Prokaryotic Nitrogen Fixation: A Model System for the Analysis of a Biological Process Chapter Abstracts
Chapter 1
The Nitrogen Cycle
Daniel J. Arp
The nitrogen cycle is a series of primarily microbially mediated transformations of inorganic N. The three primary processes involved in the N cycle are nitrogen fixation, nitrification, and denitrification. In nitrogen fixation, N2 serves as a nitrogen source for bacteria. In nitrification, ammonia or nitrite serves as the source of reductant and energy for chemolithotrophic growth. In denitrification, N oxides serve as terminal electron acceptors to support microbial respiration, usually in the absence of O2. Because the products of one process become the substrates for another process, the three processes are linked into a biogeochemical cycle. The environmental and economic significance of the cycle is profound. The activities of humans have dramatically altered the flux of N through the cycle. The enzymes that catalyze the individual steps in the N cycle and the genes that code for these enzymes have provided new insights into the mechanisms by which microorganisms can utilize various inorganic compounds and respond to the presence of these compounds in the environment.
Chapter 2
Ecology and Molecular Biology of Trichodesmium
Jonathan P. Zehr and Douglas G. Capone
Although fixed inorganic nitrogen concentrations are low in large areas of the world's oceans, few diazotrophic microorganisms have been identified or cultivated from the water column. Recent biogeochemical studies indicate that nitrogen fixation is very important in the nitrogen flux of the upper water column of oligotrophic oceans. The most obvious diazotrophic microorganism that contributes to this nitrogen fixation is the filamentous, nonheterocystous cyanobacterium Trichodesmium. Trichodesmium is a visually prominent aggregate-forming cyanobacterium, which is unusual in that it fixes nitrogen only during the day, with no obvious temporal or spatial separation of oxygen-sensitive nitrogen fixation from the oxygen evolved by photosynthesis. Studies have shown that the daily pattern of nitrogen fixation is cued by a circadian rhythm, and that expression of the photosynthetic apparatus is also under circadian control. Cloning and sequencing of twelve genes of the nitrogen fixation apparatus shows that the nitrogen fixation genes are similar to heterocystous and nonheterocystous cyanobacterial nitrogenase genes, but may be most similar to the genes of the vegetative nitrogenase of Anabaena variabilis. Trichodesmium is an important and intriguing nitrogen-fixing microorganism in oligotrophic waters. Studies using a DNA amplification approach indicate that other, less obvious diazotrophs are also present in the marine environment, and may provide interesting new microorganisms for the study of nitrogen fixation.
Chapter 3
Introduction To Nitrogenases
Robert H. Burris
Although Boussingault reported N2 fixation by leguminous plants in 1838, others were skeptical of his observations until 1886 when Hellriegel and Wilfarth reported convincing experiments and associated the process with specific bacteria in root nodules. Studies on the biochemistry of fixation were initiated by Meyerhof and Burk and extended by P. W. Wilson and associates. The use of 15N as a tracer aided in establishing that the key intermediate in N2 fixation was ammonia, and that this was assimilated promptly into glutamic acid. It was shown that adenosine triphosphate was essential in the fixation process, and that fixation was achieved by the combined action of MoFe and Fe units of the nitrogenase enzyme system. The tertiary structure of these units has been established, and the genetics and control of the nitrogenase system have been worked out in some detail.
Chapter 4
Structure of the Nitrogenase Protein Components
James Bryant Howard and Douglas C. Rees
This chapter summarizes and provides an overview of the three dimensional structures, as determined by x-ray crystallography, of the two nitrogenase protein components, the Fe-protein and the MoFe-protein. In addition, the structure of an intermediate in the enzymic catalytic turnover, the Fe-protein-ADP.AlF4-MoFe-protein complex, is discussed in terms of the enzyme mechanism.
Chapter 5
Biosynthesis of the Iron-Molybdenum and Iron-vanadium Cofactors of the nif- and vnf-Encoded Nitrogenases
Priya Rangaraj, Carmen Rüttimann-Johnson, Vinod K. Shah and Paul W. Ludden
The iron-molybdenum cofactor (FeMo-co) of nitrogenase is a unique Mo-Fe-S cluster that has been proposed as the site of substrate reduction in dinitrogenase. At least six different nif-gene products, NifQ, V, B, H, N and E, are known to be involved in the biosynthesis of FeMo-co in the bacterium Klebsiella pneumoniae. Although the structure of FeMo-co has been solved, little is known about the biosynthetic pathway of the cofactor. An in vitro FeMo-co synthesis system which requires the products of nifB, H, N and E in the presence of homocitrate, ATP, Mo, and reductant has been described and this system has proved to be invaluable in the identification of other components that play a role in the synthesis of the cofactor. In this chapter, we summarize some of the salient features and relatively new developments concerning FeMo-co biosynthesis. We also discuss what is known about the recently discovered V-nitrogenase, and the many aspects that are common to V-nitrogenase and the conventional Mo-containing enzyme are reviewed.
Chapter 6
Time Resolved Infra-red Spectroscopy of Functioning Nitrogenase
Roger N.F. Thorneley and Simon J. George
Stopped-flow Fourier transform infra-red spectroscopy (SF-FTIR) has great potential as a dynamic probe of small molecules binding to metal sites in proteins and their concomitant chemistry. In this chapter we describe some of the technical issues concerning measurement and analysis of SF-FTIR data. We also describe our current SF-FTIR apparatus recently installed at the John Innes Centre. We detail the application of the technique to the inhibition of nitrogenase by carbon monoxide (CO) and present the first IR spectra of a small molecule; CO, bound to functioning nitrogenase. This spectrum is dependent on the concentration of CO. Under limiting CO conditions only a single v(CO) band is initially seen whereas under excess CO a complex spectrum is observed indicating more than one binding site. In both cases, after several minutes the IR spectrum changes to indicate a novel CO bound species with a v(CO) stretch at the low energy of 1715 cm-1. The future direction of our work on small molecule activation at metal sites in enzymes is also outlined.
