Effective symbiosis between Rhizobium etli and Phaseolus vulgaris requires the alarmone ppGpp

The symbiotic interaction between Rhizobium etli and Phaseolus vulgaris, the common bean plant, ultimately results in the formation of nitrogen-fixing nodules. Many aspects of the intermediate and late stages of this interaction are still poorly understood. The R. etli relA gene was identified through a genome-wide screening for R. etli symbiotic mutants. RelA has a pivotal role in cellular physiology, as it catalyzes the synthesis of (p)ppGpp, which mediates the stringent response in bacteria. The synthesis of ppGpp was abolished in an R. etli relA mutant strain under conditions of amino acid starvation. Plants nodulated by an R. etli relA mutant had a strongly reduced nitrogen fixation activity (75% reduction). Also, at the microscopic level, bacteroid morphology was altered, with the size of relA mutant bacteroids being increased compared to that of wild-type bacteroids. The expression of the sigma(N)-dependent nitrogen fixation genes rpoN2 and iscN was considerably reduced in the relA mutant. In addition, the expression of the relA gene was negatively regulated by RpoN2, the symbiosis-specific sigma(N) copy of R. etli. Therefore, an autoregulatory loop controlling the expression of relA and rpoN2 seems operative in bacteroids. The production of long- and short-chain acyl-homoserine-lactones by the cinIR and raiIR systems was decreased in an R. etli relA mutant. Our results suggest that relA may play an important role in the regulation of gene expression in R. etli bacteroids and in the adaptation of bacteroid physiology.     

Genome sequence of Rhizobium etli CNPAF512, a nitrogen-fixing symbiont isolated from bean root nodules in Brazil

Rhizobium etli is a Gram-negative soil-dwelling alphaproteobacterium that carries out symbiotic biological nitrogen fixation in close association with legume hosts. R. etli strains exhibit high sequence divergence and are geographically structured, with a potentially dramatic influence on the outcome of symbiosis. Here, we present the genome sequence of R. etli CNPAF512, a Brazilian isolate from bean nodules. We anticipate that the availability of genome sequences of R. etli strains from distinctly different areas will provide valuable new insights into the geographic mosaic of the R. etli pangenome and the evolutionary dynamics that shape it.     

Rhizobium etli taxonomy revised with novel genomic data and analyses

The taxonomic position of Phaseolus vulgaris rhizobial strains with available sequenced genomes was examined. Phylogenetic analyses with concatenated conserved genomic fragments accounting for over half of each genome showed that Rhizobium strains CIAT 652, Ch24-10 (newly reported genome) and CNPAF 512 constituted a well-supported group independent from Rhizobium etli CFN 42(T). DNA-DNA hybridization results indicated that CIAT 652, Ch24-10 and CNPAF 512 could correspond to R. etli, although the hybridization values were at the borderline that distinguishes different species. In contrast, experimental hybridization results were higher (over 80%) with Rhizobium phaseoli type strain ATCC 14482(T) in congruence to phylogenetic and ANIm analyses. The latter criterion allowed the reclassification of R. etli strains 8C-3 and Brasil5 as R. phaseoli. It was therefore concluded, based on all the evidence, that the CIAT 652, Ch24-10, and CNPAF 512 strains should be reclassified as R. phaseoli in spite of several common features linking them to R. etli. The R. phaseoli and R. etli speciation process seems to be a more recent event than the speciation that has occurred among other sister species, such as R. leguminosarum-R. etli or R. rhizogenes-R. tropici.     

The Rhizobium etli gene iscN is highly expressed in bacteroids and required for nitrogen fixation

Sequence analysis of the rpoN (2)- fixA intergenic region in the genome of Rhizobium etli CNPAF512 has uncovered three genes involved in nitrogen fixation, namely nifU, nifS and nifW. These genes are preceded by an ORF that is highly conserved among nitrogen-fixing bacteria. It encodes a putative gene product of 105 amino acids, belonging to the HesB-like protein family. A phylogenetic analysis of members of the HesB-like protein family showed that the R. etli HesB-like protein clusters with polypeptides encoded by ORFs situated upstream of the nifUS nitrogen fixation regions in the genomes of other diazotrophs. The R. etli ORF that encodes the HesB-like protein was designated iscN. iscN is co-transcribed with nifU and nifS, and is preferentially expressed under free-living microaerobic conditions and in bacteroids. Expression is regulated by the alternative sigma factor RpoN and the enchancer-binding protein NifA. A R. etli iscN mutant displays a reduction in nitrogen fixation capacity of 90% compared to the wild-type strain. This Nif(-) phenotype could be complemented by the introduction of intact copies of R. etli iscN.     

