RESUMO
BACKGROUND: Certain legume plants produce a plethora of AMP-like peptides in their symbiotic cells. The cationic subgroup of the nodule-specific cysteine-rich (NCR) peptides has potent antimicrobial activity against gram-negative and gram-positive bacteria as well as unicellular and filamentous fungi. FINDINGS: It was shown by scanning and atomic force microscopies that the cationic peptides NCR335, NCR247 and Polymyxin B (PMB) affect differentially on the surfaces of Sinorhizobium meliloti bacteria. Similarly to PMB, both NCR peptides caused damages of the outer and inner membranes but at different extent and resulted in the loss of membrane potential that could be the primary reason of their antimicrobial activity. CONCLUSIONS: The primary reason for bacterial cell death upon treatment with cationic NCR peptides is the loss of membrane potential.
Assuntos
Peptídeos Catiônicos Antimicrobianos/farmacologia , Membrana Celular/efeitos dos fármacos , Potenciais da Membrana/efeitos dos fármacos , Proteínas de Plantas/farmacologia , Sinorhizobium meliloti/efeitos dos fármacos , Peptídeos Catiônicos Antimicrobianos/metabolismo , Membrana Celular/ultraestrutura , Medicago truncatula/fisiologia , Microscopia de Força Atômica , Microscopia Eletrônica de Varredura , Proteínas de Plantas/metabolismo , Polimixina B/farmacologia , Nódulos Radiculares de Plantas/fisiologia , Sinorhizobium meliloti/crescimento & desenvolvimento , Sinorhizobium meliloti/ultraestruturaRESUMO
Type IVb pili in enteropathogenic bacteria function as a host colonization factor by mediating tight adherence to host cells, but their role in bacterium-plant symbiosis is currently unknown. The genome of the symbiotic soil bacterium Sinorhizobium meliloti contains two clusters encoding proteins for type IVb pili of the Flp (fimbrial low-molecular-weight protein) subfamily. To establish the role of Flp pili in the symbiotic interaction of S. meliloti and its host, Medicago sativa, we deleted pilA1, which encodes the putative pilin subunit in the chromosomal flp-1 cluster and conducted competitive nodulation assays. The pilA1 deletion strain formed 27% fewer nodules than the wild type. Transmission electron microscopy revealed the presence of bundle-forming pili protruding from the polar and lateral region of S. meliloti wild-type cells. The putative pilus assembly ATPase CpaE1 fused to mCherry showed a predominantly unilateral localization. Transcriptional reporter gene assays demonstrated that expression of pilA1 peaks in early stationary phase and is repressed by the quorum-sensing regulator ExpR, which also controls production of exopolysaccharides and motility. Binding of acyl homoserine lactone-activated ExpR to the pilA1 promoter was confirmed with electrophoretic mobility shift assays. A 17-bp consensus sequence for ExpR binding was identified within the 28-bp protected region by DNase I footprinting analyses. Our results show that Flp pili are important for efficient symbiosis of S. meliloti with its plant host. The temporal inverse regulation of exopolysaccharides and pili by ExpR enables S. meliloti to achieve a coordinated expression of cellular processes during early stages of host interaction.
