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Globally, 800 million people are malnourished. Heavily subsidised farmers in rich countries produce sufficient surplus food to feed the hungry, but not at a price the poor can afford. Even donating the rich world's surplus to the poor would not solve the problem. Most poor people earn their living from agriculture, so a deluge of free food would destroy their livelihoods. Thus, the only answer to world hunger is to safeguard and improve the productivity of farmers in poor countries. Diets of subsistence level farmers in Africa and Latin America often contain sufficient carbohydrates (through cassava, corn/maize, rice, wheat, etc.), but are poor in proteins. Dietary proteins can take the form of scarce animal products (eggs, milk, meat, etc.), but are usually derived from legumes (plants of the bean and pea family). Legumes are vital in agriculture as they form associations with bacteria that 'fix-nitrogen' from the air. Effectively this amounts to internal fertilisation and is the main reason that legumes are richer in proteins than all other plants. Thousands of legume species exist but more common beans (Phaseolus vulgaris L.) are eaten than any other. In some countries such as Mexico and Brazil, beans are the primary source of protein in human diets. As half the grain legumes consumed worldwide are common beans, they represent the species of choice for the study of grain legume nutrition. Unfortunately, the yields of common beans are low even by the standards of legumes, and the quality of their seed proteins is sub-optimal. Most probably this results from millennia of selection for stable rather than high yield, and as such, is a problem that can be redressed by modern genetic techniques. We have formed an international consortium called 'Phaseomics' to establish the necessary framework of knowledge and materials that will result in disease-resistant, stress-tolerant, high-quality protein and high-yielding beans. Phaseomics will be instrumental in improving living conditions in deprived regions of Africa and the Americas. It will contribute to social equity and sustainable development and enhance inter- and intra-cultural understanding, knowledge and relationships. A major goal of Phaseomics is to generate new common bean varieties that are not only suitable for but also desired by the local farmer and consumer communities. Therefore, the socio-economic dimension of improved bean production and the analysis of factors influencing the acceptance of novel varieties will be an integral part of the proposed research (see Figure 1). Here, we give an overview of the economic and nutritional importance of common beans as a food crop. Priorities and targets of current breeding programmes are outlined, along with ongoing efforts in genomics. Recommendations for an international coordinated effort to join knowledge, facilities and expertise in a variety of scientific undertakings that will contribute to the overall goal of better beans are given. To be rapid and effective, plant breeding programmes (i.e., those that involve crossing two different 'parents') rely heavily on molecular 'markers'. These genetic landmarks are used to position important genes (e.g. for resistance to particular pests, for yield, etc.) on a chromosome and ensure that they can be 'crossed in' to another plant. There are several ways of obtaining molecular markers but the project will establish partial sequences of messenger RNA's extracted from tissues of interest (e.g. developing pods). These so-called expressed sequence-tags (ESTs), can be used like milestones on a chromosome, to position these and other genes. These efforts will complement current studies on other legumes such as Lotus japonicus and Medicago truncatula as well as the EST projects in soybean by providing a framework for comparative genomics between legumes. Complete sequencing and molecular analysis of the bean genome will follow. Individual laboratories will be encouraged to internally finance or find additional funding for the construction of cDNA libraries and the sequencing of thousands ESTs. Funds donated to the consortium will be used primarily for sequencing the genome and to co-ordinate the consortium's activities. As sequence and expression data become available it will provide an elaborate framework for plant geneticists to 'design' new, improved common bean lines. Amongst these lines will be higher-yielding varieties, cultivars that are resistant to drought, pests and so on. It will also be possible to enhance the content of essential amino acids, minerals and vitamins in the seeds and so improve the nutrition and health of countless people who consume beans. By considering the socio-economic implications of common bean improvement from the outset, this project should lead to sustainable development, to increased social equity, and to greater use of beans in international trade. The added value in this innovative approach to common beans as model food legumes lies in the combination of existing and novel genetic approaches with socio-economic criteria that will efficiently target the end users.

