RESUMEN

Indole-3-acetic acid (IAA) is one of the most important molecules produced by Azospirillum sp., given that it affects plant growth and development. Azospirillum brasilense strains Sp245 and Az39 (pFAJ64) were pre-incubated in MMAB medium plus 100 mg/mL L-tryptophan and treated with or exposed to the following (a) abiotic and (b) biotic stress effectors: (a) 100 mM NaCl or Na2SO4, 4.0% (w/v) PEG6000, 0.5 mM H2O2, 0.1 mM abscisic acid, 0.1 mM 1-aminocyclopropane 1-carboxylic acid, 45 °C or daylight, and (b) 4.0% (v/v) filtered supernatant of Pseudomonas savastanoi (Ps) or Fusarium oxysporum (Fo), 0.1 mM salicylic acid (SA), 0.1 mM methyl jasmonic acid (MeJA), and 0.01% (w/v) chitosan (CH). After 30 and 120 min of incubation, biomass production, cell viability, IAA concentration (µg/mL), and ipdC gene expression were measured. Our results show that IAA production increases with daylight or in the presence of PEG6000, ABA, SA, CH, and Fo. On the contrary, exposure to 45 °C or treatment with H2O2, NaCl, Na2SO4, ACC, MeJA, and Ps decrease IAA biosynthesis. In this report, growth and IAA biosynthesis in A. brasilense under biotic and abiotic stress conditions are discussed from the point of view of their role in bacterial lifestyle and their potential application as bioproducts.

Asunto(s)

Azospirillum brasilense/genética , Regulación Bacteriana de la Expresión Génica , Ácidos Indolacéticos/metabolismo , Reguladores del Crecimiento de las Plantas/biosíntesis , Azospirillum brasilense/metabolismo , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Medios de Cultivo/química , Medios de Cultivo/metabolismo , Triptófano/metabolismo

RESUMEN

We present the complete genome sequence of Azospirillum brasilense Az39, isolated from wheat roots in the central region of Argentina and used as inoculant in extensive and intensive agriculture during the last four decades. The genome consists of 7.39 Mb, distributed in six replicons: one chromosome, three chromids, and two plasmids.

RESUMEN

The auxin-producing bacterium Azospirillum brasilense Sp245 can promote the growth of several plant species. The model plant Arabidopsis thaliana was chosen as host plant to gain an insight into the molecular mechanisms that govern this interaction. The determination of differential gene expression in Arabidopsis roots after inoculation with either A. brasilense wild-type or an auxin biosynthesis mutant was achieved by microarray analysis. Arabidopsis thaliana inoculation with A. brasilense wild-type increases the number of lateral roots and root hairs, and elevates the internal auxin concentration in the plant. The A. thaliana root transcriptome undergoes extensive changes on A. brasilense inoculation, and the effects are more pronounced at later time points. The wild-type bacterial strain induces changes in hormone- and defense-related genes, as well as in plant cell wall-related genes. The A. brasilense mutant, however, does not elicit these transcriptional changes to the same extent. There are qualitative and quantitative differences between A. thaliana responses to the wild-type A. brasilense strain and the auxin biosynthesis mutant strain, based on both phenotypic and transcriptomic data. This illustrates the major role played by auxin in the Azospirillum-Arabidopsis interaction, and possibly also in other bacterium-plant interactions.

Asunto(s)

Arabidopsis/genética , Arabidopsis/microbiología , Azospirillum brasilense/metabolismo , Ácidos Indolacéticos/metabolismo , Raíces de Plantas/anatomía & histología , Raíces de Plantas/microbiología , Análisis por Conglomerados , Perfilación de la Expresión Génica , Regulación de la Expresión Génica de las Plantas , Ontología de Genes , Mutación/genética , Análisis de Secuencia por Matrices de Oligonucleótidos , Fenotipo , Raíces de Plantas/genética , Raíces de Plantas/crecimiento & desarrollo , Transducción de Señal/genética