Assuntos
Fímbrias Bacterianas/metabolismo , Regulação Bacteriana da Expressão Gênica , Medicago sativa/microbiologia , Percepção de Quorum , Sinorhizobium meliloti/genética , Simbiose , Fatores de Transcrição/metabolismo , Aderência Bacteriana , Proteínas de Bactérias/biossíntese , Fímbrias Bacterianas/ultraestrutura , Deleção de Genes , Perfilação da Expressão Gênica , Microscopia Eletrônica de Transmissão , Nodulação , Sinorhizobium meliloti/fisiologia , Sinorhizobium meliloti/ultraestruturaRESUMO
Cerium oxide (CeO(2)) and zinc oxide (ZnO) nanoparticles (NPs) are extensively used in a variety of instruments and consumer goods. These NPs are of great concern because of potential toxicity towards human health and the environment. The present work aimed to assess the toxic effects of 10nm CeO(2) and ZnO NPs towards the nitrogen fixing bacterium Sinorhizobium meliloti. Toxicological parameters evaluated included UV/Vis measurement of minimum inhibitory concentration, disk diffusion tests, and dynamic growth. Ultra high-resolution scanning transmission electron microscopy (STEM) and infrared spectroscopy (FTIR) were utilized to determine the spatial distribution of NPs and macromolecule changes in bacterial cells, respectively. Results indicate that ZnO NPs were more toxic than CeO(2) NPs in terms of inhibition of dynamic growth and viable cells counts. STEM images revealed that CeO(2) and ZnO NPs were found on bacterial cell surfaces and ZnO NPs were internalized into the periplasmic space of the cells. FTIR spectra showed changes in protein and polysaccharide structures of extra cellular polymeric substances present in bacterial cell walls treated with both NPs. The growth data showed that CeO(2) NPs have a bacteriostatic effect, whereas ZnO NPs is bactericidal to S. meliloti. Overall, ZnO NPs were found to be more toxic than CeO(2) NPs.
Assuntos
Cério/toxicidade , Medicago sativa/microbiologia , Nanopartículas , Sinorhizobium meliloti/efeitos dos fármacos , Poluentes do Solo/toxicidade , Simbiose , Óxido de Zinco/toxicidade , Testes de Sensibilidade a Antimicrobianos por Disco-Difusão , Medicago sativa/fisiologia , Testes de Sensibilidade Microbiana , Microscopia Eletrônica de Varredura , Nanopartículas/toxicidade , Fixação de Nitrogênio/efeitos dos fármacos , Nodulação/efeitos dos fármacos , Raízes de Plantas/microbiologia , Raízes de Plantas/fisiologia , Polissacarídeos Bacterianos/metabolismo , Sinorhizobium meliloti/crescimento & desenvolvimento , Sinorhizobium meliloti/ultraestrutura , Espectroscopia de Infravermelho com Transformada de FourierRESUMO
Nitrogen fixation by legumes is very sensitive to salinity stress, which can severely reduce the productivity of legume crops and their soil-enriching capacity. Salinity is known to cause oxidative stress in the nodule by generating reactive oxygen species (ROS). Flavodoxins are involved in the response to oxidative stress in bacteria and cyanobacteria. Prevention of ROS production by flavodoxin overexpression in bacteroids might lead to a protective effect on nodule functioning under salinity stress. Tolerance to salinity stress was evaluated in alfalfa nodules elicited by an Ensifer meliloti strain that overexpressed a cyanobacterial flavodoxin compared with nodules produced by the wild-type bacteria. Nitrogen fixation, antioxidant and carbon metabolism enzyme activities were determined. The decline in nitrogenase activity associated to salinity stress was significantly less in flavodoxin-expressing than in wild-type nodules. We detected small but significant changes in nodule antioxidant metabolism involving the ascorbate-glutathione cycle enzymes and metabolites, as well as differences in activity of the carbon metabolism enzyme sucrose synthase, and an atypical starch accumulation pattern in flavodoxin-containing nodules. Salt-induced structural and ultrastructural alterations were examined in detail in alfalfa wild-type nodules by light and electron microscopy and compared to flavodoxin-containing nodules. Flavodoxin reduced salt-induced structural damage, which primarily affected young infected tissues and not fully differentiated bacteroids. The results indicate that overexpression of flavodoxin in bacteroids has a protective effect on the function and structure of alfalfa nodules subjected to salinity stress conditions. Putative protection mechanisms are discussed.
