Common bean (Phaseolus vulgaris) has become a cosmopolitan crop, but was originally domesticated in the Americas and has been grown in Latin America for several thousand years. Consequently an enormous diversity of bean nodulating bacteria have developed and in the centers of origin the predominant species in bean nodules is R. etli. In some areas of Latin America, inoculation, which normally promotes nodulation and nitrogen fixation is hampered by the prevalence of native strains. Many other species in addition to R. etli have been found in bean nodules in regions where bean has been introduced. Some of these species such as R. leguminosarum bv. phaseoli, R. gallicum bv. phaseoli and R. giardinii bv. phaseoli might have arisen by acquiring the phaseoli plasmid from R. etli. Others, like R. tropici, are well adapted to acid soils and high temperatures and are good inoculants for bean under these conditions. The large number of rhizobia species capable of nodulating bean supports that bean is a promiscuous host and a diversity of bean-rhizobia interactions exists. Large ranges of dinitrogen fixing capabilities have been documented among bean cultivars and commercial beans have the lowest values among legume crops. Knowledge on bean symbiosis is still incipient but could help to improve bean biological nitrogen fixation.

Diversity of Rhizobium-Phaseolus vulgaris symbiosis: overview and perspectives

More than 300 catalase sequences are now available, divided among monofunctional catalases (> 225), bifunctional catalase-peroxidases (> 50) and manganese-containing catalases (> 25). When combined with the recent appearance of crystal structures from at least two representatives from each of these groups (nine from the monofunctional catalases), valuable insights into the catalatic reaction mechanism in its various forms and into catalase evolution have been gained. The structures have revealed an unusually large number of modifications unique to catalases, a result of interacting with reactive oxygen species. Biochemical and physiological characterization of catalases from many different organisms has revealed a surprisingly wide range of catalatic efficiencies, despite similar sequences. Catalase gene expression in micro-organisms generally is controlled either by sensors of reactive oxygen species or by growth phase regulons, although the detailed mechanisms vary considerably.

Diversity of structures and properties among catalases.

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

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Beans (Phaseolus spp.) - model food legumes

The scarcity of usable nitrogen frequently limits plant growth. A tight metabolic association with rhizobial bacteria allows legumes to obtain nitrogen compounds by bacterial reduction of dinitrogen (N2) to ammonium (NH4+). We present here the annotated DNA sequence of the alpha-proteobacterium Sinorhizobium meliloti, the symbiont of alfalfa. The tripartite 6.7-megabase (Mb) genome comprises a 3.65-Mb chromosome, and 1.35-Mb pSymA and 1.68-Mb pSymB megaplasmids. Genome sequence analysis indicates that all three elements contribute, in varying degrees, to symbiosis and reveals how this genome may have emerged during evolution. The genome sequence will be useful in understanding the dynamics of interkingdom associations and of life in soil environments.

http://science.sciencemag.org/content/sci/293/5530/668.full.pdf

The composite genome of the legume symbiont Sinorhizobium meliloti.

Eukaryotes often form symbioses with microorganisms. Among these, associations between plants and nitrogen-fixing bacteria are responsible for the nitrogen input into various ecological niches. Plants of many different families have evolved the capacity to develop root or stem nodules with diverse genera of soil bacteria. Of these, symbioses between legumes and rhizobia (Azorhizobium, Bradyrhizobium, Mesorhizobium, and Rhizobium) are the most important from an agricultural perspective. Nitrogen-fixing nodules arise when symbiotic rhizobia penetrate their hosts in a strictly controlled and coordinated manner. Molecular codes are exchanged between the symbionts in the rhizosphere to select compatible rhizobia from pathogens. Entry into the plant is restricted to bacteria that have the “keys” to a succession of legume “doors”. Some symbionts intimately associate with many different partners (and are thus promiscuous), while others are more selective and have a narrow host range. For historical reasons, narrow host range has been more intensively investigated than promiscuity. In our view, this has given a false impression of specificity in legume-Rhizobium associations. Rather, we suggest that restricted host ranges are limited to specific niches and represent specialization of widespread and more ancestral promiscuous symbioses. Here we analyze the molecular mechanisms governing symbiotic promiscuity in rhizobia and show that it is controlled by a number of molecular keys.