RESUMEN

The rhizosphere bacterium Azospirillum brasilense produces the auxin indole-3-acetic acid (IAA) through the indole-3-pyruvate pathway. As we previously demonstrated that transcription of the indole-3-pyruvate decarboxylase (ipdC) gene is positively regulated by IAA, produced by A. brasilense itself or added exogenously, we performed a microarray analysis to study the overall effects of IAA on the transcriptome of A. brasilense. The transcriptomes of A. brasilense wild-type and the ipdC knockout mutant, both cultured in the absence and presence of exogenously added IAA, were compared.Interfering with the IAA biosynthesis/homeostasis in A. brasilense through inactivation of the ipdC gene or IAA addition results in much broader transcriptional changes than anticipated. Based on the multitude of changes observed by comparing the different transcriptomes, we can conclude that IAA is a signaling molecule in A. brasilense. It appears that the bacterium, when exposed to IAA, adapts itself to the plant rhizosphere, by changing its arsenal of transport proteins and cell surface proteins. A striking example of adaptation to IAA exposure, as happens in the rhizosphere, is the upregulation of a type VI secretion system (T6SS) in the presence of IAA. The T6SS is described as specifically involved in bacterium-eukaryotic host interactions. Additionally, many transcription factors show an altered regulation as well, indicating that the regulatory machinery of the bacterium is changing.

Asunto(s)

Azospirillum brasilense/genética , Azospirillum brasilense/metabolismo , Perfilación de la Expresión Génica , Ácidos Indolacéticos/metabolismo , Rizosfera , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Regulación Bacteriana de la Expresión Génica , Datos de Secuencia Molecular

RESUMEN

The phytostimulatory properties of Azospirillum inoculants, which entail production of the phytohormone indole-3-acetic acid (IAA), can be enhanced by genetic means. However, it is not known whether this could affect their interactions with indigenous soil microbes. Here, wheat seeds were inoculated with the wild-type strain Azospirillum brasilense Sp245 or one of three genetically modified (GM) derivatives and grown for one month. The GM derivatives contained a plasmid vector harboring the indole-3-pyruvate/phenylpyruvate decarboxylase gene ipdC (IAA production) controlled either by the constitutive promoter PnptII or the root exudate-responsive promoter PsbpA, or by an empty vector (GM control). All inoculants displayed equal rhizosphere population densities. Only inoculation with either ipdC construct increased shoot biomass compared with the non-inoculated control. At one month after inoculation, automated ribosomal intergenic spacer analysis (ARISA) revealed that the effect of the PsbpA construct on bacterial community structure differed from that of the GM control, which was confirmed by 16S rDNA-based denaturing gradient gel electrophoresis (DGGE). The fungal community was sensitive to inoculation with the PsbpA construct and especially the GM control, based on ARISA data. Overall, fungal and bacterial communities displayed distinct responses to inoculation of GM A. brasilense phytostimulators, whose effects could differ from those of the wild-type.

Asunto(s)

Azospirillum brasilense/metabolismo , Biodiversidad , Ácidos Indolacéticos/metabolismo , Raíces de Plantas/microbiología , Microbiología del Suelo , Triticum/microbiología , Azospirillum brasilense/genética , Azospirillum brasilense/crecimiento & desarrollo , Proteínas Bacterianas/genética , Biomasa , Vías Biosintéticas/genética , Carboxiliasas/genética , Dermatoglifia del ADN/métodos , ADN Espaciador Ribosómico/genética , Electroforesis en Gel de Poliacrilamida , Dosificación de Gen , Ingeniería Genética , Metagenoma , Desnaturalización de Ácido Nucleico , Organismos Modificados Genéticamente/genética , Organismos Modificados Genéticamente/crecimiento & desarrollo , Organismos Modificados Genéticamente/metabolismo , Brotes de la Planta/crecimiento & desarrollo , Plásmidos

