Role of polyhydroxybutyrate and glycogen as carbon storage compounds in pea and bean bacteroids

Rhizobium leguminosarum synthesizes polyhydroxybutyrate and glycogen as its main carbon storage compounds. To examine the role of these compounds in bacteroid development and in symbiotic efficiency, single and double mutants of R. legumosarum bv. viciae were made which lack polyhydroxybutyrate synthase (phaC), glycogen synthase (glgA), or both. For comparison, a single phaC mutant also was isolated in a bean-nodulating strain of R. leguminosarum bv. phaseoli. In one large glasshouse trial, the growth of pea plants inoculated with the R. leguminosarum bv. viciae phaC mutant were significantly reduced compared with wild-type-inoculated plants. However, in subsequent glasshouse and growth-room studies, the growth of pea plants inoculated with the mutant were similar to wildtype-inoculated plants. Bean plants were unaffected by the loss of polyhydroxybutyrate biosynthesis in bacteroids. Pea plants nodulated by a glycogen synthase mutants or the glgA/phaC double mutant, grew as well as the wild type in growth-room experiments. Light and electron micrographs revealed that pea nodules infected with the glgA mutant accumulated large amounts of starch in the II/III interzone. This suggests that glycogen may be the dominant carbon storage compound in pea bacteroids. Polyhydroxybutyrate was present in bacteria in the infection thread of pea plants but was broken down during bacteroid formation. In nodules infected with a phaC mutant of R. leguminosarum bv. viciae, there was a drop in the amount of starch in the II/III interzone, where bacteroids form. Therefore, we propose a carbon burst hypothesis for bacteroid formation, where polyhydroxybutyrate accumulated by bacteria is degraded to fuel bacteroid differentiation.

Effects of forage source and soybean and marine algal oil supplementation on milk fatty acid composition in ewes

The objective of the present studies was to determine effects of basal dietary forage source on the response of milk fatty acid composition to an oil supplement based (2:1, respectively, w/w) on soybean oil and marine algae biomass oil high in cis-9, cis-12 C18:2n − 3 and C22:6n − 3, respectively. In Study 1, Hampshire × Dorset ewes (48) were randomly assigned to one of four treatments and 12 pens in a completely randomized design blocked on the basis of lambing date and number of lambs suckled. Control rations (60:40 forage:concentrate, dry matter (DM) basis) based on alfalfa pellets (AP) or corn silage (CS) were fed from lambing. Beginning at 22 days postpartum, three pens of ewes fed AP and three pens of ewes fed CS were supplemented with oil (30 g/kg of ration DM) in place of corn meal. Average ewe DM intake (DMI) and average daily gain (ADG) were measured weekly. Milk yield and composition were measured at 42 days postpartum. DMI was lower (P<0.02) for CS and for oil, but milk yield was not affected by forage source or oil supplementation. Milk fat content was higher for oil (P<0.10) and milk protein content was higher for AP (P<0.04). Total CLA concentration (g/100 g fatty acids) increased (P<0.01) with CS and oil, and the response to oil was greater for AP (P<0.04). Similarly, total trans-C18:1 and C22:6ω−3 concentrations were higher for CS and oil, but the response to oil was greater for CS (P<0.06 and P<0.01, respectively). In Study 2, the experiment was repeated using alfalfa haylage (AH) instead of AP. The DMI decreased (P<0.05) with oil feeding, but was not affected by forage source. Milk yield was decreased by feeding oil with AH, but not by feeding oil with CS (P<0.03). Milk fat content tended to be increased by feeding oil with AH, but tended to be decreased by feeding oil with CS (P<0.08). Total CLA concentration was increased (P<0.01) for AH versus CS and by oil, and the response to oil supplementation was greater for AH (P<0.01). In contrast, total trans-C18:1 concentration was higher for CS versus AH, with a greater response to oil for CS (P<0.05). Feeding marine oil increased the C22:6ω−3 (P<0.01) concentration, and the response was greater for AH (P<0.04). To further characterize the response of milk fat composition to dietary oil in ewes, a third study used six pens of three ewes each assigned to either the control CS diet used for Study 2 or the same diet supplemented with 45 g/kg (DM basis) of the oil mixture. Feeding oil had no effect on DMI, milk yield or milk fat concentration, but again increased (P<0.001) total trans-C18:1 and C22:6ω−3 concentrations and numerically increased (114%) total CLA concentration. Milk fatty acid composition responses to supplemental vegetable and marine oils were affected by forage source. Milk trans-C18:1 concentration was higher when CS was fed in Studies 1 and 2, but the effect of forage species on CLA concentration differed between studies, which may reflect differences in diet PUFA content and consumption, as well as amounts of dietary starch and fiber consumed. Despite large increases in trans-C18:1 concentration, milk fat content was not decreased by feeding unsaturated oils to ewes, even at diet levels of 45 g/kg of ration DM.