Chapter 7
Use of Amino Acid Substitutions to Study the Functional Properties of the Nitrogenase MoFe Protein
Jason Christiansen, Jeannine M. Chan, Lance C. Seefeldt, and Dennis R. Dean
Site-directed mutagenesis methods and facile gene-replacement techniques have permitted the construction of many mutant strains of Azotobacter vinelandii that carry known amino acid substitutions within the nitrogenase MoFe protein. The goal of such studies is to gain insight into how the MoFe protein elicits Fe protein MgATP hydrolysis in order to promote intercomponent electron transfer, the role of the P-cluster in nitrogenase catalysis, and where and how substrates are bound to the active site. Preliminary evidence that the MoFe protein a- and b-subunit Phe125 residues are involved in some aspect of intercomponent interaction that leads to MgATP hydrolysis are described. Experiments are described which indicate that electron and proton transfer could be coupled during nitrogenase catalysis. Evidence for a role for the P-cluster in the nitrogenase catalytic mechanism was obtained by analysis of an altered MoFe protein that has the redox-dependent P-cluster ligand, b-Ser188, substituted by cysteine. The altered MoFe protein exhibits an EPR signal originating from the P-cluster in the dithionite-reduced, resting state. This signal disappears under turnover conditions and reappears when the MoFe protein returns to the resting state. Efforts to identify the substrate-binding site have been unsuccessful. Nevertheless, characterization of an altered form of the MoFe protein for which the a-His195 residue was substituted by glutamine has provided some insight into MoFe protein function. This altered MoFe protein is able to bind N2 but is not able to reduce it. Although N2 is not a substrate for the altered MoFe protein, it is a strong inhibitor of both acetylene and proton reduction, both of which are otherwise effectively reduced by the altered MoFe protein. Contrary to the suggestion of Dilworth et al (Biochemistry 37, 17495-17505), these results argue for a common or shared site for N2, acetylene, and proton binding. N2 uncouples MgATP hydrolysis from proton reduction catalyzed by the altered MoFe protein but does so without lowering the overall rate of MgATP hydrolysis. Thus, the flow of electrons from the Fe protein to the MoFe protein is controlled, in part, by the substrate serving as an effective electron sink.
Chapter 8
Roles for Nucleotides in Nitrogenase Catalysis
Jeannine M. Chan, Jennifer E. Huyett, and Lance C. Seefeldt
The roles for nucleotides in nitrogenase catalyzed reduction of N2 to NH3 are reviewed. MgATP was first identified as a co-substrate for all nitrogenase catalyzed reactions nearly forty years ago. Nucleotides are today known to participate at several different levels in the nitrogenase mechanism, which include: (i) binding to the Fe protein component of nitrogenase, (ii) hydrolysis by the nitrogenase Fe protein-MoFe protein complex, (iii) coupling of MgATP hydrolysis to electron transfer and substrate reduction by the nitrogenase complex, (iv) regulation of the affinity for association of the Fe protein and the MoFe protein, and (v) biosynthesis of metal clusters of nitrogenase. The state of knowledge for the roles of nucleotides at each of the first four levels is reviewed here, while the last role is reviewed in another chapter of this book.
Chapter 9.
Control of Nitrogen Fixation Genes in Klebsiella pneumoniae
Timothy R. Hoover
Expression of the nitrogen fixation (nif) genes in Klebsiella pneumoniae is tightly controlled in response to oxygen and fixed nitrogen through a sophisticated hierarchy of regulatory mechanisms. Transcription of the nif genes requires a form of RNA polymerase holoenzyme that has the alternative sigma factor, σ54. To initiate transcription from the nif promoters, σ54-holoenzyme requires the transcriptional activator NifA . NifA binds to sites that are generally 100-200 bp upstream of the transcriptional start site and contacts σ54-holoenzyme bound at the promoter through DNA looping to activate transcription. At several of the nif promoter regions, productive interactions between NifA and σ54-holoenzyme are facilitated by an auxiliary DNA-binding protein, the integration host factor. NifA activates transcription by catalyzing the isomerization of a closed complex between σ54-holoenzyme and the promoter to an open complex that is competent to initiate transcription. NifA must couple energy released from the hydrolysis of either ATP or GTP to catalyze this isomerization. The activity of NifA is controlled by NifL in response to oxygen and fixed nitrogen. The levels of NifA inside the cell are also regulated at the transcriptional level, which is mediated through the global nitrogen regulatory system of K. pneumoniae.
Chapter 10.
Regulation of Nitrogenase Activity in Phototrophic Bacteria by Reversible Covalent Modification
Stefan Nordlund
In Rhodospirillum rubrum, some other photosynthetic bacteria, and in some species of Azospirillum nitrogenase is regulated on the metabolic level in addition to the transcriptional control operating in all diazotrophs studied. This metabolic control is manifested as a decrease in nitrogenase activity, the "switch-off" effect, when ammonium, or some other compounds, is added to a nitrogen fixing culture of R. rubrum. Subjecting the culture to darkness has the same effect and in A. brasilense changing the conditions to anaerobicity also leads to "switch-off". The loss of nitrogenase activity is due to reversible ADP-ribosylation of a specific arginine on dinitrogenase reductase which makes it inactive in transferring electrons to dinitrogenase, the other protein in the nitrogenase complex. The enzymes catalyzing modification/demodification, called DRAT and DRAG, have been purified and characterized.
The major question remaining in this metabolic regulation is the identity of the signal(s) from the externally added "switch-off" effectors to DRAT and DRAG and how it is transduced. This issue is discussed and the properties of the enzymes involved are reviewed.
Chapter 11
Regulation of nitrogen fixation genes in phototrophs: new mechanisms of bacterial gene activation.