RESUMEN

Strains Sp7 and Cd of Azospirillum brasilense, a plant growth-promoting rhizobacterium, differ in synthesis of carotenoids. While colonies of strain Sp7 have a white-cream colour on plates, colonies of strain Cd are orange-pink coloured because of the synthesis of carotenoids. Screening of a mini-Tn5 mutant library of A. brasilense Sp7 revealed two orange-pink-coloured mutants that produced carotenoids. Cloning and sequencing of the Tn5 flanking region in both the carotenoid-producing mutants of Sp7 revealed insertion of Tn5 in an ORF encoding anti-sigma factor, a ChrR-like protein. The upstream region of the Tn5-mutated ORF contained another ORF that encoded an extra-cytoplasmic function (ECF)-class sigma factor (sigma(E), RpoE). When the nucleotide sequences of the corresponding ORFs from the carotenoid-producing strain Cd were analysed, the sequence of the Cd sigma(E) was identical to that of the carotenoid non-producing strain Sp7, but the Cd anti-sigma(E) ORF had a deletion that caused frame shifting and creation of a stop codon. This resulted in the premature termination of the protein, which was about 7 kDa smaller than the Sp7 anti-sigma(E). Cloning of Sp7 anti-sigma(E) in a broad-host-range expression vector and expression in A. brasilense Cd and in the anti-sigma(E) knockout mutant of A. brasilense Sp7 resulted in the inhibition of carotenoid synthesis. Similarly, cloning and overexpression of A. brasilense Sp7 sigma(E) in A. brasilense Sp7 resulted in the production of carotenoids. These observations clearly indicate that carotenoid synthesis in A. brasilense is controlled by sigma(E) with its cognate anti-sigma(E).

Asunto(s)

Azospirillum brasilense/metabolismo , Proteínas Bacterianas/antagonistas & inhibidores , Proteínas Bacterianas/metabolismo , Carotenoides/metabolismo , Citoplasma/metabolismo , Regulación Bacteriana de la Expresión Génica , Factor sigma/antagonistas & inhibidores , Factor sigma/metabolismo , Secuencia de Aminoácidos , Azospirillum brasilense/química , Azospirillum brasilense/genética , Proteínas Bacterianas/química , Secuencia de Bases , Clonación Molecular , Citoplasma/química , Citoplasma/genética , Datos de Secuencia Molecular , Mutagénesis Insercional , Mutación , Regiones Promotoras Genéticas , Alineación de Secuencia , Factor sigma/química

RESUMEN

Three transposon mutants of Rhizobium tropici CIAT899 affected in lipopolysaccharide (LPS) biosynthesis were characterized and their maize rhizosphere and endophytic root colonization abilities were evaluated. The disrupted genes coded for the following putative products: the ATPase component of an O antigen ABC-2 type transporter (wzt), a nucleotide-sugar dehydratase (lpsbeta2) and a bifunctional enzyme producing GDP-mannose (noeJ). Electrophoretic analysis of affinity purified LPS showed that all mutants lacked the smooth LPS bands indicating an O antigen minus phenotype. In the noeJ mutant, the rough LPS band migrated faster than the parental band, suggesting a truncated LPS core. When inoculated individually, the wzt and noeJ mutants colonize the rhizosphere and root to a lower extent than the parental strain while no differences were observed between the lpsbeta2 mutant and the parental strain. All mutants were impaired in competitive rhizosphere and root colonization. Pleiotropic effects of the mutations on known colonization traits such as motility and growth rate were observed, but they were not sufficient to explain the colonization behaviours. It was found that the LPS mutants were sensitive to the maize antimicrobial 6-methoxy-2-benzoxazolinone (MBOA). Only the combined effects of altered growth rate and susceptibility to maize antimicrobials could account for all the observed colonization phenotypes. The results suggest an involvement of the LPS in protecting R. tropici against maize defence response during rhizosphere and root colonization.

