RESUMO
The nodABC genes of rhizobia are essential for the synthesis of lipo-oligosaccharidic (N-acylated chitin oligomers) nodulation signals. nodC gene products from Rhizobium, Bradyrhizobium, and Azorhizobium exhibit extensive homology with chitin synthases, suggesting that the NodC proteins are involved in the synthesis of the chitin oligomer backbone by catalyzing the beta-1,4-linkage between N-acetyl-D-glucosamine residues.
Assuntos
Proteínas de Bactérias/genética , Quitina Sintase/genética , N-Acetilglucosaminiltransferases , Rhizobiaceae/genética , Sequência de Carboidratos , Genes Bacterianos , Homologia de Sequência de Aminoácidos , Leveduras/genéticaRESUMO
The ability of Azorhizobium caulinodans ORS571 and other diazotrophic bacteria to internally colonize roots of Arabidopsis thaliana has been studied. Strains tagged with lacZ or gusA reporter genes were used, and the principal colonization sites were found to be the points of emergence of lateral roots, lateral root cracks (LRCs). High frequencies of colonization were found; 63 to 100% of plants were colonized by ORS571, and 100% of plants were colonized by Herbaspirillum seropedicae. After LRCs were colonized, bacteria moved into intercellular spaces between the cortical and endodermal cell layers. Specific flavonoids, naringenin and daidzein, at 5 x 10(-5) M, significantly promoted colonization by ORS571. By using a nodC and a nodD mutant of ORS571, it was shown that neither Nod factors nor NodD are involved in colonization or flavonoid stimulation of colonization. Flavonoids did not appear to be stimulating LRC colonization by their activity as nutritional factors. LRC and intercellular colonization by H. seropedicae was stimulated by naringenin and daidzein at the same concentration that stimulated colonization by ORS571.
Assuntos
Arabidopsis/microbiologia , Flavanonas , Flavonoides/farmacologia , Rhizobiaceae/efeitos dos fármacos , Rhizobiaceae/crescimento & desenvolvimento , Genes Bacterianos , Isoflavonas/farmacologia , Mutação , Fixação de Nitrogênio/genética , Raízes de Plantas/microbiologia , SimbioseRESUMO
The establishment of the nitrogen-fixing symbiosis between rhizobia and legumes requires an exchange of signals between the two partners. In response to flavonoids excreted by the host plant, rhizobia synthesize Nod factors (NFs) which elicit, at very low concentrations and in a specific manner, various symbiotic responses on the roots of the legume hosts. NFs from several rhizobial species have been characterized. They all are lipo-chitooligosaccharides, consisting of a backbone of generally four or five glucosamine residues N-acylated at the non-reducing end, and carrying various O-substituents. The N-acyl chain and the other substituents are important determinants of the rhizobial host specificity. A number of nodulation genes which specify the synthesis of NFs have been identified. All rhizobia, in spite of their diversity, possess conserved nodABC genes responsible for the synthesis of the N-acylated oligosaccharide core of NFs, which suggests that these genes are of a monophyletic origin. Other genes, the host specific nod genes, specify the substitutions of NFs. The central role of NFs and nod genes in the Rhizobium-legume symbiosis suggests that these factors could be used as molecular markers to study the evolution of this symbiosis. We have studied a number of NFs which are N-acylated by alpha,beta-unsaturated fatty acids. We found that the ability to synthesize such NFs does not correlate with taxonomic position of the rhizobia. However, all rhizobia that produce NFs such nodulate plants belonging to related tribes of legumes, the Trifolieae, Vicieae, and Galegeae, all of them being members of the so-called galegoid group. This suggests that the ability to recognize the NFs with alpha-beta-unsaturated fatty acids is limited to this group of legumes, and thus might have appeared only once in the course of legume evolution, in the galegoid phylum.