Robert G. Kranz
Rhodobacter capsulatus has been the best studied anoxygenic photosynthetic bacterium with respect to regulation of nitrogen fixation (nif) genes. Studies on this organism's nif regulatory circuitry have led to the discovery of a new type of enhancer-binding activator in bacteria, called RcNtrC. The properties that make RcNtrC unique are discussed. Like nif genes, the genes for photosynthesis are only expressed under anaerobic conditions in R. capsulatus. The regulatory circuitry for photosynthesis genes has been established in the last few years with results suggesting that oxygen-control mechanisms are also different than in the better-studied enteric bacteria. Historical questions concerning any regulatory overlap between nitrogen fixation and photosynthesis can now be addressed at the molecular level. Recent results on possible links are presented. Finally, Rhodobacter is a high GC organism, like most members of the α proteobacteria, and some recent results suggest that the RNA polymerase from this organism may have capabilities not found in RNA polymerase from enteric bacteria.
Chapter 12
Phylogeny of Root- and Stem-Nodule Bacteria Associated with Legumes
En Tao Wang and Esperanza Martínez-Romero
Root- and stem-nodule bacteria associated with legumes have been classified into 27 species within 6 genera. These species form four deep branches within the -subclass of Proteobacteria, Azorhizobium, Bradyrhizobium, Mesorhizobium, and Allorhizobium-Rhizobium-Sinorhizobium. These four branches are also distinguishable by phenotypic features. Non-symbiotic relatives exist in each of these four branches, indicating common ancestors between rhizobial species and other parasite or soil-borne bacteria. Relationships different from 16S gene phylogeny are revealed in the phylogenies of symbiotic genes (nif and nod) in some cases. The possibility that rhizobia originated by acquiring symbiotic gene clusters via lateral gene transfer is discussed based on the comparison of phylogenies of 16S rRNA and symbiotic genes and on other evidence.
Chapter 13
Molecular Evolution of Interactions between Rhizobia and their Legume Hosts
Zewdu Terefework*, Gilles Lortet*, Leena Suominen and Kristina Lindström
* Both authors contributed equally to the work.
Closely interacting organisms are supposed to follow a common history where one of the associates tracks the other in an evolutionary timescale. In the rhizobia-legume symbiosis, phylogenies from ribosomal sequences do not show parallel divergence with plant taxonomy. The molecular machinery of the symbiosis has been shown to be shaped among others by gene duplication, lateral transfer and recombination. These genetic events render most genes and gene products unsuitable for studying the codiverging pattern of the symbiosis. Some of the genes involved in the nodulation process could have been acquired by different genera of rhizobia by lateral transfer. Their evolution, however, could have been then under the plant functional constraint, with the Nod factors as a target for this selection pressure. The nodA phylogeny reveals a common ancestor for all the rhizobia producing Nod factors acylated by polyunsaturated fatty acids. In a similar way, the legume phylogeny also displays a common origin for the host plants of these rhizobia. Based on these data, two strategies can be defined for the recognition of Nod factors by the plant, and a model has been built to try to clarify the evolutionary relationships between them.
Chapter 14
Evolution of the Actinorhizal Plant Symbiosis
David R. Benson and Michael L. Clawson
The Frankia-actinorhizal plant root nodule symbiosis occurs in eight families of angiosperms. Phylogenetic analyses show that actinorhizal plants define three out of four major lineages within a "nitrogen-fixing clade" of angiosperms, and infective Frankia strains group in three clades. Although a close relationship exists between the plant and bacterial groups, the phylogenies of the symbionts are not congruent. Given the general order of appearance of the plants in the fossil record, an older lineage of actinorhizal plants is proposed to have served as a reservoir of infective Frankia that more recently differentiated plants have tapped on at least three additional occasions. Diversification of the host and symbionts can be viewed in light of a geographic mosaic model of coevolution.
Chapter 15
Nitrogen Fixation by Termite Symbionts
Deborah A. Waller
Bacterial symbionts in the hindguts of many termite species fix atmospheric nitrogen that is incorporated into termite tissues. Two diazotrophs, both facultative anaerobes, have been cultured from termite hindguts, but it is likely that many more species will be revealed by using a greater variety of culturing techniques. Genetic probing for nifH sequences in one termite species has isolated dozens of clones representing diverse phylogentic lineages, although there is no information on whether these microbes fix nitrogen in vivo. The source of the hindgut diazotrophs is unknown: they may be endemic to the termite host and passed exclusively through trophallaxis (social feeding) among nestmates, or they may be acquired incidentally from the environment. Nitrogen fixation rates vary widely among termite species, different castes and environmental conditions. Long-term studies focused on the microbial communities of different termite species will be valuable in understanding the evolution and significance of this unique association.
Chapter 16
Rhizobial Motility and Chemotaxis: Molecular Biology and Ecological Role
Christopher K. Yost and Michael F. Hynes
Rhizobia are motile bacteria, with pronounced chemotactic responses to a wide variety of metabolites likely to be found in soil, in the rhizosphere, and in plant exudates. There is convincing experimental evidence that both motility and chemotaxis can play a significant role in the autecology of rhizobial strains, in that mutants impaired in either process have reduced competiveness or nodulation efficiency. This chapter reviews some of the progress made in understanding the processes of chemotaxis and motility in the rhizobia, with emphasis on
the molecular mechanisms involved, and the ecology of the process.
Chapter 17
Solving the Competition Problem: Genetic and Field Approaches to Enhance the Effectiveness of the Rhizobium-Legume Symbiosis
Alexandra J Scupham, Eduardo A. Robleto, and Eric W. Triplett
This chapter summarizes current efforts to improve the ability of Rhizobium inoculum strains to limit infection of legume roots by indigenous rhizobia. These efforts require the identification and isolation of those genetic determinants that confer increased nodulation competitiveness followed by their transfer and stable expression in inoculum strains. To date, this approach has been used successfully in the development of new inoculum strains that possess the ability to produce a potent anti-rhizobial peptide called trifolitoxin. These new strains have significantly higher nodule occupancy under agricultural conditions compared to either isogenic strains that lack trifolitoxin production or indigenous rhizobia. This system is being further improved by the addition of yield-enhancing uptake hydrogenase genes to inoculum strains along with trifolitoxin production and resistance genes.