Asunto(s)

Proteínas Bacterianas/genética , Lipopolisacáridos/biosíntesis , Mutación , Raíces de Plantas/microbiología , Rhizobium tropici/crecimiento & desarrollo , Rhizobium tropici/genética , Microbiología del Suelo , Zea mays/microbiología , Antibacterianos/farmacología , Lipopolisacáridos/química , Lipopolisacáridos/aislamiento & purificación , Pruebas de Sensibilidad Microbiana , Datos de Secuencia Molecular , Phaseolus/microbiología , Rhizobium tropici/efectos de los fármacos , Rhizobium tropici/metabolismo , Análisis de Secuencia de ADN

RESUMEN

Azospirillum brasilense belongs to the plant growth-promoting rhizobacteria with direct growth promotion through the production of the phytohormone indole-3-acetic acid (IAA). A key gene in the production of IAA, annotated as indole-3-pyruvate decarboxylase (ipdC), has been isolated from A. brasilense, and its regulation was reported previously (A. Vande Broek, P. Gysegom, O. Ona, N. Hendrickx, E. Prinsen, J. Van Impe, and J. Vanderleyden, Mol. Plant-Microbe Interact. 18:311-323, 2005). An ipdC-knockout mutant was found to produce only 10% (wt/vol) of the wild-type IAA production level. In this study, the encoded enzyme is characterized via a biochemical and phylogenetic analysis. Therefore, the recombinant enzyme was expressed and purified via heterologous overexpression in Escherichia coli and subsequent affinity chromatography. The molecular mass of the holoenzyme was determined by size-exclusion chromatography, suggesting a tetrameric structure, which is typical for 2-keto acid decarboxylases. The enzyme shows the highest kcat value for phenylpyruvate. Comparing values for the specificity constant kcat/Km, indole-3-pyruvate is converted 10-fold less efficiently, while no activity could be detected with benzoylformate. The enzyme shows pronounced substrate activation with indole-3-pyruvate and some other aromatic substrates, while for phenylpyruvate it appears to obey classical Michaelis-Menten kinetics. Based on these data, we propose a reclassification of the ipdC gene product of A. brasilense as a phenylpyruvate decarboxylase (EC 4.1.1.43).

Asunto(s)

Azospirillum brasilense/enzimología , Carboxiliasas/metabolismo , Ácidos Indolacéticos/metabolismo , Secuencia de Aminoácidos , Azospirillum brasilense/genética , Proteínas Bacterianas/metabolismo , Carboxiliasas/clasificación , Carboxiliasas/genética , Regulación Bacteriana de la Expresión Génica , Regulación Enzimológica de la Expresión Génica , Cinética , Datos de Secuencia Molecular , Filogenia , Reacción en Cadena de la Polimerasa , Proteínas Recombinantes/metabolismo , Especificidad por Sustrato

RESUMEN

In this paper the problem of reliable and accurate parameter estimation for unstructured models is considered. It is illustrated how a theoretically optimal design can be successfully translated into a practically feasible, robust, and informative experiment. The well-known parameter estimation problem of Monod kinetic parameters is used as a vehicle to illustrate our approach. As known for a long time, noisy batch measurements do not allow for unique and accurate estimation of the kinetic parameters of the Monod model. Techniques of optimal experiment design are, therefore, exploited to design informative experiments and to improve the parameter estimation accuracy. During the design process, practical feasibility has to be kept in mind. The designed experiments are easy to implement in practice and do not require additional monitoring equipment. Both design and experimental validation of informative fed batch experiments are illustrated with a case study, namely, the growth of the nitrogen-fixing bacteria Azospirillum brasilense.

Asunto(s)

Azospirillum brasilense/crecimiento & desarrollo , Azospirillum brasilense/metabolismo , Reactores Biológicos/microbiología , Malatos/metabolismo , Modelos Biológicos , Proliferación Celular , Simulación por Computador , Cinética