Molecular Basis of Symbiotic Promiscuity

Rhizobium leguminosarum bv. phaseoli CFN42 contains six plasmids (pa to pf), and pd has been shown to be the symbiotic plasmid. To determine the participation of the other plasmids in cellular functions, we used a positive selection scheme to isolate derivatives cured of each plasmid. These were obtained for all except one (pe), of which only deleted derivatives were recovered. In regard to symbiosis, we found that in addition to pd, pb is also indispensable for nodulation, partly owing to the presence of genes involved in lipopolysaccharide synthesis. The positive contribution of pb, pc, pe, and pf to the symbiotic capacity of the strain was revealed in competition experiments. The strains that were cured (or deleted for pe) were significantly less competitive than the wild type. Analysis of the growth capacity of the cured strains showed the participation of the plasmids in free-living conditions: the pf- strain was unable to grow on minimal medium, while strains cured of any other plasmid had significantly reduced growth capacity in this medium. Even on rich medium, strains lacking pb or pc or deleted for pe had a diminished growth rate compared with the wild type. Complementation of the cured strains with the corresponding wild-type plasmid restored their original phenotypes, thus confirming that the effects seen were due only to loss of plasmids. The results indicate global participation of the Rhizobium genome in symbiotic and free-living functions.

Different plasmids of Rhizobium leguminosarum bv. phaseoli are required for optimal symbiotic performance.

Rhizobium species are gram-negative soil bacteria that are able to fix nitrogen and to establish a symbiotic relationship with leguminous plants. Besides the chromosome, their genome is usually constituted by large plasmids which carry genetic material relevant for very diverse functions, such as utilization of plant metabolites, aromatic compounds, and diverse sugars (14), in addition to nodulation and nitrogen fixation, which is the best-studied role of rhizobial plasmids.

Rhizobium etli CFN42 induces the formation of nitrogen fixing nodules in roots of Phaseolus vulgaris (bean) plants. In this strain, the genomic material is distributed between one chromosome and six plasmids (p42a to p42f). One of these plasmids, p42d, has been identified as the symbiotic plasmid (pSym), because it carries most of the genes required for nodulation and nitrogen fixation (43). Some functional characteristics have previously been ascribed to these plasmids (4). The conjugative abilities of the six plasmids have been evaluated by using an Agrobacterium tumefaciens strain lacking the Ti plasmid as a recipient. It was found that p42a is self-transmissible at a high frequency (10−2). Transfer of pSym was also detected but was found to be fully dependent on the presence of p42a. Thus, p42a functions as a helper for the transfer of the pSym, which is a mobilizable but not self-transmissible plasmid. The mechanism for pSym transfer requires its cointegration with p42a, and this cointegration is apparently accomplished through two mechanisms: one dependent on and another independent of recA. Transfer of the other plasmids of R. etli CFN42 has not been detected (5).

Distribution of symbiotic plasmids is a fundamental issue in the Rhizobium soil population. Some transfer genes have been found in rhizobial plasmids such as pNGR234a, the symbiotic plasmid of Rhizobium sp. strain NGR234. These genes were identified through homology to the conjugal transfer genes of Agrobacterium Ti plasmids (13); however, clear data regarding the transfer frequency of this plasmid have not been published. The horizontal transfer of genetic material between bacteria of the genus Rhizobium is a frequent event and has been demonstrated previously under laboratory conditions. Indeed, self-transmissible symbiotic plasmids have been observed in Rhizobium leguminosarum (28). Indirect evidence also suggests pSym transfer in soil (16), and recently, several studies have demonstrated the presence of similar plasmids in different genomic backgrounds (31). An identical organization of plasmid-borne symbiotic genes was found in isolates belonging to four distinct 16S ribosomal DNA species (22). These studies suggest the occurrence of horizontal transfer during the diversification of natural populations of rhizobia. From an applied perspective, it has been possible to increase the effectiveness of the Rhizobium-legume symbiosis with an R. leguminosarum strain through selective transfer of symbiotic plasmids (10).

In Agrobacterium, another genus of the Rhizobiaceae, the tumor-inducing plasmid (pTi) can be transferred between bacterial populations, which remain in the soil after infection of the plant tissues. Opines produced during the infection by the plant are the signal that turns on the expression of the genes required for their utilization. Additionally, they also induce the expression of the transcriptional regulator of the transfer process, traR, which activates traI expression. This regulatory effect is mediated by the AccR repressor (2, 20). The TraR and TraI proteins are LuxR-LuxI-type regulators that activate the expression of target genes in a quorum-sensing-dependent manner. The TraI protein is an acylated homoserine lactone (acyl-HSL) synthase that synthesizes 3-oxo-C8-HSL, and TraR is the autoinducer-responsive transcriptional regulator (40, 25). Both control the expression of at least five promoters of genes involved in plasmid transfer, and consequently, pTi transfer is quorum sensing dependent. A third regulatory protein, TraM, antagonizes TraR activity (26, 35).

