Azospirillum represents the best characterized genus of plant growth-promoting rhizobacteria. Other free-living diazotrophs repeatedly detected in association with plant roots, include Acetobacter diazotrophicus, Herbaspirillum seropedicae, Azoarcus spp. and Azotobacter. Four aspects of the Azospirillum–plant root interaction are highlighted: natural habitat, plant root interaction, nitrogen fixation and biosynthesis of plant growth hormones. Each of these aspects is dealt with in a comparative way. Azospirilla are predominantly surface-colonizing bacteria, whereas A. diazotrophicus, H. seropedicae and Azoarcus sp. are endophytic diazotrophs. The attachment of Azospirillum cells to plant roots occurs in two steps. The polar flagellum, of which the flagellin was shown to be a glycoprotein, mediates the adsorption step. An as yet unidentified surface polysaccharide is believed to be essential in the subsequent anchoring phase. In Azoarcus sp. the attachment process is mediated by type IV pili. Nitrogen fixation structural genes (nif) are highly conserved among all nitrogen-fixing bacteria, and in all diazotrophic species of the class of proteobacteria examined, the transcriptional activator NifA is required for expression of other nif genes in response to two major environmental signals (oxygen and fixed N). However, the mechanisms involved in this control can vary in different organisms. In Azospirillum brasilense and H. seropedicae (α- and β-subgroup, respectively), NifA is inactive in conditions of excess nitrogen. Activation of NifA upon removal of fixed N seems to involve, either directly or indirectly, the signal transduction protein PII. The presence of four conserved cysteine residues in the NifA protein might be an indication that NifA is directly sensitive to oxygen. In Azotobacter vinelandii (γ-subgroup) nifA is cotranscribed with a second gene nifL. The nifL gene product inactivates NifA in response to high oxygen tension and cellular nitrogen-status. NifL was found to be a redox-sensitive flavoprotein. The relief of NifL inhibition on NifA activity, in response to N-limitation, is suggested to involve a PII-like protein. Moreover, nitrogenase activity is regulated according to the intracellular nitrogen and O2 level. In A. brasilense and Azospirillum lipoferum posttranslational control of nitrogenase, in response to ammonium and anaerobiosis, involves ADP-ribosylation of the nitrogenase iron protein, mediated by the enzymes DraT and DraG. At least three pathways for indole-3-acetic acid (IAA) biosynthesis in A. brasilense exist: two Trp-dependent (the indole-3-pyruvic acid and presumably the indole-3-acetamide pathway) and one Trp-independent pathway. The occurrence of an IAA biosynthetic pathway not using Trp (tryptophan) as precursor is highly unusual in bacteria. Nevertheless, the indole-3-pyruvate decarboxylase encoding ipdC gene is crucial in the overall IAA biosynthesis in Azospirillum. A number of genes essential for Trp production have been isolated in A. brasilense, including trpE(G) which codes for anthranilate synthase, the key enzyme in Trp biosynthesis. The relevance of each of these four aspects for plant growth promotion by Azospirillum is discussed.

Azospirillum, a free-living nitrogen-fixing bacterium closely associated with grasses: genetic, biochemical and ecological aspects

The plant hormones, auxins and cytokinins, are involved in several stages of plant growth and development such as cell elongation, cell division, tissue differentiation, and apical dominance. The biosynthesis and the underlying mechanism of auxins and cytokinins action are subjects of intense investigation. Not only plants but also microorganisms can synthesize auxins and cytokinins. The role of phytohormone biosynthesis by microorganisms is not fully elucidated: in several cases of pathogenic fungi and bacteria these compounds are involved in pathogenesis on plants; auxin and cytokinin production may also be involved in root growth stimulation by beneficial bacteria and associative symbiosis. The genetic mechanism of auxin biosynthesis and regulation by Pseudomonas, Agrobacterium, Rhizobium, Bradyrhizobium, and Azospirillum, are well studied; in these bacteria several physiological effects have been correlated to the bacterial phytohormones biosynthesis. The pathogenic bacteria Pseudomonas and Agrobacterium produce indole-3-acetic acid via the indole-3-acetamide pathway, for which the genes are plasmid borne. However, they do possess also the indole-3-pyruvic acid pathway, which is chromosomally encoded. In addition, they have genes that can conjugate free auxins or hydrolyze conjugated forms of auxins and cytokinins. In Agrobacterium there are also several genes, located near the auxin and cytokinin biosynthetic genes, that are involved in the regulation of auxins and cytokinins sensibility of the transformed plant tissue. Symbiotic bacteria Rhizobium and Bradyrhizobium synthesize indole-3-acetic acid via indole-3-pyruvic acid; also the genetic determinants for the indole-3-acetamide pathway have been detected, but their activity has not been demonstrated. In the plant growth-promoting bacterium Azospirillum, as in Agrobacterium and Pseudomonas, both the indole-3-pyruvic acid and the indole-3-acetamide pathways are present, although in Azospirillum the indole-3-pyruvic acid pathway is of major significance. In addition, biochemical evidence for a tryptophan-independent indole-3-acetic acid pathway in Azospirillum has been presented.

