D. Allaway

Amino-acid cycling drives nitrogen fixation in the legume-Rhizobium symbiosis

The biological reduction of atmospheric N2 to ammonium (nitrogen fixation) provides about 65% of the biospheres available nitrogen. Most of this ammonium is contributed by legume–rhizobia symbioses, which are initiated by the infection of legume hosts by bacteria (rhizobia), resulting in formation of root nodules. Within the nodules, rhizobia are found as bacteroids, which perform the nitrogen fixation: to do this, they obtain sources of carbon and energy from the plant, in the form of dicarboxylic acids. It has been thought that, in return, bacteroids simply provide the plant with ammonium. But here we show that a more complex amino-acid cycle is essential for symbiotic nitrogen fixation by Rhizobium in pea nodules. The plant provides amino acids to the bacteroids, enabling them to shut down their ammonium assimilation. In return, bacteroids act like plant organelles to cycle amino acids back to the plant for asparagine synthesis. The mutual dependence of this exchange prevents the symbiosis being dominated by the plant, and provides a selective pressure for the evolution of mutualism.

Carbon and nitrogen metabolism in Rhizobium

One of the paradigms of symbiotic nitrogen fixation has been that bacteroids reduce N2 to ammonium and secrete it without assimilation into amino acids. This has recently been challenged by work with soybeans showing that only alanine is excreted in 15N2 labelling experiments. Work with peas shows that the bacteroid nitrogen secretion products during in vitro experiments depend on the experimental conditions. There is a mixed secretion of both ammonium and alanine depending critically on the concentration of bacteroids and ammonium concentration. The pathway of alanine synthesis has been shown to be via alanine dehydrogenase, and mutation of this enzyme indicates that in planta there is likely to be mixed secretion of ammonium and alanine. Alanine synthesis directly links carbon catabolism and nitrogen assimilation in the bacteroid. There is now overwhelming evidence that the principal carbon sources of bacteroids are the C4-dicarboxylic acids. This is based on labelling and bacteroid respiration data, and mutation of both the dicarboxylic acid transport system (dct) and malic enzyme. L-malate is at a key bifurcation point in bacteroid metabolism, being oxidized to oxaloacetate and oxidatively decarboxylated to pyruvate. Pyruvate can be aminated to alanine or converted to acetyl-CoA where it either enters the TCA cycle by condensation with oxaloacetate or forms polyhydroxybutyrate (PHB). Thus regulation of carbon and nitrogen metabolism are strongly connected. Efficient catabolism of C4-dicarboxylates requires the balanced input and removal of intermediates from the TCA cycle. The TCA cycle in bacteroids may be limited by the redox state of NADH/NAD+ at the 2-ketoglutarate dehydrogenase complex, and a number of pathways may be involved in bypassing this block. These pathways include PHB synthesis, glutamate synthesis, glycogen synthesis, GABA shunt and glutamine cycling. Their operation may be critical in maintaining the optimum redox poise and carbon balance of the TCA cycle. They can also be considered to be overflow pathways since they act to remove or add electrons and carbon into the TCA cycle. Optimum operation of the TCA cycle has a major impact on nitrogen fixation.

Identification of alanine dehydrogenase and its role in mixed secretion of ammonium and alanine by pea bacteroids