RESUMEN

Phenylpyruvate decarboxylase (PPDC) of Azospirillum brasilense, involved in the biosynthesis of the plant hormone indole-3-acetic acid and the antimicrobial compound phenylacetic acid, is a thiamine diphosphate-dependent enzyme that catalyses the nonoxidative decarboxylation of indole- and phenylpyruvate. Analogous to yeast pyruvate decarboxylases, PPDC is subject to allosteric substrate activation, showing sigmoidal v versus [S] plots. The present paper reports the crystal structure of this enzyme determined at 1.5 A resolution. The subunit architecture of PPDC is characteristic for other members of the pyruvate oxidase family, with each subunit consisting of three domains with an open alpha/beta topology. An active site loop, bearing the catalytic residues His112 and His113, could not be modelled due to flexibility. The biological tetramer is best described as an asymmetric dimer of dimers. A cysteine residue that has been suggested as the site for regulatory substrate binding in yeast pyruvate decarboxylase is not conserved, requiring a different mechanism for allosteric substrate activation in PPDC. Only minor changes occur in the interactions with the cofactors, thiamine diphosphate and Mg2+, compared to pyruvate decarboxylase. A greater diversity is observed in the substrate binding pocket accounting for the difference in substrate specificity. Moreover, a catalytically important glutamate residue conserved in nearly all decarboxylases is replaced by a leucine in PPDC. The consequences of these differences in terms of the catalytic and regulatory mechanism of PPDC are discussed.

Asunto(s)

Azospirillum brasilense/enzimología , Carboxiliasas/química , Carboxiliasas/metabolismo , Modelos Moleculares , Sitio Alostérico/genética , Sustitución de Aminoácidos/genética , Azospirillum brasilense/genética , Carboxiliasas/genética , Catálisis , Clonación Molecular , Cristalización , Cristalografía por Rayos X , Dimerización , Activación Enzimática/genética , Ácido Glutámico/genética , Leucina/genética , Modelos Químicos , Especificidad por Sustrato/genética

RESUMEN

Plant growth promoting rhizobacteria such as Azospirillum brasilense are agronomically important as they are frequently used for crop inoculation. But adverse factors such as increasing soil salinity limit their survival, multiplication and phytostimulatory effect. In order to understand the role of the genes involved in the adaptation of A. brasilense Sp7 to salt stress, a mutant library (6,800 mutants) was constructed after random integration of a mini-Transposon Tn5 derivative containing a promoterless gusA and oriV. The library was screened for salt stress inducible Gus activity on minimal malate agar medium containing NaCl and 5-bromo-4-chloro-3-indolyl-beta-D: -glucuronide. Salt stress responsiveness of the promoters was estimated by quantifying GusA activity in the presence and absence of NaCl stress using p-nitrophenyl-beta-D: -glucuronide as a substrate. In 11 mutants showing high levels of gusA expression in the presence of salt-stress, the partial nucleotide sequence of the DNA region flanking the site of Tn5 insertion was determined and analysed using the NCBI-BLAST programs. Similarity searches revealed that 10 out of the 11 genes sequenced showed notable similarity with genes involved in functions related to modulation in the composition of exopolysaccharides, capsular polysaccharides, lipopolysaccharides, peptidoglycan and lipid bilayer of the cell envelope. Induction of cell envelope related genes in response to salt stress and salt sensitive phenotype of several mutants in A. brasilense indicate a prominent role of cell envelope in salt-stress adaptation.

Asunto(s)

Azospirillum/metabolismo , Elementos Transponibles de ADN/genética , Regulación de la Expresión Génica de las Plantas , Mutagénesis , Sales (Química)/química , Biblioteca de Genes , Glucuronidasa/química , Membrana Dobles de Lípidos/química , Lipopolisacáridos/química , Mutación , Plásmidos/metabolismo , Polisacáridos/química , Regiones Promotoras Genéticas , Cloruro de Sodio/química , Factores de Tiempo

RESUMEN

Here we report on the presence of sulfated lipopolysaccharide molecules in Azospirillum brasilense, a plant growth-promoting rhizosphere bacterium. Chemical analysis provided structural data on the O-antigen composition and demonstrated the possible involvement of the nodPQ genes in O-antigen sulfation.

