Jozef Vanderleyden

PHYTOSTIMULATORY EFFECT OF AZOSPIRILLUM BRASILENSE WILD TYPE AND MUTANT STRAINS ALTERED IN IAA PRODUCTION ON WHEAT

Auxin production by Azospirillum is believed to play a major role in the observed plant growth promoting effect. By using different genetically modified strains, the contribution of auxin biosynthesis by A. brasilense in altering root morphology was evaluated in a plate assay. Inoculation with the wild type strains A. brasilense Sp245 and Sp7 resulted in a strong decrease in root length and increase in root hair formation. This effect was abolished when inoculating with an ipdC mutant of A. brasilense. The ipdC gene encodes a key enzyme in the IPyA pathway of IAA synthesis by A. brasilense. On the other hand, the observed auxin effect was further enhanced by adding tryptophan, a precursor of IAA, to the plates and could be mimicked by replacing the Azospirillum cells by a particular concentration of IAA. Furthermore, particular mutants (rpoN, scrp) and transconjugants (extra copy of ipdC) of A. brasilense were tested in the plate assay. Together, these results confirm the important role of IAA produced by Azospirillum in altering root morphology and illustrate the power of combining genetic tools and bioassays to elucidate the mechanism of a beneficial Azospirillum-plant interaction.

Rhizosphere Bacterial Signalling: A Love Parade Beneath Our Feet

Plant roots support the growth and activities of a wide variety of microorganisms that may have a profound effect on the growth and/or health of plants. Among these microorganisms, a high diversity of bacteria have been identified and categorized as deleterious, beneficial, or neutral with respect to the plant. The beneficial bacteria, termed plant growth-promoting rhizobacteria (PGPR), are widely studied by microbiologists and agronomists because of their potential in plant production. Azospirillum, a genus of versatile PGPR, is able to enhance the plant growth and yield of a wide range of economically important crops in different soils and climatic regions. Plant beneficial effects of Azospirillum have mainly been attributed to the production of phytohormones, nitrate reduction, and nitrogen fixation, which have been subject of extensive research throughout the years. These elaborate studies made Azospirillum one of the best-characterized genera of PGPR. However, the genetic and molecular determinants involved in the initial interaction between Azospirillum and plant roots are not yet fully understood. This review will mainly highlight the current knowledge on Azospirillum plant root interactions, in the context of preceding and ongoing research on the association between plants and plant growth-promoting rhizobacteria.

Auxin and Plant-Microbe Interactions

Microbial synthesis of the phytohormone auxin has been known for a long time. This property is best documented for bacteria that interact with plants because bacterial auxin can cause interference with the many plant developmental processes regulated by auxin. Auxin biosynthesis in bacteria can occur via multiple pathways as has been observed in plants. There is also increasing evidence that indole-3-acetic acid (IAA), the major naturally occurring auxin, is a signaling molecule in microorganisms because IAA affects gene expression in some microorganisms. Therefore, IAA can act as a reciprocal signaling molecule in microbe-plant interactions. Interest in microbial synthesis of auxin is also increasing in yet another recently discovered property of auxin in Arabidopsis. Down-regulation of auxin signaling is part of the plant defense system against phytopathogenic bacteria. Exogenous application of auxin, e.g., produced by the pathogen, enhances susceptibility to the bacterial pathogen.

The C‐terminal sequence conservation between OmpA‐related outer membrane proteins and MotB suggests a common function in both Gram‐positive and Gram‐negative bacteria, possibly in the interaction of these domains with peptidoglycan