Assuntos
Flavodoxina/genética , Medicago sativa/microbiologia , Fixação de Nitrogênio , Nitrogênio/metabolismo , Nódulos Radiculares de Plantas/microbiologia , Sinorhizobium meliloti/fisiologia , Antioxidantes/metabolismo , Flavodoxina/metabolismo , Medicago sativa/efeitos dos fármacos , Medicago sativa/fisiologia , Medicago sativa/ultraestrutura , Nitrogenase/metabolismo , Estresse Oxidativo , Nódulos Radiculares de Plantas/fisiologia , Nódulos Radiculares de Plantas/ultraestrutura , Salinidade , Tolerância ao Sal , Sinorhizobium meliloti/química , Sinorhizobium meliloti/ultraestrutura , Cloreto de Sódio/farmacologia , Estresse Fisiológico , SimbioseRESUMO
BACKGROUND: Rhizobium leguminosarum bv. viciae mutants unable to transport branched-chain amino acids via the two main amino acid ABC transport complexes AapJQMP and BraDEFGC produce a nitrogen starvation phenotype when inoculated on pea (Pisum sativum) plants [1], [2]. Bacteroids in indeterminate pea nodules have reduced abundance and a lower chromosome number. They reduce transcription of pathways for branched-chain amino acid biosynthesis and become dependent on their provision by the host. This has been called "symbiotic auxotrophy". METHODOLOGY/PRINCIPAL FINDINGS: A region important in solute specificity was identified in AapQ and changing P144D in this region reduced branched-chain amino acid transport to a very low rate. Strains carrying P144D were still fully effective for N(2) fixation on peas demonstrating that a low rate of branched amino acid transport in R. leguminosarum bv. viciae supports wild-type rates of nitrogen fixation. The importance of branched-chain amino acid transport was then examined in other legume-Rhizobium symbioses. An aap bra mutant of R. leguminosarum bv. phaseoli also showed nitrogen starvation symptoms when inoculated on French bean (Phaseolus vulgaris), a plant producing determinate nodules. The phenotype is different from that observed on pea and is accompanied by reduced nodule numbers and nitrogen fixation per nodule. However, an aap bra double mutant of Sinorhizobium meliloti 2011 showed no phenotype on alfalfa (Medicago sativa). CONCLUSIONS/SIGNIFICANCE: Symbiotic auxotrophy occurs in both determinate pea and indeterminate bean nodules demonstrating its importance for bacteroid formation and nodule function in legumes with different developmental programmes. However, only small quantities of branched chain amino acids are needed and symbiotic auxotrophy did not occur in the Sinorhizobium meliloti-alfalfa symbiosis under the conditions measured. The contrasting symbiotic phenotypes of aap bra mutants inoculated on different legumes probably reflects altered timing of amino acid availability, development of symbiotic auxotrophy and nodule developmental programmes.
Assuntos
Aminoácidos/metabolismo , Fabaceae/microbiologia , Rhizobium/fisiologia , Simbiose , Transporte Biológico , Fabaceae/crescimento & desenvolvimento , Fabaceae/metabolismo , Interações Hospedeiro-Patógeno , Microscopia Eletrônica de Transmissão , Mutação , Phaseolus/crescimento & desenvolvimento , Phaseolus/metabolismo , Phaseolus/microbiologia , Rhizobium/genética , Rhizobium/ultraestrutura , Rhizobium leguminosarum/genética , Rhizobium leguminosarum/fisiologia , Rhizobium leguminosarum/ultraestrutura , Sinorhizobium meliloti/genética , Sinorhizobium meliloti/fisiologia , Sinorhizobium meliloti/ultraestrutura , Especificidade da EspécieRESUMO
We systematically studied the origin of surface patterns observed on single Sinorhizobium meliloti bacterial cells by comparing the complementary techniques atomic force microscopy (AFM) and scanning electron microscopy (SEM). Conditions ranged from living bacteria in liquid to fixed bacteria in high vacuum. Stepwise, we applied different sample modifications (fixation, drying, metal coating, etc.) and characterized the observed surface patterns. A detailed analysis revealed that the surface structure with wrinkled protrusions in SEM images were not generated de novo but most likely evolved from similar and naturally present structures on the surface of living bacteria. The influence of osmotic stress to the surface structure of living cells was evaluated and also the contribution of exopolysaccharide and lipopolysaccharide (LPS) by imaging two mutant strains of the bacterium under native conditions. AFM images of living bacteria in culture medium exhibited surface structures of the size of single proteins emphasizing the usefulness of AFM for high resolution cell imaging.