Legumes regulate Rhizobium bacteroid development and persistence by the supply of branched-chain amino acids

One of the largest contributions to biologically available nitrogen comes from the reduction of N-2 to ammonia by rhizobia in symbiosis with legumes. Plants supply dicarboxylic acids as a carbon source to bacteroids, and in return they receive ammonia. However, metabolic exchange must be more complex, because effective N-2 fixation by Rhizobium leguminosarum bv viciae bacteroids requires either one of two broad-specificity amino acid ABC transporters (Aap and Bra). It was proposed that amino acids cycle between plant and bacteroids, but the model was unconstrained because of the broad solute specificity of Aap and Bra. Here, we constrain the specificity of Bra and ectopically express heterologous transporters to demonstrate that branched-chain amino acid (LIV) transport is essential for effective N-2 fixation. This dependence of bacteroids on the plant for LIV is not due to their known down-regulation of glutamate synthesis, because ectopic expression of glutamate dehydrogenase did not rescue effective N-2 fixation. Instead, the effect is specific to LIV and is accompanied by a major reduction in transcription and activity of LIV biosynthetic enzymes. Bacteroids become symbiotic auxotrophs for LIV and depend on the plant for their supply. Bacteroids with aap bra null mutations are reduced in number, smaller, and have a lower DNA content than wild type. Plants control LIV supply to bacteroids, regulating their development and persistence. This makes it a critical control point for regulation of symbiosis. MICROBIOLOGY

Characterization of a gamma-aminobutyric acid transport system of rhizobium leguminosarum bv. viciae 3841

Spontaneous mutants of Rhizobium leguminosarum bv. viciae 3841 were isolated that grow faster than the wild type on gamma-aminobutyric acid (GABA) as the sole carbon and nitrogen source. These strains (RU1736 and RU1816) have frameshift mutations (gtsR101 and gtsR102, respectively) in a GntR-type regulator (GtsR) that result in a high rate of constitutive GABA transport. Tn5 mutagenesis and quantitative reverse transcription-PCR showed that GstR regulates expression of a large operon (pRL100242 to pRL100252) on the Sym plasmid that is required for GABA uptake. An ABC transport system, GtsABCD (for GABA transport system) (pRL100248-51), of the spermidine/putrescine family is part of this operon. GtsA is a periplasmic binding protein, GtsB and GtsC are integral membrane proteins, and GtsD is an ATP-binding subunit. Expression of gtsABCD from a lacZ promoter confirmed that it alone is responsible for high rates of GABA transport, enabling rapid growth of strain 3841 on GABA. Gts transports open-chain compounds with four or five carbon atoms with carboxyl and amino groups at, or close to, opposite termini. However, aromatic compounds with similar spacing between carboxyl and amino groups are excellent inhibitors of GABA uptake so they may also be transported. In addition to the ABC transporter, the operon contains two putative mono-oxygenases, a putative hydrolase, a putative aldehyde dehydrogenase, and a succinate semialdehyde dehydrogenase. This suggests the operon may be involved in the transport and breakdown of a more complex precursor to GABA. Gts is not expressed in pea bacteroids, and gtsB mutants are unaltered in their symbiotic phenotype, suggesting that Bra is the only GABA transport system available for amino acid cycling.