Plant roots support the growth and activities of a wide variety of microorganisms that may have a profound effect on the growth and/or health of plants. Among these microorganisms, a high diversity of bacteria have been identified and categorized as deleterious, beneficial, or neutral with respect to the plant. The beneficial bacteria, termed plant growth-promoting rhizobacteria (PGPR), are widely studied by microbiologists and agronomists because of their potential in plant production. Azospirillum, a genus of versatile PGPR, is able to enhance the plant growth and yield of a wide range of economically important crops in different soils and climatic regions. Plant beneficial effects of Azospirillum have mainly been attributed to the production of phytohormones, nitrate reduction, and nitrogen fixation, which have been subject of extensive research throughout the years. These elaborate studies made Azospirillum one of the best-characterized genera of PGPR. However, the genetic and molecular determinants involved in the initial interaction between Azospirillum and plant roots are not yet fully understood. This review will mainly highlight the current knowledge on Azospirillum plant root interactions, in the context of preceding and ongoing research on the association between plants and plant growth-promoting rhizobacteria.

of the Azospirillum brasilense ipdC gene, encoding an indole-3-pyruvate decarboxylase, a key enzyme in the production of indole-3-acetic acid (IAA) in this bacterium, is upregulated by IAA. Here, we demonstrate that the ipdC gene is the promoter proximal gene in a bicistronic operon. Database searches revealed that the second gene of this operon, named iaaC, is well conserved evolutionarily and that the encoded protein is homologous to the Escherichia coli protein SCRP-27A, the zebrafish protein ES1, and the human protein KNP-I/GT335 (HES1), all of unknown function and belonging to the DJ-1/PfpI superfamily. In addition to this operon structure, iaaC is also transcribed monocistronically. Mutation analysis of the latter gene indicated that the encoded protein is involved in controlling IAA biosynthesis but not ipdC expression. Besides being upregulated by IAA, expression of the ipdC-iaaC operon is pH dependent and maximal at acidic pH. The ipdC promoter was studied using a combination of deletion analyses and site-directed mutagenesis. A dyadic sequence (ATTGTTTC(GAAT)GAAACAAT), centered at -48 was demonstrated to be responsible for the IAA inducibility. This bacterial auxin-responsive element does not control the pH-dependent expression of ipdC-iaaC.

To unravel the biological function of the widely used probiotic bacterium Lactobacillus rhamnosus GG, we compared its 3.0-Mbp genome sequence with the similarly sized genome of L. rhamnosus LC705, an adjunct starter culture exhibiting reduced binding to mucus. Both genomes demonstrated high sequence identity and synteny. However, for both strains, genomic islands, 5 in GG and 4 in LC705, punctuated the colinearity. A significant number of strain-specific genes were predicted in these islands (80 in GG and 72 in LC705). The GG-specific islands included genes coding for bacteriophage components, sugar metabolism and transport, and exopolysaccharide biosynthesis. One island only found in L. rhamnosus GG contained genes for 3 secreted LPXTG-like pilins ( spaCBA ) and a pilin-dedicated sortase. Using anti-SpaC antibodies, the physical presence of cell wall-bound pili was confirmed by immunoblotting. Immunogold electron microscopy showed that the SpaC pilin is located at the pilus tip but also sporadically throughout the structure. Moreover, the adherence of strain GG to human intestinal mucus was blocked by SpaC antiserum and abolished in a mutant carrying an inactivated spaC gene. Similarly, binding to mucus was demonstrated for the purified SpaC protein. We conclude that the presence of SpaC is essential for the mucus interaction of L. rhamnosus GG and likely explains its ability to persist in the human intestinal tract longer than LC705 during an intervention trial. The presence of mucus-binding pili on the surface of a nonpathogenic Gram-positive bacterial strain reveals a previously undescribed mechanism for the interaction of selected probiotic lactobacilli with host tissues.