Other approaches described here include the identification of other antibiosis genes and other phenotypes that may contribute to competitiveness such as motility and cell surface characteristics. In addition, the effects of non-rhizobial strains on the competitiveness of rhizobial inoculum strains are discussed as are efforts to develop Rhizobium strain-specific host genotypes.
Chapter 18
Competition for Nodulation in the Soybean/Bradyrhizobium Symbiosis
Michael Jay Sadowsky
Bradyrhizobium japonicum is the nitrogen-fixing, root nodule symbiont of soybeans. A major emphasis of recent dinitrogen fixation research with B. japonicum has been the development of genetic engineering techniques for the construction of bradyrhizobial strains with enhanced nitrogen (N2) fixation capacity. Establishing these strains in a significant proportion of soybean (Glycine max) nodules requires that the inoculated strains can compete for nodulation with indigenous strains. The inability to introduce engineered or selected bradyrhizobia into soils containing indigenous populations of Bradyrhizobium japonicum is referred to as "The Competition Problem". Competition limits nodulation of soybean by highly efficient inoculant-quality strains, and in the future, will likely limit nodulation by strains selected or engineered for superior N2-fixation capacity. Several studies have shown that host and abiotic soil factors influence the survival and competitiveness of soil bradyrhizobial populations. In this chapter, I focus on the biotic and abiotic factors influencing survival and the competitiveness of B. japonicum strains in soil, and on the genetics of host and microsymbiont shown to influence competition for nodulation.
Chapter 19
Unravelling the Infection Process in the Rhizobium-Legume Symbiosis
by Microscopy
Frank B. Dazzo and Judith L. Wopereis
This chapter documents by example how microscopy has vividly revealed many of the cellular and molecular events of the nitrogen-fixing Rhizobium-legume symbiosis. The discussion starts by highlighting the develop of improvements in microscopical technique applied to studies of the symbiosis, then the developmental morphology of the infection process beginning with rhizobial motility in the external root environment, later primary host infection and nodule invasion, and finally to release of bacteria at nodule senescence. The chapter also covers applications of in situ molecular microscopy and computer-assisted microscopy applied to studies of the Rhizobium-legume symbiosis. This information provides a richly illustrated overview of the spatio-temporal development of this important prokaryotic-eukaryotic symbiosis. Discussions of the infection process are mainly focused on the legume hosts white clover (Trifolium repens), Lotus japonicus, and neptunia (Neptunia natans) with their corresponding rhizobial symbiont, Rhizobium leguminosarum bv. trifolii, Mesorhizobium loti, and Allorhizobium undicola. These represent three significantly different symbiotic systems (two temperate, terrestrial legumes with primary infection and nodule invasion via infection threads plus distinctions of indeterminate and determinant nodulation, and a tropical aquatic legume with primary infections and nodule invasion via crack entry, followed by infection thread dissemination within the nodule). Indeed, microscopy has proven repeatedly to be a powerful, indispensable tool that will continue to help unravel the infection process in the nitrogen-fixing Rhizobium-legume symbiosis.
Chapter 20
Biosynthesis and Release of Rhizobial Nodulation Gene Inducers by Legumes
Donald A. Phillips
Symbiotic N2-fixing bacteria in the Rhizobiaceae respond to structurally diverse plant compounds by transcribing genes required for root nodule formation. Natural nodulation gene inducers occur as flavonoids (chalcones, flavanones, flavones, flavonols, anthocyanidins, isoflavonoids) and as non-flavonoids, including betaines (stachydrine, trigonelline) and aldonic acids (erythronic acid, tetronic acid). These molecular signals are released from roots and/or from seed coats during germination. In some cases, the same compounds regulate other microbial processes, including chemotaxis, growth, and mycorrhizal spore germination. These phenomena are ecologically important because they show that very low concentrations of plant-derived compounds in the rhizosphere signal the presence of the plant and promote beneficial responses in bacteria and fungi.
Chapter 21
Structure and function of nod factors
Cristina Pacios-Bras, Herman P. Spaink, and Nico Stuurman
Nod factors are signal molecules secreted by rhizobia. When secreted into the rhizosphere of a host leguminous plant, nod factors trigger a series of morphological changes on the plant root that culminate in the formation of symbiotic nitrogen fixing structures. The nod factors are major determinants of the correct development of this symbiosis. In this chapter we will summarise the current knowledge about the synthesis and structure of these bacterial signal molecules.
Chapter 22
Signal Exchange Involved in the Establishment of the Bradyrhizobium-Legume Symbiosis.
Day, R. Bradley, Loh, John T., Cohn, Jonathan, and Stacey, G.
Bradyrhizobium and other members of the Rhizobiaceae are capable of establishing a host-specific symbiotic association with leguminous plants, resulting in the formation of a new plant organ, the nodule. The onset of this association is marked by the exchange of signals between the invading rhizobia and the legume root. Perception of host-specific flavonoids by the bacteria trigger the induction of the rhizobial nodulation (nod) genes, which mediate host-specificity through the synthesis and secretion of modified lipo-chitin Nod signals. This review focuses on those unique aspects of rhizobial-plant signaling found in symbioses with Bradyrhizobium species. For example, B. japonicum has novel regulatory mechanisms controlling nod gene expression that involve several distinct regulatory proteins, such as NolA, NodVW, and NodD. Similarly, investigations of the Bradyrhizobium-soybean symbiosis are providing novel insights into how the legume host recognizes the lipo-chitin Nod signal. For example, this work supports that hypothesis that legumes possess two, distinct Nod signal recognition pathways. Work is now focusing on identifying the protein receptors that mediate Nod signal recognition. It is clear that research into the symbioses between Bradyrhizobium species and their legume hosts is adding fundamental new knowledge to our understanding of the mechanisms and diversity of rhizobial-legume interactions.