Assuntos
Fabaceae/microbiologia , Fabaceae/fisiologia , Genes Bacterianos , Lipopolissacarídeos/metabolismo , Rhizobium/genética , Rhizobium/fisiologia , Evolução Biológica , Fabaceae/genética , Fixação de Nitrogênio , Filogenia , Transdução de Sinais , Simbiose/genética , Simbiose/fisiologiaAssuntos
Fabaceae/embriologia , Lipopolissacarídeos/metabolismo , Plantas Medicinais , Rhizobium/metabolismo , Transdução de Sinais , Simbiose , Sequência de Carboidratos , Fabaceae/genética , Fabaceae/metabolismo , Fabaceae/microbiologia , Lipopolissacarídeos/biossíntese , Dados de Sequência Molecular , Fixação de NitrogênioRESUMO
Mu cts 62 a thermo-inductible mutant of phage Mu was integrated in E. coli in to the broad host range RP4 plasmid. The hybrid plasmid RP4::Mu cts 62 was then transferred by mating to the dinitrogen-fixing strain K. pneumoniae M5al. In Klebsiella Mu cts 62 is still heat inducible and the phage production is similar to that observed in E. coli. Mu Should be a useful tool for the genetic analysis of nitrogen fixation.
Assuntos
Bacteriófagos/crescimento & desenvolvimento , Klebsiella pneumoniae , Engenharia Genética , Klebsiella pneumoniae/metabolismo , Mutação , Fixação de Nitrogênio , Plasmídeos , Replicação ViralRESUMO
To analyse the regulation of the nodulation (nod) genes of Rhizobium meliloti RCR2011 we have isolated lacZ gene fusions to a number of common, host-range and regulatory nod genes, using the mini-Mu-lac bacteriophage transposon MudII1734. Common (nodA, nodC, nod region IIa) and host-range (nodE, nodG, nodH) genes were found to be regulated similarly. They were activated (i) by the regulatory nodD1 gene in the presence of flavones such as chrysoeriol, luteolin and 7,3',4'-trihydroxyflavone, (ii) by nodD2 in the presence of alfalfa root exudate but not with the NodD1-activating flavones, and (iii) by the regulatory genes syrM-nodD3 even in the absence of plant inducers. Thus common and host-range nod genes belong to the same regulon. In contrast to the nodD1 gene, the regulatory nodD3 gene was not expressed constitutively and exhibited a complex regulation. It required syrM for expression, was activated by nodD1 in the presence of luteolin and was positively autoregulated.
Assuntos
Regulação Bacteriana da Expressão Gênica , Genes Bacterianos , Genes Reguladores , Rhizobium/genética , DNA Bacteriano/genética , DNA Bacteriano/isolamento & purificação , Escherichia coli/genética , Óperon , Plasmídeos , Proteínas Recombinantes de Fusão/metabolismo , Mapeamento por Restrição , beta-Galactosidase/genética , beta-Galactosidase/metabolismoRESUMO
Rhizobium, Bradyrhizobium, and Azorhizobium strains, collectively referred to as rhizobia, elicit on their leguminous hosts, in a specific manner, the formation of nodules in which they fix nitrogen. Rhizobial nod genes, which determine host specificity, infection, and nodulation, are involved in the exchange of low molecular weight signal molecules between the plant and the bacteria as follows. Transcription of the nod operons is under the control of NodD regulatory proteins, which are specifically activated by plant flavonoid signals. The common and species-specific structural nod genes are involved in turn in the synthesis of specific lipo-oligosaccharides that signal back to the plant to elicit root-hair deformations, cortical-cell divisions, and nodule-meristem formation.
Assuntos
Fabaceae/genética , Fabaceae/microbiologia , Variação Genética , Fixação de Nitrogênio , Plantas Medicinais , Rhizobiaceae/genéticaRESUMO
Multicopy plasmid RSF1010 and four of its in vitro-constructed derivatives were mobilized by the self-transmissible RP4 plasmid into Azotobacter vinelandii UW. Modifications of the Escherichia coli transformation procedure of Cohen et al. (Proc. Natl. Acad. Sci. U.S.A. 69:2110-2114, 1972) allowed transformation of A. vinelandii strains UW and ATCC 12837 with purified RP4 or RSF1010 deoxyribonucleic acid.