Assuntos
Artefatos , Membrana Celular/ultraestrutura , Microscopia de Força Atômica/métodos , Microscopia Eletrônica de Varredura/métodos , Sinorhizobium meliloti/ultraestrutura , Técnicas Histológicas/métodosRESUMO
Free-living Sinorhizobium meliloti lpxXL and acpXL mutants lack lipid A very-long-chain fatty acids (VLCFAs) and have reduced competitiveness in alfalfa. We demonstrate that LpxXL and AcpXL play important but distinct roles in bacteroid development and that LpxXL is essential for the modification of S. meliloti bacteroid lipid A with VLCFAs.
Assuntos
Proteínas de Bactérias/fisiologia , Ácidos Graxos/metabolismo , Medicago sativa/microbiologia , Sinorhizobium meliloti/crescimento & desenvolvimento , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Técnicas de Inativação de Genes , Lipídeo A/metabolismo , Sinorhizobium meliloti/genética , Sinorhizobium meliloti/metabolismo , Sinorhizobium meliloti/ultraestruturaRESUMO
Sinorhizobium meliloti enters into a symbiotic relationship with legume host plants, providing fixed nitrogen in exchange for carbon and amino acids. In S. meliloti, exoR and the exoS-chvI two-component system regulate the biosynthesis of succinoglycan, an exopolysaccharide important for host invasion. It was previously reported that a loss-of-function mutation in exoR and a gain-of-function mutation in exoS cause overproduction of succinoglycan and loss of motility, indicating that ExoR negatively regulates and ExoS-ChvI positively regulates downstream genes. However, a relationship between exoR and exoS-chvI has never been clearly established. By identification and detailed characterization of suppressor strains, we provide genetic evidence that exoR and exoS-chvI control many similar phenotypes. These include succinoglycan production, symbiosis, motility, and previously uncharacterized prototrophy and biofilm formation, all of which are co-ordinately restored by suppressors. We further demonstrate that ExoR is located in the periplasm, suggesting that it functions to regulate downstream genes in a novel manner. In pathogenic bacteria closely related to S. meliloti, exoS-chvI homologues are required for virulence and the regulation of cell envelope composition. Our data suggest that periplasmically localized ExoR and ExoS-ChvI function together in a unique and critical regulatory system associated with both free-living and symbiotic states of S. meliloti.
Assuntos
Proteínas de Bactérias/metabolismo , Periplasma/metabolismo , Sinorhizobium meliloti/metabolismo , Fatores de Transcrição/metabolismo , Acetileno/metabolismo , Proteínas de Bactérias/genética , Biofilmes/crescimento & desenvolvimento , Cinamatos/farmacologia , Farmacorresistência Bacteriana , Flagelos/metabolismo , Flagelos/ultraestrutura , Regulação Bacteriana da Expressão Gênica/efeitos dos fármacos , Higromicina B/análogos & derivados , Higromicina B/farmacologia , Microscopia Eletrônica de Transmissão , Modelos Biológicos , Fixação de Nitrogênio , Periplasma/efeitos dos fármacos , Periplasma/ultraestrutura , Fosforilação/efeitos dos fármacos , Polissacarídeos Bacterianos/biossíntese , Nódulos Radiculares de Plantas/crescimento & desenvolvimento , Nódulos Radiculares de Plantas/metabolismo , Nódulos Radiculares de Plantas/microbiologia , Sinorhizobium meliloti/genética , Sinorhizobium meliloti/ultraestrutura , Fatores de Transcrição/genéticaRESUMO
Sinorhizobium meliloti differentiates from rod-shaped, free-living cells into pleomorphic, non-dividing, N(2)-fixing bacteroids within alfalfa root nodules. Here, the role of the minCDE genes in bacteroid differentiation and in free-living cell division is examined. Disruption of the minE gene resulted in large, swollen and branched free-living cells, and in symbiosis a minE mutation resulted in a defect in nitrogen fixation with activity reduced by approximately 70 % compared to the wild-type. It has been demonstrated that the minCDE genes form an operon driven by a promoter located 173 bp upstream of minC. The minCDE genes were expressed in free-living cells and in both the infection zone and the symbiotic zone of alfalfa nodules; however, no changes in the free-living cell morphology, growth or symbiotic N(2) fixation were detected as a result of deletion of these genes. Induced production of individual or combinations of Min proteins in S. meliloti altered its rod-shaped cell morphology. Moreover, cell morphologies resulting from the overexpression of the S. meliloti Min proteins in Escherichia coli suggested similar functions for the E. coli and S. meliloti min genes. These data suggest that there is greater redundancy in the roles of cell division genes in S. meliloti compared with E. coli.