Chapter 23
Rhizobium lipopolysaccharide and its role in symbiosis
K. Dale Noel and Dominik M. Duelli
Among legume-nodulating bacteria, lipopolysaccharide (LPS) is best understood in Rhizobium leguminosarum and R. etli. A complete structure of the lipid A and LPS core oligosaccharide of these species has been determined from R. etli CE3 and two mutant derivatives. Much of lipid A biosynthesis and three steps in core synthesis have been demonstrated in vitro. The biosynthetic pathway diverges from that of enteric bacteria after the formation of an intermediate known as Kdo2-lipid IVA. Mutations that affect LPS map in at least six genetic loci in these species. Associated phenotypes indicate that normal abundance and certain structural features of the LPS O-polysaccharide (OPS) are required for successful infection of host legumes. Depending on the host, aberrant consequences of OPS deficiency include marked host defense responses, defective release from the infection thread, severely reduced proliferation of bacteroids, complete blockage early in infection thread formation, stunted nodule anatomical development, and reduced frequency of nodule emergence. As another indication of its role in adapting to the plant and other environments, LPS structure is altered in response to factors such as low pH, low oxygen, and host anthocyanins. The symbiotic role of these alterations is under active investigation.
Chapter 24
The Role of Rhizobial Extracellular Polysaccharides (EPS) in the Sinorhizobium meliloti Alfalfa Symbiosis
Anke Becker, Karsten Niehaus and Alfred Pühler
Sinorhizobium meliloti is able to enter the host plant root via a sophisticated infection mechanism, induce a new plant organ, the root nodule, and differentiate within the infected plant cells into nitrogen fixing bacteroids. There is growing evidence that surface carbohydrates, namely the extracellular polysaccharides EPS I (succinoglycan) and EPS II (galactoglucan) play an active role in the infection of the host plant. Mutants defective in the EPS biosynthesis are not able to infect the host plant and induce the plant defense system. The S. meliloti Medicago sativa or Medicago truncatula symbiosis serves as a model system to analyse the biosynthesis and function of EPS. A large gene cluster involved in EPS I biosynthesis (exo-genes) comprising at least 27 kb was identified on the megaplasmid 2 of S. meliloti. Functions were assigned to most of the exo genes by the analysis of lipid-linked biosynthetic intermediates that accumulated in exo mutants. Genes responsible for the biosynthesis of EPS II (exp genes) were identified on megaplasmid 2 separated from the exo genes by approx. 200 kb. Since EPS defective mutants of S. meliloti induce the defense system of the host plant, it was speculated that EPS has a function in the suppression of plant defense reactions against the symbiont. In accordance with the proposed function, low molecular weight EPS I is able to suppress the elicitor induced oxidative burst in alfalfa cell cultures. One of the emerging questions to be answered is whether surface carbohydrates are signal molecules, specifically recognized by the host plant.
Chapter 25
Control of Root Nodule Organogenesis
Eva Kondorosi and Adam Kondorosi
Symbiosis between rhizobia and leguminous plants results in the formation of nitrogen-fixing root nodules. The competence of root cells for nodule development is determined by environmental and physiological conditions while nodule organogenesis is triggered by the rhizobial Nod factors acting as host-specific morphogens. Nod factors are likely perceived by specific receptors and transduction of the signal in the epidermal and cortical cell layers results in different responses; entry of bacteria into the plant cells via formation of the infection threads in the root hairs and activation of the cell cycle in the cortical cells. Cell division in the root cortex leads to the formation of the nodule meristem from which cells differentiate to various types of nodule cells. Development of infected cells necessitates multiple rounds of endocycles and a gradual increase in the cell volume. This review focuses on the early events of nodule development, summarising the present knowledge on the perception and transduction of Nod signals and particularly on the integration of the cell cycle control during development of the Sinorhizobium meliloti-Medicago symbiosis.
Chapter 26
Nodulins: Nodule-Specific Host Gene Products, their Induction and Function in Root Nodule Symbiosis
Desh Pal S. Verma
A successful interaction between Rhizobium and a legume plant leeds to the establishment of intracellular symbiosis between these two organisms inside root nodules. Development of this unique organ requires interactions between a number of host and bacterial genes leading to creation and support of an environment that allows bacteria to fix nitrogen for the host. The infected cells of the symbiotic zone of root nodules are hypoxic, hyper-osmotic and may have higher subcellular pH. Hence, the metabolism of this tissue would require new alleles of host genes adapted for functioning in this environment. Most of the plant genes expressed in root nodules are also expressed in other tissues at a much lower level, but their expression is enhanced in root nodules. The nodule-specific alleles of various genes may have evolved as a result of gene duplication. Thus, not only the structure of these genes but also their mode of regulation is altered. It is interesting to note that the entire developmental program of root nodules appears to be under the control of nod factors secreted by Rhizobium that act as morphogens. Finally, the symbiotic state is maintained by the exchange of carbon and nitrogen, two different currencies of respective value to each organism.
Chapter 27
Uptake Hydrogenases in Root Nodule Bacteria
T. Ruiz-Argüeso, J. Imperial and J. M. Palacios
Uptake hydrogenases in diazotrophic root nodule bacteria (rhizobia, Frankia) can recycle the H2 generated as a by-product of the nitrogenase reaction and have a potential to improve the energy efficiency of the symbiosis. This is especially relevant in view of the fact that uptake hydrogenases are uncommon in many of the rhizobia used as legume inoculants. The Hup (Hydrogen uptake) system has been studied in depth only in two species of root nodule bacteria, Rhizobium leguminosarum bv. viciae and Bradyrhizobium japonicum. In these organisms, a multigenic (18-24 genes) cluster responsible for synthesis of an active hydrogenase has been isolated. The current status of research on characterization of the functions of their gene products and the regulation of their expression is reviewed. This information, together with available gene transfer technology, opens the door to biotechnological exploitation of the Hup system for the design and generation of more energy efficient rhizobial inoculants.