Assuntos
Azotobacter/genética , Plasmídeos , Transformação Bacteriana , Escherichia coli/genéticaRESUMO
Rhizobia elicit on their specific leguminous hosts the formation of new organs, called nodules, in which they fix nitrogen. The rhizobial nodulation genes specify the synthesis of lipo-chitooligosaccharide signals, the Nod factors (NFs). Each rhizobial species has a characteristic set of nodulation genes that specifies the length of the chitooligosaccharide backbone and the type of substitutions at both ends of the molecule, thus making the NFs specific for a given plant host. At extremely low concentrations, purified NFs are capable of eliciting on homologous legume hosts many of the plant developmental responses characteristic of the bacteria themselves, including cell divisions, and the triggering of a plant organogenic program. This review summarizes our current knowledge on the biosynthesis, structure, and function of this new class of signaling molecules. Finally we discuss the possibility that these signals could be part of a new family of plant lipo-chitooligosaccharide growth regulators.
Assuntos
Lipopolissacarídeos/metabolismo , Fixação de Nitrogênio , Sinais Direcionadores de Proteínas/metabolismo , Rhizobium/metabolismo , Sequência de Carboidratos , Lipopolissacarídeos/química , Dados de Sequência Molecular , Fixação de Nitrogênio/genética , Reguladores de Crescimento de Plantas/metabolismo , Sinais Direcionadores de Proteínas/biossíntese , Sinais Direcionadores de Proteínas/química , Rhizobium/genéticaRESUMO
Large plasmids of molecular weight varying from 90 to around 200 x 10(6) have earlier been detected in most Rhizobium meliloti strains using an alkaline denaturation - phenol extraction procedure. With a less destructive method (Eckardt 1978) it was possible additionally to detect one plasmid of molecular weight clearly greater than 300 x 10(6) (= megaplasmid) in all of twenty-seven R. meliloti strains of various geographical origins and nodulation groupings investigated. Four strains (RCR 2011, A145, S26 and CC2013) were found to carry one megaplasmid and no smaller plasmids. Hybridization experiments with Klebsiella pneumoniae and R. meliloti cloned nitrogenase structural genes D and H showed that these genes are located on the megaplasmid and not on the smaller plasmids. All of the ten independent spontaneous non-nodulating derivatives of three strains of R. meliloti were shown to have suffered a deletion in the nifDH region of the megaplasmid. These results indicate that a gene controlling an early step in nodule formation is located in the nifDH region of the megaplasmid. This indicates that the same replicon carries genes controlling early and late functions in symbiosis.
Assuntos
Genes Bacterianos , Plasmídeos , Rhizobium/genética , Simbiose , Mapeamento Cromossômico , Peso Molecular , Fenótipo , Tumores de Planta/microbiologiaRESUMO
Rhizobium meliloti nodulation (nod) genes required for specific infection and nodulation of alfalfa have been cloned. Transposon Tn5 mutagenesis defined three nod regions spanning 16 kilobases of the pSym megaplasmid. Genetic and cytological studies of 62 nodulation-defective mutants allowed the assignment of symbiotic developmental phenotypes to common and specific nod loci. Root hair curling was determined by both common (region I) and specific (region III) nod transcription units; locus IIIb (nodH gene) positively controlled curling on the homologous host alfalfa, whereas loci IIIa (nodFE) and IIIb (nodH) negatively controlled curling on heterologous hosts. Region I (nodABC) was required for bacterial penetration and infection thread initiation in shepherd's crooks, and the nodFE transcription unit controlled infection thread development within the alfalfa root hair. In contrast, induction of nodule organogenesis, which can be triggered from a distance, seemed to be controlled by common nodABC genes and not to require specific nod genes nodFE and nodH. Region II affected the efficiency of hair curling and infection thread formation.