Assuntos
Proteínas de Bactérias/metabolismo , Proteínas de Ciclo Celular/metabolismo , Medicago sativa/microbiologia , Mutação , Sinorhizobium meliloti/ultraestrutura , Simbiose , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Sequência de Bases , Proteínas de Ciclo Celular/química , Proteínas de Ciclo Celular/genética , Regulação Bacteriana da Expressão Gênica , Dados de Sequência Molecular , Fixação de Nitrogênio , Raízes de Plantas/microbiologia , Sinorhizobium meliloti/citologia , Sinorhizobium meliloti/genética , Sinorhizobium meliloti/crescimento & desenvolvimentoRESUMO
The flagella of the soil bacterium Sinorhizobium meliloti differ from the enterobacterial paradigm in the complex filament structure and modulation of the flagellar rotary speed. The mode of motility control in S. meliloti has a molecular corollary in two novel periplasmic motility proteins, MotC and MotE, that are present in addition to the ubiquitous MotA/MotB energizing proton channel. A fifth motility gene is located in the mot operon downstream of the motB and motC genes. Its gene product was originally designated MotD, a cytoplasmic motility protein having an unknown function. We report here reassignment of MotD as FliK, the regulator of flagellar hook length. The FliK gene is one of the few flagellar genes not annotated in the contiguous flagellar regulon of S. meliloti. Characteristic for its class, the 475-residue FliK protein contains a conserved, compactly folded Flg hook domain in its carboxy-terminal region. Deletion of fliK leads to formation of prolonged flagellar hooks (polyhooks) with missing filament structures. Extragenic suppressor mutations all mapped in the cytoplasmic region of the transmembrane export protein FlhB and restored assembly of a flagellar filament, and thus motility, in the presence of polyhooks. The structural properties of FliK are consistent with its function as a substrate specificity switch of the flagellar export apparatus for switching from rod/hook-type substrates to filament-type substrates.
Assuntos
Proteínas de Bactérias/genética , Proteínas de Bactérias/fisiologia , Flagelos/fisiologia , Flagelos/ultraestrutura , Sinorhizobium meliloti/genética , Sinorhizobium meliloti/fisiologia , Sequência de Aminoácidos , Proteínas de Bactérias/química , Flagelos/genética , Deleção de Genes , Proteínas de Membrana/genética , Proteínas de Membrana/fisiologia , Proteínas Motores Moleculares , Dados de Sequência Molecular , Movimento , Estrutura Terciária de Proteína/genética , Homologia de Sequência de Aminoácidos , Sinorhizobium meliloti/ultraestrutura , Supressão GenéticaRESUMO
During the course of the development of nitrogen-fixing root nodules induced by Sinorhizobium meliloti on the model plant Medicago truncatula, tubules called infection threads are cooperatively constructed to deliver the bacterial symbiont from the root surface to cells in the interior of the root and developing nodule. Three-dimensional reconstructions of infection threads inside M. truncatula nodules showed that the threads formed relatively simple, tree-like networks. Some characteristics of thread networks, such as branch length, branch density, and branch surface-to-volume ratios, were remarkably constant across nodules in different stages of development. The overall direction of growth of the networks changed as nodules developed. In 5-d-old nodules, the overall growth of the network was directed inward toward the root. However, well-defined regions of these young networks displayed an outward growth bias, indicating that they were likely in the process of repolarizing their direction of development in response to the formation of the outward-growing nodule meristem. In 10- and 30-d-old nodules, the branches of the network grew outward toward the meristem and away from the roots on which the nodules developed.
