Anne Van Dommelen

Characterization of a sugar-binding protein from Azospirillum brasilense mediating chemotaxis to and uptake of sugars

Our approach to the isolation of plant‐inducible bacterial genes of Azospirillum brasilense, based on the analysis of protein patterns of bacteria grown in the presence and in the absence of plant root exudates, led to the identification of an acidic 40 kDa protein. Cloning and sequencing analysis of the corresponding coding DNA region revealed the presence of two open reading frames transcribed in the same orientation. The deduced ORF1 protein, which corresponds to the 40 kDa protein, is very similar to the periplasmic ChvE protein, identified in Agrobacterium tumefaciens and involved in enhanced virulence. The deduced ORF2 protein shows homology to members of the LysR family of transcriptional regulators. The function of the ChvE‐like protein in A. brasilense was investigated further. The protein, designated as SbpA (sugar binding protein A), is involved in the uptake of D‐galactose and functions in the chemotaxis of A. brasilense towards several sugars, including D‐galactose, L‐arabinose and D‐fucose. Expression of the sbpA gene requires the presence of the same sugars in the growth medium and is enhanced further in combination with carbon starvation of A. brasilense cells.

Associative Nitrogen Fixation

Publisher Summary This chapter provides a short overview of the best-studied plant-associated N2-fixing bacteria. Because the tissue is not yet differentiated at the root tip, they may reach the site of the developing stele before the formation of the endodermis, which will later constitute a thick-walled boundary. This explains how these bacteria are able to invade xylem vessels. The chapter describes in detail colonization by Azospirillum. The N2 fixation phenotype is dependent on the plant species and can even differ according to the cultivar. Another example of some kind of specificity is the limited host range found for Azoarcus species. Presently, eight Azospirillum species have been described: A. brasilense, A. lipoferum, A. halopraeferens, A. irakense, A. largimobile, A. doebereinerae, A. oryzae, and A. amazonense. Azospirillum has been shown to positively influence plant growth, crop yields, and the N content of the plant. Herbaspirillum spp. are the aggressive colonizers of the root interior, establishing themselves not only in the cortex and vascular tissues of roots but also systemically in the whole plant. In endophytic associations where the substantial amounts of fixed N are transferred to the plant (e.g. sugarcane), it would be interesting to find out which factors influence the release of fixed N2.

Phenotypic Changes Resulting from Distinct Point Mutations in the Azospirillum brasilense glnA Gene, Encoding Glutamine Synthetase

ABSTRACT Sequencing the glnA genes of two chemically induced Azospirillum brasilense glutamine synthetase mutants revealed an Arg→Cys mutation, corresponding to the glutamate binding site, in one mutant and an Asp→Asn mutation, corresponding to the ammonium binding site, in the second mutant. The phenotypic changes in these mutants are discussed in relation to their genotypes.

Involvement of glnB, glnZ, and glnD Genes in the Regulation of Poly-3-Hydroxybutyrate Biosynthesis by Ammonia in Azospirillum brasilense Sp7

ABSTRACT The role of three key nitrogen regulatory genes, glnB (encoding the PII protein), glnZ (encoding the Pz protein), and glnD (encoding the GlnD protein), in regulation of poly-3-hydroxybutyrate (PHB) biosynthesis by ammonia in Azospirillum brasilense Sp7 was investigated. It was observed that glnB glnZ and glnD mutants produce substantially higher amounts of PHB than the wild type produces during the active growth phase. glnB and glnZ mutants have PHB production phenotypes similar to that of the wild type. Our results indicate that the PII-Pz system is apparently involved in nitrogen-dependent regulation of PHB biosynthesis in A. brasilense Sp7.

Wheat growth promotion through inoculation with an ammonium-excreting mutant of Azospirillum brasilense

Azospirillum brasilense is a diazotrophic bacterium and one of the best studied plant-growth-promoting bacterium living in close association with several agronomically important crops. The production of plant-growth-regulating substances is a main mechanism of plant growth stimulation, although other mechanisms have also been proposed. Nitrogen transfer from the bacterium to the plant is one among the other possible mechanisms of plant growth stimulation. In this study, we investigated, by means of a greenhouse trial with winter wheat inoculation, the effect of a point mutation in the ammonium binding site of the A. brasilense glutamine synthetase. The glutamine synthetase is one of the main ammonium-assimilating enzymes and mutations in this enzyme generally result in the release of ammonium from the bacterium to its environment. The ammonium-excreting mutant used in this study was shown to perform better than the wild-type A. brasilense strain with respect to wheat growth parameters and yield. In the greenhouse conditions used, this effect was independent of the way fertilizer was applied.

Ammonium transport: unifying concepts and unique aspects

Ammonium uptake by cells has been studied for more than a century, but only recently a family of ammonium transporters (Mep/Amt) with 10-12 transmembrane domains has been defined. These proteins are probably ubiquitous, since homologues have been found in the major kingdoms of living organisms. Plants as well as yeast and some archaebacteria have multiple Mep/Amt paralogues, which can be distinguished by their affinity for ammonium and the ammonium analogue methylammonium. Most ammonium transporters are induced in nitrogen-starving conditions, both in prokaryotes and plants. In Saccharomyces cerevisiae, Escherichia coli and Azospirillum brasilense Mep/Amt proteins where shown to be necessary for growth when the external concentration of the diffusive ammonium form (NH 3 ) becomes limiting. Ammonium transporters also play an important role in pseudohyphal differentiation in yeast and efficient symbiotic interaction between Rhizobium etli and its host plant. In most bacteria, NH 4 + transport appears to be a uniport mechanism driven by the membrane potential, but, depending on the organism, a different mode of ammonium uptake may be operating. Current knowledge offers the basis to investigate further the physiological role of ammonium transporters in the natural habitat of organisms and their importance in plant--bacteria interactions.

Identification of the glutamine synthetase adenylyltransferase of Azospirillum brasilense

Glutamine synthetase, a key enzyme in nitrogen metabolism of both prokaryotes and eukaryotes, is strictly regulated. One means of regulation is the modulation of activity through adenylylation catalyzed by adenylyltransferases. Using PCR primers based on conserved sequences in glutamine synthetase adenylyltransferases, we amplified part of the glnE gene of Azospirillum brasilense Sp7. The complete glnE sequence of A. brasilense Sp245 was retrieved from the draft genome sequence of this organism (http://genomics.ornl.gov/research/azo/). Adenylyltransferase is a bifunctional enzyme consisting of an N-terminal domain responsible for deadenylylation activity and a C-terminal domain responsible for adenylylation activity. Both domains are partially homologous to each other. Residues important for catalytic activity were present in the deduced amino acid sequence of the A. brasilense Sp245 glnE sequence. A glnE mutant was constructed in A. brasilense Sp7 by inserting a kanamycin resistance cassette between the two active domains of the enzyme. The resulting mutant was unable to adenylylate the glutamine synthetase enzyme and was impaired in growth when shifted from nitrogen-poor to nitrogen-rich medium.

Azospirillum-Cereals: An Intriguing Partnership

Azospirillum is probably the best studied example of beneficial plant rhizosphere bacteria. Studies in our laboratory focus on the identification of bacterial genes and gene products that are of importance in the physical and metabolic interaction of Azospirillum brasilense with plant roots. Here we report for Azospirillum brasilense, flagellation, motility, the physical interaction with plant roots, the synthesis of indole-3-acetic acid, the expression of nif genes in plant-root associated bacteria, and the induction of gene expression with plant root exudates.