Assuntos
Genes Bacterianos , Fixação de Nitrogênio , Rhizobium/genética , Simbiose , Clonagem Molecular , Elementos de DNA Transponíveis , Teste de Complementação Genética , Medicago sativa/microbiologia , Fenótipo , Plasmídeos , Rhizobium/fisiologia , Especificidade da EspécieRESUMO
Rhizobia nodulation (nod) genes are involved in the synthesis of symbiotic signals, the Nod factors, which are mono-N-acylated chito-oligosaccharides. Nod factors elicit, in a specific manner, various plant responses on legume roots. In this report we address the question of the role of nodFEG genes in the synthesis of the acyl moiety of Rhizobium meliloti Nod factors. In a Nod factor-overproducing strain with the wild-type nod region, in addition to the delta 2,9-C16:2 and delta 2, 4,9-C16:3 acyl groups already described, delta 9-C16:1 was also found, together with a series of C18 to C26 (omega-1)-hydroxylated fatty acids. A deletion of nodE resulted in the absence of C16:2 and C16:3 fatty acids, which were replaced by vaccenic acid (delta 11-C18:1), but did not change the proportion of (omega-1)-hydroxylated fatty acids. A nodF deletion, non-polar with respect to nodE, resulted in the same alterations in the Nod factor N-acyl composition, showing that both nodF and nodE are required for the synthesis of the C16 polyunsaturated chains. In contrast, nodG mutations did not result in a detectable change in the Nod factor N-acyl moiety. When a plasmid carrying the nodFE genes of Rhizobium leguminosarum bv. viciae was introduced into R. meliloti nodFE- and nodFEG-deleted strains, Nod factors with polyunsaturated C18 fatty acids (C18:2, C18:3, and C18:4) could be detected. These results provide evidence that the molecular basis of allelic variation between the R. meliloti and R. leguminosarum bv. viciae host range nodFE genes lies in the fact that the two nodFE alleles specify the synthesis of unsaturated fatty acid substituents with a different carbon length.
Assuntos
Aciltransferases , Proteínas de Bactérias/metabolismo , Genes Bacterianos , Lipopolissacarídeos/metabolismo , Proteínas de Membrana , Sinorhizobium meliloti/genética , Sinorhizobium meliloti/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/isolamento & purificação , Configuração de Carboidratos , Sequência de Carboidratos , Clonagem Molecular , DNA Bacteriano/genética , DNA Bacteriano/metabolismo , Escherichia coli/genética , Ácidos Graxos Insaturados/análise , Deleção de Genes , Teste de Complementação Genética , Lipopolissacarídeos/química , Lipopolissacarídeos/isolamento & purificação , Espectrometria de Massas , Dados de Sequência Molecular , Plasmídeos , Mapeamento por RestriçãoRESUMO
Phage Mu-1 and a thermoinducible derivative, Mu-1 cts 62 were inserted into the broad host range R factor RP4. These hybrid plasmids were transferred by conjugation to a phytopathogenic bacterium Pseudomonas solanacearum GMI 1000 and a legume-root nodule bacterium Rhizobium meliloti 2011. The Mu genome is transcribed and tranlated in these new hosts: P. solanacearum (RP4:Mu cts) cultures have a spontaneous production of about 5 X 10(5) plaque-forming units ml-1 which is similar to the frequency of spontaneous Mu production in E. coli; the Mu production of R. meliloti is lower (about 10(2) plaque-forming units ml-1).
Assuntos
Colífagos , Resistência Microbiana a Medicamentos , Pseudomonas , Fatores R , Rhizobium , Antibacterianos/farmacologia , Colífagos/crescimento & desenvolvimento , Conjugação Genética , Escherichia coli/efeitos dos fármacos , Lisogenia , Pseudomonas/efeitos dos fármacos , Rhizobium/efeitos dos fármacos , Replicação ViralRESUMO
The pSym megaplasmid of Rhizobium meliloti 2011 mobilized by plasmid RP4, or plasmid pGMI42, an RP4-prime derivative which carries a 290-kilobase pSym fragment including nitrogenase and nod genes, was introduced into Agrobacterium tumefaciens. The resulting transconjugants induced root deformations specifically on the homologous hosts Medicago sativa and Melilotus alba and not on the heterologous hosts Trifolium pratense and Trifolium repens. The root deformations were shown to be genuine nodules by physiological and cytological studies. Thus, host specificity nodulation genes are located on the pSym megaplasmid. Host nodulation specificity did not seem to require recognition at the root hair level since no infection threads could be detected in the root hairs. Cytological observations indicated that bacteria penetrated only the superficial layers of the host root tissue by an atypical infection process. The submeristematic zone and the central tissue of the nodules were bacteria free. Thus, nodule organogenesis was probably triggered from a distance by the bacteria. Agrobacterium transconjugants carrying pSym induced the formation of more numerous and larger nodules than those carrying the RP4-prime plasmid pGMI42, suggesting that some genes influencing nodule organogenesis are located in a pSym region(s) outside that which has been cloned into pGMI42.