Bacterial cells can produce and sense signal molecules, allowing the whole population to initiate a concerted action once a critical concentration (corresponding to a particular population density) of the signal has been reached, a phenomenon known as quorum sensing. One of the possible quorum sensing-regulated phenotypes is swarming, a flagella-driven movement of differentiated swarmer cells (hyperflagellated, elongated, multinucleated) by which bacteria can spread as a biofilm over a surface. The glycolipid or lipopeptide biosurfactants thereby produced function as wetting agent by reducing the surface tension. Quorum sensing systems are almost always integrated into other regulatory circuits. This effectively expands the range of environmental signals that influence target gene expression beyond population density. In this review, we first discuss the regulation of AHL-mediated surface migration and the involvement of other low-molecular-mass signal molecules (such as the furanosyl borate diester AI-2) in biosurfactant production of different bacteria. In addition, population density-dependent regulation of swarmer cell differentiation is reviewed. Also, several examples of interspecies signalling are reported. Different signal molecules either produced by bacteria (such as other AHLs and diketopiperazines) or excreted by plants (such as furanones, plant signal mimics) might influence the quorum sensing-regulated swarming behaviour in bacteria different from the producer. On the other hand, specific bacteria can reduce the local available concentration of signal molecules produced by others. In the last part, the role and regulation of a surface-associated movement in biofilm formation is discussed. Here we also describe how quorum sensing may disperse existing biofilms and control the interaction between bacteria and higher organisms (such as the Rhizobium-bean symbiosis).

Azospirillum represents the best characterized genus of plant growth-promoting rhizobacteria. Other free-living diazotrophs repeatedly detected in association with plant roots, include Acetobacter diazotrophicus, Herbaspirillum seropedicae, Azoarcus spp. and Azotobacter. Four aspects of the Azospirillum–plant root interaction are highlighted: natural habitat, plant root interaction, nitrogen fixation and biosynthesis of plant growth hormones. Each of these aspects is dealt with in a comparative way. Azospirilla are predominantly surface-colonizing bacteria, whereas A. diazotrophicus, H. seropedicae and Azoarcus sp. are endophytic diazotrophs. The attachment of Azospirillum cells to plant roots occurs in two steps. The polar flagellum, of which the flagellin was shown to be a glycoprotein, mediates the adsorption step. An as yet unidentified surface polysaccharide is believed to be essential in the subsequent anchoring phase. In Azoarcus sp. the attachment process is mediated by type IV pili. Nitrogen fixation structural genes ( nif) are highly conserved among all nitrogen-fixing bacteria, and in all diazotrophic species of the class of proteobacteria examined, the transcriptional activator NifA is required for expression of other nif genes in response to two major environmental signals (oxygen and fixed N). However, the mechanisms involved in this control can vary in different organisms. In Azospirillum brasilense and H. seropedicae (α- and β-subgroup, respectively), NifA is inactive in conditions of excess nitrogen. Activation of NifA upon removal of fixed N seems to involve, either directly or indirectly, the signal transduction protein P II. The presence of four conserved cysteine residues in the NifA protein might be an indication that NifA is directly sensitive to oxygen. In Azotobacter vinelandii (γ-subgroup) nifA is cotranscribed with a second gene nifL. The nifL gene product inactivates NifA in response to high oxygen tension and cellular nitrogen-status. NifL was found to be a redox-sensitive flavoprotein. The relief of NifL inhibition on NifA activity, in response to N-limitation, is suggested to involve a P II-like protein. Moreover, nitrogenase activity is regulated according to the intracellular nitrogen and O 2 level. In A. brasilense and Azospirillum lipoferum posttranslational control of nitrogenase, in response to ammonium and anaerobiosis, involves ADP-ribosylation of the nitrogenase iron protein, mediated by the enzymes DraT and DraG. At least three pathways for indole-3-acetic acid (IAA) biosynthesis in A. brasilense exist: two Trp-dependent (the indole-3-pyruvic acid and presumably the indole-3-acetamide pathway) and one Trp-independent pathway. The occurrence of an IAA biosynthetic pathway not using Trp (tryptophan) as precursor is highly unusual in bacteria. Nevertheless, the indole-3-pyruvate decarboxylase encoding ipdC gene is crucial in the overall IAA biosynthesis in Azospirillum. A number of genes essential for Trp production have been isolated in A. brasilense, including trpE(G) which codes for anthranilate synthase, the key enzyme in Trp biosynthesis. The relevance of each of these four aspects for plant growth promotion by Azospirillum is discussed.