Chapter 28
Heme Biosynthesis and Function in the Rhizobium-Legume Symbiosis
Mark R. O'Brian
Nitrogen fixation is an energy-intensive process that requires specialized adaptations by the plant and bacterial partners of symbiotic systems. This energy demand is accommodated, in part, by the induction of plant hemoglobin and bacterial cytochromes within root nodules. These heme proteins allow efficient respiration of the prokaryote within a hypoxic milieu. Biosynthesis of the heme prosthetic group involves a multistep pathway that must be regulated in accordance with cellular function. Herein, recent progress in elucidating the mechanisms and control of heme biosynthesis are described in the Rhizobium-legume symbiosis. These findings are integrated, where appropriate, with general problems of heme biosynthesis and function in other systems.
Chapter 29
Phosphorus Assimilation and Regulation in the Rhizobia
Timothy R. McDermott
With the exception of nitrogen, phosphorus is typically the limiting nutrient for plant growth in most soils. Phosphorus bioavailability has been shown to have a significant impact on both the establishment and the functioning of the Rhizobium-legume symbiosis. Nodule number and mass, plant dry matter, whole plant nitrogenase activity, and whole plant N concentration are all significantly reduced when phosphorus is limiting. Few details about phosphorus metabolism in either partner of this symbiosis have been available, but recently our understanding of phosphorus metabolism in the microbial partner has advanced significantly and is the subject of this chapter. Available information about phosphate transport, alkaline and acid phosphatases, Pho regulation, and global regulatory effects of phosphate stress on metabolism in free-living Sinorhizobium meliloti and Rhizobium tropici is summarized. Bacteroid phosphorus metabolism is discussed from the perspective of bacteroid phosphorus nutrition and overall nodule metabolism. This includes summarizing our current understanding of phosphorus allocation to alfalfa bacteroids, speculation as to how phosphorus may be provided to bacteroids of other symbioses, particularly bean, and possible implications for phosphorus utilization and movement within the symbiosis.
Chapter 30
Transport of Metabolites to and from Symbiosomes and Bacteroids
James K. Waters and David W. Emerich
The symbiotic nitrogen fixation process occurs in specialized plant structures called nodules in which the prokaryotic endophyte is physically separated from the plant cytoplasm. This separation requires the transport of carbon metabolites to the bacteria and reduced nitrogen metabolites to the plant. Dicarboxylic acids, primarily malate, are actively transported to the bacteroids and, until recently, ammonia was believed to be the primary nitrogen metabolite, which moved to the plant via diffusion. Recent evidence demonstrated that alanine is the nitrogen metabolite released by the nitrogen-fixing bacteroids from soybeans. The rapid and efficient transport of alanine to the plant portion of the nodule implies that a nutrient exchange cycle may exist within soybean root nodules facilitating the exchange of carbon and nitrogen metabolites.
Chapter 31
Ureide Synthesis in Legume Nodules
Craig Atkins and Penny Smith
These constitute the principal form of N translocated in xylem to the rest of the plant and serve as the source of N for amino acid and protein synthesis. The synthesis of ureides from ammonia exported from the bacteroids is complex and involves both the surrounding infected plant cell and adjacent uninfected cells. Ammonia is assimilated in the infected cells through glutamine synthetase and glutamate synthase to provide glutamine, glycine and aspartate to a pathway of de novo purine nucleotide synthesis that is expressed in both plastids and mitochondria. A number of the pur genes encoding the enzymes of purine synthesis have been cloned and characterized from cowpea and soybean nodules. Their cDNA's all have presequences consistent with organelle targeting but in a number of cases only a single gene has been detected suggesting that a single transcript is dual targeted. The mechanisms that allow this dual targeting have yet to be discovered. The product of the purine pathway in nodules, inosine monophosphate (IMP), is oxidised via IMP and xanthine dehydrogenases expressed in the cytosol of the infected cell to yield uric acid. Uric acid is not metabolized in these cells but is oxidized to allantoin by urate oxidase confined to enlarged microbodies in the adjacent uninfected cell. The very high levels of expression of the pathways of purine synthesis and oxidation in nodules are dependent on the flux of fixed N from bacteroids. Specific instances of post-translational and transcriptional regulation have been demonstrated for a few of the component enzymes but the effectors have yet to be identified. Despite the significance of ureides in the N economy of this group of legumes, reasons for the expression of such a complex assimilatory mechanism (compared to the synthesis of amides in nodules of temperate legumes) for fixed N remain elusive.
Chapter 32
Amide Biosynthesis in Root Nodules of Temperate Legumes
Carroll P. Vance
Most temperate legumes assimilate symbiotically fixed nitrogen (N) into the amides asparagine (Asn) and glutamine (Gsn) for export from root nodules and transport to other plant organs. Alfalfa root nodules serve as an excellent model system to study biochemical and molecular regulation of amide biosynthesis. Here we report on the subcellular location and genetic control of glutamine synthetase (GS), NADH-glutamate synthase (NADH-GOGAT), aspartate aminotransferase (AAT), asparagine synthetase (AS), phosphoenolpyruvate carboxylase (PEPC), and malate dehydrogenase (MDH). Nitrogen assimilation in alfalfa root nodules is closely coupled to carbon metabolism. Amyloplasts play a key role in N assimilation being the location of both NADH-GOGAT and AAT. A nodule enhanced form of MDH may also occur in amyloplasts. Immunocytochemical localization, in situ hybridization, and mRNA analysis indicate that NADH-GOGAT plays a critical role in regulation of amide biosynthesis in alfalfa root nodules.
Chapter 33
The Genetic System of Alfalfa (Medicago Sativa L.) as an Aid for Isolation of Symbiotic Genes, and for Structural, Comparative and Functional Genomics of Legumes
György B. Kiss and Gabriella Endre
This review gives a brief summary of the recent status of the diploid and tetraploid alfalfa subspecies in the Medicago sativa complex concerning mainly genetics and other properties relevant to genomics research. We discuss to what extent alfalfa can be used as a complementary model plant to study symbiotic nitrogen fixation and mycorrhizal interactions. Although diploid and tetraploid alfalfa species have slightly larger genomes and are out-crossing plants, they expand in many ways the potential of Medicago truncatula that has been selected recently as the main model legume with indeterminate nodule structure. The highly saturated genetic map and valuable mutant derivatives impaired in symbiotic nitrogen fixation and mycorrhizal symbiosis are the most relevant attributes by which alfalfa, a major crop plant, can contribute to the structural, comparative and functional genomics of M. truncatula.