The major outer membrane protein OprF from Pseudomonas species displays strong homology to several outer membrane proteins from unrelated species, including OmpA from enteric bacteria (De Mot et ai, 1992, Mol Gen Genef 231: 489-493). However, this homology is confined to the respective C-terminal regions (about 100-140 residues), and there is no obvious similarity between the A/-terminal regions. Such remarkable intergeneric sequence conservation presumably reflects a similar, but as yet unidentified, function of this domain. For both OmpA of Escherichia coii, and OprF of Pseudomonas aeruginosa, a structural role in stabilizing the outer membrane has been proposed (Gotoh et ai, 1989, J Bacterioi 171: 983-990; Woodruff and Hancock, 1989, J Bacterioi 171: 3304-3309). In addition, pore-forming activity has been demonstrated for both proteins (Nikaido et ai., 1991, J Bioi Chem 266: 770-779; Sugawara and Nikaido, 1992, J Bioi Chem 267: 2507-2511). The extended C-terminal homology is also found in lipoproteins that are tightly, but non-covalently bound to peptidogiycan. These peptidoglycan-associated lipoproteins (PALs) are important structural elements for the cell envelope (Lazzaroni and Portalier, 1992, Mol Microbiol 6: 735-742). The functions of the other outer membrane proteins in this family are poorly characterized. However, it is noteworthy that for several of them strong, non-covalent association with peptidogiycan has been described (Lugtenberg and van Alphen, 1983, Biochim Biophys Acta 737: 51-115; Hancock ef ai, 1990, Moi Microbioi 4: 1069-1075), although the protein domains Interacting with the peptidogiycan layer remain to be identified.

Synergistic Enhancement of the Antifungal Activity of Wheat and Barley Thionins by Radish and Oilseed Rape 2S Albumins and by Barley Trypsin Inhibitors.

Although thionins and 2S albumins are generally considered as storage proteins, both classes of seed proteins are known to inhibit the growth of pathogenic fungi. We have now found that the wheat (Triticum aestivum L.) or barley (Hordeum vulgare L.) thionin concentration required for 50% inhibition of fungal growth is lowered 2- to 73-fold when combined with 2S albumins (at sub- or noninhibitory concentrations) from radish (Raphanus sativus L.) or oilseed rape (Brassica napus L.). Furthermore, the thionin antifungal activity is synergistically enhanced (2- to 33-fold) by either the small subunit or the large subunit of the radish 2S albumins. Three other 2S albumin-like proteins, the barley trypsin inhibitor and two barley Bowman-Birk-type trypsin inhibitor isoforms, also act synergistically with the thionins (2- to 55-fold). The synergistic activity of thionins combined with 2S albumins is restricted to filamentous fungi and to some Gram-positive bacteria, whereas Gram-negative bacteria, yeast, cultured human cells, and erythrocytes do not show an increased sensitivity to thionin/albumin combinations (relative to the sensitivity to the thionins alone). Scanning electron microscopy and measurement of K+ leakage from fungal hyphae revealed that 2S albumins have the same mode of action as thionins, namely the permeabilization of the hyphal plasmalemma. Moreover, 2S albumins and thionins act synergistically in their ability to permeabilize fungal membranes.

The functions of Ca2+ in bacteria: a role for EF-hand proteins?

In bacteria, Ca(2+) is implicated in a wide variety of cellular processes, including the cell cycle and cell division. Dedicated influx and efflux systems tightly control the low cytoplasmic Ca(2+) levels in prokaryotes. Additionally, the growing number of proteins containing various Ca(2+)-binding motifs supports the importance of Ca(2+), which controls various protein functions by affecting protein stability, enzymatic activity or signal transduction. The existence of calmodulin-like proteins (containing EF-hand motifs) in bacteria is a long-standing hypothesis. Analysis of the prokaryotic protein sequences available in the databases has revealed the presence of several calmodulin-like proteins containing two or more authentic EF-hand motifs, suggesting that calmodulin-like proteins could be involved in Ca(2+) regulation in bacteria.

A metabolic node in action: chorismate-utilizing enzymes in microorganisms.

The shikimate pathway has been described as a metabolic tree with many branches that led to the synthesis of an extensive range of products. This pathway is present only in bacteria, fungi, and plants. While there is only little difference in the sequence of the chemical reactions of the pathway, significant differences exist in terms of organization and regulation. In the main trunk of the shikimate pathway, D-erythrose 4-phosphate and phosphoenolpyruvate are converted via shikimate to chorismate. Chorismate is the common precursor for the biosynthesis of the aromatic amino acids, phenylalanine, tyrosine, and tryptophan, but also for other products as diverse as folate cofactors, benzoid and naphthoid coenzymes, phenazines, and siderophores. Five chorismate-utilizing enzymes have been characterized in microorganisms: chorismate mutase, anthranilate synthase, aminodeoxychorismate synthase, isochorismate synthase, and chorismate pyruvate-lyase. In this review these enzymes are discussed in terms of the corresponding gene structures and regulation, nucleotide and protein sequences, protein structures, and reaction mechanisms. The main emphasis is on transcriptional and posttranslational regulatory mechanisms, in view of how a microbial cell exploits its chorismate pool in diverse anabolic pathways. Comparison of the chorismate-utilizing enzymes has shown that some of them share sequence similarity, suggesting divergent evolution and commonality in reaction mechanisms. However, other chorismate-utilizing enzymes are examples of convergent evolution toward similar reaction capabilities.

