called Nod-factors trigger root hair curling in the host which entraps root hair-attached bacteria. From there rhizobia enter the root of host plants via infection threads. These are plant-derived structures which guide the bacteria into the root cortex where they are released into plant cells. Eventually, rhizobia will differentiate into nitrogen-fixing bacteroids and exchange the fixed nitrogen for reduced carbon sources and other nutrients. Within infection threads rhizobia are enclosed completely by plant tissue and hence, all nutrients needed for growth must be provided by the host. Likewise, rhizobia growing in the rhizosphere receive most of their nutrients from root exudates. So far, the microscopic nature of early infection stages has hampered biochemical experiments and the physico-chemical nature of these important structures remains unknown. A novel experiment using INseq has revealed genes of the pea symbiont Rhizobium leguminosarum bv. viciae 3841 which are essential or advantageous during the infection process. Here, we describe mutations in such metabolic genes and their effect on the symbiotic efficiency of R. leguminosarum