Assuntos
Medicago truncatula/microbiologia , Sinorhizobium meliloti/ultraestrutura , Simulação por Computador , Medicago truncatula/crescimento & desenvolvimento , Modelos Moleculares , Raízes de Plantas/crescimento & desenvolvimento , Raízes de Plantas/microbiologia , Sinorhizobium meliloti/crescimento & desenvolvimento , Sinorhizobium meliloti/metabolismo , SimbioseRESUMO
In Medicago nodules, endoreduplication cycles and ploidy-dependent cell enlargement occur during the differentiation of bacteroid-containing nitrogen-fixing symbiotic cells. These events are accompanied by the expression of ccs52A, a plant ortholog of the yeast and animal cdh1/srw1/fzr genes, acting as a substrate-specific activator of the anaphase-promoting complex (APC) ubiquitin ligase. Because CCS52A is involved in the transition of mitotic cycles to endoreduplication cycles, we investigated the importance of somatic endoploidy and the role of the M. truncatula ccs52A gene in symbiotic cell differentiation. Transcription analysis and ccs52A promoter-driven beta-glucuronidase activity in transgenic plants showed that ccs52A was dispensable for the mitotic cycles and nodule primordium formation, whereas it was induced before nodule differentiation. The CCS52A protein was present in the nucleus of endoreduplication-competent cells, indicating that it may activate APC constitutively during the endoreduplication cycles. Downregulation of ccs52A in transgenic M. truncatula plants drastically affected nodule development, resulting in lower ploidy, reduced cell size, inefficient invasion, and the maturation of symbiotic cells, accompanied by early senescence and finally the death of both the bacterium and plant cells. Thus, ccs52A expression is essential for the formation of large highly polyploid symbiotic cells, and endoreduplication is an integral part of normal nodule development.
Assuntos
Diferenciação Celular/genética , Medicago/genética , Proteínas de Plantas/genética , Simbiose/genética , Complexos Ubiquitina-Proteína Ligase/genética , Ciclossomo-Complexo Promotor de Anáfase , Apoptose/fisiologia , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Diferenciação Celular/fisiologia , Divisão Celular/genética , Divisão Celular/fisiologia , Regulação da Expressão Gênica no Desenvolvimento , Regulação da Expressão Gênica de Plantas , Glucuronidase/genética , Glucuronidase/metabolismo , Medicago/microbiologia , Medicago/ultraestrutura , Microscopia Eletrônica , Fixação de Nitrogênio/genética , Fixação de Nitrogênio/fisiologia , Proteínas de Plantas/metabolismo , Plantas Geneticamente Modificadas , Poliploidia , Sinorhizobium meliloti/crescimento & desenvolvimento , Sinorhizobium meliloti/ultraestrutura , Simbiose/fisiologia , Complexos Ubiquitina-Proteína Ligase/metabolismo , Ubiquitina-Proteína Ligases/genética , Ubiquitina-Proteína Ligases/metabolismoRESUMO
Rhizobium meliloti (ATCC 55340) was grown at different specific growth rates in a chemostat apparatus. Metabolic products, relating to the Embden-Meyerhof-Parnas (EMP) pathway and the tricarboxylic acid (TCA) cycle, were measured and quantified to probe the influence of specific growth rate on the distribution of important metabolites. The detection of propionate in the fermentation broth implies that the imbalance of reducing equivalents of FADH(2) and NADH + H(+) resulted in a partially reductive operation of the TCA cycle. Additionally, experimental results show that the specific growth rate plays an essential role in modulating the biomass concentration, the specific substrate uptake rate, the cell length, the specific exopolysaccharide (EPS) production rate, the distribution of EPS molecular weight, and the profiles of carbohydrate and organic acid. The specific EPS production rate (varying from 13.3 to 111 mg EPS/g-DW/h) follows a growth-associated pattern at the specific growth rate ranging from 0.06 to 0.20 h(-1) and switches into non-growth-associated mode when the specific growth rate is over 0.20 h(-1).