Chapter 34
Melilotus alba Desr., White Sweetclover, a Mellifluous Model Legume
Ann M. Hirsch, Michelle R. Lum, Rebecca S.N. Krupp, Weigang Yang, and Wojciech M. Karlowski
Melilotus alba (white sweetclover) is a small-seeded, self-fertilizing diploid (n = 8) that goes from seed to seed within 3 months. Sweetclover is nodulated by Sinorhizobium meliloti and exhibits a close relationship to alfalfa (Medicago sativa), but appears to have a simple genome in terms of symbiotically-associated genes when compared to medics. The U389 line and its dwarf derivative U390 are highly mutable, and a large number of morphological and symbiotic, especially non-nodulating, mutants have already been obtained. A linkage map has been constructed, and transformation experiments are in progress. Both U389 and U390 are prolific seed producers considering their sizes, and the seeds are easy to harvest. Moreover, sweetclover seeds are exceptionally long-lived, an advantage for genetic studies. The U390 line is smaller in stature than Arabidopsis and is proposed as an excellent system for screening large numbers of plants in greenhouses, growth cabinets, or petri dishes.
Chapter 35
The Use of the Genus Trifolium for the Study of Plant-Microbe Interactions, Root Development and Plant Defence Responses
Barry G. Rolfe, Ulrike Mathesius, Michael A. Djordjevic, Jeremy J. Weinman, Nelson Guerreiro, Siria Natera, Angela C. Morris
Historically, the clovers have been valuable model systems for the investigation of Rhizobium leguminosarum bv. trifolii symbioses in practical agriculture and in the laboratory. These studies have now been supplemented by applying many recent molecular and plant biology techniques, such as the construction of transgenic plants and the use of rooted leaf bioassays. These techniques have enabled extensive research into the interactions involved in nodule initiation, signal transduction, plant development, the study of root morphogenesis and the analysis of clover defence systems (pathogenesis-related (PR) protein genes) during microbial infection. Studies using clovers have generated significant insights into plant-microbe interactions and have provided some valuable contrasts with other legume species. This is especially true where alternative model systems have not yet permitted studies to elucidate particular signal transduction pathways. An expanding area of current and future research makes use of Proteome analysis to examine gene expression in both rhizobia and plant roots.
Chapter 36
Nitrogen Fixation in Methanogensthe Archaeal Perspective
John A. Leigh
The methanogenic Archaea bring a broadened perspective to the field of nitrogen fixation. Biochemical and genetic studies show that nitrogen fixation in Archaea is evolutionarily related to nitrogen fixation in Bacteria and operates by the same fundamental mechanism. At least six nif genes present in Bacteria (nif H, D, K, E, N and X) are also found in the diazotrophic methanogens. Most nitrogenases in methanogens are probably of the molybdenum type. However, differences exist in gene organization and regulation. All six known nif genes of methanogens, plus two homologues of the bacterial nitrogen sensor-regulator glnB, occur in a single operon in Methanococcus maripaludis. nif gene transcription in methanogens is regulated by what appears to be a classical prokaryotic repression mechanism. At least one aspect of regulation, post-transcriptional ammonia switch-off, involves novel members of the glnB family. Phylogenetic analysis suggests that nitrogen fixation may have originated in a common ancestor of the Bacteria and the Archaea.
Chapter 37
Genomics of Rhizobium etli
Guillermo Dávila, Susana Brom, Julio Collado, Georgina Hernández, Jaime Mora, Rafael Palacios, and David Romero
Rhizobium etli is a soil bacterium that elicits the formation of nitrogen-fixing nodules on Phaseolus vulgaris roots. The genome of this bacterium is partitioned between the chromosome and several large plasmids that may represent half of the genome. Another characteristic of the Rhizobium genome, is the existence of a large amount of reiterated DNA sequences that participate in the generation of genomic diversity mediated by homologous recombination. In R. etli, as in other Rhizobium species, the majority of the genes required for nodulation and nitrogen fixation are located on a high molecular weight plasmid called the symbiotic plasmid. We have started a genome-sequencing project of CFN42, the type strain for R. etli. The whole sequence of its symbiotic plasmid is almost concluded and the comparative analysis with the pNGR234a is in progress. We are also analyzing the proteome expressed by several R. etli strains under different metabolic or ecological conditions.
Chapter 38
Physical and Genetic Analysis of the Broad Host-Range Rhizobium sp. NGR234
Xavier Perret, Virginie Viprey, and William John Broughton
Rhizobium sp. NGR234 has the broadest host-range of any known bacterium and can nodulate more than 112 genera of legumes as well as the non-legume Parasponia andersonii. Most symbiotic genes are carried by pNGR234a, a 536-kb self-transmissible plasmid. In addition to the chromosome, NGR234 possess a > 2 Mb mega-plasmid which encodes several other loci involved in nodulation and nitrogen fixation. Marked differences in the G+C contents as well as the distinct origins of the various symbiotic gene-clusters on pNGR234a, suggest that genetic recombination and lateral transfer of genes have shaped the mosaic structure of this plasmid. Combined genetic and high-resolution transcriptional analyses of pNGR234a loci confirmed that most nod-box- (19) and NifA-s54-(16) promoters are active, despite being dispersed around the replicon. Expression of a number of flavonoid-inducible and bacteroid-specific operons is nod-box and NifA-s54 independent however. Among these, are genes encoding a type three-secretion system (TTSS) that modulates protein secretion by NGR234 and with it nodulation of various tropical legumes. It thus seems possible that certain pathogens and symbionts share molecular mechanisms in interacting with their hosts.