Assuntos
Genes Bacterianos , Doenças das Plantas , Plasmídeos , Rhizobium/genética , Rhizobium/patogenicidade , Especificidade da EspécieRESUMO
The symbiosis between Rhizobium and legumes is highly specific. For example, R. meliloti elicits the formation of root nodules on alfalfa and not on vetch. We recently reported that R. meliloti nodulation (nod) genes determine the production of acylated and sulfated glucosamine oligosaccharide signals. We now show that the biochemical function of the major host-range genes, nodH and nodPQ, is to specify the 6-O-sulfation of the reducing terminal glucosamine. Purified Nod factors (sulfated or not) from nodH+ or nodH- strains exhibited the same plant specificity in a variety of bioassays (root hair deformations, nodulation, changes in root morphology) as the bacterial cells from which they were purified. These results provide strong evidence that the molecular mechanism by which the nodH and nodPQ genes mediate host specificity is by determining the sulfation of the extracellular Nod signals.
Assuntos
Lipopolissacarídeos/metabolismo , Rhizobium/genética , Sulfotransferases/metabolismo , Sequência de Aminoácidos , Clonagem Molecular , Genes Bacterianos , Medicago sativa/microbiologia , Dados de Sequência Molecular , Mapeamento por Restrição , Alinhamento de Sequência , Sulfatos , Sulfotransferases/genética , SimbioseRESUMO
Transfer of an IncP plasmid carrying the Rhizobium meliloti nodFE, nodG, and nodH genes to Rhizobium trifolii enabled R. trifolii to nodulate alfalfa (Medicago sativa), the normal host of R. meliloti. Using transposon Tn5-linked mutations and in vitro-constructed deletions of the R. meliloti nodFE, nodG, and nodH genes, we showed that R. meliloti nodH was required for R. trifolii to elicit both root hair curling and nodule initiation on alfalfa and that nodH, nodFE, and nodG were required for R. trifolii to elicit infection threads in alfalfa root hairs. Interestingly, the transfer of the R. meliloti nodFE, nodG, and nodH genes to R. trifolii prevented R. trifolii from infecting and nodulating its normal host, white clover (Trifolium repens). Experiments with the mutated R. meliloti nodH, nodF, nodE, and nodG genes demonstrated that nodH, nodF, nodE, and possibly nodG have an additive effect in blocking infection and nodulation of clover.
Assuntos
Genes Bacterianos , Fixação de Nitrogênio/genética , Rhizobium/genética , Deleção Cromossômica , Elementos de DNA Transponíveis , Escherichia coli/genética , Genótipo , Cinética , Plantas/microbiologia , Plasmídeos , Mapeamento por Restrição , Especificidade da EspécieRESUMO
The Rhizobium meliloti nodH gene is involved in determining host range specificity. By comparison with the wild-type strain, NodH mutants exhibit a change in host specificity. That is, although NodH mutants lose the ability to elicit root hair curling (Hac-), infection threads (Inf-), and nodule meristem formation (Nod-) on the homologous host alfalfa, they gain the ability to be Hac+ Inf+ Nod+ on a nonhomologous host such as common vetch. Using root hair deformation (Had) bioassays on alfalfa and vetch, we have demonstrated that sterile supernatant solutions of R. meliloti cultures, in which the nod genes had been induced by the plant flavone luteolin, contained symbiotic extracellular signals. The wild-type strain produced at least one Had signal active on alfalfa (HadA). The NodH- mutants did not produce this signal but produced at least one factor active on vetch (HadV). Mutants altered in the common nodABC genes produced neither of the Had factors. This result suggests that the nodABC operon determines the production of a common symbiotic factor which is modified by the NodH product into an alfalfa-specific signal. An absolute correlation was observed between the specificity of the symbiotic behavior of rhizobial cells and the Had specificity of their sterile filtrates. This indicates that the R. meliloti nodH gene determines host range by helping to mediate the production of a specific extracellular signal.