Glycoproteins in prokaryotes

Rather recently it has become clear that prokaryotes (Archaea and Bacteria) are able to glycosylate proteins. A literature survey revealed the different types of glycoproteins. They include mainly surface layer (S-layer) proteins, flagellins, and polysaccharide-degrading enzymes. Only in a few cases is structural information available. Many different structures have been observed that display much more variation than that observed in eukaryotes. A few studies have given evidence for the function of the prokaryotic glycoprotein glycans. Also from the biosynthetic point of view, information is rather scarce. Due to their different cell structure, prokaryotes have to use mechanisms different from those found in eukaryotes to glycosylate proteins. However, from the fragmented data available for the prokaryotic glycoproteins, similarities with the eukaryotic system can be noticed.

Unraveling the Secret Lives of Bacteria: Use of In Vivo Expression Technology and Differential Fluorescence Induction Promoter Traps as Tools for Exploring Niche-Specific Gene Expression

SUMMARY A major challenge for microbiologists is to elucidate the strategies deployed by microorganisms to adapt to and thrive in highly complex and dynamic environments. In vitro studies, including those monitoring genomewide changes, have proven their value, but they can, at best, mimic only a subset of the ensemble of abiotic and biotic stimuli that microorganisms experience in their natural habitats. The widely used gene-to-phenotype approach involves the identification of altered niche-related phenotypes on the basis of gene inactivation. However, many traits contributing to ecological performance that, upon inactivation, result in only subtle or difficult to score phenotypic changes are likely to be overlooked by this otherwise powerful approach. Based on the premise that many, if not most, of the corresponding genes will be induced or upregulated in the environment under study, ecologically significant genes can alternatively be traced using the promoter trap techniques differential fluorescence induction and in vivo expression technology (IVET). The potential and limitations are discussed for the different IVET selection strategies and system-specific variants thereof. Based on a compendium of genes that have emerged from these promoter-trapping studies, several functional groups have been distinguished, and their physiological relevance is illustrated with follow-up studies of selected genes. In addition to confirming results from largely complementary approaches such as signature-tagged mutagenesis, some unexpected parallels as well as distinguishing features of microbial phenotypic acclimation in diverse environmental niches have surfaced. On the other hand, by the identification of a large proportion of genes with unknown function, these promoter-trapping studies underscore how little we know about the secret lives of bacteria and other microorganisms.

Effects of Azospirillum brasilense indole-3-acetic acid production on inoculated wheat plants

The production of phytohormones by plant-growth promoting rhizobacteria is considered to be an important mechanism by which these bacteria promote plant growth. In this study the importance of indole-3-acetic acid (IAA) produced by Azospirillum brasilense Sp245 in the observed plant growth stimulation was investigated by using Sp245 strains genetically modified in IAA production. Firstly wild-type A. brasilense Sp245 and an ipdC knock-out mutant which produces only 10% of wild-type IAA levels (Vande Broek et al., J Bacteriol 181:1338–1342, 1999) were compared in a greenhouse inoculation experiment for a number of plant parameters, thereby clearly demonstrating the IAA effect in plant growth promotion. Secondly, the question was addressed whether altering expression of the ipdC gene, encoding the key enzyme for IAA biosynthesis in A. brasilense, could also contribute to plant growth promotion. For that purpose, the endogenous promoter of the ipdC gene was replaced by either a constitutive or a plant-inducible promoter and both constructs were introduced into the wild-type strain. Based on a greenhouse inoculation experiment it was found that the introduction of these recombinant ipdC constructs could further improve the plant-growth promoting effect of A. brasilense. These data support the possibility of constructing Azospirillum strains with better performance in plant growth promotion.