Symbiotic performance of some modified Rhizobium etli strains in assays with Phaseolus vulgaris beans that have a high capacity to fix N2

Rhizobium etli and R. tropici form nitrogen-fixing nodules on Phaseolus vulgaris (common bean). In the hope that R. etli strains with additional citrate synthase genes have better carbon economies, merodiploid strains were constructed. Previously, one such construct was shown to have an increased nodulation capacity in the standard bean cultivar Negro Xamapa. In the present work, derivatives from different R. etli strains carrying the R. tropici plasmid-borne or chromosomal citrate synthase gene were constructed and tested for nodulation in bean cultivars selected for their high capacity to fix nitrogen. Nodule numbers were dependent on the strain and the cultivar used. Differences in nodule number were not reflected in plant biomass.     

Conservation of plasmid-encoded traits among bean-nodulating Rhizobium species

Rhizobium etli type strain CFN42 contains six plasmids. We analyzed the distribution of genetic markers from some of these plasmids in bean-nodulating strains belonging to different species (Rhizobium etli, Rhizobium gallicum, Rhizobium giardinii, Rhizobium leguminosarum, and Sinorhizobium fredii). Our results indicate that independent of geographic origin, R. etli strains usually share not only the pSym plasmid but also other plasmids containing symbiosis-related genes, with a similar organization. In contrast, strains belonging to other bean-nodulating species seem to have acquired only the pSym plasmid from R. etli.     

Evidence of an American origin for symbiosis-related genes in Rhizobium lusitanum

Randomly amplified polymorphic DNA (RAPD) analysis was used to investigate the diversity of 179 bean isolates recovered from six field sites in the Arcos de Valdevez region of northwestern Portugal. The isolates were divided into 6 groups based on the fingerprint patterns that were obtained. Representatives for each group were selected for sequence analysis of 4 chromosomal DNA regions. Five of the groups were placed within Rhizobium lusitanum, and the other group was placed within R. tropici type IIA. Therefore, the collection of Portuguese bean isolates was shown to include the two species R. lusitanum and R. tropici. In plant tests, the strains P1-7, P1-1, P1-2, and P1-16 of R. lusitanum nodulated and formed nitrogen-fixing symbioses both with Phaseolus vulgaris and Leucaena leucocephala. A methyltransferase-encoding nodS gene identical with the R. tropici locus that confers wide host range was detected in the strain P1-7 as well as 24 others identified as R. lusitanum. A methyltransferase-encoding nodS gene also was detected in the remaining isolates of R. lusitanum, but in this case the locus was that identified with the narrow-host-range R. etli. Representatives of isolates with the nodS of R. etli formed effective nitrogen-fixing symbioses with P. vulgaris and did not nodulate L. leucocephala. From sequence data of nodS, the R. lusitanum genes for symbiosis were placed within those of either R. tropici or R. etli. These results would support the suggestion that R. lusitanum was the recipient of the genes for symbiosis with beans from both R. tropici and R. etli.     

Role of the ruvB gene in homologous and homeologous recombination in Rhizobium etli

The Rhizobium etli ruvA and ruvB genes were cloned through a PCR-based approach, using degenerate primers matching conserved sectors in the amino acid sequences of RuvB from eight bacterial species. Comparative analysis of the predicted polypeptides for RuvA and RuvB of R. etli showed highly conserved blocks with the corresponding homologs in other bacteria; RuvB depicts characteristic motifs for DNA helicases (ATP-binding and DEXH-box motifs). An R. etli ruvB::loxP Sp mutant was constructed by interposon mutagenesis. This mutant was highly sensitive to DNA-damaging agents, such as methyl methanesulfonate and nitrofurantoin, implying a deficiency in DNA repair. Homologous and homeologous conjugational recombination was reduced almost tenfold in the ruvB::loxP Sp mutant; a recombination defect was also observed in assays employing recombination between small plasmids, albeit at a smaller magnitude. Although the ruvA and ruvB genes are contiguous in R. etli, complementation studies suggest that they are expressed independently.     