Asunto(s)

Azospirillum brasilense/metabolismo , Proteínas Bacterianas/metabolismo , Lipopolisacáridos/biosíntesis , Lipopolisacáridos/química , Complejos Multienzimáticos/metabolismo , Antígenos O/metabolismo , Sulfato Adenililtransferasa/metabolismo , Azospirillum brasilense/genética , Proteínas Bacterianas/genética , Fraccionamiento Químico , Electroforesis en Gel de Poliacrilamida , Lipopolisacáridos/aislamiento & purificación , Complejos Multienzimáticos/genética , Sulfato Adenililtransferasa/genética

RESUMEN

Batch and fed batch cultures of Azospirillum brasilense Sp245 were conducted in a bioreactor. Growth response, IAA biosynthesis and the expression of the ipdC gene were monitored in relation to the environmental conditions (temperature, availability of a carbon source and aeration). A. brasilense can grow and produce IAA in batch cultures between 20 and 38 degrees C in a standard minimal medium (MMAB) containing 2.5 gl(-1)l-malate and 50 microgml(-1) tryptophan. IAA synthesis requires depletion of the carbon source from the growth medium in batch culture, causing growth arrest. No significant amount of IAA can be detected in a fed batch culture. Varying the concentration of tryptophan in batch experiments has an effect on both growth and IAA synthesis. Finally we confirmed that aerobic growth inhibits IAA synthesis. The obtained profile for IAA synthesis coincides with the expression of the indole-3-pyruvate decarboxylase gene (ipdC), encoding a key enzyme in the IAA biosynthesis of A. brasilense.

Asunto(s)

Azospirillum brasilense/crecimiento & desarrollo , Azospirillum brasilense/metabolismo , Regulación Bacteriana de la Expresión Génica , Ácidos Indolacéticos/metabolismo , Aerobiosis , Azospirillum brasilense/genética , Reactores Biológicos , Carboxiliasas/análisis , Medios de Cultivo/química , Compuestos Orgánicos/metabolismo , Temperatura , Triptófano/farmacología

RESUMEN

The plant growth-promoting soil bacterium Azospirillum brasilense enhances growth of economically important crops, such as wheat, corn and rice. In order to improve plant growth, a close bacterial association with the plant roots is needed. Genes encoded on a 90-MDa plasmid, denoted pRhico plasmid, present in A. brasilense Sp7, play an important role in plant root interaction. Sequencing, annotation and in silico analysis of this 90-MDa plasmid revealed the presence of a large collection of genes encoding enzymes involved in surface polysaccharide biosynthesis. Analysis of the 90-MDa plasmid genome provided evidence for its essential role in the viability of the bacterial cell.

Asunto(s)

Azospirillum brasilense/genética , Proteínas de la Membrana Bacteriana Externa/genética , Genoma Bacteriano , Raíces de Plantas/microbiología , Plásmidos/genética , Azospirillum brasilense/metabolismo , Proteínas Bacterianas/genética , Flagelos/fisiología , Complejos Multienzimáticos/genética , Polisacáridos Bacterianos/biosíntesis , Microbiología del Suelo , Sulfato Adenililtransferasa/genética

RESUMEN

Azospirillum brasilense shows chemotaxis to a variety of nutrients and oxygen. Genes encoding the central signal transduction pathway in chemotaxis were identified by phenotypic complementation of generally non-chemotactic mutants. Sequencing of a DNA fragment, which complemented two different mutants, revealed a region of five open reading frames translated in one direction and encoding homologs of known genes comprising excitation and adaptation pathways for chemotaxis in other bacterial species. The major chemotaxis gene cluster appears to be essential for all known behavioral responses that direct swimming motility in A. brasilense. Phylogenetic and genomic analysis revealed three groups of chemotaxis operons in alpha-proteobacterial species and assigned the A. brasilense operon to one of them. Interestingly, operons that are shown to be major regulators of behavior in several alpha-proteobacterial species are not orthologous.