Assuntos
Proteínas de Bactérias/metabolismo , Reatores Biológicos/microbiologia , Técnicas de Cultura de Células/métodos , Polissacarídeos Bacterianos/metabolismo , Sinorhizobium meliloti/crescimento & desenvolvimento , Sinorhizobium meliloti/metabolismo , Divisão Celular/fisiologia , Peso Molecular , Polissacarídeos Bacterianos/química , Sinorhizobium meliloti/química , Sinorhizobium meliloti/ultraestruturaRESUMO
Seventeen arginine auxotrophic mutants of Sinorhizobium meliloti Rmd201 were isolated by random transposon Tn5 mutagenesis using Tn5 delivery vector pGS9. Based on intermediate feeding studies, these mutants were designated as argA/argB/argC/argD/argE (ornithine auxotrophs), argF/argI, argG and argH mutants. The ornithine auxotrophs induced ineffective nodules whereas all other arginine auxotrophs induced fully effective nodules on alfalfa plants. In comparison to the parental strain induced nodule, only a few nodule cells infected with rhizobia were seen in the nitrogen fixation zone of the nodule induced by the ornithine auxotroph. TEM studies showed that the bacteroids in the nitrogen fixation zone of ornithine auxotroph induced nodule were mostly spherical or oval unlike the elongated bacteroids in the nitrogen fixation zone of the parental strain induced nodule. These results indicate that ornithine or an intermediate of ornithine biosynthesis, or a chemical factor derived from one of these compounds is required for the normal development of nitrogen fixation zone and transformation of rhizobial bacteria into bacteroids during symbiosis of S. meliloti with alfalfa plants.
Assuntos
Arginina/metabolismo , Elementos de DNA Transponíveis , Medicago sativa/microbiologia , Sinorhizobium meliloti/isolamento & purificação , Sinorhizobium meliloti/fisiologia , Simbiose/fisiologia , Mutagênese , Mutação , Plasmídeos , Sinorhizobium meliloti/ultraestruturaRESUMO
Twenty three pyrimidine auxotrophs of Sinorhizobium meliloti Rmd201 were generated by random mutagenesis with transposon Tn5. On the basis of biochemical characters these auxotrophic mutants were classified into car, pyrC and pyrE/pyrF categories. All auxotrophs induced white nodules which were ineffective in nitrogen fixation. Light and electron microscopic studies revealed that the nodules induced by pyrC mutants were more developed than the nodules of car mutants. Similarly the nodules induced by pyrE/pyrF mutants had more advanced structural features than the nodules of pyrC mutants. The nodule development in case of pyrE/pyrF mutants was not to the extent observed in the parental strain. These results indicated that some of the intermediates and/or enzymes of pyrimidine biosynthetic pathway of S. meliloti play a key role in bacteroidal transformation and nodule development.
Assuntos
Medicago sativa/microbiologia , Sinorhizobium meliloti/fisiologia , Medicago sativa/metabolismo , Medicago sativa/ultraestrutura , Microscopia Eletrônica , Mutagênese , Fixação de Nitrogênio , Raízes de Plantas/metabolismo , Raízes de Plantas/microbiologia , Raízes de Plantas/ultraestrutura , Pirimidinas/metabolismo , Sinorhizobium meliloti/genética , Sinorhizobium meliloti/ultraestrutura , SimbioseRESUMO
Symbiotic nitrogen fixation involves the development of specialized organs called nodules within which plant photosynthates are exchanged for combined nitrogen of bacterial origin. To determine the importance of bacterial nitrogen metabolism in symbiosis, we have characterized a key regulator of this metabolism in Rhizobium meliloti, the uridylylatable P(II) protein encoded by glnB. We have constructed both a glnB null mutant and a point mutant making nonuridylylatable P(II). In free-living conditions, P(II) is required for expression of the ntrC-dependent gene glnII and for adenylylation of glutamine synthetase I. P(II) is also required for efficient infection of alfalfa but not for expression of nitrogenase. However alfalfa plants inoculated with either glnB mutant are nitrogen-starved in the absence of added combined nitrogen. We hypothesize that P(II) controls expression or activity of a bacteroid ammonium transporter required for a functional nitrogen-fixing symbiosis. Therefore, the P(II) protein affects both Rhizobium nitrogen metabolism and alfalfa nodule development.