N2‐fixation by Rhizobium‐legume symbionts is of major ecological and agricultural importance, responsible for producing a substantial fraction of the biospheres nitrogen. On the basis of 15N‐labelling studies, it had been generally accepted that ammonium is the sole secretion product of N2‐fixation by the bacteroid and that the plant is responsible for assimilating it into amino acids. However, this paradigm has been challenged in a recent 15N‐labelling study showing that soybean bacteroids only secrete alanine. Hitherto, nitrogen secretion has only been assessed from in vitro15N‐labelling studies of isolated bacteroids. We show that both ammonium and alanine are secreted by pea bacteroids. The in vitro partitioning between them will depend on whether the system is open or closed, as well as the ammonium concentration and bacteroid density. To overcome these limitations we identified and mutated the gene for alanine dehydrogenase (aldA) and demonstrate that AldA is the primary route for alanine synthesis in isolated bacteroids. Bacteroids of the aldA mutant fix nitrogen but only secrete ammonium at a significant rate, resulting in lower total nitrogen secretion. Peas inoculated with the aldA mutant are green and healthy, demonstrating that ammonium secretion by bacteroids can provide sufficient nitrogen for plant growth. However, plants inoculated with the mutant are reduced in biomass compared with those inoculated with the wild type. The labelling and plant growth studies suggest that alanine synthesis and secretion contributes to the efficiency of N2‐fixation and therefore biomass accumulation.

Rhizobium leguminosarum Has a Second General Amino Acid Permease with Unusually Broad Substrate Specificity and High Similarity to Branched-Chain Amino Acid Transporters (Bra/LIV) of the ABC Family

Amino acid uptake by Rhizobium leguminosarum is dominated by two ABC transporters, the general amino acid permease (Aap) and the branched-chain amino acid permease (Bra(Rl)). Characterization of the solute specificity of Bra(Rl) shows it to be the second general amino acid permease of R. leguminosarum. Although Bra(Rl) has high sequence identity to members of the family of hydrophobic amino acid transporters (HAAT), it transports a broad range of solutes, including acidic and basic polar amino acids (L-glutamate, L-arginine, and L-histidine), in addition to neutral amino acids (L-alanine and L-leucine). While amino and carboxyl groups are required for transport, solutes do not have to be alpha-amino acids. Consistent with this, Bra(Rl) is the first ABC transporter to be shown to transport gamma-aminobutyric acid (GABA). All previously identified bacterial GABA transporters are secondary carriers of the amino acid-polyamine-organocation (APC) superfamily. Also, transport by Bra(Rl) does not appear to be stereospecific as D amino acids cause significant inhibition of uptake of L-glutamate and L-leucine. Unlike all other solutes tested, L-alanine uptake is not dependent on solute binding protein BraC(Rl). Therefore, a second, unidentified solute binding protein may interact with the BraDEFG(Rl) membrane complex during L-alanine uptake. Overall, the data indicate that Bra(Rl) is a general amino acid permease of the HAAT family. Furthermore, Bra(Rl) has the broadest solute specificity of any characterized bacterial amino acid transporter.

A Monocarboxylate Permease of Rhizobium leguminosarum Is the First Member of a New Subfamily of Transporters

Amino acid transport by Rhizobium leguminosarum is dominated by two ABC transporters, the general amino acid permease (Aap) and the branched-chain amino acid permease (Bra). However, mutation of these transporters does not prevent this organism from utilizing alanine for growth. An R. leguminosarum permease (MctP) has been identified which is required for optimal growth on alanine as a sole carbon and nitrogen source. Characterization of MctP confirmed that it transports alanine (K(m) = 0.56 mM) and other monocarboxylates such as lactate and pyruvate (K(m) = 4.4 and 3.8 micro M, respectively). Uptake inhibition studies indicate that propionate, butyrate, alpha-hydroxybutyrate, and acetate are also transported by MctP, with the apparent affinity for solutes demonstrating a preference for C3-monocarboxylates. MctP has significant sequence similarity to members of the sodium/solute symporter family. However, sequence comparisons suggest that it is the first characterized permease of a new subfamily of transporters. While transport via MctP was inhibited by CCCP, it was not apparently affected by the concentration of sodium. In contrast, glutamate uptake in R. leguminosarum by the Escherichia coli GltS system did require sodium, which suggests that MctP may be proton coupled. Uncharacterized members of this new subfamily have been identified in a broad taxonomic range of species, including proteobacteria of the beta-subdivision, gram-positive bacteria, and archaea. A two-component sensor-regulator (MctSR), encoded by genes adjacent to mctP, is required for activation of mctP expression.