Synthesis of phytohormones by plant-associated bacteria

Publisher Summary The chapter presents a discussion on plant growth-regulating (PGR) substances in the rhizosphere, with emphasis on microbial production and functions. The chapter discusses the rhizosphere as a site of plant-microbe interactions; plant growth-regulating substances and their sources; biochemistry of microbial production of PGRs; production of PGRs by rhizosphere microorganisms; metabolism of PGRs in soil; and ecological significance of PGRs produced in the rhizosphere. The chapter provides a better understanding of the mechanisms of actions of microbially derived PGRs and their interactions with plants. Moreover, development of hormone-deficient plant mutants can provide opportunities to clearly define the role of microbially produced PGRs in plan-microbe interactions. An understanding of these aspects can aid in the utilization of microbial PGRs for the betterment and benefit of sustainable agriculture.

Plant growth-regulating substances in the rhizosphere: microbial production and functions

Section A: Interaction of Bacterial Pathogens with Plants. Section B: Rhizobium--Plant Symbiotic Interactions. Section C: Interaction of Fungi with Plants. Section D: Isolation of Plant Disease Resistance Genes. Section E: Plant Responses to Pathogens and Resistance Mechanisms. Section F: Engineered Resistance to Plant Pathogens. Index.

Advances in molecular genetics of plant-microbe interactions : proceedings of the 7th International Symposium on the Molecular Plant-Microbe Interactions, Edinburgh, U.K., June 1994

Involvement of indole-3-acetic acid (IAA), produced by the microalgae-growth-promoting bacteria Azospirillum brasilens and A. lipoferum, in promoting growth of the microalga Chlorella vulgaris Beij. was studied. Four wildtype strains of Azospirillum and their IAA-deficient mutants were co-immobilized with C. vulgaris in alginate beads. Cultures were grown in synthetic growth medium supplemented with tryptophan. Growth promotion of microalgae and production of exogenous IAA by Azospirillum spp. were monitored. All wildtype Azospirillum spp. produced significant but varying amounts of IAA, while their mutant forms produced significantly less. The results demonstrated a significant growth promotion in Chlorella cultures when immobilized with the four wildtype strains of Azospirillum, while very low or no enhanced growth was induced by the four IAA-deficient mutants, compared to when C. vulgaris is immobilized alone. A complementation experiment, where an IAA-attenuated mutant (A. brasilense SpM7918) was supplemented with IAA produced by its parental wildtype strain (A. brasilense Sp6), restored growth promotion in the microalgae-mutant culture.

Involvement of indole-3-acetic acid produced by the growth-promoting bacterium azospirillum spp. in promoting growth of chlorella vulgaris(1).

Involvement of a 120-MDa plasmid of Azospirillum brasilense Sp245 in the production of lipopolysaccharides.

Indole acetic acid (IAA) production in Azospirillum brasilense strain Sp245 is controlled by a 85 MDa plasmid naturally in this bacterium. In the presence of l-tryptophan, anthranilic acid production and almost no IAA production occurs in a derivative strain harbouring a Tn5-Mob insertion in the 85 MDa plasmid. Agrobacterium tumefaciens strain GM19023, upon transfer of Tn5-Mob labelled 85 MDa plamid of A. brasilense Sp245, gains the ability to produce antranilic acid.