Diversity, phylogeny and distribution of bean rhizobia in salt-affected soils of North-West Morocco

Different molecular methods: BOX-PCR fingerprinting, R-FLP-PCR and sequencing of the 16S rDNA as well as the symbiotic genes nodC and nifH, were used to study the genetic diversity within a collection of nodulating bean rhizobia isolated from five soils of North-West Morocco. BOX fingerprints analysis of 241 isolates revealed 19 different BOX profiles. According to the PFLP-PCR and sequencing of 16S rDNA carried out on 45 representative isolates, 5 genotypes were obtained corresponding to the species Rhizobium etli, R. tropici, R. gallicum, R. leguminosarum and Sinorhizobium meliloti. The most abundant species were R. etli and R. tropici (61% and 24%, respectively). A high intraspecific diversity was observed among the R. etli isolates, while the R. tropici group was homogeneous. Most of the rhizobia studied belong to species known to nodulate common bean, while 2 species were unconventional microsymbionts: R. leguminosarum biovar viciae and S. meliloti. Our results, especially the nodulation promiscuity of common bean and the relation between the predominance of some species of rhizobia in particular soils and the salt content of these soils, indicate that there is a real need for a better understanding of the distribution of common bean rhizobia species in the soils of Morocco before any inoculation attempt.

The genome of Rhizobium leguminosarum has recognizable core and accessory components

Background: Rhizobium leguminosarum is an alpha-proteobacterial N-2-fixing symbiont of legumes that has been the subject of more than a thousand publications. Genes for the symbiotic interaction with plants are well studied, but the adaptations that allow survival and growth in the soil environment are poorly understood. We have sequenced the genome of R. leguminosarum biovar viciae strain 3841. Results: The 7.75 Mb genome comprises a circular chromosome and six circular plasmids, with 61% G+C overall. All three rRNA operons and 52 tRNA genes are on the chromosome; essential protein-encoding genes are largely chromosomal, but most functional classes occur on plasmids as well. Of the 7,263 protein-encoding genes, 2,056 had orthologs in each of three related genomes ( Agrobacterium tumefaciens, Sinorhizobium meliloti, and Mesorhizobium loti), and these genes were overrepresented in the chromosome and had above average G+C. Most supported the rRNA-based phylogeny, confirming A. tumefaciens to be the closest among these relatives, but 347 genes were incompatible with this phylogeny; these were scattered throughout the genome but were over-represented on the plasmids. An unexpectedly large number of genes were shared by all three rhizobia but were missing from A. tumefaciens. Conclusion: Overall, the genome can be considered to have two main components: a 'core', which is higher in G+C, is mostly chromosomal, is shared with related organisms, and has a consistent phylogeny; and an 'accessory' component, which is sporadic in distribution, lower in G+C, and located on the plasmids and chromosomal islands. The accessory genome has a different nucleotide composition from the core despite a long history of coexistence.

Resistance is costly: trade-offs between immunity, fecundity and survival in the pea aphid

Parasitoids are among the most important natural enemies of insects in many environments. Acyrthosiphon pisum, the pea aphid, is a common pest of the leguminous crops in temperate regions. Pea aphids are frequently attacked by a range of endoparasitic wasps, including the common aphidiine, Aphidius ervi. Immunity to parasitoid attack is thought to involve secondary symbiotic bacteria, the presence of which is associated with the death of the parasitoid egg. It has been suggested that there is a fecundity cost of resistance, as individuals carrying the secondary symbionts associated with parasitoid resistance have fewer offspring. Supporting this hypothesis, we find a positive relationship between fecundity and susceptibility to parasitoid attack. There is also a negative relationship between fecundity and off-plant survival time (which positively correlates with resistance to parasitoid attack). Taken together, these results suggest that the aphids can either invest in defence (parasitoid resistance, increased off-plant survival time) or reproduction, and speculate that this may be mediated by changes in the aphids' endosymbiont fauna. Furthermore, there is a positive relationship between aphid size and resistance, suggesting that successful resistance to parasitoid attack may involve physical, as well as physiological, defences.

De novo alanine synthesis by bacteroids of Mesorhizobium loti is not required for nitrogen transfer in the determinate nodules of Lotus corniculatus

Deletion of both alanine dehydrogenase genes (aldA) in Mesorhizobium loti resulted in the loss of AldA enzyme activity from cultured bacteria and bacteroids but had no effect on the symbiotic performance of Lotus corniculatus plants. Thus, neither indeterminate pea nodules nor determinate L. corniculatus nodules export alanine as the sole nitrogen secretion product.