Improvement of drought tolerance and grain yield in common bean by overexpressing trehalose-6-phosphate synthase in rhizobia

Improving stress tolerance and yield in crops are major goals for agriculture. Here, we show a new strategy to increase drought tolerance and yield in legumes by overexpressing trehalose-6-phosphate synthase in the symbiotic bacterium Rhizobium etli. Phaseolus vulgaris (common beans) plants inoculated with R. etli overexpressing trehalose-6-phosphate synthase gene had more nodules with increased nitrogenase activity and higher biomass compared with plants inoculated with wild-type R. etli. In contrast, plants inoculated with an R. etli mutant in trehalose-6-phosphate synthase gene had fewer nodules and less nitrogenase activity and biomass. Three-week-old plants subjected to drought stress fully recovered whereas plants inoculated with a wild-type or mutant strain wilted and died. The yield of bean plants inoculated with R. etli overexpressing trehalose-6-phosphate synthase gene and grown with constant irrigation increased more than 50%. Macroarray analysis of 7,200 expressed sequence tags from nodules of plants inoculated with the strain overexpressing trehalose-6-phosphate synthase gene revealed upregulation of genes involved in stress tolerance and carbon and nitrogen metabolism, suggesting a signaling mechanism for trehalose. Thus, trehalose metabolism in rhizobia is key for signaling plant growth, yield, and adaptation to abiotic stress, and its manipulation has a major agronomical impact on leguminous plants.     

Natural endophytic association between Rhizobium etli and maize (Zea mays L.)

Maize (Zea mays) and bean (Phaseolus vulgaris) have been traditionally grown in association for thousands of years in Mesoamerica. From surface sterilized maize roots, we have isolated over 60 Rhizobium strains that correspond to Rhizobium etli bv. phaseoli (the main symbiont of bean) on the basis of 16S rRNA gene restriction patterns, metabolic enzyme electropherotypes, organization of nif genes, and the ability to nodulate beans. The colonization capacity of some of the isolates was tested with an unimproved maize cultivar and with 30 maize land races. Increases in plant dry weight upon R. etli inoculation were recorded with some of the land races, and these increases may be related to plant growth promotion effects. Additionally, from within maize grown in monoculture we have also recovered R. etli isolates recognizable by their 16S rRNA gene types, which lack nif genes and are incapable of nodulating bean. These strains are presumed to correspond to the earlier described non-symbiotic R. etli obtained from bean rhizosphere.     

Regulatory role of Rhizobium etli CNPAF512 fnrN during symbiosis

The Rhizobium etli CNPAF512 fnrN gene was identified in the fixABCX rpoN(2) region. The corresponding protein contains the hallmark residues characteristic of proteins belonging to the class IB group of Fnr-related proteins. The expression of R. etli fnrN is highly induced under free-living microaerobic conditions and during symbiosis. This microaerobic and symbiotic induction of fnrN is not controlled by the sigma factor RpoN and the symbiotic regulator nifA or fixLJ, but it is due to positive autoregulation. Inoculation of Phaseolus vulgaris with an R. etli fnrN mutant strain resulted in a severe reduction in the bacteroid nitrogen fixation capacity compared to the wild-type capacity, confirming the importance of FnrN during symbiosis. The expression of the R. etli fixN, fixG, and arcA genes is strictly controlled by fnrN under free-living microaerobic conditions and in bacteroids during symbiosis with the host. However, there is an additional level of regulation of fixN and fixG under symbiotic conditions. A phylogenetic analysis of the available rhizobial FnrN and FixK proteins grouped the proteins in three different clusters.     