Asunto(s)

Alphaproteobacteria/genética , Azospirillum brasilense/genética , Proteínas Bacterianas/genética , Quimiotaxis/genética , Genes Bacterianos , Operón , Alphaproteobacteria/fisiología , Azospirillum brasilense/fisiología , Proteínas Bacterianas/metabolismo , Secuencia de Bases , Quimiotaxis/fisiología , Regulación Bacteriana de la Expresión Génica , Prueba de Complementación Genética , Datos de Secuencia Molecular , Familia de Multigenes , Mutación , Filogenia , Análisis de Secuencia de ADN , Transducción de Señal

RESUMEN

To visualize rice root colonization by two Azospirillum species, A. irakense KBC1 was equipped with a plasmid expressing the enhanced green fluorescent protein (EGFP) and A. brasilense Sp7 was equipped with a plasmid expressing the enhanced yellow fluorescent protein (EYFP). In both cases, intensive fluorescence was observed under the epifluorescent microscope. Striking differences for association with roots of rice seedlings were observed between the two species: (i) A. irakense cells attached faster than A. brasilense to rice roots following inoculation; (ii) A. irakense attached to rice roots as vibroid cells, while A. brasilense occurred as rounded cyst-like cells; (iii) A. irakense cells were mainly found on root hairs, whereas A. brasilense cells were mainly concentrated on root surfaces. The two Azospirillum species obviously do not compete with each other for colonization of rice roots. These results demonstrate that the two Azospirillum species differ in their mode of rice root colonization. Asthe two Azospirillum species are extensively studied for unravelling mechanisms of plant root colonization and plant growth promotion, labelling with fluorescent protein is a useful additional tool for these studies.

RESUMEN

Bacteria of the genus Azospirillum are able to colonize plant roots. Using the beta-glucuronidase (GUS) reporter system, various Azospirillum mutants, including mutants affected in chemotactic motility or extracellular polysaccharide biosynthesis, were investigated for their capacity to initiate wheat root colonization at the root hair zones. Only non-flagellated mutants and a generally non-chemotactic mutant exhibited a strongly reduced colonization ability as compared to the wild-type. No role of the Azospirillum calcofluor-binding polysaccharide in primary wheat root colonization could be observed. This is the first report demonstrating directly, by using different motility mutants, the requirement of bacterial motility in the establishment of the Azospirillum-plant root association.

Asunto(s)

Azospirillum brasilense/fisiología , Azospirillum brasilense/patogenicidad , Triticum/microbiología , Azospirillum brasilense/genética , Adhesión Bacteriana/genética , Adhesión Bacteriana/fisiología , Movimiento Celular/genética , Movimiento Celular/fisiología , Quimiotaxis/genética , Quimiotaxis/fisiología , Genes Bacterianos , Genes Reporteros , Glucuronidasa/genética , Mutación , Raíces de Plantas/microbiología , Plásmidos/genética , Polisacáridos Bacterianos/biosíntesis , Polisacáridos Bacterianos/genética

RESUMEN

Adhesion of Azospirillum brasilense to glass and polystyrene was investigated by bringing the cells into contact with the support by sedimentation. Adhesion depended on time and temperature: lower adhesion densities were observed when the contact time was only 2 h or 6 h, as compared to 24 h, or when the test was performed at 4 -C, as compared to 30 °. The influence of cell physiology was further demonstrated by the effect of tetracycline, which inhibited adhesion. Scanning electron microscopy showed that cells produced extracellular material when left in contact with a support for 24 h. The surface elemental composition of cells and of polystyrene supports after cell adhesion and subsequent detachment was determined by X-ray photoelectron spectroscopy; this provided information on the relative concentrations of proteins and polysaccharides at the surface. The protein concentration at the surface of a cell sediment increased as a function of time at 30 °, correlating with an increase of adhesion density. A similar correlation between protein concentration and adhesion density was found when comparing exponentialphase cells with stationary-phase cells. The surface composition of polystyrene supports examined after cell detachment was found to be rich in proteins, indicating that proteins are the major constituent at the support surface. Lowering the contact time, or performing adhesion under unfavourable metabolic conditions (4 °) or in the presence of tetracycline, resulted in a decrease in protein concentration at the support surface, which was correlated with a decrease in adhesion density. The correlation between protein concentration at the cell surface or at the support surface and adhesion density, under different experimental conditions, provides a direct demonstration of the involvement of extracellular proteins in the adhesion of A. brasilense to inert surfaces.