Role of polyhydroxybutyrate and glycogen as carbon storage compounds in pea and bean bacteroids

Rhizobium leguminosarum synthesizes polyhydroxybutyrate and glycogen as its main carbon storage compounds. To examine the role of these compounds in bacteroid development and in symbiotic efficiency, single and double mutants of R. legumosarum bv. viciae were made which lack polyhydroxybutyrate synthase (phaC), glycogen synthase (glgA), or both. For comparison, a single phaC mutant also was isolated in a bean-nodulating strain of R. leguminosarum bv. phaseoli. In one large glasshouse trial, the growth of pea plants inoculated with the R. leguminosarum bv. viciae phaC mutant were significantly reduced compared with wild-type-inoculated plants. However, in subsequent glasshouse and growth-room studies, the growth of pea plants inoculated with the mutant were similar to wildtype-inoculated plants. Bean plants were unaffected by the loss of polyhydroxybutyrate biosynthesis in bacteroids. Pea plants nodulated by a glycogen synthase mutants or the glgA/phaC double mutant, grew as well as the wild type in growth-room experiments. Light and electron micrographs revealed that pea nodules infected with the glgA mutant accumulated large amounts of starch in the II/III interzone. This suggests that glycogen may be the dominant carbon storage compound in pea bacteroids. Polyhydroxybutyrate was present in bacteria in the infection thread of pea plants but was broken down during bacteroid formation. In nodules infected with a phaC mutant of R. leguminosarum bv. viciae, there was a drop in the amount of starch in the II/III interzone, where bacteroids form. Therefore, we propose a carbon burst hypothesis for bacteroid formation, where polyhydroxybutyrate accumulated by bacteria is degraded to fuel bacteroid differentiation.

Comparison of the euryarchaeal microbial community in guts and food-soil of the soil-feeding termite Cubitermes fungifaber across different soil types

Termites are an important component of tropical soil communities and have a significant affect on the structure and nutrient content of soil. Digestion in termites is related to gut structure, gut physico-chemical conditions and gut symbiotic microbiota. Here we describe the use of 16S rRNA gene sequencing and Terminal-restriction Fragment Length Polymorphism (T-RFLP) analysis to examine methanogenic Archaea (MA) in the guts and food-soil of the soil-feeder Cubitermes fungifaber Sjostedt across a range of soil types. If they are strictly vertically inherited, then MA in guts should be the same in all individuals even if the soils differ across sites. In contrast, gut MA should reflect what is present in soil if populations are merely a reflection of what is ingested as the insects forage. We show clear differences between the euryarchaeal communities in termite guts and in food-soils from five different sites. Analysis of 16S rRNA gene clones indicated little overlap between the gut and soil communities. Gut clones were related to a termite-derived Methanomicrobiales cluster, to Methanobrevibacter and, surprisingly, to the haloalkaliphile Natronococcus. Soil clones clustered with Methanosarcina, Methanomicrococcus or Rice Cluster I. T-RFLP analysis indicated that the archaeal communities in the soil samples differed from site to site, whereas those in termite guts were similar between sites. There was some overlap between the gut and soil communities but these may represent transient populations in either guts or soil. Our data does not support the hypothesis that termite gut MA are derived from their food soil but also does not support a purely vertical transmission of gut microflora.

Comparison of Euryarchaea strains in the guts and food-soil of the soil-feeding termite Cubitermes fungifaber across different soil types

Termites are an important component of tropical soil communities and have a significant effect on the structure and nutrient content of soil. Digestion in termites is related to gut structure, gut physicochemical conditions, and gut symbiotic microbiota. Here we describe the use of 16S rRNA gene sequencing and terminal-restriction fragment length polymorphism (T-RFLP) analysis to examine methanogenic archaea (MA) in the guts and food-soil of the soil-feeder Cubitermes fungifaber Sjostedt across a range of soil types. If these MA are strictly vertically inherited, then the MA in guts should be the same in all individuals even if the soils differ across sites. In contrast, gut MA should reflect what is present in soil if populations are merely a reflection of what is ingested as the insects forage. We show clear differences between the euryarchaeal communities in termite guts and in food-soils from five different sites. Analysis of 16S rRNA gene clones indicated little overlap between the gut and soil communities. Gut clones were related to a termite-derived Methanomicrobiales cluster, to Methanobrevibacter and, surprisingly, to the haloalkaliphile Natronococcus. Soil clones clustered with Methanosarcina, Methanomicrococcus, or rice cluster I. T-RFLP analysis indicated that the archaeal communities in the soil samples differed from site to site, whereas those in termite guts were similar between sites. There was some overlap between the gut and soil communities, but these may represent transient populations in either guts or soil. Our data do not support the hypothesis that termite gut MA are derived from their food-soil but also do not support a purely vertical transmission of gut microflora.

