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.

Auxins were discovered early in the twentieth century as plant-regulating substances. Indole-3-acetic acid (IAA) is a naturally occurring auxin with broad physiological effects. Although many plant genes that are transcriptionally regulated by IAA have been characterized in recent years, our understanding of the auxin signal transduction pathway(s) in plants is still incomplete. IAA biosynthesis in plants can occur via different pathways 1 Bartel B. Auxin biosynthesis. Annu. Rev. Plant Physiol. Plant Mol. Biol. 1997; 48: 49-64 Crossref Scopus (269) Google Scholar , which are classified according to their intermediates: indole-3- acetamide (IAM), indole-3-pyruvate (IPyA), tryptamine, and indole-3-acetonitrile 2 Patten C.L. Glick B.R. Bacterial biosynthesis of indole-3-acetic acid. Can. J. Microbiol. 1996; 42: 207-220 Crossref PubMed Scopus (824) Google Scholar . To further our understanding of plant growth and development in nature, microbially released auxins, which can have a pronounced effect on plants, should be considered. The two most common routes for IAA biosynthesis in bacteria are the IAM and the IPyA pathways.

The trpBA gene cluster of Azospirillum brasilense Sp7 was isolated by complementation of an Escherichia coli trpBA mutant. Both genes code for the two subunits of tryptophan synthase, which catalyzes the last step in tryptophan biosynthesis. No structural features indicating transcriptional regulation could be identified. Upstream of the trpBA cluster an open reading frame encoding a putative periplasmic binding protein, involved in amino acid transport, was identified. Analysis of the downstream region of the trpBA cluster revealed the presence of a putative open reading frame encoding a subunit of the acetyl-coenzyme A carboxylase carboxyl transferase complex.

The trpBA ene cluster of Azospirillum brasilense Sp7 was isolated by complementation of an Escherichia coli trpBA mutant. Both genes code for the two subunits of tryptophan synthase, which catalyzes the last step in tryptophan biosynthesis. No structural features indicating transcriptional relation could be identified. Upstream of the trpBA cluster an open reading frame encoding a putative periplasmic binding protein, involve in amino acid transport, was identified. Analysis of the downstream region of the trpBA cluster revealed the presence of a utative open reading frame encodin a subunit of the acetl-coenzyme A carboxylase carboxyl transferase complex.

ABSTRACT Transcription of the Azospirillum brasilense ipdC gene, encoding an indole-3-pyruvate decarboxylase involved in the biosynthesis of indole-3-acetic acid (IAA), is induced by IAA as determined by ipdC-gusA expression studies and Northern analysis. Besides IAA, exogenously added synthetic auxins such as 1-naphthaleneacetic acid, 2,4-dichlorophenoxypropionic acid, and p -chlorophenoxyacetic acid were also found to upregulate ipdC expression. No upregulation was observed with tryptophan, acetic acid, or propionic acid or with the IAA conjugates IAA ethyl ester and IAA- l -phenylalanine, indicating structural specificity is required for ipdC induction. This is the first report describing the induction of a bacterial gene by auxin.

The gfp gene, encoding the green fluorescent protein, was combined with the gusA gene, coding for the β-glucuronidase enzyme, in mini-Tn5 transposon derivatives for use in Gram-negative bacteria. These mini-Tn5 elements allow simultaneously monitoring of gene expression and localization of the marked bacteria. Introduction of the resultant mini-Tn5 transposons into Rhizobium etli, Azospirillum brasilense and Pseudomonas stutzeri allowed us to visualise the interaction of these bacteria with their host plant. The dual-marker mini-Tn5 transposons constitute a powerful new tool for studying gene expression and ecology of bacteria in the environment and during the interaction with plants.