Assuntos
Proteínas de Bactérias/metabolismo , Medicago sativa/crescimento & desenvolvimento , Medicago sativa/microbiologia , Fixação de Nitrogênio , Sinorhizobium meliloti/metabolismo , Proteínas de Bactérias/genética , Primers do DNA/genética , Genes Bacterianos , Medicago sativa/metabolismo , Microscopia Eletrônica , Modelos Biológicos , Mutação , Nitrogênio/metabolismo , Nitrogenase/metabolismo , Proteínas PII Reguladoras de Nitrogênio , Sinorhizobium meliloti/genética , Sinorhizobium meliloti/ultraestrutura , SimbioseRESUMO
Heme-deficient mutants of Rhizobium and Bradyrhizobium have been found to exhibit diverse phenotypes with respect to symbiotic interactions with plant hosts. We observed that R. meliloti hemA mutants elicit nodules that do not contain intracellular bacteria; the nodules contain either no infection threads ("empty" nodule phenotype) or aberrant infection threads that failed to release bacteria (Bar- phenotype). These mutant nodules expressed nodulin genes associated with nodules arrested at an early stage of development, including ENOD2, Nms-30, and four previously undescribed nodulin genes. These nodules also failed to express any of six late nodulin genes tested by hybridization, including leghemoglobin, and twelve tested by in vitro translation product analysis which are not yet correlated with specific cloned genes. We observed that R. meliloti leucine and adenosine auxotrophs induced invaded Fix- nodules that expressed late nodulin genes, suggesting that it is not auxotrophy per se that causes the hemA mutants to elicit Bar- or empty nodules. Because R. meliloti hemA mutants elicit nodules that do not contain intracellular bacteria, it is not possible to decide whether or not the Fix- phenotype of these nodules is a direct consequence of the failure of R. meliloti to supply the heme moiety of hololeghemoglobin. Our results demonstrate the importance of establishing the stage in development at which a mutant nodule is arrested before conclusions are drawn about the role of small metabolite exchange in the symbiosis.
Assuntos
Heme/genética , Medicago sativa/microbiologia , Proteínas de Membrana , Fixação de Nitrogênio , Proteínas de Plantas/genética , Sinorhizobium meliloti/genética , Northern Blotting , DNA Bacteriano/genética , Expressão Gênica , Glucanos/biossíntese , Microscopia Eletrônica , Polissacarídeos Bacterianos/biossíntese , Biossíntese de Proteínas , Sinorhizobium meliloti/crescimento & desenvolvimento , Sinorhizobium meliloti/metabolismo , Sinorhizobium meliloti/ultraestruturaRESUMO
Among the genes of Rhizobium meliloti SU47 that affect nitrogen-fixing symbiosis with alfalfa are nod genes, in which mutations block nodule induction, and exo genes, in which mutations allow nodule formation but block rhizobial exopolysaccharide production as well as nodule invasion and nitrogen fixation. To investigate whether an exo+ bacterium can "help" (that is, reverse the symbiotic defect of) an exo mutant in trans, we have coinoculated alfalfa with pairs of rhizobia of different genotypes. Coinoculant genotypes were chosen so that the exo+ helper strain was nif while the exo "indicator" strain was nif+, and thus any fixation observed was carried out by the exo coinoculant. We find that a nod exo+ coinoculant can help an exo mutant both to invade nodules and to fix nitrogen. However, a nod+ exo+ coinoculant cannot help an exo mutant: Few exo bacteria are recovered from nodules, some bacteroids differentiate into bizarre aberrant forms, and the nodules fail to fix nitrogen. In a triple coinoculation, the effect of nod+ helper supersedes that of nod helper. Implications of these results for interaction of nod and exo gene products are discussed.