Involvement of Azopirillum brasilense plasmid DNA in the productio of indole acetic acid

Results of genetic analysis of three derivatives of Azospirillum brasilense Sp245 (strains BK570, SK051, and SK248) carrying cointegrates of plasmids 85-MDa and pJFF350 (the vector for omegon mutagenesis), which manifest abnormalities in flagellation and motility, are presented. It was shown for the first time that the integration of the suicide vector into one of Azospirillum resident plasmids is accompanied by the formation of various fusion products and changes in flagellation and motility of these bacteria, such as the loss of the polar (Fla) and lateral (Laf) flagella in SK051; inactivation of Fla and Laf in SK248; and Fla-dependent acceleration of expansion in semiliquid media in BK570.

Effect of the Integration of Vector pJFF350 into Plasmid 85-MDa of Azospirillum brasilense Sp245 on Bacterial Flagellation and Motility

An Azospirillum brasilense Sp245 spontaneous mutant, Sp245.5, lacking indigenous Plasmids of 85 (p85), 120 and 130 Mda produced about two times less indole-3-acetic acid (IAA) in the presence of tryptophan (Trp) than the wild type strain. Positive influence of p85-pSUP5011 cointegrate molecule transfer into A. brasilense Sp245.5 on IAA production in the transconjugants obtained was distinguishable only in suboptimal for IAA biosynthesis conditions. On the contrary, IAA production in Agrobacterium tumefaciens GMI9023 (p85-pSUP5011) increased several times in comparison with A. tumefaciens GMI9023 and was even greater than in the donor A. brasilense Sp245.160 mutant or in the wild type Sp245 strains. Transfer of p85-pSUP5011 into Pseudomonas putida 53a resulted in increase of IAA excretion and decrease of anthranilic acid (Ant) release. A suggestion about the dependence of an hypothetical IAA pathway probably encoded by p85 on the presence of unidentified products of Trp breakdown through Ant or of Ant itself is given.

Genetical aspects of indole acetate production in Azospirillum brasilense Sp245

The sequencing data were analyzed for two regions of the 120-MDa plasmid (p120) ofAzospirillum brasilense Sp245. The 2420-bp region I, which flanks the omegon insertion in the SK048 mutant defective in production of the polar flagellum (Fla–) and swarming (Swa–), was shown to contain a cluster of two open reading frames (orf) that possess properties of coding sequences (CDSs). The NtrA (σ54) boxes were found in their upstream regions. The products of orf1 and orf2 are 16.5 and 15.5 kDa in molecular weight and consist of 151 and 152 amino-acid residues, respectively. The ORF1 polypeptide was found to contain a region homologous to the cysteine- and glycine-rich zinc-binding domain of the DnaJ heat-shock protein. ORF2 showed a homology to Haemophilus ducreyi pilin, fragments of Streptomycesand Mycobacterium integral membrane proteins, and eukaryotic transcriptional regulators. The omegon proved to be inserted into orfX1/X2 which possibly has a deletion and shows a GC content untypical forA. brasilense genes. The deduced ORFX2 polypeptide is homologous to fragments of arsenite-translocating ATPase and signal-transducing histidine kinase of archaebacteria. Possible causes of the Fla– Swa–phenotype of the A. brasilense SK048 mutant are considered. One coding orf was identified in the 1194-bp region II located approximately 4 kb away from the omegon insertion. The N-terminal region of the deduced product of this partly sequenced orf was shown to contain a signal sequence typical for secreted proteins.

E. I. Katzy

Papers

Involvement of a 120-MDa plasmid of Azospirillum brasilense Sp245 in the production of lipopolysaccharides.

Involvement of Azopirillum brasilense plasmid DNA in the productio of indole acetic acid

Effect of the Integration of Vector pJFF350 into Plasmid 85-MDa of Azospirillum brasilense Sp245 on Bacterial Flagellation and Motility

Genetical aspects of indole acetate production in Azospirillum brasilense Sp245

Characterization of Genes Identified in the 120-MDa Plasmid of an Azospirillum brasilense Sp245 Mutant Defective in Polar Flagellation and Swarming