Sir, Auxins are a major class of plant growth regulators known to be involved in diverse processes at the whole plant level and at the cellular level. Regulation of these processes by auxin is thought to be the result of modified gene expression. A number of early auxin-induced plant genes have recently been cloned and characterized (Napier and Venis, 1995, New Phytol129: 167–201). A common short sequence element TGTCTC (or the degenerate version (G/T)GTCCCAT), termed auxin-responsive element (AuxRE), has been identified in the promoters of some of these auxin-regulated genes, including the soybean GH3 and SAUR 15A genes, and the pea PS-IAA4/5 gene (Abel et al., 1996, BioEssays18: 647–654; Guilfoyle et al., 1998, Plant Physiol118: 341–347). In naturally occurring auxin-responsive promoters, these AuxREs have been found to function with a coupling element overlapping with or adjacent to the TGTCTC half-site. Within these composite cis-acting elements, the coupling element confers tissue-specific or development-specific expression to the auxin-regulated promoter, and the TGTCTC half-site acts to repress this expression when auxin levels are low. Derepression then follows increasing auxin levels. Experiments with synthetic AuxREs have shown that direct repeats or palindromes of the conserved half-site are preferred in conferring high inducibility by auxin to a minimal promoter construct (Ulmasov et al., 1997, Plant Cell9: 1963–1971). A TGTCTC palindrome was subsequently identified in the pea PS-IAA4/5 promoter. The 'coupling model' of plant hormone response complexes, in which general transcriptional regulators are adjacent to sites that confer hormone inducibility, is valid for most plant hormones, e.g. gibberellin (Lanahan et al., 1992, Plant Cell4: 203–211), abscisic acid (Shen and Ho, 1995, Plant Cell7: 295–307) and ethylene (Mason et al., 1993, Plant Cell5: 241–251) (Fig. 1). The modular composition of hormone-inducible promoter elements is apparently conserved over the plant and animal kingdoms, as similarities between TGTCTC AuxREs and TGTTCT glucocorticoid or steroid hormone response elements (GREs or HREs) have been noted (Ulmasov et al., 1998, Science276: 1865–1868). Both contain the conserved response element and a second DNA binding site for an activator protein (Fig. 1). . Comparison of plant and animal hormone-responsive elements to the auxin-responsive ipdC sequence element of Azospirillum brasilense. HRE, hormone response element; OS2, opaque-2 DNA binding site; GARE, gibberellic acid response element; ABRE, abscisic acid response element; CE1, coupling element; ERE, ethylene response element; AP-1, activator protein-1. The biosynthesis of plant growth-regulating substances is not restricted to plants, and biosynthetic pathways have also been identified in many micro-organisms, such as fungi, phytopathogenic and plant growth-stimulating bacteria. Azospirillum brasilense, a plant growth-promoting rhizobacterium, produces gibberellins, cytokinins and the auxin indole-3-acetic acid (IAA). IAA biosynthesis in this bacterium occurs through different biosynthetic pathways (Prinsen et al., 1993, Mol. Plant-Microbe Interact6: 609–615), one of which is the indole-3-pyruvate (IPyA) pathway. In the IPyA pathway, the IPyA decarboxylase is responsible for the conversion of IPyA to indole-3-acetaldehyde, and the ipdC gene encoding this enzyme has been cloned and characterized in A. brasilense (Costacurta et al., 1994, Mol Gen Genet243: 463–472). Surprisingly, transcription of the A. brasilenseipdC gene, analysed by means of an ipdC promoter–gusA translational fusion and by Northern analysis, is specifically induced by IAA and by other compounds that are known as synthetic auxins in plant physiology (Vande Broek et al., 1999, J Bacteriol181: 1338–1342). Upstream of the ipdC gene, a DNA motif can be recognized: a consensus sequence for a σ54-dependent promoter (Fig. 1) that partially overlaps at the 3′ end with a TGTCCC element, reminiscent of the AuxREs in plants. This sequence could thus represent a new type of operator. On the basis of this observation, we propose that the combination of the coupling element (in this case the σ54 consensus sequence) and the TGTCCC element, similar to the modular build-up of hormone responsive promoters in plants and animals, is responsible for conferring auxin inducibility to the ipdC promoter. In plants, auxin response factors (ARFs) were discovered that bind to AuxREs with the consensus sequence TGTCNC. ARF1 was the first of those transcription factors to be identified in Arabidopsis (Ulmasov et al., 1998, ibid.). ARFs are thought to act as repressors or activators depending on the binding of other ARFs, the binding of transcriptional factors for the coupling elements or through interactions with another class of transcriptional regulators, the Aux/IAA proteins. All of these interactions could somehow be mediated by auxin. Auxin-binding proteins and transcription factors remain to be identified in A. brasilense. It has already been suggested that a repressor/activator may work by decreasing/increasing the concentration of RNA polymerase at a promoter capable of forming an open complex (Müller-Hill, 1998, Mol Microbiol29: 13–18). This strategy is called increase in local concentration or recruitment. It could well be that a new type of A. brasilense transcription factors, binding at the TGTCCC consensus site, is able to influence the formation of the RNA polymerase complex through synergistic recruitment of additional proteins for transcriptional activation. These interactions could be directly or indirectly mediated by auxin. If this hypothesis is confirmed experimentally, the molecular mechanism of conferring hormone inducibility to genes in eukaryotes might have originated in prokaryotes. The authors gratefully acknowledge Dr Rene De Mot for critical reading of the manuscript.

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.

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.

Regarding direct plant growth promotion, the ability of Azospirillum brasilense to produce indole-3-acetic acid (IAA) is now intensively studied. IAA biosynthesis in A. brasilense proved to be surprisingly complex, since at least three biosynthetic pathways are present (Prinsen et al, 1993). For one biosynthesis pathway, we have cloned and sequenced the A. brasilense ipdC gene, encoding the indole-3-pyruvic acid decarboxylase (Costacurta et al, 1994).

The genus Azospirillum comprises free-living N2 fixing rhizosphere bacteria that have been isolated from different soil types and from the roots of numerous wild and cultivated plants all over the world. Field trials, carried out at different locations, have demonstrated that under certain environmental and soil conditions, inoculation with Azospirillum has beneficial effects on plant yields. Bacterial phytohormone biosynthesis has often been proposed as being responsible for the observed plant growth promotion upon Azospirillum inoculation.