Abstract Azospirillum represents the best characterized genus of plant growth-promoting rhizobacteria. Other free-living diazotrophs repeatedly detected in association with plant roots, include Acetobacter diazotrophicus, Herbaspirillum seropedicae, Azoarcus spp. and Azotobacter. Four aspects of the Azospirillum–plant root interaction are highlighted: natural habitat, plant root interaction, nitrogen fixation and biosynthesis of plant growth hormones. Each of these aspects is dealt with in a comparative way. Azospirilla are predominantly surface-colonizing bacteria, whereas A. diazotrophicus, H. seropedicae and Azoarcus sp. are endophytic diazotrophs. The attachment of Azospirillum cells to plant roots occurs in two steps. The polar flagellum, of which the flagellin was shown to be a glycoprotein, mediates the adsorption step. An as yet unidentified surface polysaccharide is believed to be essential in the subsequent anchoring phase. In Azoarcus sp. the attachment process is mediated by type IV pili. Nitrogen fixation structural genes (nif) are highly conserved among all nitrogen-fixing bacteria, and in all diazotrophic species of the class of proteobacteria examined, the transcriptional activator NifA is required for expression of other nif genes in response to two major environmental signals (oxygen and fixed N). However, the mechanisms involved in this control can vary in different organisms. In Azospirillum brasilense and H. seropedicae (α- and β-subgroup, respectively), NifA is inactive in conditions of excess nitrogen. Activation of NifA upon removal of fixed N seems to involve, either directly or indirectly, the signal transduction protein PII. The presence of four conserved cysteine residues in the NifA protein might be an indication that NifA is directly sensitive to oxygen. In Azotobacter vinelandii (γ-subgroup) nifA is cotranscribed with a second gene nifL. The nifL gene product inactivates NifA in response to high oxygen tension and cellular nitrogen-status. NifL was found to be a redox-sensitive flavoprotein. The relief of NifL inhibition on NifA activity, in response to N-limitation, is suggested to involve a PII-like protein. Moreover, nitrogenase activity is regulated according to the intracellular nitrogen and O2 level. In A. brasilense and Azospirillum lipoferum posttranslational control of nitrogenase, in response to ammonium and anaerobiosis, involves ADP-ribosylation of the nitrogenase iron protein, mediated by the enzymes DraT and DraG. At least three pathways for indole-3-acetic acid (IAA) biosynthesis in A. brasilense exist: two Trp-dependent (the indole-3-pyruvic acid and presumably the indole-3-acetamide pathway) and one Trp-independent pathway. The occurrence of an IAA biosynthetic pathway not using Trp (tryptophan) as precursor is highly unusual in bacteria. Nevertheless, the indole-3-pyruvate decarboxylase encoding ipdC gene is crucial in the overall IAA biosynthesis in Azospirillum. A number of genes essential for Trp production have been isolated in A. brasilense, including trpE(G) which codes for anthranilate synthase, the key enzyme in Trp biosynthesis. The relevance of each of these four aspects for plant growth promotion by Azospirillum is discussed.

Because of their ability to transform atmospheric N 2 into ammonia that can be used by the plant, researchers were originally very optimistic about the potential of associative diazotrophic bacteria to promote the growth of many cereals and grasses. However, multiple inoculation experiments during recent decades failed to show a substantial contribution of Biological Nitrogen Fixation (BNF) to plant growth in most cases. It is now clear that associative diazotrophs exert their positive effects on plant growth directly or indirectly through (a combination of) different mechanisms. Apart from fixing N 2 , diazotrophs can affect plant growth directly by the synthesis of phytohormones and vitamins, inhibition of plant ethylene synthesis, improved nutrient uptake, enhanced stress resistance, solubilization of inorganic phosphate and mineralization of organic phosphate. Indirectly, diazotrophs are able to decrease or prevent the deleterious effects of pathogenic microorganisms, mostly through the synthesis of antibiotics and/or fungicidal compounds, through competition for nutrients (for instance, by siderophore production) or by the induction of systemic resistance to pathogens. In addition, they can affect the plant indirectly by interacting with other beneficial microorganisms, for example, Azospirillum increasing nodulation of legumes by rhizobia. The further elucidation of the different mechanisms involved will help to make associative diazotrophs a valuable partner in future agriculture.