Chapter 39
The Symbiosis Island of a Mesorhizobium Strain that Nodulates Lotus
J. T. Sullivan J. R. Trzebiatowski, F. J de Bruijn and C. W. Ronson
The symbiosis island of Mesorhizobium strain ICMP3153 is a 500-kb chromosomally integrated transferable element containing almost the entire complement of genes required for the bacterial component of the symbiosis. It transfers to nonsymbiotic mesorhizobia in the environment and in the laboratory. The island integrates into a phenylalanine tRNA gene, reconstructing the gene at one (left) end of the island and producing a 17-bp direct repeat of the 3' end of the tRNA gene at the other end. An integrase of the P4 phage family located within the left end of the island is required for excision of the island as a circle. The island is representative of a new class of elements termed fitness islands which, in a single acquisition event, can confer a complex adaptive phenotype on the host bacterial strain.
Chapter 40
Assessment of Bacterial Nitrogen Fixation in Grass Species
Robert M. Boddey, Lucia G. da Silva, Verônica Reis, Bruno J.R. Alves and Segundo Urquiaga
A consensus now appears to be forming that the best strategy to develop a technology whereby cereal or grass crops will benefit significantly from plant associated biological nitrogen fixation (BNF) seems to lie in the improvement of existing plant/diazotroph associations. However, only a limited amount of research has focused on examining existing associations of N2-fixing micro-organisms with grasses or cereals for BNF inputs and ecological studies of the bacteria that are associated with field-grown plants. In this review, we outline the techniques available to quantify BNF contributions to such associations, the problems associated with their application and discuss the existing evidence that indicates in which associations there are benefits of BNF which can be considered agronomically significant. We also briefly discuss the techniques available to enumerate populations of N2-fixing bacteria within plant tissues, giving an example from our studies
using the ELISA (enzyme-linked immuno-absorbent assay) technique. These results show very clearly that direct viable counts in semi-solid culture media seriously
under-estimates
actual bacterial numbers.
Chapter 41
Analysis of the Presence and Diversity of Diazotrophic Endophytes
Anton Hartmann, Marion Stoffels, Barbara Eckert, Gudrun Kirchhof and Michael Schloter
The intial isolation step of endophytic diazotrophs is a surface disinfection, followed by extensive washing and maceration of the tissue. Alternatively, bacteria can also be isolated from axenically derived plant sap, using vacuum devices or centrifugation steps. These materials are used to inoculate semisolid, nitrogen-free enrichment media. In addition, specific antisera for certain groups of diazotrophic bacteria can be applied to specifically enrich endophytic bacteria in a fast and efficient immunoadsorption procedure. The diversity of diazotrophic bacteria is certainly far from being approximately revealed, since new endophytic diazotrophs are being continuously discovered. Since the initial isolation step bears uncertaincies of a true endophytic origin of the bacteria, inoculation and/or direct in situ localization studies are necessary to prove the endophytic character of the diazotrophs in a certain plant. New whole-cell binding 16S rRNA-directed oligonucleotide probes have been developed and are available for in situ localization studies of Herbaspirillum spp. and Azospirillum spp Highly specific polyclonal or monoclonal antibodies are additional important tools for the in situ identification of bacteria with or without combination with the oligonucleotide probes. Using fluorescently labeled probes or bacteria, which are tagged with the gfp- or gus-marker, confocal laser scanning microscopy provides a clear in situ identification of bacteria in optical sections down to a depth of about 30µm. In addition, immunogold-labelling and electron microscopy enables a localization with high resolution.
Chapter 42
Genetic Analysis of Nitrogen Fixation and Plant-Growth Stimulating Properties of Acetobacter diazotrophicus, an Endophyte of Sugarcane
Myrna Sevilla and Christina Kennedy
The discovery in 1988 of Acetobacter diazotrophicus, a nitrogen fixing bacterium inhabiting the interior of roots, stems, and leaves of sugarcane, opened a new avenue of research in nitrogen fixation and plant-microbe interactions. The endophytic colonization of sugarcane by A. diazotrophicus represents a model system for beneficial association between a monocot and a diazotrophic bacterial species. While a few other identified nitrogen-fixing bacteria are apparently endophytic, none but A. diazotrophicus has been shown to contribute fixed N for growth of its plant host. The evidence for this including the potential of A. diazotrophicus to colonize and benefit the growth of other grasses is presented in this review. Results of our analysis of bacterial genes involved in nitrogen fixation and their regulation in A. diazotrophicus are also discussed. In addition, other aspects of the A. diazotrophicus-sugarcane association are described.
Chapter 43
Azospirillum-Plant Root Interactions: Signaling and Metabolic Interactions
Ann Vande Broek, Sofie Dobbelaere, Jos Vanderleyden and Anne Vandommelen
From the extensive genetic, biochemical and applied studies over the past two decades, Azospirillum can be considered as one of the best-studied examples of associative plant growth promoting rhizobacteria. In this review, three aspects of the Azospirillum-plant root association, that is, the biosynthesis of phytohormones, nitrogen fixation and assimilation, and the use of Azospirillum as inoculum, are highlighted.
Chapter 44
Diazotrophic Endophytes Associated with Maize
Marisa K. Chelius and Eric W. Triplett
Diazotrophs have been cultured from both the plant interior and root environment of maize and acetylene reduction and stable isotope (15N) incorporation imply that bacteria in association with maize are actively fixing nitrogen. This evidence suggests that diazotroph-maize associations have potential benefits to agriculture if the yield of maize can be sustained in the absence of high N fertilizer inputs. As yet, data in support of plant growth benefits to maize accrued via nitrogenase activity are lacking. Investigations on the mechanisms that affect diazotroph-maize associations, such as plant genotype and cultivation conditions, and bacterial community structure and population dynamics of associating diazotrophs should illuminate those interactions that can be further explored and manipulated to yield viable, productive associations of agronomic significance.
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