The tumor-inducing plasmid pTiC58 of A. tumefaciens seems to be phylogenetically close to p42a. Previous studies from our laboratory have shown that these plasmids are incompatible (15). In addition, Bittinger et al. have identified a region in p42a that contains genes with similarity to vir genes of pTi (3).

Recent data suggest that the cinI locus, belonging to the LuxI family, is involved in conjugative transfer of pRL1JI in R. leguminosarum. Surprisingly, insertion mutations in cinI in both the donor and the recipient are required to decrease conjugation frequency (34). BisR and TriR, two LuxR-type transcriptional regulators, seem to participate in conjugal transfer gene expression, through an elaborate regulatory cascade (53). Also, two quorum-sensing systems have been identified in Sinorhizobium meliloti (36). Based on sequence homologies, those authors propose that a traR-traM locus is involved in conjugation. Nevertheless, data regarding the transfer mechanism for Rhizobium plasmids are still scarce.

In this paper we present the structural and functional characterization of the mobilization region of p42a of R. etli. Although the arrangement of the mobilization region is highly similar to that of pTi, there are important differences among the regulatory mechanisms. Knowledge regarding the regulation of p42a transfer will be an important factor to better understand the distribution of symbiotic information, due to its fundamental role in the conjugative transfer of this plasmid.

Conjugative transfer of p42a from rhizobium etli CFN42, which is required for mobilization of the symbiotic plasmid, is regulated by quorum sensing.

One remarkable characteristic of the genomes of some Rhizobium species is the frequent occurrence of rearrangements. In some instances these rearrangements alter the symbiotic properties of the strains. However, no detailed molecular mechanisms have been proposed for the generation of these rearrangements. To understand the mechanisms involved in the formation of rearrangements in the genome of Rhizobium phaseoli, we have designed a system which allows the positive selection for amplification and deletion events. We have applied this system to investigate the stability of the symbiotic plasmid of R. phaseoli. High-frequency amplification events were detected which increase the copy number of a 120-kb region carrying nodulation and nitrogen fixation genes two to eight times. Deletion events that affect the same region were also found, albeit at a lower frequency. Both kinds of rearrangements are generated by recombination between reiterated nitrogenase (nifHDK) operons flanking the 120-kb region. Images

Amplification and deletion of a nod-nif region in the symbiotic plasmid of Rhizobium phaseoli.

Repeated DNA sequences are a general characteristic of eucaryotic genomes. Although several examples of DNA reiteration have been found in procaryotic organisms, only in the case of the archaebacteria Halobacterium halobium and Halobacterium volcanii [C. Sapienza and W. F. Doolittle, Nature (London) 295:384-389, 1982], has DNA reiteration been reported as a common genomic feature. The genomes of two Rhizobium phaseoli strains, one Rhizobium meliloti strain, and one Agrobacterium tumefaciens strain were analyzed for the presence of repetitive DNA. Rhizobium and Agrobacterium spp. are closely related soil bacteria that interact with plants and that belong to the taxonomical family Rhizobiaceae. Rhizobium species establish a nitrogen-fixing symbiosis in the roots of legumes, whereas Agrobacterium species is a pathogen in different plants. The four strains revealed a large number of repeated DNA sequences. The family size was usually small, from 2 to 5 elements, but some presented more than 10 elements. Rhizobium and Agrobacterium spp. contain large plasmids in addition to the chromosomes. Analysis of the two Rhizobium strains indicated that DNA reiteration is not confined to the chromosome or to some plasmids but is a property of the whole genome. Images

Susana Brom

Papers

Different plasmids of Rhizobium leguminosarum bv. phaseoli are required for optimal symbiotic performance.

Conjugative transfer of p42a from rhizobium etli CFN42, which is required for mobilization of the symbiotic plasmid, is regulated by quorum sensing.

Amplification and deletion of a nod-nif region in the symbiotic plasmid of Rhizobium phaseoli.

Reiterated DNA sequences in Rhizobium and Agrobacterium spp.

In Rhizobium etli symbiotic plasmid transfer, nodulation competitivity and cellular growth require interaction among different replicons.