Assuntos
Genes Bacterianos , Rhizobium/genética , Escherichia coli/genética , Genótipo , Fenótipo , Plantas/microbiologia , Plasmídeos , Rhizobium/fisiologia , Especificidade da EspécieRESUMO
Rhizobia are symbiotic bacteria that elicit the formation on leguminous plants of specialized organs, root nodules, in which they fix nitrogen. In various Rhizobium species, such as R. leguminosarum and R. meliloti, common and host-specific nodulation (nod) genes have been identified which determine infection and nodulation of specific hosts. Common nodABC genes as well as host-specific nodH and nodQ genes were shown recently, using bioassays, to be involved in the production of extracellular Nod signals. Using R. meliloti strains overproducing symbiotic Nod factors, we have purified the major alfalfa-specific signal, NodRm-1, by gel permeation, ion exchange and C18 reverse-phase high performance liquid chromatography. From mass spectrometry, nuclear magnetic resonance, (35)S-labelling and chemical modification studies, NodRm-1 was shown to be a sulphated beta-1,4-tetrasaccharide of D-glucosamine (Mr 1,102) in which three amino groups were acetylated and one was acylated with a C16 bis-unsaturated fatty acid. This purified Nod signal specifically elicited root hair deformation on the homologous host when added in nanomolar concentration.
Assuntos
Aciltransferases , Medicago sativa/microbiologia , N-Acetilglucosaminiltransferases , Oligossacarídeos/metabolismo , Rhizobium/metabolismo , Simbiose , Acilação , Proteínas da Membrana Bacteriana Externa/genética , Proteínas de Bactérias/genética , Sequência de Carboidratos , Genes Bacterianos , Espectroscopia de Ressonância Magnética , Espectrometria de Massas , Dados de Sequência Molecular , Estrutura Molecular , Mutação , Oligossacarídeos/genética , Plasmídeos , Rhizobium/genética , Transdução de Sinais , Sulfatos/metabolismo , TransfecçãoRESUMO
Our objectives were to identify substances produced by plant roots that might act as nutritional mediators of specific plant-bacterium relationships and to delineate the bacterial genes responsible for catabolizing these substances. We discovered new compounds, which we call calystegins, that have the characteristics of nutritional mediators. They were detected in only 3 of 105 species of higher plants examined: Calystegia sepium, Convolvulus arvensis (both of the Convolvulaceae family), and Atropa belladonna. Calystegins are abundant in organs in contact with the rhizosphere and are not found, or are observed only in small quantities, in aerial plant parts. Just as the synthesis of calystegins is infrequent in the plant kingdom, their catabolism is rare among rhizosphere bacteria that associate with plants and influence their growth. Of 42 such bacteria tested, only one (Rhizobium meliloti 41) was able to catabolize calystegins and use them as a sole source of carbon and nitrogen. The calystegin catabolism gene(s) (cac) in this strain is located on a self-transmissible plasmid (pRme41a), which is not essential to nitrogen-fixing symbiosis with legumes. We suggest that under natural conditions calystegins provide an exclusive carbon and nitrogen source to rhizosphere bacteria which are able to catabolize these compounds. Calystegins (and the corresponding microbial catabolic genes) might be used to analyze and possibly modify rhizosphere ecology.
Assuntos
Alcaloides/metabolismo , Extratos Vegetais/metabolismo , Plantas/microbiologia , Plasmídeos , Rhizobium/genética , Fixação de Nitrogênio , Plantas/metabolismo , Microbiologia do Solo , Simbiose , TropanosRESUMO
Rhizobium meliloti produces lipochitooligosaccharide nodulation NodRm factors that are required for nodulation of legume hosts. NodRm factors are O-acetylated and N-acylated by specific C16-unsaturated fatty acids. nodL mutants produce non-O-acetylated factors, and nodFE mutants produce factors with modified acyl substituents. Both mutants exhibited a significantly reduced capacity to elicit infection thread (IT) formation in alfalfa. However, once initiated, ITs developed and allowed the formation of nitrogen-fixing nodules. In contrast, double nodF/nodL mutants were unable to penetrate into legume hosts and to form ITs. Nevertheless, these mutants induced widespread cell wall tip growth in trichoblasts and other epidermal cells and were also able to elicit cortical cell activation at a distance. NodRm factor structural requirements are thus clearly more stringent for bacterial entry than for the elicitation of developmental plant responses.