The Rhizobium etli opt operon is required for symbiosis and stress resistance

Rhizobium etli is a Gram-negative root-colonizing soil bacterium capable of fixing nitrogen while living in symbiosis with its leguminous host Phaseolus vulgaris. A genome-wide screening for R. etli symbiotic mutants revealed a R. etli operon encoding an oligopeptide ABC-transporter (Opt), two redA homologous genes and one redB gene. Expression analysis showed this opt operon to be transcribed both under free-living and symbiotic conditions and expression levels were demonstrated to be growth-phase-dependent. Plants nodulated by R. etli opt mutants showed a reduced symbiotic nitrogen fixation activity (approximately 50% reduction). Growth experiments with opt mutants in the presence of oligopeptides as the sole nitrogen source confirmed the involvement of the opt genes in oligopeptide uptake. Further phenotypic analysis of the opt mutants revealed them to display an enhanced resistance to the oligopeptide antibiotic bacitracin, an increased susceptibility to the beta-lactam antibiotic ampicillin and a decreased osmotolerance. In conclusion, our results demonstrate that the opt operon plays a crucial role during symbiosis and stress resistance.     

Purification and mass spectrometry of six lipid A species from the bacterial endosymbiont Rhizobium etli. Demonstration of a conserved distal unit and a variable proximal portion

Lipid A of Rhizobium etli CE3 differs dramatically from that of other Gram-negative bacteria. Key features include the presence of an unusual C28 acyl chain, a galacturonic acid moiety at position 4', and an acylated aminogluconate unit in place of the proximal glucosamine. In addition, R. etli lipid A is reported to lack phosphate and acyloxyacyl residues. Most of these remarkable structural claims are consistent with our recent enzymatic studies. However, the proposed R. etli lipid A structure is inconsistent with the ability of the precursor (3-deoxy-D-manno-octulosonic acid)(2)-4'-(32)P-lipid IV(A) to accept a C28 chain in vitro (Brozek, K. A., Carlson, R. W., and Raetz, C. R. H. (1996) J. Biol. Chem. 271, 32126-32136). To re-evaluate the structure, CE3 lipid A was isolated by new chromatographic procedures. CE3 lipid A is now resolved into six related components. Aminogluconate is present in D-1, D-2, and E, whereas B and C contain the typical glucosamine disaccharide seen in lipid A of most other bacteria. All the components possess a peculiar acyloxyacyl moiety at position 2', which includes the ester-linked C28 chain. As judged by mass spectrometry, the distal glucosamine units of A through E are the same, but the proximal units are variable. As described in the accompanying article (Que, N. L. S., Ribeiro, A. A., and Raetz, C. R. H. (2000) J. Biol. Chem. 275, 28017-28027), the discovery of component B suggests a plausible enzymatic pathway for the biosynthesis of the aminogluconate residue found in species D-1, D-2, and E of R. etli lipid A. We suggest that the unusual lipid A species of R. etli might be essential during symbiosis with leguminous host plants.     

Three genes encoding for putative methyl- and acetyltransferases map adjacent to the wzm and wzt genes and are essential for O-antigen biosynthesis in Rhizobium etli CE3

The elucidation of the structure of the O-antigen of Rhizobium etli CE3 predicts that the R. etli CE3 genome must contain genes encoding acetyl- and methyltransferases to confer the corresponding modifications to the O-antigen. We identified three open reading frames (ORFs) upstream of wzm, encoding the membrane component of the O-antigen transporter and located in the lps alpha-region of R. etli CE3. The ORFs encode two putative acetyltransferases with similarity to the CysE-LacA-LpxA-NodL family of acetyltransferases and one putative methyltransferase with sequence motifs common to a wide range of S-adenosyl-L-methionine-dependent methyltransferases. Mutational analysis of the ORFs encoding the putative acetyltransferases and methyltransferase revealed that the acetyl and methyl decorations mediated by these specific enzymes are essential for O-antigen synthesis. Composition analysis and high performance anion exchange chromatography analysis of the lipopolysaccharides (LPSs) of the mutants show that all of these LPSs contain an intact core region and lack the O-antigen polysaccharide. The possible role of these transferases in the decoration of the O-antigen of R. etli is discussed.