Abstract Current data from bacterial pathogens of animals and from bacterial symbionts of plants support some of the more general proposed functions for lipopolysaccharides (LPS) and underline the importance of LPS structural versatility and adaptability. Most of the structural heterogeneity of LPS molecules is found in the O-antigen polysaccharide. In this review, the role and mechanisms of this striking flexibility in molecular structure of the O-antigen in bacterial pathogens and symbionts are illustrated by some recent findings. The variation in O-antigen that gives rise to an enormous structural diversity of O-antigens lies in the sugar composition and the linkages between monosaccharides. The chemical composition and structure of the O-antigen is strain-specific (interstrain LPS heterogeneity) but can also vary within one bacterial strain (intrastrain LPS heterogeneity). Both LPS heterogeneities can be achieved through variations at different levels. First of all, O-polysaccharides can be modified non-stoichiometrically with sugar moieties, such as glucosyl and fucosyl residues. The addition of non-carbohydrate substituents, i.e. acetyl or methyl groups, to the O-antigen can also occur with regularity, but in most cases these modifications are again non-stoichiometric. Understanding LPS structural variation in bacterial pathogens is important because several studies have indicated that the composition or size of the O-antigen might be a reliable indicator of virulence potential and that these important features often differ within the same bacterial strain. In general, O-antigen modifications seem to play an important role at several (at least two) stages of the infection process, including the colonization (adherence) step and the ability to bypass or overcome host defense mechanisms. There are many reports of modifications of O-antigen in bacterial pathogens, resulting either from altered gene expression, from lysogenic conversion or from lateral gene transfer followed by recombination. In most cases, the mechanisms underlying these changes have not been resolved. However, in recent studies some progress in understanding has been made. Changes in O-antigen structure mediated by lateral gene transfer, O-antigen conversion and phase variation, including fucosylation, glucosylation, acetylation and changes in O-antigen size, will be discussed. In addition to the observed LPS heterogeneity in bacterial pathogens, the structure of LPS is also altered in bacterial symbionts in response to signals from the plant during symbiosis. It appears to be part of a molecular communication between bacterium and host plant. Experiments ex planta suggest that the bacterium in the rhizosphere prepares its LPS for its roles in symbiosis by refining the LPS structure in response to seed and root compounds and the lower pH at the root surface. Moreover, modifications in LPS induced by conditions associated with infection are another indication that specific structures are important. Also during the differentiation from bacterium to bacteroid, the LPS of Rhizobium undergoes changes in the composition of the O-antigen, presumably in response to the change of environment. Recent findings suggest that, during symbiotic bacteroid development, reduced oxygen tension induces structural modifications in LPS that cause a switch from predominantly hydrophilic to predominantly hydrophobic molecular forms. However, the genetic mechanisms by which the LPS epitope changes are regulated remain unclear. Finally, the possible roles of O-antigen variations in symbiosis will be discussed.

Radish seeds have previously been shown to contain two homologous, 5-kD cysteine-rich proteins designated Raphanus sativus-antifungal protein 1 (Rs-AFP1) and Rs-AFP2, both of which exhibit potent antifungal activity in vitro. We now demonstrate that these proteins are located in the cell wall and occur predominantly in the outer cell layers lining different seed organs. Moreover, Rs-AFPs are preferentially released during seed germination after disruption of the seed coat. The amount of released proteins is sufficient to create a microenvironment around the seed in which fungal growth is suppressed. Both the cDNAs and the intron-containing genomic regions encoding the Rs-AFP preproteins were cloned. Transcripts (0.55 kb) hybridizing with an Rs-AFP1 cDNA-derived probe were present in near-mature and mature seeds. Such transcripts as well as the corresponding proteins were barely detectable in healthy uninfected leaves but accumulated systemically at high levels after localized fungal infection. The induced leaf proteins (designated Rs-AFP3 and Rs-AFP4) were purified and shown to be homologous to seed Rs-AFPs and to exert similar antifungal activity in vitro. A chimeric Rs-AFP2 gene under the control of the constitutive cauliflower mosaic virus 35S promoter conferred enhanced resistance to the foliar pathogen Alternaria longipes in transgenic tobacco. The term \"plant defensins\" is proposed to denote these defense-related proteins