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

The biological reduction of atmospheric N-2 to ammonium (nitrogen fixation) provides about 65% of the biosphere's available nitrogen. Most of this ammonium is contributed by legume rhizobia symbioses(1), 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(2,3). 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.

Making sense of supply chain management: acomparative study of aerospace and construction

Current recipes for learning across business sectors too often fail to recognize the embedded and contextual nature of management practice. The existing literature gives little emphasis to the symbiotic relationship between supply chain management and the broader dynamics of context. The aerospace and construction sectors are selected for comparison on the basis that they are so different. The UK aerospace sector has undergone extensive consolidation as a result of the imperatives of global competitive pressures. In contrast, the construction industry has experienced decades of fragmentation and remains highly localized. An increasing proportion of output in the aerospace sector occurs within a small number of large, globally orientated firms. In contrast, construction output is dominated by a plethora of small firms with high levels of subcontracting and a widespread reliance on self-employment. These differences have fundamental implications for the way that supply chain management is understood and implemented in the two sectors. Semi-structured interviews with practitioners from both sectors support the contention that supply chain management is more established in aerospace than construction. The introduction of prime contracting and the increasing use of framework agreements within the construction sector potentially provide a much more supportive climate for supply chain management than has traditionally prevailed. However, progress depends upon an improved continuity of workload under such arrangements.

Dietary manipulation of fatty acid composition in lamb meat and its effect on the volatile aroma compounds of grilled lamb

The effect on lamb muscle of five dietary supplements high in polyunsaturated fatty acids (PUFA) was measured. The supplements were linseed oil, fish oil, protected lipid (high in linoleic acid (C18:2 n-6) and alpha-linolenic acid (C18:3 n-3)), fish oil/marine algae (1:1), and protected lipid/marine algae (1:1). Eicosapentaenoic acid (C20:5 n-3) and docosahexaenoic acid (C22:6 n-3) were found in the highest amounts in the meat from lambs fed diets containing algae. Meat from lambs fed protected lipid had the highest levels of C18:2 n-6 and C18:3 n-3, due to the effectiveness of the protection system. In grilled meat from these animals, volatile compounds derived from n-3 fatty acids were highest in the meat from the lambs fed the fish oil/algae diet, whereas compounds derived from n-6 fatty acids were highest in the meat from the lambs fed the protected lipid diet. (C) 2004 Elsevier Ltd. All rights reserved.

Human-machine symbiosis overview

The interface between humans and technology is a rapidly changing field. In particular as technological methods have improved dramatically so interaction has become possible that could only be speculated about even a decade earlier. This interaction can though take on a wide range of forms. Indeed standard buttons and dials with televisual feedback are perhaps a common example. But now virtual reality systems, wearable computers and most of all, implant technology are throwing up a completely new concept, namely a symbiosis of human and machine. No longer is it sensible simply to consider how a human interacts with a machine, but rather how the human-machine symbiotic combination interacts with the outside world. In this paper we take a look at some of the recent approaches, putting implant technology in context. We also consider some specific practical examples which may well alter the way we look at this symbiosis in the future. The main area of interest as far as symbiotic studies are concerned is clearly the use of implant technology, particularly where a connection is made between technology and the human brain and/or nervous system. Often pilot tests and experimentation has been carried out apriori to investigate the eventual possibilities before human subjects are themselves involved. Some of the more pertinent animal studies are discussed briefly here. The paper however concentrates on human experimentation, in particular that carried out by the authors themselves, firstly to indicate what possibilities exist as of now with available technology, but perhaps more importantly to also show what might be possible with such technology in the future and how this may well have extensive social effects. The driving force behind the integration of technology with humans on a neural level has historically been to restore lost functionality in individuals who have suffered neurological trauma such as spinal cord damage, or who suffer from a debilitating disease such as lateral amyotrophic sclerosis. Very few would argue against the development of implants to enable such people to control their environment, or some aspect of their own body functions. Indeed this technology in the short term has applications for amelioration of symptoms for the physically impaired, such as alternative senses being bestowed on a blind or deaf individual. However the issue becomes distinctly more complex when it is proposed that such technology be used on those with no medical need, but instead who wish to enhance and augment their own bodies, particularly in terms of their mental attributes. These issues are discussed here in the light of practical experimental test results and their ethical consequences.