Solute-binding protein-dependent ABC transporters are responsible for solute efflux in addition to solute uptake.

The ATP‐binding cassette (ABC) transporter superfamily is one of the most widespread of all gene families and currently has in excess of 1100 members in organisms ranging from the Archaea to man. The movement of the diverse solutes of ABC transporters has been accepted as being strictly unidirectional, with recent models indicating that they are irreversible. However, contrary to this paradigm, we show that three solute‐binding protein‐dependent (SBP) ABC transporters of amino acids, i.e. the general amino acid permease (Aap) and the branched‐chain amino acid permease (Bra) of Rhizobium leguminosarum and the histidine permease (His) of Salmonella typhimurium, are bidirectional, being responsible for efflux in addition to the uptake of solutes. The net solute movement measured for an ABC transporter depends on the rates of uptake and efflux, which are independent; a plateau is reached when both are saturated. SBP ABC transporters promote active uptake because, although the Vmax values for uptake and efflux are not significantly different, there is a 103−104 higher affinity for uptake of solute compared with efflux. Therefore, the SBP ABC transporters are able to support a substantial concentration gradient and provide a net uptake of solutes into bacterial cells.

Regulation of l-Alanine Dehydrogenase in Rhizobium leguminosarum bv. viciae and Its Role in Pea Nodules

Alanine dehydrogenase (AldA) is the principal enzyme with which pea bacteroids synthesize alanine de novo. In free-living culture, AldA activity is induced by carboxylic acids (succinate, malate, and pyruvate), although the best inducer is alanine. Measurement of the intracellular concentration of alanine showed that AldA contributes to net alanine synthesis in laboratory cultures. Divergently transcribed from aldA is an AsnC type regulator, aldR. Mutation of aldR prevents induction of AldA activity. Plasmid-borne gusA fusions showed that aldR is required for transcription of both aldA and aldR; hence, AldR is autoregulatory. However, plasmid fusions containing the aldA-aldR intergenic region could apparently titrate out AldR, sometimes resulting in a complete loss of AldA enzyme activity. Therefore, integrated aldR::gusA and aldA::gusA fusions, as well as Northern blotting, were used to confirm the induction of aldA activity. Both aldA and aldR were expressed in the II/III interzone and zone III of pea nodules. Overexpression of aldA in bacteroids did not alter the ability of pea plants to fix nitrogen, as measured by acetylene reduction, but caused a large reduction in the size and dry weight of plants. This suggests that overexpression of aldA impairs the ability of bacteroids to donate fixed nitrogen that the plant can productively assimilate. We propose that the role of AldA may be to balance the alanine level for optimal functioning of bacteroid metabolism rather than to synthesize alanine as the sole product of N(2) reduction.

Optical traps: shedding light on biological processes

Optical traps exploit the radiation forces of laser light to manipulate microscopic particles. The ability to manipulate biological material and quantify the force required has been exploited in the biosciences; from the isolation of single cells to kinetic measurements of single motor molecules. This review describes the theory of optical trapping and using recent publications gives examples of how it has been employed across a broad spectrum of biological research.

Poly-β-Hydroxybutyrate and Glycogen Metabolism in Rhizobium leguminosarum

The role of the carbon storage compounds poly-β-hydroxybutyrate and glycogen in bacteroids is not fully understood. Both compounds are synthesised in bacteroids however the overall pool sizes vary considerably. Furthermore, mutation of the biosynthetic pathways has been shown to effect the efficiency of symbiotic nitrogen fixation in beans (Cevallos, et al., 1996; Marroqui et al., 2001). Therefore the pathways appear to be involved in the regulation of bacteroid metabolism. Here we have constructed single mutants in phaC (PHB synthase) and glgA (glycogen synthase), and a double mutant, to assess their impact on the R. leguminosarum bv. viciae symbiosis. Summary